Rep 268
Rep 268
Rep 268
FACULTAD DE QUÍMICA
OPTIMIZACIÓN DE METODOLOGÍAS
ANALÍTICAS PARA LA DETERMINACIÓN DE
CONTAMINANTES EMERGENTES EN AGUAS
DE ABASTECIMIENTO Y RESIDUALES
TESIS DOCTORAL
FACULTAD DE QUÍMICA
OPTIMIZACIÓN DE METODOLOGÍAS
ANALÍTICAS PARA LA DETERMINACIÓN DE
CONTAMINANTES EMERGENTES EN AGUAS
DE ABASTECIMIENTO Y RESIDUALES
Autorizan:
A todos los amigos que desde el inicio me acogieron y apoyaron, muchas gracias (Jorge,
Noelia, María F., Néstor, Iria, Paula, Rebe, Pili, ….). A los compañeros, colegas y
amigos que en la distancia me apoyaron (Judith, Cati, Víctor, Eloina, Alma, Soledad,
Inés, Richard, Pepe, José Antonio, Alejandro,…), gracias.
Mi más sincero agradecimiento a todos los amigos y amigas que siempre me motivaron y
colaboraron para lograr mi objetivo. Y también, a los que ya no están.
Gracias, Compostela por tus piedras mojadas y tu siempre verde para contemplar.
Agradezco a mis padres, su inmenso apoyo desde el inicio de esta interminable odisea de
aprender.
A Sandy, mi esposa,
Por su amor
Por su cariño
Por su existencia
Por su incondicionalidad
Por estar siempre conmigo
Por ser y estar ahora y aquí
Por contar siempre con su apoyo, ánimo y coraje
"Il faut faire de la vie un rêve et faire d'un rêve une réalité"
Pierre Curie
Índice
Indice
Índice
Resumen.…………...………………………………………………………..... I
Resumo………….………………………………………………………..….... III
Abstract………………………..………………………………………..……… V
I Justificación y Objetivos…...………………………………………....…... 3
Bibliografía……………………..……………….…….……………….…........ 6
II Introducción…………………………………………..…………………..….. 9
1 Contextualización………………………………………………………... 9
Bibliografía……………………..……………….…….……………….…... 13
2 Contaminantes orgánicos emergentes……………………….……... 14
Bibliografía……………………..……………….…………….…….……... 18
3 Fragancias Alérgenas…………………………………………….…….. 20
3.1 Clasificación de las fragancias alérgenas……………................. 24
3.2 Aplicaciones…………………………………………………………... 27
3.3 Uso potencial como pesticidas……………………………………... 27
3.4 Exposición de las fragancias alérgenas en los seres humanos… 28
3.5 Distribución……………………………………………….…............. 31
3.6 Evaluación de la alergenicidad de las fragancias….……………. 33
3.7 Toxicidad…………………………………………………….............. 43
Bibliografía……………………..……………………………………..……. 54
4 Contaminantes con elevado volumen de producción………..…… 67
Bibliografía…………………………………………………………….…… 76
5 Técnicas analíticas aplicadas a la determinación de
contaminantes emergentes en agua…………………….……….…... 80
5.1 Extracción en fase líquida………………………………….…...….. 81
5.2 Extracción en fase sólida……………………….…….………...….. 82
5.3 Microextracción en fase sólida……………………….……...…….. 83
5.4 Microextracción emulsificación asistida por ultrasonidos ……… 87
Bibliografía…………………………………………………….…………… 97
Indice
Indice
Indice
Resumen
________________________________________________________________
I
de los casos. Los procedimientos se aplicaron a muestras acuosas de
diversos origenes.
II
Resumo
________________________________________________________________
III
casos. Os procedementos aplicáronse a mostras acuosas de diversas
procedencias.
IV
Abstract
Water sources have been used to supply the populations with water for
human consumption, as well as to eliminate their own waste, which over
time has caused that the amount and type of wastes released into water
courses have increased and diversified, with the consequent
deterioration of the original quality of the water resource. Currently, there
is a growing interest in "emerging contaminants" such as
pharmaceuticals and personal care products, surfactants, industrial
additives, pesticides, steroids and hormones, as well as disinfection by-
products. It has been shown that these compounds incorporate the
hydrological cycle, due to their persistence. These contaminants are, in
most cases, unregulated pollutants, which may be candidates for future
regulation depending on research on their potential effects on health and
the monitoring data with respect to their impact on the environment.
Many of these contaminants are not removed through conventional
systems of wastewater treatment and water potabilisation.
________________________________________________________________
V
During the development and implementation of the methodologies,
principles of the "green analytical chemistry” were intended to be
enforced and sustainable analytical procedures for their implementation
to the context of emerging countries as Mexico are proposed.
VI
I. Justificación y Objetivos
Justificación y Objetivos
I. Justificación y Objetivos
3
Justificación y Objetivos
4
Justificación y Objetivos
5
Justificación y Objetivos
Bibliografía
6
II. Introducción
Introducción
II. Introducción
1. Contextualización
9
Introducción
10
Introducción
11
Introducción
12
Introducción
Bibliografía
13
Introducción
14
Introducción
Clases de
Ejemplos
compuestos
Fármacos Paracetamol, carbamazepina, diazepam, iopamidol
Esteroides y
Estradiol, estrona, estriol, dietistilbestrol
hormonas
Drogas de abuso Anfetamina, cocaína, tetrahidrocanabinol
15
Introducción
16
Introducción
así que es difícil predecir qué efectos en la salud pueden tener en los
seres humanos y los organismos acuáticos (Barceló, 2003).
17
Introducción
Bibliografía
18
Introducción
Pal A., Gin K. Y., Lin A. Y., Reinhard M. (2010) Impacts of emerging
organic contaminants on freshwater resources: Review of recent
occurrences, sources, fate and effects, Sci Total Environ, 408 (24),
6062-6069.
Peck A. M. (2006) Analytical methods for the determination of persistent
ingredients of personal care products in environmental matrices, Anal
Bioanal Chem, 386, 907-939.
Slack R. J., Gronow J. R., Hall D. H., Voulvoulis N. (2007) Household
hazardous waste disposal to landfill: Using LandSim to model leachate
migration, Environ Pollut, 146 (2) 501-509.
Stackelberg, P. E., Furlong E. T., Meyer, M. T., Zaugg, S. D.,
Henderson A. K., Reissman D. B. (2004) Persistence of pharmaceutical
compounds and other organic wastewater contaminants in a
conventional drinking-water-treatment plant, Sci Total Environ, 329 (1-
3), 99-113.
Ternes T.A., Joss A., Siegrist H. (2004) Scrutinizing pharmaceuticals
and personal care products in wastewater treatment, Environ Sci
Technol, 38, 392A–399A.
von der Ohe P. C., Dulio V., Slobodnik J., De Deckere E., Kühne R.,
Ralf-Uwe E., Ginebreda A., De Cooman W., Schüürmann G., Brack W.
(2011) A new risk assessment approach for the prioritization of 500
classical and emerging organic microcontaminants as potential river
basin specific pollutants under the European Water Framework
Directive, Sci Total Environ, 409 (11), 2064-2077.
19
Introducción
3. Fragancias Alérgenas
20
Introducción
21
Introducción
22
Introducción
23
Introducción
Geraniol Linalol
24
Introducción
Hidroxi-citronelal Hexil cinamaldehído Į-isometilionona
® ®
Lilial Lyral
Esteres y lactonas:
25
Introducción
Fenoles y Éteres:
OH
Hidrocarburos cíclicos:
Limoneno y pineno
(d)-Limoneno Į-Pineno
26
Introducción
3.2 Aplicaciones
27
Introducción
28
Introducción
29
Introducción
30
Introducción
3.5 Distribución
31
Introducción
32
Introducción
33
Introducción
34
Introducción
35
Tabla II.4. Descripción, clasificación, restricciones, producción y uso de las fragancias alérgenas.
Nombre del Clasificación Grupo Potencial Valores Restricciones de acuerdo Clasificación y Volumen de Uso principal
compuesto/ por SCCNFP alérgeno alérgeno umbral para con la Directiva de etiquetado (6)/datos producción
número CAS (1) (Schnuch) (2) (IKW/IVK) (3) humanos Cosméticos (5) sobre ecotoxicidad(7) HPVC(Alto)/
ȝg/cm2 (4) LPVC (Reducido) (8)
Amil cinamal Lista A III (0.1) Muy débil 23622 – Irrita los ojos, las vías R Jabón, productos de
122-40-7 respiratorias y la piel; limpieza
Posibilidad de
sensibilización en contacto
con la piel; WGK2
Alcohol bencílico Lista A III (0.3) Poco frecuente sin datos Uso autorizado en Nocivo por inhalación y A Conservante,
100-51-6 disolventes, perfumes y por ingestión; WJK1; disolvente
aromatizantes en una Peces, Daphnia, Algas,
concentración máxima del Bacterias
1% en el producto terminado
Cinamil alcohol Lista A II (0.6) No es muy 4724 Nivel máximo de 0,8% en el Nocivo por ingestión: R Cosméticos,
104-54-1 potente producto terminado Posibilidad de detergentes para
sensibilización en contacto ropa, jabón
con la piel Irrita los ojos,
las vías respiratorias y la
piel; WGK2
Citral Lista A II (0.6) Muy débil 775 Sólo en combinación con el Irrita la piel; Posibilidad de A Cosméticos,
5392-40-5 25% de su peso de D- sensibilización en contacto saborizante en
limoneno, o mezclado con con la piel; WGK1; Peces, alimentos
terpenos cítricos o alfa- Daphnia, Algas, Bacterias
pineno.
Eugenol Lista A II (0.4) Muy débil 5905 – Nocivo por ingestión; Irrita R Pasta de dientes,
97-53-0 los ojos, las vías enjuague bucal,
respiratorias y la piel; perfume, jabón,
Posibilidad de antisépticos
sensibilización por
inhalación y contacto con
la piel; WGK1;
Peces
Hidroxicitronelal Lista A I (1.3) No es muy 2953 Nivel máximo de 1% en el Irrita la piel; Riesgo de R Cosméticos,
107-75-5 potente producto terminado lesiones oculares graves; perfumes, jabones,
WGK1 insecticidas,
Daphnia, Algas antisépticos
Nombre del Clasificación Grupo Potencial Valores Restricciones de acuerdo Clasificación y Volumen de Uso principal
compuesto/ por SCCNFP alérgeno alérgeno umbral para con la Directiva de etiquetado (6)/datos producción
número CAS (1) (Schnuch) (2) (IKW/IVK) (3) humanos Cosméticos (5) sobre ecotoxicidad(7) HPVC(Alto)/
ȝg/cm2 (4) LPVC (Reducido) (8)
Isoeugenol Lista A I (1.1) Potente 250 Nivel máximo de 0.02% en Nocivo por ingestión; Irrita R Aceites esenciales,
97-54-1 el producto terminado los ojos, vías respiratorias perfumes, cosméticos,
y la piel; Posibilidad de jabones, detergentes
sensibilización en contacto
con la piel; WGK2;
Daphnia
Alcohol cinamílico Lista A II (0.3) Poco frecuente 23622 – Irrita los ojos; WGK3 n.d. Perfumes, jabones,
101-85-9 — cosméticos
Salicilato de Lista A III (0.1) Poco frecuente sin datos – Irrita los ojos, las vías A Cosméticos, aceites
bencilo respiratorias y la piel; esenciales
118-58-1 WGK2
—
Cinamaldehído Lista A I (1.0) Potente 591 Sólo en combinación con el Irrita los ojos, las vías A Cosméticos
104-55-2 50% de su peso de eugenol respiratorias y la piel;
o d-limoneno u otras Posibilidad de
sustancias para prevención sensibilización en contacto
de la sensibilización con la piel; WGK1;
peces
Cumarina Lista A II (0.4) Poco frecuente sin datos – Nocivo por inhalación, en R Cosméticos,
91-64-5 contacto con la piel y por perfumes, pesticidas
ingestión; Irrita los ojos,
las vías respiratorias y la
piel; WGK1;
Peces, Daphnia
Nombre del Clasificación Grupo Potencial Valores Restricciones de acuerdo Clasificación y Volumen de Uso principal
compuesto/ por SCCNFP alérgeno alérgeno umbral para con la Directiva de etiquetado (6)/datos producción
número CAS (1) (Schnuch) (2) (IKW/IVK) (3) humanos Cosméticos (5) sobre ecotoxicidad(7) HPVC(Alto)/
ȝg/cm2 (4) LPVC (Reducido) (8)
Geraniol Lista A II (0.4) Poco frecuente 3875 – Irrita los ojos, las vías A Perfumes, cuidado de
106-24-1 respiratorias y la piel la piel
Posibilidad de
sensibilización en contacto
con la piel; Tóxico para los
organismos acuáticos,
puede provocar a largo
plazo efectos negativos en
el medio ambiente
acuático; Irrita los ojos,
las vías respiratorias y la
piel; WGK1;
Peces
Farnesol Lista B I (0.9) Muy débil 2755 Como mínimo 96 % de WGK2 n.d. Desodorantes,
4602-84-0 pureza Peces, Daphnia
Nombre del Clasificación Grupo Potencial Valores Restricciones de acuerdo Clasificación y Volumen de Uso principal
compuesto/ por SCCNFP alérgeno alérgeno umbral para con la Directiva de etiquetado (6)/datos producción
número CAS (1) (Schnuch) (2) (IKW/IVK) (3) humanos Cosméticos (5) sobre ecotoxicidad(7) HPVC(Alto)/
ȝg/cm2 (4) LPVC (Reducido) (8)
Lilial® Lista B II (0.3) Muy débil 29525 – Nocivo por ingestión; Irrita A Cosméticos,
80-54-6 la piel; Tóxico para los productos de lavado
organismos acuáticos, (colada)
puede provocar a largo
plazo efectos negativos en
el medio ambiente
acuático; WGK2
Peces, Daphnia, Bacterias
Linalool Lista B III (0.2) Poco frecuente 13793 – Irrita los ojos, las vías A Perfume
78-70-6 respiratorias y la piel;
WGK1
Peces, Daphnia, Algas,
Bacterias
Benzoato de Lista B III (0.0) Poco frecuente 20690 Concentración máxima Nocivo por ingestión; A insecticida
bencilo autorizada 0.5% (calculada WGK2 antiparasitario,
120-51-4 como ácido benzoico) en el Peces, Daphnia, Bacterias perfumes, plastificante
producto terminado
Citronelol Lista B II (0.5) Poco frecuente sin datos – Posibilidad de A Aceites esenciales,
106-22-9 sensibilización en contacto desodorantes, aerosol
con la piel; Tóxico para los para mosquitos
organismos acuáticos,
puede provocar a largo
plazo efectos negativos en
el medio ambiente
acuático; Irrita los ojos,
las vías respiratorias y la
piel; WGK1;
Peces, Daphnia, Algas,
Bacterias
Hexilcinamaldehído Lista B III (0.1) Poco frecuente 23622 – Irrita los ojos, las vías A Aceites esenciales
101-86-0 respiratorias y la piel;
WGK2
Provoca quemaduras
—
Nombre del Clasificación Grupo Potencial Valores Restricciones de acuerdo Clasificación y Volumen de Uso principal
compuesto/ por SCCNFP alérgeno alérgeno umbral para con la Directiva de etiquetado (6)/datos producción
número CAS (1) (Schnuch) (2) (IKW/IVK) (3) humanos Cosméticos (5) sobre ecotoxicidad(7) HPVC(Alto)/
ȝg/cm2 (4) LPVC (Reducido) (8)
Limoneno Lista B III (0.1) sin datos sin datos Para ser utilizado Inflamable; Irrita la piel; A Cosméticos,
5989-27-5 conjuntamente con Posibilidad de aromatizantes,
antioxidantes, el límite sensibilización en contacto insecticidas botánicos,
máximo del contenido de con la piel; Tóxico para los productos de limpieza
peróxidos es 20 mmoles/litro organismos acuáticos,
puede provocar a largo
plazo efectos negativos en
el medio ambiente
acuático; WGK2;
Peces, Daphnia
Octanoato de 2- Lista B III (0.2) Muy débil sin datos – Nocivo por ingestión; Irrita n.d. Productos de lavado
metilo los ojos, las vías (colada)
111-12-6 respiratorias y la piel;
WGK1
Į-Isometil ionona Lista B III (0.1) Poco frecuente 70866 – Nocivo por inhalación, en n.d. Cosméticos, perfumes
127-51-5 contacto con la piel y por
ingestión;
Metileugenol III n.d Saborizante, trampas
93-15-2
para insectos
(Adaptada de Klaschka, 2010)
(n.d. = no hay datos)
(-) No hay datos experimentales disponibles de las pruebas estándar
(1) Clasificación por SCCNFP. Lista A: fragancias, que de acuerdo a los conocimientos existentes son comunes y bien conocidos como alérgenos. Lista B: fragancias, que son menos
frecuentes y por lo tanto menos documentados como alérgenos (SCCNFP, 1999).
(2) Grupos de alérgenos (Schnuch et al., 2007) Grupo I: alérgenos importantes. Grupo II: Claramente alérgenos, pero menos importante en términos de frecuencia de sensibilización. Grupo III:
(Muy) poco frecuentes sensibilizadores o incluso no son sensibilizadores. () Frecuencias de sensibilización.
(3) Clasificación del potencial alérgeno de la IKW (German Cosmetic, Toiletry, Perfumery and Detergent Association): potente, no muy potente, débil, muy débil, poco frecuente (Pleschka, 2007).
(4) Umbrales Humanos de sensibilización para alérgenos obtenida por la combinación de datos de bases de datos de los umbrales humanos y ensayos de ganglios linfáticos EC3 (en ȝg/cm2)
(Basketter et al., 2008; Wijnhoven et al., 2008).
(5) Algunas fragancias están sujetas a restricciones de acuerdo con la Directiva Cosmética (EEC, 1976; SCCNFP, 2000).
(6) Clasificación y etiquetado. Comentarios sobre riesgos de acuerdo con la Directiva 67/548/EEC (EEC, 1967) como se indica en las Hojas de Seguridad de los productores.
(7) Datos ecotoxicológicos disponibles para: peces, Daphnia, algas y bacterias son datos experimentales de pruebas estándar disponbles. Clasificación de Riesgos para el Agua WGK (Water
Hazard Classification) de acuerdo con German Administrative Regulation on the Classification of Substances Hazardous to Waters into Water Hazard Classes (Anon, 2005).
(8) Productos químicos de alto volumen de producción (HPVC, más de 1000 toneladas por año en la UE); sustancias químicas de bajo volumen de producción (LPVC, menos de 1000 toneladas
por año en la UE), de acuerdo con los datos de European Chemicals Bureau.
Introducción
Efectos alérgenicos
41
Introducción
42
Introducción
3.7 Toxicidad
43
Introducción
bencilo pteronyssinus
-2 Acaro/Dermatophagoides Wongkamchai et al.,
50 mg m
pteronyssinus 2005
Salicilato de bencilo 2230 mg/Kg Rata RIFM, 1970
44
Introducción
45
Introducción
46
Introducción
47
Introducción
48
Introducción
49
Introducción
50
Introducción
51
Introducción
52
Introducción
Efectos fototóxicos
53
Introducción
Bibliografía
Abdo K.M., Cunningham M. L., Snell M. L., Herbert R. A., Travlos G. S.,
Eldridge S.R., Bucher J. R. (2001) 14-week toxicity and cell proliferation
of methyleugenol administered by gavage to F344 rats and B6C3F1
mice, Food Chem Toxicol, 39, 303-316.
Abramovici A., Wolf R., Sandbank M. (1983) Sebaceous glands
changes following topical application of citral, Acta Derm Venereol,
63(5), 428-31.
Adany I., Yazlovitskaya E. M., Haug J. S., Voziyan P. A., Melnykovych
G. (1994) Differences in sensitivity to farnesol toxicity between
neoplastically- and non-neoplastically-derived cells in culture, Cancer
Lett, 79(2), 175-9.
Andrejak M., Gersberg M., Sgro C., Decocq G., Hamel J. D., Morin M.,
Gras V. (1998) French pharmacovigilance survey evaluating the hepatic
toxicity of coumarin, Pharmacoepidem Dr S, 7 (Suppl. 1, European
Society of Pharmacovigilance, Annual Meeting, 1997).
Anon (2005) The Administrative Regulation on the Classification of
Substances Hazardous to Waters into Water Hazard Classes
(Verwaltungsvorschrift wassergefährdende Stoffe–VwVwS.
Antignac E., Nohynek G. J., Re T., Clouzeau J., Toutain H. (2011)
Safety of botanical ingredients in personal care products/cosmetics,
Food Chem Toxicol, 49 (2), 324-341.
Babich H., Stern A., Borenfreund E. (1993) Eugenol cytotoxicity
evaluated with continuous cell lines, Toxicol in Vitro, 7(2), 105-9.
Bakkali F., Averbeck S., Averbeck D., Idaomar M. (2008) Biological
effects of essential oils – A review, Food Chem Toxicol, 46 (2), 446-
475.
BASF (2008) Safety data sheet, Hydroxycitronellal, pp4.
http://www.basf.co.kr/02_products/04_finechemicals/cosmetics/data/MS
DS_%20Hydroxycitronellal_EN_20040603.pdf (consultada el 25 de
noviembre de 2011)
Basketter, D., Gerberick, F., Kimber I. (2008). Appendix A: LLNA/EC3
Validation–Submission ICCVAM Draft Background Review Document
Use of the Murine Local Lymph Node Assay (LLNA) to Determine Skin
Sensitization Potency Categories.
Benda G. I., Hiller J. L., Reynolds J. W. (1986) Benzyl alcohol toxicity:
impact on neurologic handicaps among surviving very low birth weight
infants, Pediatrics, 77(4), 507-12.
54
Introducción
Bickers D., Calow P., Greim H., Hanifin J.M, Rogers A.E., Saurat J.H.,
Sipes I.G., Smith R.L., Tagami H. (2005) A toxicologic and dermatologic
assessment of cinnamyl alcohol, cinnamaldehyde and cinnamic acid
when used as fragrance ingredients: The RIFM expert panel, Food
Chem Toxicol, 43 (6), 799-836.
Bickers D.R., Calow P., Greim H. A., Hanifin J. M., Rogers A. E., Saurat
J. H., Sipes I. G., Smith R. L., Tagami H. (2003) The safety assessment
of fragrance materials, Regul Toxicol Pharm, 37, 218–273.
Bidevkina M. V., Guglya E. B., Ivanov N. G., Migukina N. V. (2003)
Isoeugenol [1-hydroxy-2-methoxy-4-(prop-1yl)benzene]- mixture of cis-
and trans-isomers, Toksikologicheskii Vestnik, (6), 51-52.
Born S. L., Caudill D., Smith B. J., Lehman-McKeeman L. D. (2000) In
vitro kinetics of coumarin 3,4-epoxidation: application to species
differences in toxicity and carcinogenicity, Toxicol Sci, 58(1), 23-31.
Born S. L., Fix A. S., Caudill D., Lehman-McKeeman L. D. (1998)
Selective Clara cell injury in mouse lung following acute administration
of coumarin, Toxicol Appl Pharm, 151(1), 45-56.
Boverhof D. R., Wiescinski C. M., Botham P., Lees D., Debruyne E.,
Repetto-Larsay M., Ladics G., Hoban D., Gamer A., Remmele M.,
Wang-Fan W., Ullmann L.G., Mehta J., Billington R., Woolhiser M.R.
(2008) Interlaboratory validation of 1% pluronic l92 surfactant as a
suitable, aqueous vehicle for testing pesticide formulations using the
murine local lymph node assay, Toxicol Sci, 105(1), 79-85.
Buckley D.A. (2007) Fragrance ingredient labelling in products on sale
in the U.K., Brit J Dermatol, 157, 295-300.
Carlton B. D., Aubrun J. C., Simon G. S. (1996) Effects of coumarin
following perinatal and chronic exposure in Sprague-Dawley rats and
CD-1 mice, Fundam Appl Toxicol, 30(1), 145-51.
Catanzaro I., Avellone G., Marci G., Saverini M., Scalici L., Sciandrello
G., Palmisano L. (2011) Biological effects and photodegradation by
TiO2 of terpenes present in industrial wastewater, J Hazard Mater,
185(2-3), 591-7.
Caujolle F., Meynier D. (1960) The toxicity of methyl eugenol, the
methyl isoeugenols, and of methyl dihydroeugenol, Compt Rend, 250,
1148-9.
Chipinda I., Anderson S. E., Butterworth L. F., Beezhold D., Siegel P. D.
(2009) Increased cell proliferation in spleen and lymph nodes peripheral
to contact allergen application site, Toxicology, 257(3), 113-6.
55
Introducción
56
Introducción
57
Introducción
George J. D., Price C. J., Marr M. C., Myers C. B., Jahnke G. D. (2001)
Evaluation of the developmental toxicity of isoeugenol in Sprague-
Dawley (CD) rats, Toxicol Sci, 60(1), 112-120.
Gerberick G. F., Cruse L. W., Miller C. M., Sikorski E. E., Ridder G. M.
(1997) Selective modulation of T cell memory markers CD62L and
CD44 on murine draining lymph node cells following allergen and irritant
treatment, Toxicol Appl Pharmacol, 146(1), 1-10.
Gerberick G. F., Cruse L. W., Ryan C. A., Hulette B. C., Chaney J. G.,
Skinner R. A., Dearman R. J., Kimber I. (2002) Use of a B cell marker
(B220) to discriminate between allergens and irritants in the local lymph
node assay, Toxicol Sci, 68(2), 420-8.
Ghimire B., Ramaswamy K., Pasha A. (2011) Insecticidal and fungicidal
activity of phosphorothioates of eugenol, J Pure Appl Microbiol, 5(1),
307-311.
Gilpin S., Maibach H. (2010) Allergic contact dermatitis caused by
farnesol: clinical relevance, Cutan Ocul Toxicol, 29(4), 278-287.
Giordano-Labadie F. (2011) Composants parfumés. Nouvelles sources
d’exposition aux allergènes de la batterie standard Fragrances, Rev Fr
Allergol, 51 (3), 306-309.
Gladden J. N., Brainard B. M., Shelton J. L., Camus A. C., Divers S. J.
(2010) Evaluation of isoeugenol for anesthesia in koi carp (Cyprinus
carpio), Am J Vet Res, 71(8), 859-866.
Gonzalez-Audino P., Picollo M. I., Gallardo A., Toloza A., Vassena C.,
Mougabure-Cueto G. (2011) Comparative toxicity of oxygenated
monoterpenoids in experimental hydroalcoholic lotions to
permethrinresistant adult head lice, Arch Dermatol Res, 303(5), 361-
366.
Goulet F., Vachon P., Helie P. (2011) Evaluation of the toxicity of
eugenol at anesthetic doses in African clawed frogs (Xenopus laevis),
Toxicolc Pathol, 39(3), 471-477.
Gowder S., Devaraj H. (2008) Food flavor cinnamaldehyde-induced
biochemical and histological changes in the kidney of male albino wistar
rat, Environ Toxicol Phar, 26(1), 68-74.
Gowder S., Devaraj H. (2010) A review of the nephrotoxicity of the food
flavor cinnamaldehyde, Curr Bioact Compd, 6(2), 106-117.
Griffiths, D. E. (2005) Psi-Screen, an in vitro toxicity test system:
Applications in the bioassay of perfumes and fragrance chemicals,
Altern Lab Anim, 33(5), 471-486.
58
Introducción
Groux, H., Cottrez, F. (2011) Gene expression markers and their use in
evaluating the sensitizing or irritant potential of a compound, Fr.
Demande, FR 2957089 A1 20110909.
Haugaard J. (1995) Environmentally hazardous substances. D-
Limonene, Dansk Kemi, 76(4), 7. Edited By Beek, Bernd.
Hierro I., Valero A., Diaz V., Gonzalez J. M., Navarro M. C. (2004)
Larvicide activity of geraniol (monoterpenic derivate) against L3 larvae
of Anisakis simplex s.l Multidisciplinarity for Parasites, Vectors and
Parasitic Diseases, Proceedings of the European Multicolloquium of
Parasitology, 9th, Valencia, Spain, July 18-23, 2004, 2, 263-266. Edited
By:Mas-Coma, Santiago.
Hiller J. L., Benda G. I., Rahatzad M., Allen J. R., Culver D. H., Carlson
C. V., Reynolds J. W. (1986) Benzyl alcohol toxicity: impact on mortality
and intraventricular hemorrhage among very low birth weight infants,
Pediatrics, 77(4), 500-6.
Hogg C.L., Svoboda K.P., Hampson J.B., Brocklehurst S. (2001)
Investigation into the composition and bioactivity of essential oil from
lovage (Levisticum officinale W.D.J. Koch), Int J Aromather, 11, 144–
151.
Huchel U., Bunn R., Materne M., Faber W., Smyrek H., Pierik T. T.,
Rittler F, Bauer A, Dischmann M., Sauf S. et al (2010) Lilial substitutes
for fragrances, Ger. Offen., DE 102009001570 A1 20100923.
IFF (2011) Material Safety Data Sheet, INTERNATIONAL FLAVORS &
FRAGRANCES, 7.
IFRA (2011) International Fragrance Association
< http://www.ifraorg.org/ >
Jenner, P. M., Hagan, E. C., Taylor J. M., Cook E. L., Fitzhugh O.
G. (1964) Food flavorings and compounds of related structure. I.
Acute oral toxicity, Food Cosmet Toxicol, 2(3), 327-43.
Johansen, J. D. (2003) Fragrance Contact Allergy: A Clinical Review,
Am J Clin Dermatol, 4(11), 789-798.
Kasugai S., Hasegawa N., Ogura H. (1990) A simple in vitro cytotoxicity
test using the MTT [3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] colorimetric assay: analysis of eugenol toxicity on dental pulp
cells (RPC-C2A), Jpn J Pharmacol, 52(1), 95-100.
Kaufman P. E., Mann R. S., Butler J. F. (2010) Evaluation of
semiochemical toxicity to Aedes aegypti, Ae. albopictus and Anopheles
quadrimaculatus (Diptera: Culicidae), Pest Manag Sci, 66(5), 497-504.
59
Introducción
60
Introducción
61
Introducción
62
Introducción
Ress N. B., Bristol D. W., Bucher J. R., Hailey J. R., Haseman J. K.,
Herbert R. A., Maronpot R. R., Orzech D. P., Peddada S. D., Rao G. N.,
Roycroft J. H. Smith C. S., Travlos G. S., Witt K. L. (2003a) NTP
toxicology and carcinogenesiss studies of citral (microencapsulated)
(CAS No. 5392-40-5) in F344/N rats and B6C3F1 mice (feed studies),
Natl Toxicol Program Tech Rep Ser (505), 1-268.
Ress N. B., Hailey J. R., Maronpot R. R., Bucher J. R., Travlos G. S.,
Haseman J. K., Orzech D. P., Johnson J. D., Hejtmancik M. R. (2003b)
Toxicology and Carcinogenesis Studies of Microencapsulated Citral in
Rats and Mice, Toxicol Sci, 71(2), 198-206.
RIFM (1970) (Research Institute for Fragrance Material, Inc.), Acute
oral toxicity studies in rats. RIFM report number 2734, July 1 (RIFM,
Woodcliff Lake, NJ, USA).
Rozman V., Kalinovic I., Korunic Z. (2007) Toxicity of naturally occurring
compounds of Lamiaceae and Lauraceae to three stored-product
insects, J Stored Prod Res, 43(4), 349-355.
Saverini M., Catanzaro I., Sciandrello G., Avellone G., Indelicato S.,
Marci G., Palmisano L., (2012) Genotoxicity of citrus wastewater in
prokaryotic and eukaryotic cells and efficiency of heterogeneous
photocatalysis by, TiO2, J. Photochem. Photobiol. B: Biol, 108, 8-15.
SCCNFP (2000) Opinion Concerning the 1st update of the Inventory of
Ingredients Employed in Cosmetic Products. Section II: Perfume and
Aromatic Materials. SCCNFP/0389/00/Final.
SCCNFP, (1999) Opinion Concerning Fragrance Allergy in consumers.
A Review of the Problem. Analysis of the Need for Appropriate
Consumer Information and Identification of Consumer Allergens.
SCCNFP 0017/98
Schnuch A., Uter W., Geier J., Lessmann H., Frosch P. (2007)
Sensitization to 26 allergens to be labelled according to current
European regulation. Results of the IVDK and review of the literature,
Contact Dermatitis 57, 1–10.
Sciencelab (2005) Material Safety Data Sheet, Cinnamyl Alcohol
MSDS, Sciencelab, Inc. 1.
Seefeld, F. (2006) Chemical detection of damage to honey bees caused
by pesticides, Nachrichtenblatt des Deutschen Pflanzenschutzdienstes
(Braunschweig, Germany), 58(2), 59-66.
Simonich S. L., Begley W.M., Debaere G., Eckhoff W. (2000) Trace
Analysis of Fragrance Materials in Wastewater and treated Wastewater,
Environ Sci Technol, 34, 959–965.
63
Introducción
Singh R., Koul O., Rup P. J. Jindal J. (2009) Toxicity of some essential
oil constituents and their binary mixtures against Chilo partellus
(lepidoptera: pyralidae), Int J Trop Insect Sci, 29(2), 93-101.
Sober H. A., Hollander F., Sober E. K. (1950) Toxicity of Eugenol:
Determination of LD50 on Rats, Proc Soc Exp Biol Med, 73, 148-151.
Sporn, A., Dinu I. Stanciu V. (1965) The toxicity of cinnamaldehyde,
Igiena, 14(6), 339-46.
Stadler M., Mayer A., Anke H., Sterner O. (1994) Fatty acids and other
compounds with nematocidal activity from cultures of Basidiomycetes,
Planta Med, 60(2), 128-32.
Stroh J., Wan M. T., Isman M. B., Moul D. J. (1998) Evaluation of the
acute toxicity to juvenile Pacific Coho salmon and rainbow trout of some
plant essential oils, a formulated product, and the carrier, Bull Environ
Contam Toxicol, 60(6), 923-930.
TCI (2005) Material Safety Data Sheet, 2.
https://www.vwrsp.com/msds/10/TCH/TCH0685-025ML.pdf (consultado
25 de noviembre de 2011).
Thompson D. C., Barhoumi R., Burghard R. C. (1998) Comparative
toxicity of eugenol and its quinone methide metabolite in cultured liver
cells using kinetic fluorescence bioassays, Toxicol Appl Pharm, 149(1),
55-63.
Thompson D. C., Constantin-Teodosiu D., Moldéus P. (1991)
Metabolism and cytotoxicity of eugenol in isolated rat hepatocytes,
Chem-Biol Interact, 77(2), 137-47.
Thompson D., Constantin-Teodosiu D., Norbeck K., Svensson B.,
Moldeus P. (1989a) Metabolic activation of eugenol by
myeloperoxidase in polymorphonuclear leukocytes, Chem Res Toxicol,
2(3), 186-92.
Thompson D., Norbeck K., Olsson L. I., Constantin-Teodosiu D., Van
der Zee, J., Moldeus P. (1989b) Peroxidase-catalyzed oxidation of
eugenol: formation of a cytotoxic metabolite(s), J Biol Chem, 264(2),
1016-21.
Toaff M. E., Abramovici A., Sporn J., Liban E. (1979) Selective oocyte
degeneration and impaired fertility in rats treated with the aliphatic
monoterpene, Citral, J Reprod Fertil, 55(2), 347-52.
Tsuji M., Fujisaki Y., Okubo A., Arikawa Y., Noda K., Ide H., Ikeda T.
(1975) Studies on d-limonene, as gallstone solubilizer (V): Effects on
development of rat fetuses and offspring, Oyo Yakuri, 10,179-186.
64
Introducción
65
Introducción
66
Introducción
Punto de Densidad
Compuestos No.CAS 3 pKa pKow Uso
ebullición, °C g/cm
Acelerador en
Benzotiazol 95-16-9 193 1.24 1.2 2.01
vulcanización
Butóxido de Insecticida
51-03-6 180 1.05 - 4.8
piperonilo (sinérgico)
67
Introducción
2-Fenilfenol
68
Introducción
4-tert-Butilfenol
69
Introducción
Se ha demostrado que una dosis tan baja como una décima parte de la
concentración letal 50 (LC50= 6.9 mg/L) de 4-tert-butilfenol causa
alteración endocrina y cambios metabólicos en Cyprinus carpio en
menos de dos semanas (Barse et al., 2006). Por otra parte, para rata se
ha reportado una dosis letal 50 (LD50) de 4000 mg/kg oral y una
concentración efectiva media (EC50) de 3.4 mg/L en 48h para Daphnia
(OECD SIDS, 2000).
70
Introducción
71
Introducción
Butóxido de piperonilo
72
Introducción
73
Introducción
Benzotiazol
74
Introducción
75
Introducción
Bibliografía
76
Introducción
77
Introducción
Ko E. J., Kim K. W., Kang S. Y., Kim S. D., Bang S. B., Hamm S. Y.,
Kim D. W. (2007) Monitoring of environmental phenolic endocrine
disrupting compounds in treatment effluents and river waters, Korea,
Talanta, 73(4), 674-683.
Körner W., Bolz U., Sussmuth W., Hiller G., Schuller W., Hanf V.,
Hagenmaier H. (2000) Input/output balance of estrogenic active
compounds in a major municipal sewage plant in Germany,
Chemosphere, 40, 1131–1142.
Lee, H.B., Peart T.E., Svoboda M.L. (2005) Determination of endocrine-
disrupting phenols, acidic pharmaceuticals, and personal-care products
in sewage by solid-phase extraction and gas chromatography-mass
spectrometry, J Chromatogr A, 1094, 122–129.
Muguruma M., Nishimura J., Jin M., Kashida Y., Moto M., Takahashi M.,
Yokouchi Y., Mitsumori K. (2006) Molecular pathological analysis for
determining the possible mechanism of piperonyl butoxide-induced
hepatocarcinogenesis in mice, Toxicology, 228(2-3), 178-187.
Muguruma M., Unami A., Kanki M., Kuroiwa Y., Nishimura J., Dewa Y.,
Umemura T., Oishi Y., Mitsumori K. (2007) Possible involvement of
oxidative stress in piperonyl butoxide induced hepatocarcinogenesis in
rats, Toxicology, 236(1-2), 61-75.
Ni H. G., Lu F. H., Luo X. L., Tian H. Y., Zeng E.Y. (2008) Occurrence,
phase distribution, and mass loadings of benzothiazoles in riverine
runoff of the Pearl River Delta, China, Environ Sci Technol, 42, 1892–
1897.
OECD SIDS (2000) Organisation for Economic Co-Operation and
Development—Screening Information Data Sets P-TERT-
BUTYLPHENOL, Initial Assessment Report (OECD-SIDS Report) for
10th SIAM, Japan March 15–17, pp, United Nations Environment
Programme publications. (disponible en:
<http://www.chem.unep.ch/irptc/sids/OECDSIDS/98544.pdf/>).
Reddy C.M., Quinn J.G. (1997) Environmental chemistry of
benzothiazoles derived from rubber, Environ Sci Technol, 31, 2847–
2853.
Schwarzbauer J., Heim S., Brinker S., Littke R. (2002) Occurrence and
alteration of organic contaminants in seepage and leakage water from a
waste deposit landfill, Water Res, 36, 2275-2287.
Smital T., Terzic S., Zaja R., Senta I., Pivcevic B., Popovic M., Mikac I.,
Tollefsen K. E., Thomas K. V., Ahel M. (2010) Assessment of
toxicological profiles of the municipal wastewater effluents using
chemical analyses and bioassays, Ecotox Environ Safe, 74 (4), 844-
851.
78
Introducción
Spurgeon D. J., Jones Oliver A., Dorne J. L., Svendsen C., Swain S.,
Stürzenbaum S. R. (2010) Systems toxicology approaches for
understanding the joint effects of environmental chemical mixtures, Sci
Total Environ, 408(18), 3725-3734.
Tanaka T. (1992) Effects of piperonyl butoxide on F1 generation mice,l
Toxicol Lett, 60, pp. 83–90.
US EPA (2006) Eligibility Decision for Piperonyl Butoxide (PBO), United
States Environmental Protection Agency, Prevention, Pesticides and
Toxic Substances (7508C), EPA 738-R-06-005.
US EPA (2010) U.S. Environmental Protection Agency, Hazard
Characterization Document, Screening-level hazard characterization of
high production volume chemicals, Benzothiazole- and Morpholine-
Based Thiazoles Category 5 Chemicals.
Wheelock C.E., Miller J.L., Miller M.J., Gee S.J., Shan G., Hammock
B.D. (2004) Development of toxicity identification evaluation procedures
for pyrethroid detection using esterase activity, Environ Toxicol Chem,
23, 2699-2708.
Woudneh M. B., Oros D. R. (2006) Pyrethroids, pyrethrins, and
piperonyl butoxide in sediments by high-resolution gas
chromatography/high-resolution mass spectrometry, J Chomatogr A,
1135 (1), 71-77.
Zimerson E., Bruze M. (2002) Contact allergy to the monomers in p-tert-
butylphenol formaldehyde resin, Contact Dermatitis, 47, 147–153.
79
Introducción
80
Introducción
81
Introducción
82
Introducción
83
Introducción
Émbolo
Cuerpo de la jeringa
Junta
Ventana de
visualización
Indicador ajustable
de profundidad
Guía de la aguja
Septum perforado
Fibra
Recubrimiento de sílice
84
Introducción
85
Introducción
86
Introducción
87
Introducción
88
Introducción
Jeringa
Tubos de vidrio de fondo cónico
Emulsificación por
los ultrasonidos
Extracción del
disolvente
89
Introducción
90
Introducción
91
Tabla II.9. Aplicación de la técnica USAEME en agua.
PF factor de
Volumen de
Analitos Disolvente LOD Detector preconcen- REF
disolvente
tración
9 fragancias sintéticas de í1
Cloroformo 100 ȝL pg mL GC-MS 100 Regueiro et al. (2008)
almizcle, 6 ftalatos y lindano
4 parabenos, triclosan y 2 í1
1,1,1-Tricloroetano 100 ȝL 3.90 - 12.5 pg mL GC–MS/MS 100 a 200 Regueiro et al. (2009)
fenoles
25 fragancias alérgenas í1 Becerril-Bravo et al.
Cloroformo 100 ȝL 1 ng mL GC–MS
reguladas (2010)
4 ftalatos (di-butil ftalato, butil
Tetracloruro de -1
bencill ftalato, diisoctil ftalato, 20 μL 1.0 - 1.2μg L GC-FID 490- a 530 Yan et al. (2010b)
carbono
dioctil ftalato)
10 hidrocarburos aromáticos í1
Tolueno 14 ȝL 0.02–0.05 ȝg L GC-FID 1776–2714 Saleh et al. (2009)
policíclicos (PAHs)
11 hidrocarburos aromáticos Recupera 5 ȝL í1
Tween 80 y ciclohexano 0.6–62.5 ng L HPLC-FLD Cheng et al. (2011)
policiclicos (PAHs) de ciclohexano
í1
14 ng L (para PCB-153)
6 bifenilos policlorados í1
Cloroformo 200 ȝL - 30 ng L (para PCB- GC–MS Ozcan et al. (2009a)
(PCBs)
101).
4 éteres difenil polibromados
í1 í1
(PBDEs); 2ƍ,4,4ƍ-tetraBDE 2 pg mL , 2 pg mL ,
í1 í1
(BDE-47), 2,2ƍ,4,4,5- 1 pg mL , 1 pg mL para
pentaBDE (BDE-99), Cloroformo 100 ȝL BDE-47, BDE-100, BDE- GC–MS 319 Fontana et al. (2009)
2,2ƍ,4,4,6-pentaBDE (BDE- 99 y BDE-153,
100) and 2,2,4,4ƍ,5,5ƍ- respectivamente
hexaBDE (BDE-153)
PF factor de
Volumen de
Analitos Disolvente LOD Detector preconcen- REF
disolvente
tración
18 pesticidas organoclorados í1
Cloroformo 200 ȝL 0.002 - 0.016 ȝg L GC-ȝECD Ozcan et al. (2009b)
(OCPs)
150 ȝL de clorobenceno
(como disolvente de
7 pesticidas extracción) y 100 ȝL de í1
í2 0.1 y 0.3 ng mL HPLC-DAD 210 a 242 Wu C et al. (2010)
organofosforados (OPs) 1.0 × 10 mol
Lí1 Trian X-100 como
emulsificante
-1 -1
9 pesticidas 1 ng L y 2 ng L
Tolueno 20 ȝL GC-ȝECD 330 a 699 Su y Jen (2010)
organofosforados (OPPs)
Tween 20 como
6 pesticidas carbamatos
emulsificante y 150 ȝL de
(mealcarb, carbofuran, í1
clorobenceno y cloroformo CHCl3–C6H5Cl 0.1–0.3 ng mL HPLC-DAD 170 y 246 Wu Q et al. (2010)
carbaryl, pirimicarb,
como disolvente de (1:1, v/v)
isoprocarb y diethofencarb)
extracción dual
4 benzoilurea (BU)
1-hexil-3-metilimidazolium
pesticidas, diflubenzuron, -1
hexafluorofosfato 0.21-0.45 mg L HPLC Zhou y Zhang (2010)
flufenoxuron, triflumuron y
([C6MIM][PF6])
chlorfluazuron
6 piretroides: Allethrin,
Tetraclorometano como
cypermethrin, prallethrin, í1 HPLC UV-
extractante, acetona como 20 ȝL 0.11 - 0.30 ȝg L 767-1033 Yan et al. (2010c)
tetramethrin, transfluthrin, y VIS
dispersante
imiprothrin
1-Hexil-3-metilimidazolium
-1
4 herbicidas triazinas hexafluorofosfato 100 μL 0.36 - 1.41 μg L HPLC Wang et al. (2010)
([C6Mlm][PF6])
PF factor de
Volumen de
Analitos Disolvente LOD Detector preconcen- REF
disolvente
tración
1-octyl-3-metilimidazolium
-1
Residuos de diafenthiuron hexafluorofosfato 0.8 mg L LC-UV 358 Lu et al. (2010)
([OMIM]PF6) y acetonitrilo
2,4,6-tricloroanisol (2,4,6- í1 Fontana y Altamirano
Cloroformo 40 ȝL 0.2 ng L GC–MS/MS 555
TCA) (2010)
3 clorofenoles (2-clorofenol,
-1
2,4-diclorofenol y 2,6- Acetonitrilo 10 ȝL 6 - 23 ng mL HPLC Xu et al. (2007)
diclorofenol)
Diclorometano
4 ácidos fenoxiacético í1
reconstituido en mezcla 0.67 - 1.50 ng mL HPLC Lin y Fuh (2010)
clorados (CPAs)
metanol-agua (1:1)
PF
Volumen de factor de
Metal Disolvente LOD Detector REF
disolvente precon-
centración
-1
Cobre 0.76 μg L FAAS Chang et al. (2010)
1-Hexil-3-metilimidazolium -1
Cromo (VI) 0.07 ng mL ETAAS Chen et al. (2010)
hexafluorofosfato ([Hmim][PF6])
FAAS: Espectrometría de absorción atómica de flama.
ETAAS: Espectrometría de absorción atómica electrotérmica.
Tabla II.11. Aplicación de USAEME en la determinación de diversos compuestos en matrices variadas.
Volumen PF factor de
Compuestos Matriz Disolvente de LOD Detector precon- REF
disolvente centración
Introducción
Bibliografía
97
Introducción
98
Introducción
99
Introducción
100
Introducción
Wang Y., You J., Bao C., Zhang H., Yu A., Yu Y. (2010) Determination
of triazines by ultrasonic-assisted ionic liquid microextraction coupled
with high performance liquid chromatography, Chinese J Chem, 28(5),
785-790.
Wu C., Liu N., Wu Q., Wang C., Wang Z. (2010) Application of
ultrasound-assisted surfactant-enhanced emulsification microextraction
for the determination of some organophosphorus pesticides in water
samples, Anal Chim Acta, 679, 56-62.
Wu J., Xiang B., Xia J. (2009) Application of ultrasound-assisted
emulsification-microextraction combined with high performance liquid
chromatography to the determination of propoxur in environmental and
beverage samples, Microchim Acta, 166(1-2), 157-162.
Wu Q. H., Li Z., Wu C. X., Wang C., Wang Z. (2010a) Application of
ultrasound-assisted emulsification microextraction for the determination
of triazine herbicides in soil samples by high-performance liquid
chromatography, Microchim Acta, 170(1-2), 59-65.
Wu Q., Chang Q., Wu C., Rao H., Zeng X., Wang C., Wang Z. (2010b)
Ultrasound-assisted surfactant-enhanced emulsification microextraction
for the determination of carbamate pesticides in water samples by high
performance liquid chromatography, J Chromatogr A, 1217, 1773-1778.
Xu H., Liao Y., Yao J. (2007) Development of a novel ultrasound-
assisted headspace liquid-phase microextraction and its application to
the analysis of chlorophenols in real aqueous samples, J Chromatogr A,
1167, 1-8.
Yan H., Du J., Zhang X., Yang G. Row K. H., Lv Y. (2010a) Ultrasound-
assisted dispersive liquid-liquid microextraction coupled with capillary
gas chromatography for simultaneous analysis of nine pyrethroids in
domestic wastewaters, J Sep Sci, 33(12), 1829-1835.
Yan H., Liu B., Du J., Row K. H. (2010b) Simultaneous determination of
four phthalate esters in bottled water using ultrasound-assisted
dispersive liquid-liquid microextraction followed by GC-FID detection,
Analyst, 135(10), 2585-2590.
Yan H., Liu B., Du J., Yang G., Row K. H. (2010c) Ultrasound-assisted
dispersive liquid-liquid microextraction for the determination of six
pyrethroids in river water, J Chromatogr A, 1217, 5152-5157.
Zhou Q., Zhang X. (2010) Combination of ultrasound-assisted ionic
liquid dispersive liquid-phase microextraction and high performance
liquid chromatography for the sensitive determination of benzoylureas
pesticides in environmental water samples, J Sep Sci, 33(23-24), 3734-
3740.
101
Introducción
102
III. Parte Experimental y Resultados
Parte Experimental y Resultados
105
Parte Experimental y Resultados
106
Parte Experimental y Resultados
Los compuestos incluidos en este estudio (Tabla 1.1) así como los
estándares de recuperación 4-n- nonilfenoles y el estándar interno [2H16]
bisfenol A fueron adquiridos de Sigma-Aldrich (St. Louis, MO, USA). El
ácido 3,4-diclorofenoxiacetico (3,4-D) y 2,3-diclorofenoxiacetico (2,3-D)
fueron adquiridos de Riedel-de Haën (Seelze, Alemania). Los
estrógenos deuterados [2H4] estrona, [2H4]17ȕ-estradiol y [2H4]17Į-
etinilestradiol fueron obtenidos de Cambridge Isoptopes (Andover, MA,
EUA). Los agentes derivatizantes N-tert-butildimetilsilil-N-metiltri-
fluoroacetamida con 1% tert-butildimetilclorosilano (MTBSTFA) así
como N,O- bis(Trimetilsilil)trifluoroacetamida (BSTFA) con 1% de
trimetilclorosilano (TMSCI) fueron adquiridos de Sigma – Aldrich. Todos
los disolventes (agua, metanol, acetona, hexano y acetato de etilo)
fueron grado HPLC suministrados por Burdick and Jackson (Morristown,
NJ, EUA). Los cartuchos de extracción Oasis HLB fueron adquiridos de
Waters (Milford, MA, EUA).
107
Compuesto Peso
Uso Formula molecular Log Kow
(No. CAS) g/mol
Ácido clofíbrico
regulador de lípidos 2.84
Ácido 2-(4-Clorofenoxi)-2- 214.65
metabolito del clofibrato (Henschel et al., 1997)
metilpropanoico
882-09-7
Ibuprofeno
3.97
ácido 2-[4-(2-metilpropil) fenil] analgésico y antinflamatorio 206.29
(Avdeef et al., 1998)
propanoico
15687-27-1
2,4-D
2.81
ácido (2,4- diclorofenoxiacético) pesticida 221.04
(U.S. DHHS., 1993)
94-75-7
Gemfibrozil
6.72
Ácido 5-(2,5-dimetilfenoxi)-2,2- regulador de lípidos 250.33
(Hassan et al., 2004)
dimetil-pentanoico
25812-30-0
Naproxeno
3.06
Ácido (ĮS)-6-metoxi-Į-metil-2- analgésico y antinflamatorio 230.26
(Liu et al., 2004)
naftalenacético
22204-53-1
Compuesto Peso
Uso Formula molecular Log Kow
(No. CAS) g/mol
Ketoprofeno
Ácido 2-(3-benzoilfenil) 2.77
analgésico y antinflamatorio 254.28
propanoico (Liu et al., 2004)
22071-15-4
Diclofenaco
ácido 2-(2-(2,6- 4.51
antiflogístico 296.14
diclorofenilamino)fenil) acético (Avdeef et al., 1998)
15307-86-5
Pentaclorofenol 5,24
2,3,4,5,6-Pentaclorofenol pesticida 266.34 (Schellenberg et al.,
87-86-5 1984)
Triclosan
4.76
5-cloro-2-(2,4-diclorofenoxi) fenol antiséptico 289.55
(MITI, 1992)
3380-34-5
Bisfenol A
3.4
4,4'-dihidroxi-2,2-difenilpropano químico industrial 228.28
(Institute for Health, 2003)
80-05-7
Compuesto Peso
Uso Formula molecular Log Kow
(No. CAS) g/mol
Butilbencilftalato (BBP)
4,77
n-butil bencil ftalato (BBP) plastificante 312.36
(Ellington et Floyd., 1996)
85-68-7
Estrona3-hidroxi-13-metil-
6,7,8,9,11,12,13,14,15,16-
3.43
decahidrociclopenta[a]fenantreno- estrógeno natural 270.36
( Lai, et al., 2000)
17- ona
53-16-7
17- ȕ-estradiol
(8R,9S,13S,14S,17S)-13-metil-
3.94
6,7,8,9,11,12,14,15,16,17- estrógeno natural 272.39
(Lai, et al., 2000)
decahidro
ciclopenta[a]fenantreno-3,17-diol
50-28-2
17- Į-etinilestradiol
17-etinil-13-metil-
3.9
7,8,9,11,12,13,14,15,16,17- estrógeno sintético 296.41
(Holthaus, et al., 2002)
decahidro-6Hciclopenta[a]
fenantreno- 3,17-diol
57-63-6
Parte Experimental y Resultados
1.2.2 Muestreo
Agua Potable
111
Parte Experimental y Resultados
Agua Residual
112
Parte Experimental y Resultados
Muestradeagua
SPE
CartuchoOasis
EluĐióncon
Acetonabuffer(40:60)
EluĐiónconacetona
Fracción
ácida Fenoles,estrógenos
ftalatos
Extracciónen
Acetatodeetilo
Derivatización
MTBSFA
BESFA/piridina
DimetilͲtͲbutilsilil
derivados Trimetilsilil
derivados
CGͲ MS
113
Parte Experimental y Resultados
1.2.4 Extracción
Para cada muestra se acondicionó un cartucho Oasis HLB (200 mg) con
acetona (2x 5 mL) seguido por agua (5 mL). Las muestras (2000 mL de
agua de abastecimiento y 250 mL de agua residual) se pasaron a través
del cartucho a una velocidad de aproximadamente 10 mL/min usando
vacío. Al término de la extracción, los cartuchos se lavaron con agua de
grado HPLC (2 mL) y posteriormente se eluyeron con 5 mL de la
disolución 40:60 de una mezcla acetona: buffer 0.10 M de bicarbonato
de sodio (se ajustó el pH a 10 con una disolución 1.0 M de hidróxido de
sodio). Esta fracción contiene los compuestos ácidos. Los cartuchos se
lavaron con 2 mL de agua grado HPLC y se secaron bajo corriente de
114
Parte Experimental y Resultados
115
Parte Experimental y Resultados
116
Parte Experimental y Resultados
117
Parte Experimental y Resultados
Salicylic acid
gemfibrizol
ketoprofen
naproxen
clofibric acid
2,4--D
diclofenac
ibuprofen
3,4--D
2,3-D
time (mins)
bisphenol-A
abundance
d14-bisphenol-A
butylbenzylphthalate
17ȕ-estradiol
17ȕthynylestradiol
pentachlorophenol
triclosan
4--nonylphenols
DEHP
estrone
118
Parte Experimental y Resultados
119
Parte Experimental y Resultados
La elución ácida actúa como una limpieza efectiva para los disruptores
endocrinos que son remanentes en el cartucho. Esto se demostró por
una comparación en modo full scan (50-500 m/z) donde la fracción de
acetona fue mucho más limpia después de la elución previa con la
mezcla buffer – acetona (Figura 1.4).
120
Parte Experimental y Resultados
Los experimentos con estándares mostraron que era posible eluir los
disruptores endocrinos tanto con acetato de etilo como con acetona
obteniendo una muestra final limpia. Sin embargo, el metanol, es un
disolvente comúnmente usado para eluir esta clase de compuestos de
estos cartuchos.
121
Parte Experimental y Resultados
122
Parte Experimental y Resultados
2
LOD
Analito R
INS AM AR
Ácido clofibrico 0.998 0.01 0.5 100
Ibuprofeno 0.993 0.001 0.25 50
Ácido salicílico 0.999 0.005 0.25 5
2,4-D 0.994 0.01 0.5 100
Gemfibrozil 0.998 0.01 0.5 50
Naproxeno 0.993 0.005 0.25 50
Ketoprofeno 0.997 0.01 0.25 50
Diclofenaco 0.995 0.01 1 50
4-Nonilfenoles 0.996 0.025 1 50
Pentaclorofenol 0.994 0.01 0.2 20
Triclosan 0.996 0.01 0.1 10
Bisfenol-A 0.996 0.01 0.5 20
Butibencilftalato (BBP) 0.996 0.01 0.5 50
Bis-2-etilhexilftalato (DEHP) 0.997 0.01 0.5 50
Estrona 0.999 0.001 0.005 1
17ȕ-Estradiol 0.999 0.001 0.005 0.5
17Į-Etinilestradiol (EE2) 0.999 0.002 0.05 2.5
INS: instrumento; AM: agua de manantial; AR: agua residual
123
Parte Experimental y Resultados
124
Parte Experimental y Resultados
125
Parte Experimental y Resultados
126
Parte Experimental y Resultados
127
Parte Experimental y Resultados
128
Parte Experimental y Resultados
129
Parte Experimental y Resultados
130
Parte Experimental y Resultados
Ibuprofeno <LOD 4.38 (±0.62) 5.09 (±1.20) 2.2 (±1.2) 0.8 (±0.3)
Ácido salícilico 2.4 0.62 (±0.14) 29.06 (±25.18) 7.8 (±2.1) 9.6 (±12.2)
Naproxeno 0.40 15.22 (±1.76) 16.65 (±3.48) 0.8 (±0.2) 0.90 (±0.03)
Triclosan 0.13 2.04 (±0.29) 0.66 (±0.04) 1.3 (±0.2) 1.12 (±0.16)
Bisfenol-A 0.20 2.50 (±0.36) 0.77 (±0.05) 0.40 (±0.17) 0.41 (±0.21)
BBP 0.60 1.24 (±0.14) 0.71 (±0.05) 2.0 (±1.2) 1.7 (±0.4)
Estrona 0.005 0.080 (±0.013) 0.044 (±0.001) 0.17 (±0.02) 0.16 (±0.02)
17ȕ-Estradiol 0.002 0.022 (±0.004) 0.018 (±0.003) 0.020 (±0.003) 0.010 (±0.003)
* Los valores de LOD para los blancos están basados en los datos para el agua de manantial.
131
Parte Experimental y Resultados
132
Tabla 1.11. Concentración de fármacos ácidos en agua de abastecimiento de la ciudad de México (ng/L) (n=3).
Ácido Ácido
Sitio Ibuprofeno 2,4-D Gemfibrizol Naproxeno Ketoprofeno Diclofenaco
clofibrico salicílico
Influente "Berros" <LOD <LOQ 6.7 37.4 <LOD 5.22 <LOQ 3.21
Efluente "Berros" <LOD <LOD 19.7 <LOD <LOD <LOD <LOD <LOD
Presa Guadalupe <LOD 15.4 48.5 <LOD 9.84 183 <LOD 28.5
Pozo Chalco <LOD <LOD 3.4 <LOD <LOD <LOD <LOD <LOD
Pozo 310 <LOD <LOQ 8.3 <LOD <LOD <LOD <LOD <LOD
Cerro Gordo Los Reyes 1.9 2.70 1.7 <LOD <LOQ <LOD 0.88 <LOD
Cerro Gordo Cutzamala <LOD <LOD 18.1 <LOD <LOD <LOD <LOD <LOD
Cerro Gordo mezcla <LOD <LOD 14.5 <LOD <LOD <LOD <LOD <LOD
Sitio NPs Pentaclorofenol Triclosan Bisfenol-A BBP DEHP Estrona Estradiol EE2
Influente Planta Berros 94.3 <LOD 2.2 3.6 6.2 199 0.75 <LOD <LOD
Efluente Planta Berros 71 <LOD <LOQ <LOD 2.5 227 <LOD <LOD <LOD
Toma O 57 <LOD 0.6 <LOD 2.9 317 <LOD <LOD <LOD
presa Guadalupe 200 <LOD 17.1 7.1 5.4 290.5 <LOD 6.3 <LOD
Pozo Chalco 15.5 <LOD 0.5 3.6 <LOQ 101.6 <LOD <LOD <LOD
La Caldera 17.5 <LOD 0.6 <LOD 2 98.4 <LOD <LOD <LOD
Pozo 310 42.3 <LOD 0.4 <LOD <LOQ 101 <LOD <LOD <LOD
Cerro Gordo Los Reyes 36.6 <LOD <LOQ <LOD <LOD 108.1 <LOD <LOD <LOD
Cerro Gordo Cutzamala 10.6 <LOD 0.5 <LOD 14.9 96.8 <LOD <LOD <LOD
Cerro Gordo mezcla 10.1 <LOD <LOD <LOD 14.1 104.8 <LOD <LOD <LOD
Dolores 12.5 <LOD 0.4 2.2 <LOD 148.7 <LOD <LOD <LOD
Caída Borracho 9.3 0.62 0.6 2.7 <LOD 137.3 <LOD <LOD <LOD
Parte Experimental y Resultados
Bibliografia
Avdeef, A., K. Box J., Comer J. E. A., Hibbert C., Tam K.Y. (1998),
Determination of liposomal membrane-water partitioning coefficients of
ionizable drugs, Pharmaceut Res, 15(2), 209–215.
Barceló D. (2003) Emerging pollutants in water analysis, TrAC Trend
Anal Chem, 22, xiv-xvi.
Baronti C., Curini R., D'Ascenzo G., Di Corcia A., Gentili A., Samperi R.
(2000) Monitoring natural and synthetic estrogens at activated treatment
plants and in receiving river water, Environ Sci Technol, 34, 5059–5066.
Belfroid A.C., Van der Horst A., Vethaak A.D., Schafer A.J., Ris G.B.J.,
Wegener J. (1999) Analysis and occurrence of estrogenic hormones
and their glucuronides in surface water and waste water in the
Netherlands, Sci Total Environ, 225, 101–108.
Braun P., Moeder M., Schrader S., Popp P., Duschk P., Engewald W.
(2003) Trace analysis of technical nonylphenol, bisphenol A and 17 Į-
etinylestradiol in wastewater using solid-phase microextraction and gas
chromatography-mass spectrometry, J Chromatogr A, 988, 41-51.
Campbell C.G., Borglin S.E., Green G.B., Grayson A., Wozei E.,
Stringfellow W.T. (2006) Biologically directed environmental monitoring,
fate, and transport of estrogenic endocrine disrupting compounds in
water: A review, Chemosphere, 65, 1265-1280.
Carballa M., Omil F., Lema J.M., Llompart M., García-Jares C.,
Rodríguez I., Gómez M., Ternes T. (2004) Behavior of pharmaceuticals,
cosmetics and hormones in a sewage treatment plant, Water Res, 38,
2918.
Hao Ch., Zhao X., Yang P. (2007) GC-MS and HPLC-MS analysis of
bioactive pharmaceuticals and personal-care products in environmental
matrices, TrAC Trend Anal Chem, 26, 569-580.
Daughton C.G., Ternes T.A., (1999). Pharmaceuticals and personal
care products in the environment: agents of subtle change?, Environ
Health Perspect, 107, 907–938.
Ellington J. J., Floyd T. L. (1996) Octanol/Water Partition Coefficients
for Eight Phthalate Esters United States National Exposure
Environmental Protection Research Laboratory Agency Athens, GA
30605-2700 Research and Development EPA/600/S-96/006.
135
Parte Experimental y Resultados
136
Parte Experimental y Resultados
137
Parte Experimental y Resultados
138
Parte Experimental y Resultados
139
Anexo. Concentraciones de analitos reportadas por diversos autores en diferentes países.
Concentración en Concentración en plantas de tratamiento
aguas Concentración Concentración
Compuesto Caracteristicas Pais superficiales en aguas en agua Método Analítico Referencia
INFLUENTE Max- Min EFLUENTE Max-Min
Máximo-Mínimo subterráneas potable
(Media) (Media)
(media)
Canadá (μg/L) 2.40-0.21 (1.28) 0.45-0.02 (0.18) GC-MS Lee et al., (2005)
0.5-14 3-1410 (19) 0.50-2.0
USA (ƾg/L) 85-250 20-44 (19) 20-44 Campbell et al., (2006)
Estrógeno <3-230
Bisfenol A
sintético Max: 12
(μg/L) CLLE GC-MS Kolpin et al. , (2002)
Media: 0.14
España (μg/L) 3.4-0.72 (1.4) 0.98-0.14 (0.38) SPE- GC-MS Gómez, et al., (2007)
Alemania (μg/L) 3 SPE- GC-MS Braun et al., (2003)
Canadá (μg/L) 25-2.72 (14.6) 3.21-0.32 (0.70) GC-MS Lee et al., (2005)
Altera el sistema Alemania (μg/L) 2.4 SPE- GC-MS Braun et al., (2003)
4-nonilfenol
endócrino Max: 40
USA (μg/L) CLLE GC-MS Kolpin et al., (2002)
Media: 0.8
Canadá (μg/L) 1.83-0.87 (1.35) 0.36-0.05 (0.14) GC-MS Lee et al., (2005)
Agente Corea del Sur (ƾg/L) 32-1.3 (12) LC–MS/MS Kim et al. (2007)
antibacteriano y
Triclosan
fungicida, agente España (μg/L) 4.2-0.39 (1.8) 0.40-0.08 (0.2) SPE- GC-MS Gómez, et al., (2007)
desinfectante. Max: 2.3
USA (μg/L) CLLE GC-MS Kolpin et al., (2002)
Media: 0.14
Canadá (μg/L) 0.052-0.0008 (0.0016) 0.054- <0.001 (0.005) GC-MS Lee et al., (2005)
0.10-4.1
1.1-3.0
USA (ƾg/L) 0.20-0.6 Campbell, et al., (2006)
<0.1-17
<0.4-2.12
Max. 0.112
(μg/L) CLLE GC-MS Kolpin et al., (2002)
Estrona Estrógeno natural Media: 0.027
España (μg/L) 2.4 4.4 GC-MS Carballa et al., (2004)
Corea del Sur (ƾg/L) 5.0-1.7 (3.6) 36-2.2 (14) LC–MS/MS Kim et al., (2007)
7 14.5
7.2 17.7
Francia SPE- GC-MS Braun et al., (2003)
4.9 8.3
6.1 8.7
Concentración en Concentración en plantas de tratamiento
aguas Concentración Concentración
Compuesto Caracteristicas Pais superficiales en aguas en agua Método Analítico Referencia
INFLUENTE Max- Min EFLUENTE Max-Min
Máximo-Mínimo subterráneas potable
(Media) (Media)
(media)
Canadá (μg/L) 0.022-0.003 (0.009) 0.002-<0.001 (0.002) GC-MS Lee et al., (2005)
1.9- 6.0
0.15-3.6
USA (ƾg/L) 13-80 0.20-2.1 Campbell et al., (2006)
1.4-3.2
<0.1-0.7
Max: 0.2
Estrógeno natural
Media: 0.16
Estradiol (μg/L) CLLE GC-MS Kolpin et al., (2002)
Max: 0.093
Media: 0.0009
Corea del Sur (ƾg/L) < 1.0 SPE- GC-MS Lin, et al., (2005)
8.6 18.01
8.1 11.03 Mouatassim-Souali et
Francia SPE- GC-MS
7.6 11.4 al., (2003)
8.1 16.7
0.1-5.1
USA (ƾg/L) 0.15-0.50 Campbell, et al., (2006)
1.1-2.9
Max: 0.831
(μg/L) CLLE GC-MS Kolpin ,et al., (2002)
Media: 0.073
Corea del Sur (ƾg/L) 1.3 LC–MS/MS Kim, et al., (2007)
Etinilestradiol Anticonceptivo
5.2 8.7
2.9 5.7 Mouatassim-Souali et
Francia SPE- GC-MS
2 3.8 al., (2003)
5 7
Alemania (μg/L) 0.12 SPE- GC-MS Braun, et al., (2003)
Canadá (μg/L) 10.21-4010 (6.77) 2.17-0.11 (0.31) GC-MS Lee, et al., (2005)
Max: 3.4
LC-GC-MS Ternes, (1998)
Alemania (μg/L) Media: 0.37
0.1 Heberer (2002)
Analgésico Taiwan (ƾg/l) 30 SPE- GC-MS Lin, et al., (2005)
Ibuprofeno
Antiinflamatorio 5.70- 2.64 (3.69) 2.10-0.91 (1.32) GC-MS Carballa, et al., (2004)
España (μg/L)
168-34 (84) 28-0.24 (7.1) SPE- GC-MS Gómez, et al., (2007)
Corea del Sur (ƾg/L) 38-11 (28) 137-10 (65) LC–MS/MS Kim, et al., (2007)
Max: 1
USA (μg/L) SPE- GC-MS Kolpin, et al., (2002)
Media: 0.20
Concentración en Concentración en plantas de tratamiento
aguas Concentración Concentración
Compuesto Caracteristicas Pais superficiales en aguas en agua Método Analítico Referencia
INFLUENTE Max- Min EFLUENTE Max-Min
Máximo-Mínimo subterráneas potable
(Media) (Media)
(media)
Canadá (μg/L) 36.51-0.12 (0.26) 2.09-0.08 (0.19) GC-MS Lee, et al., (2005)
Max: 0.15
LC-GC-MS Ternes, (1998)
Alemania (μg/L) Media: 0.40
Gemfibrozil Regulador de lípidos 0.07 Heberer (2002)
Corea del Sur (ƾg/L) 9.1-1.8 (6.6) 17-3.9 (11.2) LC–MS/MS Kim, et al., (2007)
Max: 0.79
USA (μg/L) SPE- GC-MS Kolpin, et al., (2002)
Media: 0.048
Canadá (μg/L) 0.15-0.06 (0.08) 0.09-0.04 (0.05) GC-MS Lee, et al., (2005)
Analgésico. Max: 0.38
Ketoprofeno LC-GC-MS Ternes, (1998)
Antiinflamatorio Alemania (μg/L) Media: 0.20
0.23 Heberer, (2002)
Canadá (μg/L) 2.45-0.05 (0.17) 0.25-0.07 (0.11) GC-MS Lee, et al., (2005)
Max: 2.1
LC-GC-MS Ternes, (1998)
Alemania (μg/L) Media: 0.81
Diclofenaco Antiinflamatorio
50 (ƾg/l) 3.02 2.51 Heberer, (2002)
Corea del Sur (ƾg/L) 68-11 (3) 127-8.8 (40) LC–MS/MS Kim, et al., (2007)
España (μg/L) 3.6-0.2 (1.5) 2.2-0.14 (0.9) SPE- GC-MS Gómez, et al., (2007)
Maximo: 1.6
LC-GC-MS Ternes, (1998)
Ácido Clofibrico Regulador de lípidos Alemania (μg/L) Media 0.36
50 (ƾg/l) 0.46 0.48 Heberer, (2002)
Antiagregante Canadá (μg/L) 12.7-2.82 (6.68) 0.32-0.01 (0.14) GC-MS Lee, et al., (2005)
plaquetario Max: 0.14
Ac. Salicílilico Analgésico Media: no LC-GC-MS Ternes, (1998)
Antiinflamatorio Alemania (μg/L)
detectable
Antipirético 0.04 Heberer, (2002)
Canadá (μg/L) 6.03-1.73 (2.76) 2.54-0.36 (0.82) GC-MS Lee, et al., (2005)
Taiwan 170 SPE- GC-MS Lin, et al., (2005)
España (μg/L) 4.60-1.79 (3.28) 2.60-0.80 (1.75) GC-MS Carballa, et al., (2004)
Analgésico
Naproxeno Max: 0.52
Antiinflamatorio LC-GC-MS Ternes, (1998)
Alemania (μg/L) Media: 0.30
0.1 Heberer, (2002)
Corea del Sur (ƾg/L) 18-1.8 (11) 483-20 (128) LC–MS/MS Kim, et al., (2007)
Parte Experimental y Resultados
Por otra parte, las piscinas han sido reconocidas recientemente como
una fuente importante de exposición a contaminantes. Los estudios
________________________________________________________________
143
Parte Experimental y Resultados
144
Parte Experimental y Resultados
________________________________________________________________
145
Parte Experimental y Resultados
2.2.2 Patrones
2.2.3 Muestras
148
Parte Experimental y Resultados
________________________________________________________________
149
Parte Experimental y Resultados
Inyector Split/splitless
Temperatura 220 °C
Modo de inyección Splitless
Tiempo de splitless 2 min
Flujo de Split 20 mL min-1
Columna capilar HP5
Fase estacionaria (5% fenil, 95% dimetil)polisiloxano
Dimensiones 30 m × 0.32 mm x 0.25 μm
Programa de temperaturas
Temperatura inicial 45 °C (2 min)
-1
Rampa de temperatura 1 8 °C min hasta 100 °C
Rampa de temperatura 2 20 °C min-1 hasta 150 °C
-1
Rampa de temperatura 3 25 °C min hasta 200 °C (5 min)
-1
Rampa final 8 °C min hasta 233 °C
Tiempo total de análisis 22.5 min
Gas portador He
-1
Flujo en columna 1.0 mL min (constante)
Ionización MS
Modo de ionización Impacto electrónico (EI, +70 eV)
Modo de adquisición Full scan
Intervalo de masas 39-400 m/z
Velocidad de barrido 3 ȝscans
Temperatura interfase 280 °C
Temperatura trampa de iones 150 °C
Temperatura fuente de iones 200 °C
Temperatura manifold 40 °C
Corriente del filamento de emisión 25 ȝA
Voltaje del multiplicador 1700 V
150
Parte Experimental y Resultados
La Tabla 2.3 resume los tiempos de retención, así como los iones de
identificación y cuantificación de las fragancias.
Tiempo de Iones de
Iones de
Clave retención Compuesto cuantificación
identificación
(min) (m/z)
1 6.66 Pineno 77,93 41,77,93,121
2 8.65 Limoneno 67,93 39,67,93,136
3 8.95 Alcohol bencílico 77,79,108 77,79,108
4 9.84 Linalol 43,71,93 43,71,55,93
5 11.08 Octanoato de 2-metilo 67,79,95 67,79,93,95
6 11.36 Citronelol 41,67,69 41,67,69,81
7 11.50; 11.77 Citral 39,41,69 39,41,69,84
8 11.59 Geraniol 41,69 41,69,93,123
9 11.87 Cinamaldehído 77,103,131 51,77,103,131
10 11.92 Hidroxicitronelal 43,59,71 41,43,59,71
11 11.97 Anis alcohol 77,109,138 77,109,137,138
12 12.14 Cinamil alcohol 78,91,92 78,91,92,134
13 12.47 Eugenol 164 103,131,164
14 12.78 Metileugenol 178 147,163,178
15 12.85; 13.12 Isoeugenol 164 77,103,164
16 13.16 Cumarina 118,146 89,118,146
17 13.28 Į-isometil ionona 93,121,136 93,121,135,136
®
18 13.62 Lilial 189 131,147,189
19 14.16 Amil cinamal 129 117,129,202
®
20 14.62 Lyral 79,91,93 79,91,93,136
21 14.74 Alcohol cinamílico 91,133 91,115,129,133
22 14.94 Farnesol 41,69 41,69,81,121
23 15.32; 15.57 Hexilcinamaldehído 129,216 117,129,216
24 15.64 Benzoato de bencilo 105,194 77,91,105,194
25 16.94 Salicilato de bencilo 91 39,65,91
26 20.75 Cinamato de bencilo 91,131 91,131,192
________________________________________________________________
151
Parte Experimental y Resultados
152
Parte Experimental y Resultados
Limonene Citronellol
4 43
50 HS
HS
39
Response (cts)
Response (cts)
3
35
(x106)
(x105)
2 31
100
27
1 100
23
IM 50 IM
0 19
Temperature (°C) Extraction mode Temperature (°C) Extraction mode
Response (cts)
20 48
(x105)
100
(x103)
17 43
14 38
11 IM 33
50 50
8 28
Temperature (°C) Extraction mode Temperature (°C) Extraction mode
Response (cts)
5 100 HS
(x106)
(x104)
84
4
50 IM
3 64
IM 50
2 44
Temperature (°C) Extraction mode Temperature (°C) Extraction mode
154
Parte Experimental y Resultados
________________________________________________________________
155
Parte Experimental y Resultados
Response (cts)
HS
14 9
(x105)
(x104)
9 6 HS
4 3
IM
0 0
50 °C 100 °C 50 °C 100 °C
Temperature Temperature
Response (cts)
4
9
(x105)
3
HS (x105) IM
6
2
1 3
0 0
50 °C 100 °C 50 °C 100 °C
Temperature Temperature
Response (cts)
HS 8
1.2
(x106)
(x105)
6
0.8
4 HS
0.4 2
IM
0 0
50 °C 100 °C 50 °C 100 °C
Temperature Temperature
156
Parte Experimental y Resultados
________________________________________________________________
157
Parte Experimental y Resultados
158
Tabla 2.5. Precisión, linealidad, límites de detección y de cuantificación del método SPME.
Precisión Intra-día (%RSD, n = 3) Precisión Inter-día (%RSD, n = 5) Linealidad LOD LOQ
Compuesto í1 í1 í1 í1 í1 í1 í1
1 ng mL 5 ng mL 20 ng mL 5 ng mL 10 ng mL 20 ng mL (R) (ng mL ) (ng mLí1)
Pineno 3.6 3.8 9.7 4 11 8.6 0.9963 0.10 0.35
Limoneno 1.7 4.2 4.1 6.2 5.1 9.7 0.9993 0.006 0.021
Alcohol bencílico 2.8 5.7 1.0 9.7 13 11 0.9974 0.19 0.63
Linalol 2.2 7.8 1.0 6.4 12 4.6 0.9990 0.009 0.031
Octanoato de 2-metilo 5.2 4.1 9.6 8.6 12 8.6 0.9960 0.13 0.42
Citronelol 4.2 5.4 0.8 8.7 7.9 6.5 0.9988 0.021 0.069
Citral 0.7 0.7 0.6 1.2 3.4 2.9 0.9982 0.042 0.14
Geraniol 3.5 8.9 0.7 6.6 5.3 2.5 0.9981 0.048 0.16
Cinamaldehído 2.1 4.7 1.0 7.7 12 4.3 0.9995 0.06 0.20
Hidroxicitronelal 8.9 5.7 3.6 4.3 11 3.7 0.9944 0.20 0.67
Anis alcohol n.a. 4.7 7.1 3.6 11 6.0 0.9961 0.94 3.1
Cinamil alcohol 7.3 3.8 5.9 5.3 5.4 7.5 0.9997 0.14 0.46
Eugenol 8.9 5.5 2.8 6.5 6.6 3.9 0.9994 0.012 0.040
Metileugenol 0.9 1.4 2.7 2.5 2.7 7.3 0.9995 0.003 0.010
Isoeugenol 12 4.7 3.0 6.3 0.6 2.4 0.9963 0.038 0.13
Cumarina 2.7 4.6 4.3 7.1 7.7 3.7 0.9991 0.19 0.63
Į-isometil ionona 1.9 3.8 0.9 7.0 2.5 0.8 0.9974 0.001 0.003
®
Lilial 2.4 5.3 1.6 4.8 4.8 3.0 0.9988 0.004 0.013
Amil cinamal 0.7 0.5 2.5 4.6 8.9 3.7 0.9998 0.001 0.004
®
Lyral n.a. 7.0 1.5 11 17 3.2 0.9998 1.1 3.6
Alcohol cinamílico 4.0 4.6 0.8 5.9 5.5 4.3 0.9965 0.055 0.18
Farnesol 10 4.5 1.4 7.1 6.3 1.9 0.9996 0.30 1.0
Hexilcinamaldehído 7.8 5.2 2.5 4 8.8 3.3 0.9991 0.002 0.007
Benzoato de bencilo 5.3 5.8 3.6 8.9 7.5 2.9 0.9989 0.003 0.010
Salicilato de bencilo 6.3 3.2 7.6 11 3.2 9.2 0.9983 0.009 0.029
Cinamato de bencilo 2.3 1.3 6.8 12 15 5.3 0.9993 0.008 0.025
Parte Experimental y Resultados
160
Parte Experimental y Resultados
________________________________________________________________
161
Parte Experimental y Resultados
162
Compuesto W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12 W13 W14 W15 W16 W17 W18 W19 W20 W21 W22
Tabla 2.7. Concentración de fragancias alérgenas en muestras de agua (ng mL-1) (continuación).
Compuesto W23 W24 W25 W26 W27 W28 W29 W30 W31 W32 W33 W34 W35
Pineno
Limoneno 0.03 0.03 0.86 0.16 0.23 0.078 0.36 0.23
Alcohol bencílico
Linalol 0.051 0.21
Octanoato de 2-metilo
Citronelol 0.044a 0.032a 0.64
Citral 0.19
Geraniol
Cinamaldehído
Hidroxicitronelal 0.68 1.80
Anis alcohol
Cinamil alcohol 1.1 0.49 1.1
Eugenol 0.051
Isoeugenol 0.19 0.74
Cumarina
Į-isometil ionona 0.003 0.004 0.031 0.032 0.36 0.032 0.024 0.10 0.20 0.19
®
Lilial 0.097 0.033 0.19 0.69 0.035 0.037 0.041 0.23 0.031 0.066 0.11 0.40 0.20
Amil cinamal 0.050 0.022
®
Lyral
Farnesol 0.33a
Hexilcinamaldehído 0.051 0.071 0.087 0.028
Benzoato de bencilo 0.070 0.033 0.074 0.18
Salicilato de bencilo
Cinamato de bencilo
Celdas vacías: compuestos no detectados (<LOD).
(a) Valores estimados (<LOQ)
Parte Experimental y Resultados
1 1
0
MCounts 4 m/z: 43.0+71.0+93.0
4
2
0
MCounts m/z: 41.0+67.0+69.0 8
6
5
0
kCounts 9 m/z: 77.0+103.0+131.0
100
50
0
MCounts 10 m/z: 43.0+59.0+71.0
1.00
0.50
0.00
kCounts 12 m/z: 78.0+91.0+92.0
400
200
0
MCounts m/z: 118.0+146.0+164.0
13
2.0 16
1.0
0.0
MCounts m/z: 93.0+121.0+136.0 17
0.50
0.00
MCounts m/z: 41.0+69.0
22
5
0
kCounts m/z: 129.0+216.0 23
100
50
0
MCounts m/z: 105.0+194.0 24
4
2
0
7 8 9 10 11 12 13 14 15 Minutes
165
Parte Experimental y Resultados
Bibliografía
Bassereau M., Chaintreau A., Duperrex S., Joulain D., Leijs H., Loesing
G., Owen N., Sherlock A., Schippa C., Thorel P.J., Vey M. (2007) GC-
MS Quantification of Suspected Volatile Allergens in Fragrances. 2.
Data Treatment Strategies and Method Performances, J Agric Food
Chem, 55, 25-31.
COUNCIL DIRECTIVE of 27 July 1976 on the approximation of the laws
of the Member States relating to cosmetic products 76/768/EEC.
Dunn M.S., Vulic N., Shellie R.A., Whitehead S., Morrison P., Marriott
P.J (2006) Targeted multidimensional gas chromatography for the
quantitative analysis of suspected allergens in fragrance products, J
Chromatogr A, 1130, 122-129.
EU (2009) Regulation (EC) No. 1223/2009 of the European Parliament
and of the Council of 30 November 2009 on Cosmetic Products
(recast). Official Journal of European Union L342, 59.
García-Jares C., Llompart M., Polo M., Salgado C., Macías S., Cela R.
(2002) Optimisation of a solid-phase microextraction method for
synthetic musk compounds in water, J Chromatogr A, 963, 277-285.
Johnson J.D., Ryan M.J., Toft J.D., Graves S.W., Hejtmancik M.R.,
Cunningham M.L., Herbert R., Abdo K.M. (2000) Two-Year Toxicity and
Carcinogenicity Study of Methyleugenol in F344/N Rats and B6C3F1
Mice, J Agric Food Chem, 48, 3620-3632.
Lamas J.P., Sanchez-Prado L., Garcia-Jares C., Llompart M. (2009)
Solid-phase microextraction gas chromatography–mass spectrometry
determination of fragrance allergens in baby bathwater, Anal Bioanal
Chem, 394, 1399–1411.
Leijs H., Broekhans J., van Pelt L., Mussinan C. (2005) Quantitative
Analysis of the 26 Allergens for Cosmetic Labeling in Fragrance Raw
Materials and Perfume Oils, J Agric Food Chem, 53, 5487-5491.
Masuck I., Hutzler C., Luch A. (2010) Investigations on the emission of
fragrance allergens from scented toys by means of headspace solid-
phase microextraction gas chromatography-mass spectrometry, J
Chromatogr A, 1217, 3136-3143.
Mondello L., Casilli A., Tranchida P.Q., Sciarrone D., Dugo P., Dugo G.
(2008) Analysis of Allergens in Fragrances using Multiple Heart-cut
Multidimensional Gas Chromatography–Mass Spectrometry, LC-GC
Europe, 21, 130-140.
166
Parte Experimental y Resultados
167
Parte Experimental y Resultados
168
Parte Experimental y Resultados
169
Parte Experimental y Resultados
170
Parte Experimental y Resultados
171
Parte Experimental y Resultados
3.2.2 Patrones
3.2.3 Muestras
173
Parte Experimental y Resultados
174
Parte Experimental y Resultados
La Tabla 3.3 resume los tiempos de retención, así como los iones de
identificación y cuantificación de las fragancias.
175
Parte Experimental y Resultados
176
Parte Experimental y Resultados
177
Parte Experimental y Resultados
1.5000
A/A IS
1.0000
0.5000
0.0000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
178
Parte Experimental y Resultados
179
Parte Experimental y Resultados
180
Tabla 3.5. Relaciones F y valores de p obtenidos en el análisis de varianza.
Parte Experimental y Resultados
182
Parte Experimental y Resultados
183
Parte Experimental y Resultados
a
Limonene Benzyl alcohol
1.21 0.04 CHCl3 20
0
1.11 0.03
A/AIS
2 C2 H3 Cl3 25 50
1.01
A/AIS
2
0.02 10
0.91 25
CHCl3 50
10 0.01
0.81
20 0
C2 H3 Cl3
0.71 0
Citral Lyral
0.96 C2 H3 Cl3 0.25
CHCl3 20
0.23
20
0.86 25
10 50
A/AIS
2 0.21
A/AIS
2
50 25
10 0.19
0.76 0
0.17 0
CHCl3 C2 H3 Cl3
0.66 0.15
Time Solvent Tempera ture NaCl Time Solvent Tempera ture NaCl
(min) (°C) (%) (min) (°C) (%)
184
Parte Experimental y Resultados
A/AIS
A/AIS
0.81 0.12
0.77 0.08
0.73 0 % NaCl 0.04 0 % NaCl
0.69 0.00
CHCl3 C2 H 3 Cl3 CHCl3 C2 H 3 Cl3
185
Parte Experimental y Resultados
186
Parte Experimental y Resultados
187
Tabla 3.6. Linealidad, límites de detección (LODs) y recuperación del método USAEME.
Parte Experimental y Resultados
189
Tabla 3.7. Presencia de fragancias alérgenas en diferentes tipos de agua (ng mL-1).
Compuesto ABB1 ABB2 ABB3 ABB4 ABB5 ABB6 ABB7 ABB8 ABB9 ABB10 ABB11 ABB12 ABB13 LPR1 LPR2 LPR3 PCI1 PCI2 PCI3 PCI4 SPAA EDAR
a
Limoneno 0.620 2.64 128 0.380 4.78 1.15 2.22 7.75 4.23 5.95 3.37 7.75 18.9 0.550 0.765 4.98
Linalol 19.9 76.3 23 9.57 87.4 55.2 32.8 4.15 26.1 30.2 204 0.251
Alcohol bencílicoa 6.55 3.95 2.74 2.83 3385
Citronelol 91.8 7.59 5.23 1.69 4.29 1.82 51.1 0.987
Citral 2.11 5.33 0.190
Geraniol 23.4 9.25 11.5 2.5 2.18 5.67 11.4 21.6
Hidroxicitronelal 4.66 0.444 0.721
Cinamil alcohol 1.02
Eugenol 1.77 6.02 0.225 0.829 0.706 4.25 6.93 0.093 0.017 0.180 3.75
Metileugenol 0.146
Į-isometil ionona 2.48 4.39 >LOD >LOD 0.121
Isoeugenol 4.55
Lilial® 0.133 0.462 0.040 0.462 0.385 0.378 0.759 0.417 0.04 0.052 0.053 0.630 0.133 0.183 0.165 0.995
Lyral® 0.407
Cumarina 0.200 23.2 0.500 0.046 11.5 0.990 0.232 43.4 0.148
Hexilcinamaldehído 1.13 0.364 2.13 0.34 0.090 0.101 >LOD
Benzoato de
bencilo 0.086 2.08 0.364 0.861 8.94 0.247 0.177 >LOD 0.093 0.215 0.077 0.058
Salicilato de
bencilo 0.840 0.089 0.894 0.233 0.979 0.300 0.289 0.556 0.092
Cinamato de
bencilo 2.7
Los espacios en blanco indican valores medios por debajo de LOD
a
Los valores fueron corregidos teniendo en cuenta la eficiencia de extracción promedio
Parte Experimental y Resultados
Bibliografía
Bassereau M., Chaintreau A., Duperrex S., Joulain D., Leijs H., Loesing
G., Owen N., Sherlock A., Schippa C., Thorel P.J., Vey M. (2007) GC–
MS quantification of suspected volatile allergens in fragrances. 2. Data
treatment strategies and method performances, J Agric Food Chem, 55,
25-31.
Bossio J.P., Harry J., Kinney C.A. (2008) Application of ultrasonic
assisted extraction of chemically diverse organic compounds from soils
and sediments, Chemosphere, 70, 858–864.
Capelo J.L., Mota A.M. (2005) Ultrasonication for analytical chemistry,
Cur Anal Chem, 1, 193–201.
Chunhong J., Xiaodan Z., Li C., Min H., Pingzhong Y., Ercheng Z.
(2010) Extraction of organophosphorus pesticides in water and juice
using ultrasound-assisted emulsification-mixroextraction, J Sep Sci, 33,
244–250.
EU (2009) Regulation (EC) No. 1223/2009 of the European Parliament
and of the Council of 30 November 2009 on Cosmetic Products
(recast). Official Journal of European Union, 59.
Fontana A.R., Altamirano J.C. (2010) Sensitive determination of 2,4,6-
trichloroanisole in water samples by ultrasound assisted emulsification
microextraction prior to gas chromatography–tandem mass
spectrometry analysis, Talanta, 81, 1536–1541.
Fontana A.R., Wuilloud R.G., Martínez L.D., Altamirano J.C. (2009)
Simple approach based on ultrasound-assisted emulsification–
microextraction for determination of polibrominated flame retardants in
water samples by gas chromatography–mass spectrometry, J
Chromatogr A, 1216, 147–153.
IFRA International Fragrance Association.
<http://www.ifraorg.org/Home/page.aspx/3> (consultado junio 2010).
Lamas J.P., Sanchez-Prado L., Garcia-Jares C., Llompart M. (2010)
Determination of fragrance allergens in indoor air by active sampling
followed by ultrasound-assisted solvent extraction and gas
chromatography-mass spectrometry, J Chromatogr A, 1217, 1882–
1890.
Lamas J.P., Sanchez-Prado L., Garcia-Jares C., Llompart M. (2009)
Solid-phase microextraction gas chromatography–mass spectrometry
determination of fragrance allergens in baby bathwater, Anal Bioanal
Chem, 394, 1399–1411.
________________________________________________________________
191
Parte Experimental y Resultados
Leijs H., Broekhans J., van Pelt L., Mussinan C. (2005) Quantitative
analysis of the 26 allergens for cosmetic labeling in fragrance raw
materials and perfume oils, J Agric Food Chem, 53, 5487–5491.
Ma J.J., Du X., Zhang J.W., Li J.C., Wang L.Z. (2009) Ultrasound-
assisted emulsification–microextraction combined with flame atomic
absorption spectrometry for determination of trace cadmium in water
samples, Talanta, 80, 980-984.
Niederer M., Bollhalder R., Hohl C. (2006) Determination of fragrance
allergens in cosmetics by size-exclusion chromatography followed by
gas chromatography-mass spectrometry, J Chromatogr A, 1132, 109–
116.
Ozcan S., Tor A., Aydin M.E. (2009a) Application of ultrasound-assisted
emulsification–micro-extraction for the analysis of organochlorine
pesticides in waters, Water Res, 43, 4269–4277.
Ozcan S., Tor A., Aydin M.E. (2009b) Determination of selected
polychlorinated biphenyls in water samples by ultrasound-assisted
emulsification–microextraction and gas chromatography-mass-selective
detection, Anal Chim Acta, 647, 182–188.
Pumure I., Renton J.J., Smart R.B. (2010) Ultrasonic extraction of
arsenic and selenium from rocks associated with mountaintop
removal/valley fills coal mining: estimation of bioaccessible
concentrations, Chemosphere, 78, 1295–1300.
Rastogi S.C., Johansen J.D., Menné T., Frosch P., Bruze M., Andersen
K.E., Lepoittevin J.P., Wakelin S., White I.R. (1999) Contents of
fragrance allergens in children’s cosmetics and cosmetic-toys, Contact
Dermatitis, 41, 84–88.
Regueiro J., Llompart M., Garcia-Jares C., Garcia-Monteagudo J.C.,
Cela R., (2008) Ultrasound-assisted emulsification–microextraction of
emergent contaminants and pesticides in environmental waters, J
Chromatogr A, 1190, 27-38.
Regueiro J., Llompart M., Psillakis E., Garcia-Monteagudo J.C., Garcia-
Jares C. (2009) Ultrasound-assisted emulsification–microextraction of
phenolic preservatives in water, Talanta, 79, 1387–1397.
SCCNFP (1999), SCCNFP/0017/98 (1999) Final Fragrance Allergy in
Consumers Brussels.
Sgorbini B., Ruosi M.R., Cordero C., Liberto E., Rubiolo P., Bicchi C.
(2010) Quantitative determination of some volatile suspected allergens
in cosmetic creams spread on skin by direct contact sorptive tape
extraction-gas chromatography–mass spectrometry, J Chromatogr A,
1217, 2599-2605.
192
Parte Experimental y Resultados
________________________________________________________________
193
Parte Experimental y Resultados
194
Parte Experimental y Resultados
________________________________________________________________
195
Parte Experimental y Resultados
196
Parte Experimental y Resultados
Compuesto Proveedor
4.2.2 Patrones
4.2.3 Muestras
________________________________________________________________
197
Parte Experimental y Resultados
198
Parte Experimental y Resultados
4.2.5 Cloración
Tiempo Energía
Ión
de de Iones de identificación y
Compuesto Precursor q
retención colisión cuantificación (m/z)
(m/z)
(min) (V)
84.1 + 91.1 + 108.1 +
Benzotiazol (BTZ) 6.92 135.1 1.3 0.225
135.1
4-tert-Butilfenol(TBP) 8.93 135.1 1 0.225 95.2 + 107.1 + 135.1
2-fenilfenol (PP) 11.73 170.1 1.2 0.225 142.2 + 170. 1
Butóxido de Piperonilo 118.1 + 146.1 + 161.1 +
19.31 176.1 1.1 0.225
(PBO) 176.1
PCB-30 (IS) 13.33 186.1 1.4 0.3 151.2 + 186.1
200
Parte Experimental y Resultados
________________________________________________________________
201
Parte Experimental y Resultados
202
Parte Experimental y Resultados
Nivel
Factor Clave
Bajo Intermedio Alto
NaCl (%) A 0 15 30
Tiempo (min) B 5 10
Tipo de disolvente C Cloroformo (CLF) 1,1,1-Tricloroetano (TCE)
Reactivos derivatizantes D No Si
________________________________________________________________
203
Parte Experimental y Resultados
204
Parte Experimental y Resultados
BTZ TBP
A AD
BC D
C BC
A
CD
AC
AC C
B CD
D B
0 2 4 6 8 10 0 2 4 6 8 10
Standardized effects Standardized effects
PP PBO
D D
AD AA
A BC
AC
BC
B
BD CD
C A
B C
0 4 8 12 16 0 2 4 6 8 10
Standardized effects Standardized effects
BTZ TBP
4100 6000 Yes
30%
3800 5500 0%
TCE
10 3 *A/AIS
TCE
10 3 *A/AIS
3500 10 5000 10
Yes
5
3200 No 4500
5 CLF
CLF 30%
2900 4000
0% No
2600 3500
NaCl Time (min) Solvent Derivatizing NaCl Time (min) Solvent Derivatizing
reagents reagents
PP PBO
2400 2700
Yes Yes
2000 0% 2400 10
10 3 *A/AIS
TCE
10 3 *A/AIS
15%
1600 5 CLF TCE 2100 5
1200 10 CLF
1800
800 30% No
400 1500
No 30%
0%
0 1200
NaCl Time (min) Solvent Derivatizing NaCl Time (min) Solvent Derivatizing
reagents reagents
206
Parte Experimental y Resultados
208
Parte Experimental y Resultados
________________________________________________________________
209
Parte Experimental y Resultados
210
Parte Experimental y Resultados
________________________________________________________________
211
Parte Experimental y Resultados
0h 1h 5h
120
100
80
100xRt /R0
60
40
20
0
BTZ TBP PP PBO
120
100
Chlorine concentration:
80 0 mg/L
100xRC/R0
0.2 mg/L
60
1.5 mg/L
40 4 mg/L
20
0
TBP PP
212
Parte Experimental y Resultados
fue ligeramente más rápida que la del TBP. Estas reacciones siguen
una cinética de reacción de pseudo-primer orden. Las constantes de
velocidad de primer orden (kap) se pueden determinar como las
pendientes de las ecuaciones lineales obtenidas a partir de la regresión
lineal: lnC vs tiempo. El tiempo de vida media correspondiente a cada
compuesto se calcula utilizando la expresión siguiente: t1/2 = ln 2/kap.
Estos datos se incluyen en la Figura 4.4.
TBP
100
PP
90
80
70 R kap (h -1 ) t1/2 (h)
TBP 0.998 1.17 0.59
100xRt /R0
60
PP 0.989 1.93 0.36
50
40
30
20
10
0
0 5 10 15 20
Time (h)
________________________________________________________________
213
Parte Experimental y Resultados
Relative abundance
Counts m/z: 169.00
50
2000
141 184
Monochloro-TBP 51 63 77 89 96 105 114 129 156
1600 0
169
100 NIST MS Search 2.0 spectrum:
Relative abundance
Phenol, 2-chloro-4-(1,1-dimethylethyl)-
1200 (Match: 88%)
800 50
141 184
51 77 129
400 0
63 89 97 105 114 153
50 70 90 110 130 150 170 190
0
100 Experimental spectrum 203
Relative abundance
Counts m/z: 203.00
50
8000 Dichloro-TBP 175 218
51 63 77 89 103 123 139 150 163 191
6000 0
NIST MS Search 2.0 spectrum: 203
100
Relative abundance
Phenol, 2,6-dichloro-4-(1,1-dimethylethyl)-
(Match: 97%)
4000
50
175 218
2000 63 77 89 103
50 123 139 152 163 187
0
60 80 100 120 140 160 180 200 220
0
100 Experimental spectrum 204
Relative abundance
Counts m/z: 204.00 Monochloro-PP
1000
50
139
168
800 57 70 115
82 97 149 179 189
0
204
100 NIST MS Search 2.0 spectrum:
Relative abundance
0
100 Experimental spectrum 238
Relative abundance
168 202
8000 50
69 87 101 149 183 223
0
238
100 NIST MS Search 2.0 spectrum:
6000
Relative abundance
(1,1’-Biphenyl)-2-ol, 3,5-dichloro
(Match: 89%)
4000 50 139
168
214
Parte Experimental y Resultados
________________________________________________________________
215
Parte Experimental y Resultados
a
MonochloroTBP
DichloroTBP
100
80
100xRt /Rmax
60
40
20
0
0 5 10 15 20
Time (h)
b
MonochloroPP1
MonochloroPP2
100 DichloroPP
80
100xRt /Rmax
60
40
20
0
0 5 10 15 20
Time (h)
216
Parte Experimental y Resultados
Bibliografía
218
Parte Experimental y Resultados
________________________________________________________________
219
IV. Conclusiones
Conclusiones
IV. Conclusiones
223
Conclusiones
224
Conclusiones
225
Conclusiones
226
Conclusiones
227
ANEXO I
Anexo I: Abreviaturas y Acrónimos
BSTFA N,O-
bis(Trimetilsilil)trifluoroacetamida
BTZ Benzotiazol
CE Comisión Europea
231
Anexo I: Abreviaturas y Acrónimos
DEHP Bis-2-etilhexilfatalato
DF Distrito Federal
ETAAS Espectrometría de
absorción atómica
electrotérmica
EE2 17Į-Etinilestradiol
232
Anexo I: Abreviaturas y Acrónimos
FAAS Espectrometría de
absorción atómica de
llama
233
Anexo I: Abreviaturas y Acrónimos
234
Anexo I: Abreviaturas y Acrónimos
sustancias químicas
LC-UV
235
Anexo I: Abreviaturas y Acrónimos
min minutos
MTBSTFA N-tert-butildimetilsilil-N-
metiltri-fluoroacetamida
NOAEL No Observed
Adverse Effect Level
236
Anexo I: Abreviaturas y Acrónimos
PA Polyacrylate Poliacrilato
PP 2-Fenilfenol
PTBP-FR p-tert-Butilfenol-
formaldehído
237
Anexo I: Abreviaturas y Acrónimos
TBP 4-tert-Butilfenol
UV UltraViolet Ultravioleta
238
Anexo I: Abreviaturas y Acrónimos
239
ANEXO II
Anexo II: Listado de publicaciones
243
Anexo II: Listado de publicaciones
Otras publicaciones
244
Author's personal copy
Abstract
Although the trend in development of analytical methods for emerging contaminants is towards reduced sample preparation and increased
detector selectivity, there are still benefits from removal of matrix material during sample preparation. This paper describes a simple method
for acidic pharmaceuticals and a range of potential endocrine disrupting compounds in untreated wastewaters and spring waters. It is based on
separation of the two classes during elution from the extraction cartridge with final analysis by gas chromatography–mass spectrometry. 3,4-D
was used as the recovery standard for the acids while 4-n-nonylphenol and [2 H4 ]estrone were used for the endocrine disrupters; mean recoveries
varied between 89% and 111%. The method was also extensively validated by fortification with the target compounds. Recoveries of acids were
from 68% to 97% with relative standard deviations generally less than 10% and recoveries of endocrine disrupters were 68–109% with relative
standard deviations less than 20%. Detection limits varied from 0.005 to 1 ng/L in spring water, and from 0.5 to 100 ng/L in untreated wastewater.
Concentrations of the analytes in the wastewater ranged from 0.018 to 22.4 g/L. Values were comparable to reported data, although concentrations
were generally relatively high, probably because of a lack of treatment. Triclosan, phthalates, estrone, 17-estradiol, ibuprofen, and naproxen were
present in the spring water from aquifers recharged indirectly with this wastewater after its use for irrigation; concentrations ranged from 0.01 to
25.0 ng/L. The much lower concentrations compared to wastewater indicate effective removal processes on passage through the soil and subsoil.
© 2007 Elsevier B.V. All rights reserved.
0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2007.08.056
Author's personal copy
Table 1
Details of the use of the acids, characteristic ions, and retention times using the chromatographic conditions described
Compound Use Retention time (min) Characteristic ions (m/z)
Clofibric acid Metabolite of lipid regulator clofibrate 7.78 143 243 271
Ibuprofen Analgesic and anti-inflammatory 7.97 263 264
Salicylic acid Metabolite of the analgesic aspirin 8.55 195 309 310
2,4-D Pesticide 8.64 219 277 279
Gemfibrozil Lipid regulator 9.60 179 243 307
Naproxen Analgesic and anti-inflammatory 10.22 185 287 344
Ketoprofen Analgesic and anti-inflammatory 10.90 295 311 312
Diclofenac Antophlogistic 11.52 214 352 354
3,4-D Recovery standard 8.77 251 277 279
2,3-D Internal standard 8.88 219 277 279
and the powerful selectivity of MS–MS detection allows simple dard [2 H16 ]bisphenol-A were available from Sigma–Aldrich
preparation methods for difficult matrices such as wastewaters (St. Louis, MO, USA). 3,4-Dichlorophenoxyacetic acid
[8,9]. However, this type of instrument is still not commonly (3,4-D) and 2,3-dichlorophenoxyacetic acid (2,3-D) were
available and there are opportunities for improvements in prepa- purchased from Riedel-de Haën (Seelze, Germany). The
ration that produce cleaner samples that can then be analyzed deuterated estrogens [2 H4 ]estrone, [2 H4 ]17-estradiol and
by techniques such as gas chromatography–mass spectrometry [2 H4 ]17␣-ethinylestradiol were all obtained from Cambridge
(GC–MS). Cleaner samples also have the advantage of reduc- Isoptopes (Andover, MA, USA). The derivatization agents
ing suppression of response, especially in LC–MS systems and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTB-
increasing the number of injections possible between routine STFA) with 1% tert-butyldimethylsilylchlorane (TBDMSCI)
maintenance. An important consideration is that extra clean-up and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with
steps, which complicate preparation and add time and costs, 1% trimethylsilylchlorane (TMSCI) were also purchased from
should be avoided where possible. This paper describes the Sigma–Aldrich. All solvents used were HPLC grade, supplied
development of such a method for the analysis of acidic pharma- by Burdick and Jackson (Morristown, NJ, USA). Oasis HLB
ceuticals, potential endocrine disrupting compounds, and two extraction cartridges were purchased from Waters (Milford, MA,
pesticides in untreated wastewater and spring waters, using a USA).
simple extraction procedure with final analysis by GC–MS.
2.2. Sampling
2. Experimental
The metropolitan zone of Mexico City covers approximately
2.1. Chemicals and reagents 1450 km2 and has over 19 million inhabitants and tens of thou-
sands of industrial units. All of the domestic and industrial
All of the target acidic compounds, pesticides, and poten- wastewater, rainwater, and run-off from this area is mixed and
tial endocrine disrupters (listed in Tables 1 and 2) as well sent via three drainage channels, the Emisor Central, the Inter-
as the recovery standard 4-n-nonylphenol and internal stan- ceptor Poniente, and the Gran Canal, directly to the irrigation
Table 2
Details of the use of the potential endocrine disrupters, characteristic ions, and retention times using the chromatographic conditions described
region in the state of Hidalgo 70 km north of the city. The aver- tion with either acetone or methanol generated a sample where
age flow rate is about 50 m3 /s and there is no treatment process. an excess of matrix material caused interferences that made
Wastewater samples were taken in February and March 2007 analysis by GC–MS for some of the target compounds diffi-
from the Emisor Central, and on the same day water was col- cult because of the presence of interfering organic compounds.
lected from the Cerro Colorado spring, which first surfaced In addition it was found that the same silylating reagent was
approximately 30 years ago (locations are shown in Fig. 1). not compatible for derivatization of acids and the other ana-
Methanol (2%, v/v) was added and the samples transported sur- lytes. Using this information, the method was developed with
rounded by icepacks to the laboratory where they were stored at separation of the acids from the rest of the analytes in mind.
4 ◦ C and extracted within 24 h. Method development focussed on optimising the separation of
the acids, finding the appropriate solvent to elute the remaining
2.3. Sample preparation and method validation strategy analytes and choosing the silylating agent and conditions for
derivatization. The method was validated by fortification of the
Initial work demonstrated that simple extraction of the acids at 10, 25, and 100 ng/L in spring water, and at 10, 25, and
wastewater using a C18 or Oasis HLB cartridge followed by elu- 50 g/L in wastewater. The endocrine disrupters were fortified
The acid fraction was evaporated for 15 min under a stream 3.1. Sample preparation procedure
of nitrogen to remove a proportion of the acetone present, acid-
ified to below pH 2 with sulphuric acid, and the acids extracted The Oasis HLB cartridge is a general purpose adsorbent used
into ethyl acetate (2× 2 mL). Remnants of water were removed to isolate a wide range of organic compounds from aqueous
with anhydrous sodium sulphate, then 2,3-D added as an internal samples. The adjustment to pH 2 with sulphuric acid assured that
Author's personal copy
the acids in particular were protonated for efficient adsorption. was much cleaner after a prior buffer:acetone elution (Fig. 3).
Many extraction methods for organic compounds using this type Experiments with standards showed that it was then possible to
of adsorbent neglect to include a step to elute interferences and elute the endocrine disrupters with either ethyl acetate or ace-
do not attempt separate elution of different classes of analytes. tone, the former producing a cleaner final sample. It is of note
Although this has the advantage of simplicity, the disadvantage is that methanol, a solvent commonly used to elute these classes
the presence of more matrix material in the sample for injection of compounds from these cartridges, recovered below 90% of
in the chromatographic system. This can result in interfering several of the analytes and less than 70% of 4-nonylphenols.
peaks in the chromatogram, a more rapid deterioration in peak Acetone was finally chosen as the elution solvent because in the
shape caused by contamination in the inlet system or GC column, presence of the matrix material from untreated wastewaters less
or a faster reduction in sensitivity due to contamination of the than 50% of bisphenol-A and 4-nonylphenols were recovered
ion source of the detector. with ethyl acetate. This underlines that the behaviour of com-
Some of the samples in the study reported here were untreated pounds in cartridges (or on any other chromatography material)
wastewaters, heavily loaded with organic material. Simple can be profoundly affected by the presence of matrix material
extraction using an Oasis HLB cartridge followed by elution and development work should include work with real samples,
with acetone and analysis by GC–MS produced chromatograms not just standards. This difference may provide an explanation
at times difficult to interpret due to an excess of interferences. for a recent report of low recoveries of 4-nonylphenols from
In cases such as this, a time consuming extra clean-up step may different waters [10].
be added but it was decided to take advantage of the opportunity Although there have been many investigations of the opti-
to manipulate the sample on the cartridge, a step with little cost mum amount of reagents to use for different derivatization
in time or solvent use. Organic acids were eluted first using a processes (e.g. [11]), how these volumes affect detection limits
mixture of 0.10 M sodium hydrogencarbonate buffer at pH 10 are often not considered. For this study, low volumes of solvent
and acetone (60:40). Conversion of the acids to their ionic form and derivatizing agents were investigated (10–100 L) in order
with buffer at pH 10 was not sufficient to completely overcome to achieve detection limits relevant to environmental concen-
adsorption to the co-polymer and so a co-solvent was required. trations, particularly for spring water. Consistent results were
Preliminary studies showed that methanol was not suitable as obtained at the final volumes chosen and this approach was used
only partial elution of some acids occurred whereas 40% ace- in preference to evaporation after derivatization which risked
tone allowed recovery of all of each acid without any danger losses from volatilization. The derivatives produced by reaction
of eluting the other analytes. This acid fraction eluted most of of acidic pharmaceuticals with MTBSTFA produced better peak
the organic material (estimated by response in scan mode in the shapes and improved sensitivity in comparison to derivatization
GC–MS system), much of which was subsequently eliminated with BSTFA. Both derivatization methods were generally suit-
by the extraction step into ethyl acetate. able for the phenols and estrone, but trimethylsilyl derivatives of
The acid elution acted as an effective clean-up for the 17-estradiol and 17␣-ethinylestradiol (EE2) were more sensi-
endocrine disrupters that remained on the cartridge, demon- tive than their N-tert-butyldimethylsilyl counterparts. In addition
strated by a comparison in scan mode where the acetone fraction there is evidence that EE2 partially breaks down to estrone dur-
Fig. 3. Comparison of chromatograms (scan mode) of acetone elution (A) with prior acetone:buffer elution and (B) without prior acetone:buffer elution.
Author's personal copy
ing derivatization with MTBSTFA [12]. Hence the separation the matrix material present. The acid fraction was less clean and
not only produced a much cleaner sample of the endocrine limits were dictated partly by background response of the matrix
disrupters but permitted the flexibility to use a different deriva- material in the samples. Untreated wastewaters are much more
tization procedure to optimize response in the GC–MS system. challenging to analyze. The LODs for the acids varied between
Typical chromatography of standards are shown in Figs. 4 and 5. 5 and 100 ng/L, for the phenols between 10 and 50 ng/L, was
50 ng/L for the phthalates, and varied from 0.5 to 2.5 ng/L for
3.2. Linear correlation coefficients and limits of detection the estrogens. Limits were mostly determined by the smaller
amount of water processed and the background generated by
The linearity of the instrument was acceptable for all analytes the organic material.
with correlation coefficients always greater than 0.99 in the con- In general, LODs compare well with literature values for
centration range of the study (Table 3). The limit of detection ground, surface and wastewaters [13,14]. The simple elution
(LOD) of the method was defined as three times background with buffer:acetone is key to avoiding further clean-up of the
(signal-to-noise ratio of 3) and any peak above the LOD was endocrine disrupter fraction and allows the use of the GC–MS
quantified (Table 3). LODs for the acids in spring water were for analysis of difficult samples like untreated wastewater at
between 0.25 and 1.0 ng/L, from 0.1 to 1.0 ng/L for the phenols, environmentally relevant LODs. For example the LODs for the
0.5 ng/L for phthalates, and between 0.005 and 0.05 ng/L for the estrogens were lower in this study for untreated wastewaters than
estrogens. These waters are not overly burdened with organic reported recently for surface waters [12], lower than achieved
compounds and the limits for the phenols, phthalates and par- in a method for treated wastewaters that additionally used a
ticularly the estrogens were dictated more by the sensitivity of column clean-up [15] and comparable to that achieved using
the analytical instrument and the volume of water taken, less by GC–MS–MS for final analysis [16].
Table 3 Table 5
Limits of detection for the instrument (ng injected), spring water (ng/L) and Recovery data (% ±RSD) for target endocrine disrupting compounds (n = 3)
wastewater (ng/L), and linear correlation coefficients (R2 )
Analyte Spring water Wastewater
Analyte LOD R2 fortificationa fortificationb
IN SW WW (low) (high) (low) (high)
Table 4
Recovery data (% ±RSD) for target acidic compounds (n = 3)
Analyte Spring water fortification (ng/L) Wastewater fortification (g/L)
Clofibric acid 82 ± 5 92 ± 6 92 ± 8 68 ± 2 70 ± 2 77 ± 2
Ibuprofen 96 ± 3 97 ± 5 88 ± 8 76 ± 4 78 ± 3 85 ± 2
Salicylic acid 293 ± 39 139 ± 47 107 ± 63 114 ± 9 90 ± 8 113 ± 4
2,4-D 91 ± 5 96 ± 6 88 ± 7 81 ± 4 78 ± 3 81 ± 3
Gemfibrozil 82 ± 4 91 ± 3 87 ± 6 82 ± 3 77 ± 2 83 ± 2
Naproxen 90 ± 16 90 ± 8 83 ± 4 74 ± 7 73 ± 3 81 ± 3
Ketoprofen 78 ± 2 96 ± 9 86 ± 3 80 ± 3 76 ± 2 80 ± 2
Diclofenac 76 ± 4 87 ± 8 81 ± 2 84 ± 4 80 ± 3 94 ± 2
Author's personal copy
Spring water
Blank samples were included in each batch processed to
Recovery (%) 111 96 104 assess contamination. Initial conditioning of the cartridges with
RSD (%) 13 12 17 acetone was important to remove general contamination and 4-
n 15 15 15 nonylphenols in particular. For the acids, naproxen and salicylic
Wastewater acid were consistently present in concentrations above the LOD
Recovery(%) 89 95 94 (Table 7). The expected contamination of blank samples with 4-
RSD (%) 5 10 17 nonylphenols and phthalates, most likely from solvents and the
n 15 15 15
cartridge material, was controlled by consistent procedure. Con-
tributions of concentrations in the blanks, relevant to the spring
water but negligible compared to concentrations in the wastew-
and therefore that the method was unsuitable for PCP in wastew- ater, were accounted for by subtraction in the calculation of all
ater. This is a further indication of the differences that can occur results.
between method development with standards and real samples. As part of the validation procedure the concentrations of the
There were a wide range of compounds in the endocrine dis- analytes were measured on two occasions in the wastewater and
rupter fraction and experiments showed that there were some spring water (Table 7). All of the acids except clofibric acid
minor differences in the extent of derivatization between com- were detected in the wastewaters. Concentrations were gener-
pounds, dependent on the presence or absence of matrix material. ally within the ranges reported for influents but greater than
Although these differences were not great and the two internal reported for effluents [17,18], although values for naproxen, an
standards used were considered adequate, the result was greater analgesic widely used in Mexico, were greater than reported data
variability in results and relative standard deviations that were and salicylic acid was very variable. 4-Nonylphenols (22.4 and
acceptable but generally a little greater than for the acids. 11.0 g/L) and DEHP (702 and 356 g/L) were the most promi-
3,4-D, 4-n-nonylphenol, and [2 H4 ]estrone were proposed as nent potential endocrine disrupters in the wastewater. Mean
recovery standards to be added to all samples during future concentrations of triclosan, bisphenol-A, and BuBeP varied
routine analysis and they were also added to the validation between 0.66 and 2.50 g/L while the concentrations of estrone
samples and recoveries determined (Table 6). Mean recover- and 17-estradiol were 80 and 44 ng/L, and 22 and 17 ng/L,
ies of 3,4-D, the recovery standard for the acids, were 111% in respectively. Again these values generally fall within the ranges
spring water and 89% in wastewater. Mean recoveries of 4-n- reported for wastewaters [18,19]. It was noted that the concen-
nonylphenol were 96% and 95%, respectively, while recoveries tration of the potential endocrine disrupting chemicals in the
of [2 H4 ]estrone were 104% and 94%, respectively. Although the second sampling of wastewater were approximately half that of
recoveries were generally slightly greater than for most of the the first. First thoughts suggested a dilution of the wastewater
analytes it was concluded that these compounds were suitable by unseasonal rainfall in the city but the concentrations of acids
recovery standards for the procedure. were similar in the two samples except for diclofenac, suggest-
Table 7
Concentrations of acidic pharmaceuticals and potential endocrine disrupters in blanks, spring water and wastewater
Analyte Blanksa (ng/L) Wastewater (g/L) Spring water (ng/L)
ing some other influence. A more detailed study of the temporal government for funding this project (No. SEMARNAT-2002-
variations in concentrations in this wastewater would be needed 01-0519).
to attempt an adequate explanation.
Ibuprofen, naproxen, salicylic acid, triclosan, bisphenol-A, References
and both phthalates were detected in the spring water but at
concentrations several orders of magnitude lower than in the [1] M.D. Hernando, M. Mezcua, A.R. Fernández-Alba, D. Barceló, Talanta 69
wastewater, indicating efficient removal processes on passage (2006) 334.
[2] J.P. Sumpter, Toxicol. Lett. 102–103 (1998) 337.
through the soil. Estrone (0.17 and 0.16 ng/L) and 17-estradiol [3] K. Fent, A.A. Weston, D. Caminada, Aquat. Toxicol. 76 (2006) 122.
(0.02 and 0.01 ng/L) were also present, again at concentrations [4] G.W. Miller, Desalination 187 (2006) 65.
much less than in the wastewater. Trace concentrations of many [5] B. Jiménez-Cisneros, A. Chávez-Mejı́a, Environ. Technol. 78 (1997)
classes of emerging contaminants have been reported in ground- 721.
[6] M. Salgot, E. Huertas, S. Weber, W. Dott, J. Hollender, Desalination 187
water [20] although in two studies involving irrigation with
(2006) 29.
wastewaters most potential contaminants, including some of the [7] B. Jiménez, A. Chávez, Water Sci. Technol. 50 (2004) 269.
above, were below the LOD [21,22]. [8] M. Petrovic, M.D. Hernando, M. Silvia Dı́az-Cruz, D. Barceló, J. Chro-
matogr. A 1067 (2005) 1.
4. Conclusions [9] C. Hao, X. Zhao, P. Yang, Trends Anal. Chem. 26 (2007) 569.
[10] G. Gatidou, N.S. Thomaidis, A.S. Stasinakis, T.D. Lekkas, J. Chromatogr.
A 1138 (2007) 32.
An analytical method was developed for acidic pharma- [11] I. Rodrı́guez, J.B. Quintana, J. Carpinteiro, A.M. Carro, R.A. Lorenzo, R.
ceuticals, potential endocrine disrupters and two pesticides in Cela, J. Chromatogr. A 985 (2003) 265.
untreated wastewaters and spring waters. The method is robust, [12] Z. Yu, S. Peldszus, P.M. Huck, J. Chromatogr. A 1148 (2007) 65.
has practical limits of detection, and can be used to analyze [13] T. Ternes, Trends Anal. Chem. 20 (2001) 419.
[14] D. Fatta, A. Nikolaou, A. Achilleos, S. Meriç, Trends Anal. Chem. 26
untreated wastewater with GC–MS detection. The acid elution
(2007) 515.
proved an effective and yet simple clean-up for the endocrine [15] V. Ingrand, G. Herry, J. Beausse, M.-R. de Roubin, J. Chromatogr. A 1020
disrupters and could be useful in methods for analysis of these (2003) 99.
compounds by LC–MS, by potentially reducing the extent of [16] M. Carballa, F. Omil, J.M. Lema, M. Llompart, C. Garcı́a-Jares, I.
suppression of response. The target compounds were measured Rodrı́guez, M. Gomez, T. Ternes, Water Res. 38 (2004) 2918.
[17] S.S. Verenitch, C.J. Lowe, A. Mazumder, J. Chromatogr. A 1116 (2006)
in wastewaters from Mexico City and in spring waters from
193.
aquifers artificially recharged by use of the wastewater for irri- [18] H.-B. Lee, T.E. Peart, M.L. Svoboda, J. Chromatogr. A 1094 (2005)
gation. Concentrations of contaminants in the wastewater were 122.
comparable with reported data. Some of the analytes were [19] A.C. Johnson, H.-R. Aerni, A. Gerritsen, M. Gilbert, W. Giger, K. Hylland,
detected in the spring water although concentrations were sev- M. Jűrgens, T. Nakari, A. Pickering, M. Suter, A. Svensen, F.E. Wettstein,
Water Res. 39 (2005) 47.
eral orders of magnitude below those observed in the wastewater,
[20] T. Heberer, Toxicol. Lett. 131 (2002) 5.
suggesting that most contaminants were removed on passage of [21] M. Rabiet, A. Togola, F. Brissaud, J.-L. Seidel, H. Budzinski, F. Elbaz-
the wastewater through the soil and subsoil. Poulichet, Environ. Sci. Technol. 40 (2006) 5282.
[22] T.A. Ternes, M. Bonerz, N. Herrmann, B. Teiser, H.R. Andersen, Chemo-
Acknowledgements sphere 66 (2007) 894.
Talanta
journal homepage: www.elsevier.com/locate/talanta
a r t i c l e i n f o a b s t r a c t
Article history: Fragrance suspected allergens including those regulated by the EU Directive 76/768/EEC have
Received 25 May 2010 been determined in different types of waters using solid-phase microextraction (SPME) and gas
Received in revised form 7 September 2010 chromatography–mass spectrometry (GC–MS). The procedure was based on headspace sampling (HS-
Accepted 25 September 2010
SPME) using polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibers and has been optimized by an
Available online 28 October 2010
experimental design approach. The method performance has been studied showing good linearity
(R ≥ 0.994) as well as good intra-day and inter-day precision (RSD ≤ 12%). Detection limits (S/N = 3) ranged
Keywords:
from 0.001 to 0.3 ng mL−1 . Reliability was demonstrated through the quantitative recoveries of the com-
Suspected fragrance allergens
Solid-phase microextraction
pounds in real water samples, including baby bathwaters, swimming pool waters, and wastewaters. The
Gas chromatography–mass spectrometry absence of matrix effects allowed quantification of the compounds by external aqueous calibration. The
Water analysis analysis of 35 samples of different types of waters showed the presence of suspected allergens in all the
analyzed samples. All targets were found in the samples, with the exception of methyl eugenol and amyl
cinnamic alcohol. Highest concentrations of suspected allergens were present in baby bathwaters, con-
taining from 5 to 15 of the compounds at concentrations ranging from few pg mL−1 to several hundreds
of ng mL−1 .
© 2010 Elsevier B.V. All rights reserved.
1. Introduction usually expend long time in the bath playing with toys. During bath,
the exposure of children to chemicals is not only through dermal
Fragrances are a group of chemicals incorporated in most cos- absorption, but also inhalation and ingestion can play an important
metic and other personal care products including baby care ones. role.
The Scientific Committee on Cosmetic Products and Non-Food Swimming pools have been recently recognized as important
Products (SCCNFP), currently known as the Scientific Committee sources of exposure to pollutants. Epidemiologic studies have
on Consumer Products (SCCP), has identified 26 of these ingredi- shown increased risk of asthma both in indoor and outdoor pools
ents as likely to cause contact allergies [1,2]. Twenty-four of these [4]. In the case of suspected allergens, they can be easily transferred
suspected allergens are able to be analyzed by GC, whereas the to the pool water since they are present in all kind of cosmetics and
other two are not single compounds but very complex natural sun creams.
extracts (oak moss and tree moss) unsuitable for GC. The Euro- Some of the suspected allergens do not only pose the risk of
pean Cosmetic Directive requires an indication of the presence of causing contact allergies, but also systemic effects [5]. On the con-
potential fragrance allergens in cosmetic products if the limits of trary, methyl eugenol that had been included in 2002 in the list
0.01% and 0.001% for rinse-off and leave-on products, respectively, of forbidden substances in the EU Cosmetic Directive [6] due to
are exceeded [3]. the potential risk of inducing cancer [7] has been recently incor-
Among products for baby care, those intended for the bath such porated to the regulated list of compounds to be used as fragrance
as shampoos, bubble baths, shower gels, and soaps, contain deter- components [3].
gents that can break down the natural barrier of the skin, allowing Analytical methods for the determination of this group of
other irritants and allergens to penetrate. In the developed coun- substances are mainly based on gas chromatography–mass spec-
tries the daily baby bath is a common practice, and babies and kids trometry (GC–MS) [8–11]. To overcome difficulties on obtaining
good resolution between compounds and with other matrix com-
ponents, especially in cosmetic samples, several methods based on
multidimensional chromatography have been proposed [12,13].
∗ Corresponding author. Tel.: +34 981563100x14394; fax: +34 981595012.
The suitability of solid-phase microextraction (SPME) for the
E-mail addresses: JBecerrilB@iingen.unam.mx (E. Becerril),
analysis of suspected allergens has been recently demonstrated
juanpablo.lamas@usc.es (J.P. Lamas), lucia.sanchez@usc.es (L. Sanchez-Prado),
maria.llompart@usc.es (M. Llompart), marta.lores@usc.es (M. Lores), [14]. The proposed procedure allowed the reliable determination
carmen.garcia.jares@usc.es (C. Garcia-Jares). of 15 selected fragrance ingredients frequently found in baby bath-
0039-9140/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.talanta.2010.09.038
E. Becerril et al. / Talanta 83 (2010) 464–474 465
waters. A SPME followed by GC–MS method was also developed by nesol, mixture of isomers); 3,7-dimethylocta-2,6-dienal, 95%
Masuck et al. [15] to determine the emission of several fragrance (citral, cis/trans); 1-methyl-4-prop-1-en-2-yl-cyclohexene 97%
allergens released from scented toys into ambient air. ((R)-(+)-limonene); 4-methoxybenzene methanol, 98% (anis
The aim of the present study is to develop a general pro- alcohol); 2-methoxy-4-(1-propenyl) phenol, 98% (isoeugenol,
cedure based on solid-phase microextraction (SPME) with gas cis/trans); 2-(phenylmethylene)-heptanal, 97% (amyl cinnamalde-
chromatography–mass spectrometry (GC–MS) for the analysis of hyde); and 3-phenyl phenylmethyl ester-2-propenoic acid, 99%
fragrance suspected allergens in water samples that include all (benzyl cinnamate) were purchased from Aldrich (Sigma–Aldrich
the regulated compounds amenable by GC. Since these compounds Chemie GmbH, Steinheim, Germany).
belong to diverse chemical families with a broad range of polarities 2,6,6-Trimethylbicyclo[3.1.1]hept-2-ene, ≥99% ((−)-␣-pinene);
and volatilities, optimization of the main variables affecting the 3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one,
SPME process has been performed using an experimental design ≥85% (ionone); 3,7-dimetil-2,6-octadien-1-ol, ≥96% (geraniol);
approach. The SPME-GC–MS method has then been validated for 2-(phenylmethylene)-1-heptanol, ≥85% (amyl cinnamic alcohol);
the identification and quantification of 24 fragrance suspected 3-(4-tert-butylphenyl)-2-methylpropanal, ≥95% (lilial); 4-(4-
allergens regulated in the EU Directive [3], as well as pinene and hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde, ≥97%
methyl eugenol. The method has been applied to baby bathwaters, (lyral); and 2-hydroxy-phenylmethyl ester benzoic acid, ≥99%
swimming pool waters and wastewaters. Results demonstrated the (benzyl salicylate) were purchased from Fluka (Fluka Chemie
presence of several of the target compounds in all samples and, in GmbH, Steinheim, Germany).
the particular case of baby bathwaters at concentrations of several 2-Octynoic acid, methyl ester, ≥99% (methyl-2-octynoate);
hundreds of ng mL−1 in some of the samples. 7-hydroxy-3,7-dimethyloctanal, ≥95% (hydroxycitronel-
lal); 3-phenyl-2-propenal, ≥93% (cinnamaldehyde);
2-(phenylmethylene) octanal, ≥95% (hexylcinnamaldehyde),
2. Experimental methods
were purchased from SAFC Supply Solutions (St. Louis, USA).
Benzene methanol, 99% (benzyl alcohol); 3-phenyl-2-propen-1-
2.1. Reagents and materials
ol, 98% (cinnamyl alcohol); phenylmethyl benzoate, 98.5% (benzyl
3,7-Dimethyl-1,6-octadien-3-ol, 97% (linalool); 3,7- benzoate) was purchased from Chem Service (West Chester, USA).
dimethyloct-6-en-1-ol, 95% (-citronellol); 2-methoxy-4-prop- Table 1 shows the chemical abstract service (CAS) registry
2-enyl phenol, 99% (eugenol); 1,2-dimethoxy-4-(2-propenyl)- numbers, IUPAC names, molecular formula, as well as the main
benzene, 99% (methyl eugenol); 2H-1-benzopyran-2-one, 99% physicochemical properties of the target compounds. Molecular
(coumarin); 3,7,11-trimethyldodeca-2,6,10-trien-1-ol, 95% (far- structures are depicted in Fig. 1.
Table 1
CAS number, IUPAC names, molecular formula; and main properties of the studied allergens.
Key Compound CAS number IUPAC name Molecular Molecular log KOW Boiling Solubilitya
formula weight point (◦ C) (mg/L)
HO O
CH3
O
Linalool
CH3 Methyl-2-octynoate
CH3 OH
Pinene Benzyl alcohol
O
Citral
Limonene
O OH
O
Geraniol
OH
OH
Citronellol
OH
OH
O O
Cinnamaldehyde Hydroxycitronellal Coumarin
Cinnamyl alcohol O
Anis alcohol
O
HO
O
O O
O
OH OH Ionone
Eugenol Methyl eugenol Isoeugenol Amyl cinnamic alcohol
HO
OH
Lyral Farnesol
Lilial
O
Amylcinnamaldehyde
O
Hexylcinnamaldehyde
O O O
OH
O
O O
Methanol, ethyl acetate, and acetone were provided by Merck Sodium chloride was provided by VWR Prolabo (Fontenay-sous-
(Darmstadt, Germany). Individual stock solutions of each com- Bois, France). All solvents and reagents were of analytical grade.
pound were prepared in methanol. Further dilutions and mixtures Ultrapure water was obtained from a Milli-Q water purification
were prepared in acetone. The latter were employed for spiking system (Millipore, Billerica, MA, USA).
water samples. Working solutions were made by appropriate dilu- The SPME manual holders and 65 m polydimethylsilox-
tion and then stored in amber glass vials at −20 ◦ C. ane/divinylbenzene (PDMS/DVB) fibers were supplied by Supelco
E. Becerril et al. / Talanta 83 (2010) 464–474 467
Table 2
Retention times, quantification and identification ions of the target compounds.
Fig. 2. GC–MS chromatogram obtained by direct injection of a standard mixture of the fragrance compounds at 10 g mL−1 in ethyl acetate (see number code equivalence
in Table 1).
468 E. Becerril et al. / Talanta 83 (2010) 464–474
Table 3 (Bellefonte, PA, USA). Prior to first use, fibers were conditioned as
Optimal conditions for the microextraction mode and temperature (HS, headspace,
recommended by the manufacturer.
IM, immersion).
Water samples were collected in amber glass containers.
Key Compound Temperature (◦ C) Extraction Sodium thiosulphate (0.1 mg mL−1 ) was added to remove possible
mode
free chlorine. Samples were stored in the dark at 4 ◦ C until analysis.
1 Pinene 50.0 HS
2 Limonene 50.0 HS
3 Benzyl alcohol 50.0 IM 2.2. Gas chromatography–mass spectrometry
4 Linalool 66.8 HS
5 Methyl-2-octynoate 55.9 HS The GC–MS analysis was performed using a Varian 450-GC gas
6 Citronellol 86.3 HS
7 Citral 89.5 HS
chromatograph (Varian Chromatography Systems, Walnut Creek,
8 Geraniol 92.8 HS CA, USA) coupled to an ion trap mass spectrometer Varian 240-MS
9 Cinnamaldehyde 57.4 IM with a waveboard for multiple MS (MSn ) analysis; equipped with
10 Hydroxycitronellal 53.7 IM an automatic injector CP-8400. The system was operated by Varian
11 Anis alcohol 100 HS
MS workstation v6.9.1 software.
12 Cinnamyl alcohol 50.0 IM
13 Eugenol 75.9 IM Separation was carried out on a HP5 capillary column
14 Methyl eugenol 100 HS (30 m × 0.32 mm i.d., 0.25 m film thickness) from Agilent Tech-
15 Isoeugenol 81.7 HS nologies (Palo Alto, CA, USA). Helium (purity 99.999%) was
16 Coumarin 50.0 IM employed as carrier gas at a constant column flow of 1.0 mL min−1 .
17 Ionone 81.7 HS
18 Lilial 100 HS
The GC oven temperature was programmed from 45 ◦ C (held 2 min)
19 Amylcinnamaldehyde 100 HS to 100 ◦ C at 8 ◦ C min−1 ; to 150 ◦ C at 20 ◦ C min−1 ; to 200 ◦ C at
20 Lyral 100 IM 25 ◦ C min−1 ; (held 5 min); and a final ramp to 233 ◦ C at 8 ◦ C min−1
21 Amyl cinnamic alcohol 100 HS (total analysis time = 22.5 min). The splitless mode (held 2 min) was
22 Farnesol 100 HS
used for injection, with the split flow at 20 mL min−1 . The injector
23 Hexylcinnamaldehyde 100 HS
24 Benzyl benzoate 100 HS temperature was kept at 220 ◦ C.
25 Benzyl salicylate 81.7 HS The ion trap mass spectrometer was operated in the electron
26 Benzyl cinnamate 100 IM impact (EI) ionization mode (+70 eV) using an external ioniza-
tion configuration. Manifold, ion trap, ion source and transfer
line temperatures, were maintained at 40, 150, 200 and 280 ◦ C,
respectively.
Limonene Citronellol
4 43
50 HS
HS
39
Response(cts)
Response(cts)
3
35
(x106)
(x105)
2 31
100
27
1 100
23
IM 50 IM
0 19
Temperature (°C) Extraction mode Temperature (°C) Extraction mode
Response(cts)
20 48
(x105)
100
(x103)
17 43
14 38
11 IM 33
50 50
8 28
Temperature (°C) Extraction mode Temperature (°C) Extraction mode
Response(cts)
5 100 HS
(x106)
(x104)
84
4
50 IM
3 64
IM 50
2 44
Temperature (°C) Extraction mode Temperature (°C) Extraction mode
Response(cts)
Response(cts)
HS
14 9
(x105)
(x104)
9 6
HS
4 3
IM
0 0
50 °C 100 °C 50 °C 100 °C
Temperature Temperature
Response(cts)
4
(x105)
(x105)
3 IM
HS 6
2
1 3
0 0
50 °C 100 °C 50 °C 100 °C
Temperature Temperature
Response(cts)
HS 8
1.2
(x106)
(x105)
6
0.8
4 HS
0.4 2
IM
0 0
50 °C 100 °C 50 °C 100 °C
Temperature Temperature
In the full scan mode the mass range was varied from 39 to 3. Results and discussion
400 m/z at 3 scans, starting at 5 min and ending at 22.5 min. The
filament emission current was 25 A. The analytes were positively 3.1. Optimization of the analytical procedure
identified by comparison of their mass spectra and retention times
to those of standards. The identification and quantification ions, as Difficulties described in literature due to the complexity of fra-
well as the retention times for each target compound are listed in grance mixtures dealing with the effective separation and accurate
Table 2. determination of the 24 regulated suspected allergens [9,12,16]
led to test different oven temperature programs in order to obtain
2.3. Solid-phase microextraction a suitable chromatography of the compounds. First experiments
also allowed the selection of the quantification ions to attain the
Initial SPME conditions were optimized elsewhere [14]. In sum- maximum signal-to-noise ratio. In the GC–MS conditions summa-
mary, aliquots of 10 mL water sample are placed in 22 mL headspace rized in Section 2, as well as in Table 2, all compounds could be
vials containing sodium chloride (2 g). Then, vials are sealed with determined in less than 21 min. The GC–MS instrumental linearity
aluminium caps furnished with Teflon-faced septa and immersed was evaluated by direct injection of the target analytes at differ-
in a thermostatized water bath. Samples are let to equilibrate for ent concentrations in ethyl acetate (1–50 g mL−1 , 2 L injection
5 min before the exposition of a DVB/PDMS fiber for 20 min. In the volume, 6 calibration levels). Linearity was good in the studied con-
optimized procedure, samples are heated at 100 ◦ C and extracted in centration range, with linear regression coefficients ranging from
the headspace mode. Magnetic stirring was performed during the 0.993 to 0.999. Repeatability (n = 5) expressed in terms of relative
extraction process. Once finished the exposition period, the fiber standard deviation (RSD) was lower than 4.4%. Fig. 2 shows the
was retracted into the needle of the holder syringe and immedi- chromatogram of a standard mixture of the 26 allergen fragrances
ately inserted into the GC injector. Desorption was carried out at at a concentration of 10 g mL−1 .
220 ◦ C for 5 min. Possible carryover was checked and under the The suitability of the SPME technique for the determination of
selected conditions it was not observed. Blanks were periodically 15 fragrance allergens had been previously demonstrated, high-
run during the analysis to confirm the absence of contamination. lighting the importance of extraction mode and temperature in the
470 E. Becerril et al. / Talanta 83 (2010) 464–474
Table 4
Precision, linearity, and limits of detection and quantification of the method.
Compound Intra-day precision (%RSD, n = 3) Inter-day precision (%RSD, n = 5) Linearity (R) LOD (ng mL−1 ) LOQ (ng mL−1 )
extraction efficiency [14]. Thus, the optimization of the SPME con- Concerning the interaction between temperature and extrac-
ditions for the simultaneous extraction of the 26 targets has been tion mode, some results can be outlined (Fig. 4). Very high volatile
focused on these both parameters, and a multivariate strategy has compounds (pinene and limonene) must be extracted in the HS
been applied to account for possible second order effects. An experi- mode, and for these two compounds responses obtained at 100 ◦ C
mental screening design 3 × 2, which allowed to study temperature are clearly low and independent of the extraction mode. A very
at three levels (50, 75 and 100 ◦ C) and the two extraction modes (HS different behaviour has been followed by benzyl alcohol or cin-
and direct SPME), has been selected. Experiments were performed namaldehyde, which are better extracted in the immersion mode
using 10 mL aliquots of water spiked with the analytes at a con- but only if temperature is kept at 50 ◦ C. If 100 ◦ C is the temperature
centration of 10 ng mL−1 . Sampling time was set at 20 min, sodium selected for the extraction, then the efficiency of the extraction is
chloride was added in a proportion of 20%, and magnetic stirring higher in the HS mode. Clear examples of compounds requiring the
was used to favour mass transfer in the aqueous media. Numer-
ical analysis of data resulting from the experimental design was Table 5
made with the statistical software package Statgraphics Centurion Percent recovery of the compounds from three different water samples spiked at
XV (Manugistics, Rockville, MD, USA). 20 ng mL−1 (baby bathwater), 5 ng mL−1 (swimming pool water) and 10 ng mL−1
The optimal conditions obtained for the 26 target compounds (wastewater).
are summarized in Table 3. It can be seen that the extraction of 17 Compound Bathwater Swimming pool Wastewater
of the 26 compounds was favoured by high temperatures (>75 ◦ C).
Pinene 116 ± 11 100 ± 6 113 ± 11
The headspace extraction conditions are best suited for the most Limonene 100 ± 7 85 ± 15 112 ± 7
compounds, with only eight showing higher responses in the direct Benzyl alcohol 106 ± 7 90 ± 12 78 ± 11
SPME mode. Linalool 90 ± 1 90 ± 3 81 ± 10
Fig. 3 shows the main effect plots for several representative com- Methyl-2-octynoate 87 ± 3 115 ± 4 88 ± 7
Citronellol 83 ± 6 90 ± 3 85 ± 10
pounds. This kind of plots shows the main effects with a line drawn
Citral 85 ± 2 101 ± 4 89 ± 5
between the low and the high level of the corresponding factors. Geraniol 104 ± 5 106 ± 6 96 ± 7
The length of the line is proportional to the effect magnitude of the Cinnamaldehyde 88 ± 6 100 ± 4 103 ± 13
factor in the extraction process, and the sign of the slope indicates Hydroxycitronellal 99 ± 8 136 ± 8 110 ± 5
Anis alcohol 121 ± 5 117 ± 14 120 ± 10
the level of the factor that produces the highest response. The high
Cinnamyl alcohol 103 ± 13 78 ± 1 106 ± 1
influence on response of both extraction mode and temperature is Eugenol 103 ± 5 107 ± 2 106 ± 15
clearly appreciated for most of compounds. Methyl eugenol 79 ± 5 104 ± 2 95 ± 7
The most volatile compounds, pinene and limonene, are bet- Isoeugenol 96 ± 9 85 ± 3 80 ± 7
ter extracted at 50 ◦ C, while higher temperatures are needed as Coumarin 116 ± 1 107 ± 2 87 ± 10
Ionone 73 ± 1 91 ± 3 92 ± 1
the boiling point of the compounds increases (see Table 3). This
Lilial 87 ± 2 83 ± 10 92 ± 3
can be seen in Fig. 3, in which limonene, citronellol, isoeugenol, Amyl cinnamaldehyde 86 ± 4 n.a. 85 ± 8
and methyl eugenol are examples of this tendency. Several excep- Lyral 96 ± 4 107 ± 7 124 ± 17
tions are found regarding the extraction temperature: compounds Amyl cinnamic alcohol 88 ± 5 109 ± 9 98 ± 3
Farnesol 120 ± 12 78 ± 4 106 ± 15
such as benzyl alcohol, cinnamaldehyde, or coumarin are better
Hexylcinnamaldehyde 88 ± 3 105 ± 2 84 ± 2
extracted at the lowest temperature. Headspace extraction mode Benzyl benzoate 103 ± 6 103 ± 3 100 ± 2
was generally better for the most of compounds, with some excep- Benzyl salicylate 122 ± 12 107 ± 9 122 ± 10
tions that are better extracted in the immersion mode (see Table 3). Benzyl cinnamate 79 ± 4 101 ± 7 108 ± 7
Table 6
Concentration (ng mL−1 ) of the suspected fragrance allergens in water samples.
Compound W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12 W13 W14 W15 W16 W17 W18 W19 W20 W21 W22
Compound W23 W24 W25 W26 W27 W28 W29 W30 W31 W32 W33 W34 W35
Pinene
Limonene 0.03 0.03 0.86 0.16 0.23 0.078 0.36 0.23
Benzyl alcohol
Linalool 0.051 0.21
Methyl-2-octynoate
Citronellol 0.044a 0.032a 0.64
E. Becerril et al. / Talanta 83 (2010) 464–474
Citral 0.19
Geraniol
Cinnamaldehyde
Hydroxycitronellal 0.68 1.80
Anis alcohol
Cinnamyl alcohol 1.1 0.49 1.1
Eugenol 0.051
Isoeugenol 0.19 0.74
Coumarin
Ionone 0.003 0.004 0.031 0.032 0.36 0.032 0.024 0.10 0.20 0.19
Lilial 0.097 0.033 0.19 0.69 0.035 0.037 0.041 0.23 0.031 0.066 0.11 0.40 0.20
Amyl cinnamaldehyde 0.050 0.022
Lyral
Farnesol 0.33a
Hexylcinnamaldehyde 0.051 0.071 0.087 0.028
Benzyl benzoate 0.070 0.033 0.074 0.18
Benzyl salicylate
Benzyl cinnamate
1 1
0
MCounts 4 m/z: 43.0+71.0+93.0
4
2
0
MCounts m/z: 41.0+67.0+69.0 8
6
5
0
kCounts 9 m/z: 77.0+103.0+131.0
100
50
0
MCounts 10 m/z: 43.0+59.0+71.0
1.00
0.50
0.00
kCounts 12 m/z: 78.0+91.0+92.0
400
200
0
MCounts m/z: 118.0+146.0+164.0
13
2.0 16
1.0
0.0
MCounts m/z: 93.0+121.0+136.0 17
0.50
0.00
MCounts m/z: 41.0+69.0
22
5
0
kCounts m/z: 129.0+216.0 23
100
50
0
MCounts m/z: 105.0+194.0 24
4
2
0
7 8 9 10 11 12 13 14 15 Minutes
Fig. 5. HS-SPME-GC–MS extracted ion chromatograms for a baby bathwater sample (W13). For compound concentrations, see Table 6.
highest temperatures are amyl cinnamic alcohol and farnesol (see ertheless, its interaction graph shows that at higher temperatures
Fig. 4). This kind of compounds is extracted with very low efficiency the extraction efficiency is similar for both extraction modes. In the
at 50 ◦ C independently of the extraction mode. It can be appreciated same way, benzyl cinnamate showed optimal conditions at 100 ◦ C
that for certain compounds the difference in efficiency between the and immersion (Table 3), but the analysis of the interaction plot
direct and HS mode may be very short depending on temperature. shows that the use of both extraction modes gives the best and
Some examples are also depicted in Fig. 4. The optimal extraction quite similar responses at 100 ◦ C; direct SPME is only better than
conditions for eugenol were 75.9 ◦ C and immersion (Table 3). Nev- HS-SPME if extraction is performed at 50 ◦ C.
E. Becerril et al. / Talanta 83 (2010) 464–474 473
These all results allow concluding that the best conditions gens were found at concentrations generally below 0.1 ng mL−1 ,
for the simultaneous determination of fragrance suspected aller- although these levels can be expected to increase in summertime.
gens including all the regulated ones in waters imply HS-SPME Fig. 5 shows the HS-SPME-GC–MS extracted ion chromatograms
at 100 ◦ C. obtained for a baby bathwater sample.
The SPME-GC–MS proposed method showed good performance
3.2. Method performance study characteristics for the analysis of fragrance allergens, with low LODs
allowing the sensitive determination in waters of 26 fragrance com-
Method linearity has been evaluated performing a calibration ponents including the 24 EU regulated suspected allergens, with
study in the experimental conditions. The calibration range was low cost, simplicity and time-saving.
established from 0.01, 0.1 or 0.5 ng mL−1 (depending on the indi-
vidual limits of quantification) to 50 ng mL−1 , with 6–7 calibration 4. Conclusions
levels. The method exhibited a direct proportional relationship
between the extracted amount of each fragrance allergen and their The combination of SPME and GC–MS was shown to be a simple
initial concentration in the sample, with correlation coefficients (R) and effective procedure for the determination of fragrance com-
ranging from 0.994 to 1.000 (Table 4). pounds including 24 regulated suspected allergens in waters.
Precision of the experimental procedure was assessed at four The optimization of the extraction was carried out using experi-
concentration levels: 1, 5, 10, and 20 ng mL−1 . Results showed good mental design showing that sampling mode and temperature were
intra-day and inter-day precisions, with relative standard deviation variables that highly influenced extraction efficiency. The optimal
(RSD) values in general lower than 10% (see Table 4). experimental conditions implied the use of PDMS/DVB coating for
Limits of detection (LOD, signal-to-noise ratio of 3) and lim- the extraction in the headspace mode at 100 ◦ C.
its of quantification (LOQ, signal-to-noise ratio of 10) of the The method was validated and demonstrated to be reliable and
method are also presented in Table 4. These limits are at the linear in the concentration range of interest. LODs were satisfactory
sub-ng mL−1 , with two exceptions (anis alcohol and lyral), and (0.001–1.1 ng mL−1 ) as well as reproducibility (RSD < 12%). Quan-
therefore, the sensitivity of the proposed method can be considered titative recoveries were obtained for targets in waters including
satisfactory. wastewater (>80% for most compounds). The application of the
method to water samples including baby bathwater, swimming
3.3. Application to water samples pool waters and wastewaters, demonstrated the presence of sus-
pected allergens in all samples. In baby bathwater some compounds
The method was applied to the analysis of 35 water sam- were found at concentrations of several hundreds of ng mL−1 ,
ples including 22 baby bathwater samples (W1–W22), 4 indoor while in wastewaters the concentration levels were in the range
swimming pool water samples (W23–W26), and 9 wastewaters of those reported for other widespread fragrance components such
(W27–W35). Three samples, a baby bathwater, a swimming pool as musks.
water, and a wastewater, were selected for matrix effect and recov-
ery studies. Acknowledgements
Apparent recoveries were calculated as the ratio of the mea-
sured concentration, after subtracting the initial concentration in This research was supported by FEDER funds and project
the non-spiked sample, to the spiked concentration, and expressed CTQ2010-19831/BQU from Ministerio de Ciencia e Innovación,
as percentage. Concentrations were calculated by external cali- Spain. L.S.-P. and J.P.L. acknowledge Xunta de Galicia for a
bration using ultrapure water standards. Recoveries are shown in postdoctoral Angeles Alvariño, and an Isabel Barreto contracts,
Table 5, and ranged from 80 to 120% for most compounds. These respectively. E.B. thanks the Universidad Nacional Autónoma
recoveries can be considered quantitative, and thus, no matrix de México, Secretaría General, Dirección General de Asun-
effects were observed, allowing quantification by external water tos del Personal Académico, for the award of a research
calibration. scholarship.
The levels of the target compounds were then determined in a
wide range of water samples. Baby bathwaters were obtained at References
homes from Galicia (Northwestern Spain) during the daily bath
of children aged from few months to three years. The products [1] SCCNFP/0017/98 Final FRAGRANCE ALLERGY IN CONSUMERS (December
employed for bath preparation, as well as the quantities were 1999).
[2] Opinion concerning Oakmoss/Treemoss Extracts And Appropriate Consumer
the usual and included shampoos, bubble baths and moisturising Information adopted by the SCCNFP during the 14th plenary meeting of 24
soaps all intended for babies. Wastewaters include two samples October 2000.
taken from the spill of a collective washing place (W27, W28), [3] REGULATION (EC) No 1223/2009 OF THE EUROPEAN PARLIAMENT AND OF THE
COUNCIL of 30 November 2009 on cosmetic products (recast), Official Journal
samples taken at the influent (W29–W31) and at the effluent of European Union L342 (2009) 59.
(W32, W33) of sewage treatment plants, all in Galicia (Spain), [4] C.P. Weisel, S.D. Richardson, B. Nemery, G. Aggazzotti, E. Baraldi, R.B. Ernest III,
as well as two samples taken at the central emissary of Mex- B.C. Blount, K.-H. Carlsen, P.A. Eggleston, F.H. Frimmel, M. Goodman, G. Gordon,
S.A. Grinshpun, D. Heederik, M. Kogevinas, LaKind Judy S., M.J. Nieuwenhuijsen,
ico city (W34) and at Cerro Colorado (W35), both in Mexico, F.C. Piper, S.A. Sattar, Environ. Health Persp. 117 (2009) 500.
respectively. [5] T. Uehara, N. Kiyosawa, T. Shimizu, K. Omura, M. Hirode, T. Imazawa, Y.
Results of the analysis are shown in Table 6, and as can be Mizukawa, A. Ono, T. Miyagishima, T. Nagao, T. Urushidani, Hum. Exp. Toxicol.
27 (2008) 23.
seen, all target compounds were detected in the samples with
[6] COUNCIL DIRECTIVE of 27 July 1976 on the approximation of the laws of the
the exception of amyl cinnamic alcohol and methyl eugenol. Com- Member States relating to cosmetic products 76/768/EEC.
pounds have been found at concentrations ranging from 0.003 to [7] J.D. Johnson, M.J. Ryan, J.D. Toft, S.W. Graves, M.R. Hejtmancik, M.L. Cunning-
ham, R. Herbert, K.M. Abdo, J. Agric. Food Chem. 48 (2000) 3620.
845 ng mL−1 . It should be noticed that several bathwater samples
[8] S.C. Rastogi, J.D. Johansen, T. Menné, P. Frosch, M. Bruze, K.E. Andersen, J.P.
showed high concentrations of several compounds, reaching values Lepoittevin, S. Wakelin, I.R. White, Contact Dermatitis 41 (1999) 84.
even greater than 100 ng mL−1 . The concentration levels found in [9] H. Leijs, J. Broekhans, L. van Pelt, C. Mussinan, J. Agric. Food Chem. 53 (2005)
wastewaters are in the range of those found for other widespread 5487.
[10] M. Bassereau, A. Chaintreau, S. Duperrex, D. Joulain, H. Leijs, G. Loesing, N. Owen,
used fragrance compounds such as polycyclic musks in this kind of A. Sherlock, C. Schippa, P.J. Thorel, M. Vey, J. Agric. Food Chem. 55 (2007) 25.
waters [17,18]. In swimming pool water samples, suspected aller- [11] M. Niederer, R. Bollhalder, C. Hohl, J. Chromatogr. A 1132 (2006) 109.
474 E. Becerril et al. / Talanta 83 (2010) 464–474
[12] M.S. Dunn, N. Vulic, R.A. Shellie, S. Whitehead, P. Morrison, P.J. Marriott, J. [15] I. Masuck, C. Hutzler, A. Luch, J. Chromatogr. A 1217 (2010) 3136.
Chromatogr. A 1130 (2006) 122. [16] P.M.A.C. Robert Shellie, Flavour Frag. J. 19 (2004) 91.
[13] L. Mondello, A. Casilli, P.Q. Tranchida, D. Sciarrone, P. Dugo, G. Dugo, LC-GC [17] A. Silva, J. Nogueira, Anal. Bioanal. Chem. 396 (2010) 1853.
Europe 21 (2008) 130. [18] C. García-Jares, M. Llompart, M. Polo, C. Salgado, S. Macías, R. Cela, J. Chromatogr.
[14] J.P. Lamas, L. Sanchez-Prado, C. Garcia-Jares, M. Llompart, Anal. Bioanal. Chem. A 963 (2002) 277.
394 (2009) 1399.
Chemosphere 81 (2010) 1378–1385
Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere
a r t i c l e i n f o a b s t r a c t
Article history: A method based on ultrasound-assisted emulsification–microextraction (USAEME) and gas chromatogra-
Received 10 June 2010 phy–mass spectrometry (GC–MS) has been developed for the analysis of regulated fragrance allergens in
Received in revised form 9 September 2010 water. Extraction conditions such as the type of solvent, extraction temperature, irradiation time, and
Accepted 9 September 2010
salting-out effect were optimized using a multivariate approach. Compounds were extracted during
2 min in an acoustically emulsified media formed by 100 lL chloroform and 10 mL sample. The USAEME
process provided an efficient and exhaustive extraction (enrichment factor 100) and, after centrifuga-
Keywords:
tion, the extract was ready for GC analysis.
Fragrance allergens
Personal care products
Validation was performed using spiked ultrapure water as well as other most complex matrices such as
Water analysis sewage water. Recoveries between 75% and 110% were generally obtained, and precision was character-
Ultrasound-assisted extraction ized by RSD values <10% in most cases. The limits of detection (LODs) were at the sub-nanogram per mil-
Ultrasound-assisted emulsification– lilitre level.
microextraction The proposed procedure was applied to the determination of allergens in several real samples including
Factorial experimental design tap water, baby bathwater, recreational place water, public washing place water, and sewage water. The
presence of some of the target compounds was confirmed in all the samples excluding tap water, dem-
onstrating the ubiquity of this group of cosmetic and personal care products ingredients.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction absorbed into the body. The long-term impact due to the possible
bioaccumulation in human tissues is also cause of concern.
Most personal care products (PCPs) as well as many household Although the main route of exposition to these cosmetic ingre-
products contain fragrances among their ingredients. The Interna- dients is, in general, the direct application of cosmetics on the skin,
tional Fragrance Association (IFRA) defines fragrances as any basic the contact with water containing these fragrances should be also
substance used in the manufacture of fragrance materials for its considered. A recent study has analyzed the concentration of 15
odorous, odour enhancing or blending properties. Fragrance ingre- fragrance allergens in baby bathwater showing quite high levels
dients may be obtained by chemical synthesis from synthetic, for some of them (Lamas et al., 2009). These compounds can also
fossil or natural raw materials or by physical operations from nat- be present in recreational waters and so, skin can be extensively
ural sources (IFRA, accessed June 2010). The Scientific Committee exposed to these and other chemicals through these waters
on Cosmetic Products and Non-Food Products (SCCNFP) has (WHO, accessed June 2010).
selected 26 compounds as likely to cause contact allergies As they are important components of daily use products, aller-
(SCCNFP/0017/98, 1999). In the European Union (EU) these gen fragrances are continuously introduced into the environment
‘‘suspected allergens” require labelling on cosmetic and detergent at high quantities, mainly via urban wastewater effluents. Bath
products (EU, 2009). The presence of these fragrances must be indi- waters as well as the residual waters from many residential com-
cated in the list of ingredients when its concentration exceeds the munities enter the environment via direct spill into superficial
0.001% in leave-on products and 0.01% in rinse-off products. The waters or through disposal into the sewage treatment system, con-
most of these substances are also restricted by the IFRA (accessed tributing to water pollution and causing environmental concern.
June 2010), with the purpose of ensuring the safety of fragrance While analytical methods for fragrance allergens have been
materials. Some may have a direct impact on the skin, eyes and developed for cosmetics analysis (Rastogi et al., 1999; Leijs et al.,
mucous membranes, but they may also cross the skin and be 2005; Niederer et al., 2006; Bassereau et al., 2007) and, recently,
to evaluate the levels of several fragrances on the skin treated with
⇑ Corresponding author. Tel.: +34 981563100x14225. fortified cream (Sgorbini et al., 2010), to the best of our knowledge
E-mail address: maria.llompart@usc.es (M. Llompart). only one method has been reported for the determination of
0045-6535/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2010.09.028
E. Becerril-Bravo et al. / Chemosphere 81 (2010) 1378–1385 1379
Table 1
Physicochemical properties, retention times and selected ions of the target compounds.
Key Retention time Compound CAS number Molecular log KOW Boiling point (°C) Solubility Quantifier and
(min) weight (mg L 1) qualifier (m/z)
1 8.36 Limonene 5989-27-5 136 4.57 176 13.8 67, 93, 121
2 8.72 Benzyl alcohol 100-51-6 108 1.05 205 40 000 77, 79, 108
3 9.72 Linalol 78-70-6 154 3.28 198 1589 43, 93, 121
4 10.99 Methyl-2-octynoate 111-12-6 154 2.60 219 - 67, 79, 95
5 11.38 Citronellol 106-22-9 156 3.38 225 322 69, 81, 95
6 11.42 Citral 5392-40-5 152 3.17 229 590 39, 69, 109
11.69
7 11.59 Geraniol 106-24-1 154 3.28 229 531 41, 69, 111
8 11.82 Cinnamaldehyde 104-55-2 132 2.22 252 1420 77, 103, 131
9 11.87 Hydroxy-citronellal 107-75-5 172 1.54 240 23 800 43, 59, 81
10 12.00 Anisyl alcohol 105-13-5 138 1.10 259 2070 77, 109, 138
11 12.21 Cinnamyl alcohol 104-54-1 134 1.93 250 1800 77, 92, 134
12 12.43 Eugenol 97-53-0 164 2.20 256 <1000 103, 131, 164
13 12.71 Methyl eugenol 93-15-2 178 2.9 248 500 147, 163, 178
14 12.80 Isoeugenol 97-54-1 164 2.45 267 984 103, 131, 164
13.09
15 13.11 Coumarin 91-64-5 146 1.39 298 2500 89, 118, 146
16 13.21 Ionone 127-51-5 206 4.41 266 16 107, 135, 150
17 13.54 Lilial 80-54-6 204 4.07 279 33 131, 147, 189
18 14.36 Amyl cinnamal 122-40-7 202 4.80 289 8.5 115, 129, 202
19 14.54 Lyral 31 906-04-4 210 2.53 319 185-1045 79, 91, 136
20 14.73 Amyl cinnamyl alcohol 101-85-9 204 4.37 >200 26 91, 115, 133
21 14.54 Farnesol 4602-84-0 222 5.31 283 267 41, 69, 81
22 15.20 Hexylcinnamaldehyde 101-86-0 216 4.82 308 2.8 115, 129, 216
15.49
23 15.52 Benzyl benzoate 120-51-4 212 3.97 324 19.8 105, 194, 212
24 16.79 Benzyl salicylate 118-58-1 228 4.31 320 <1000 65, 91, 228
25 20.59 Benzyl cinnamate 103-41-3 238 3.65 371 9 91, 131, 192
equipped with an automatic injector CP-8400. The system was retention times as well as the qualification and quantification ions
operated by Varian MS workstation v6.9.1 software. of the target analytes.
Separation was carried out on a HP5 capillary column (30 m Regarding the USAEME process, the selection of a suitable
0.25 mm i.d., 0.25 lm film thickness) from Agilent Technologies extractant is limited by several characteristics that are necessary
(Palo Alto, CA, USA). Helium (purity 99.999%) was employed as car- for emulsification in the presence of ultrasonic radiation. Some of
rier gas at a constant column flow of 1.0 mL min 1. The GC oven these characteristics are a higher density than water and low water
temperature was programmed from 45 °C (held 2 min) to 100 °C solubility. The selected solvent must be compatible with the sepa-
at 8 °C min 1, to 150 °C at 20 °C min 1, to 200 °C at 25 °C min 1 ration and detection technique and, therefore, a good gas chro-
(held 5 min) and a final ramp to 225 °C (held 1 min) at 8 °C min 1. matographic behavior is another desirable characteristic. Most of
The injector was operated in the splitless mode and pro- heavy solvents are halogenated compounds; several of them, were
grammed to return to the split mode after 2 min from the begin- initially tested in order to evaluate the chromatographic perfor-
ning of a run. Split flow was set at 20 mL min 1 and the injector mance, and the possible presence of interferences during mass
temperature was kept at 260 °C. spectrometry detection. Based on previous studies (Regueiro
The ion trap mass spectrometer was operated in the electron et al., 2008, 2009), carbon tetrachloride, chloroform, and 1,1,1-tri-
impact (EI) ionization mode (+70 eV) using an external ionization con- chloroethane were initially selected. All of them demonstrated to
figuration. Manifold, ion trap, ion source and transfer line tempera- be suitable for fragrance allergens GC–MS analysis, showing simi-
tures, were maintained at 40, 150, 200 and 280 °C, respectively. lar peak resolution. In addition, the responses obtained were
In the full scan mode the mass range was varied from 39 to 400 m/ equivalent for all target analytes.
z at 3 lscans, starting at 5 min and ending at 22.50 min. The filament First USAEME experiments were performed with aliquots of
emission current was 25 lA. The analytes were positively identified 10 mL of ultrapure water and 100 lL of solvent. The US irradiation
by comparison of their mass spectra and retention times to those of time was 10 min. In all experiments, PCB-30 was used as internal
standards. The identification and quantification ions and retention standard to minimize possible problems resulting from lack of
times for each target compound are listed in Table 1. repeatability in the sedimented phase volume. Emulsification
was observed with all three solvents. Organic phases were then
3. Results and discussion separated by centrifugation and injected into the GC–MS system.
Fig. 1 compares the chromatographic responses obtained by each
3.1. Optimization of USAEME process one of the three solvents. As can be seen in the figure, the re-
sponses were equivalent for some analytes. Nevertheless, 10 of
The chromatographic method for the separation of the tar- the target compounds presented very low response with carbon
get allergens was optimized elsewhere (Lamas et al., 2010) and it tetrachloride, and, in consequence, this solvent was discarded for
is described in the Experimental section. Table 1 summarizes the further optimization.
E. Becerril-Bravo et al. / Chemosphere 81 (2010) 1378–1385 1381
2.0000
CHCl3 C2H3Cl3 CCl4
1.5000
A/A IS
1.0000
0.5000
0.0000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Fig. 1. Comparison of the chromatographic responses obtained by each one of the three solvents: chloroform (CHCl3); 1,1,1-trichloroethane (C2H3Cl3); and carbon
tetrachloride (CCl4).
3.2. Multifactor design study effect of each factor or interaction by its standard error. Vertical
line in the graphs represents the statistically significant bound at
The influence of the main variables potentially affecting the the 95% confidence level.
USAEME efficiency was evaluated by using a multifactorial screen- Other interesting graphic option provided by the statistic soft-
ing design. The study consisted of a complete factorial 2^4 design, ware is the main effects plot. Fig. 3a shows the main effects dia-
involving 16 randomized experiments and allowing 5 degrees of grams for several compounds. This kind of plots shows the main
freedom to estimate the experimental error. This design has effects with a line drawn between the low and the high level of
resolution V, which means that it is capable of evaluating all main the corresponding factors. The length of the lines is proportional
effects and all two-factor interactions. Numerical analysis of data to the effect magnitude of each factor in the extraction process,
resulting from the experimental design was made with the and the sign of the slope indicates the level of the factor that pro-
statistical software package Statgraphics-Plus v5.1 (Manugistics, duces the highest response. As commented above, solvent and con-
Rockville, MD, USA). Factorial design optimization was performed centration of sodium chloride were the most important factors and
using aliquots of water spiked with the analytes at a concentration its influence is clearly appreciated in the plots. For most com-
of 10 ng mL 1. pounds higher extraction efficiency was observed when chloro-
The selection of an appropriate solvent is essential for all li- form was used, although trichloroethane extraction was more
quid–liquid extraction (LLE) – based processes. Chloroform and tri- favourable for six analytes, such as citral (see Fig. 3a). In general,
chloroethane, preselected in previous experiments, were included the addition of NaCl affects positively the extraction of analytes
in the experimental design optimization, in an attempt to achieve (see some examples in the figure), excluding limonene (Fig. 3a),
the highest extraction efficiency for the target compounds. Extrac- hexylcinnamaldehyde, and benzyl cinnamate. Regarding tempera-
tion time is usually an important factor in most extraction proce- ture and extraction time, these factors were not significant with a
dures. The effect of this factor was examined at 2 and 10 min. few exceptions, in which case better extraction was obtained at
Temperature is other frequently important parameter, and was 25 °C and 2 min.
studied at two levels, 25 °C and 50 °C. Finally, the salting-out effect The interaction of the most important main factors, solvent and
is frequently used in LLE, SPME and liquid phase microextraction addition of salt, was also significant for several analytes, being even
(LPME) procedures. Generally, addition of salt can decrease the sol- the most important factor in some cases (e.g. benzyl alcohol,
ubility of analytes in the aqueous phase, promoting the transfer to- Fig. 2). This interaction is shown in Fig. 3b for some analytes. It
wards the organic phase. Therefore, the concentration of sodium can be appreciated how the addition of salt favours chloroform
chloride in the aqueous solution was the last factor evaluated at extraction but it does not benefit trichloroethane extraction.
two levels: 0% (no addition) and 20% (m/V). In brief, four variables In view of the results of the experimental design, the selected
were screened in this design, namely: extraction time (A), solvent general conditions for the simultaneous ultrasound-assisted emul-
(B), temperature (C), and sodium chloride concentration (D). sification–microextraction of the target compounds from water
Numerical analysis of the results leads to the ANOVA results samples were as follows: an extraction time of 2 min, chloroform
shown in Table 2. For the sake of simplicity, only the interaction as extractant, 25 °C, and 20% sodium chloride.
factors that were significant in any case are included. As can be
seen, the most important factors, with statistical significance for 3.3. Method performance: application to real samples
many of the target compounds, are the extractant and the concen-
tration of sodium chloride. The temperature and the extraction Due to the ubiquity of the target compounds, which are used in
time were in general non significant with few exceptions. Regard- all kind of cleansing and cosmetics products, the risk of sample
ing the two-factor interactions, the only important effect was the contamination is not negligible, so it is advisable to extreme
one between the solvent and the concentration of sodium chloride precautions to avoid sources of interference in the laboratory
(BD), which was statistically significant for 10 compounds. environment. Blanks must be run very frequently to warranty the
The information included in the ANOVA can by graphically plot- absence of contamination.
ted by means of the Pareto charts. In Fig. 2, some representative Analytical quality parameters were evaluated in order to assess
graphics are included. In these graphics the length of each bar is the performance of the USAEME method.
proportional to the absolute value of its associated standardized ef- Method linearity was tested by injecting chloroform standard
fect. The standardized effect is obtained by dividing the estimated solutions between 0.020 lg mL 1 and 10 lg mL 1. Determination
1382 E. Becerril-Bravo et al. / Chemosphere 81 (2010) 1378–1385
Table 2
F ratios and p values obtained in the analysis of variance.
Fig. 2. Pareto charts showing the significant factors (95%) for some selected fragrance allergens (see factor codes in Table 2).
coefficients (R2) between 0.9974 and 1.0000 were obtained for all values of 18%, 20% and 27%, respectively (Table 3). Precision of the
compounds (Table 3). experimental procedure was also evaluated at several concentra-
Recoveries were evaluated using milliQ water spiked at four tion levels by calculating the relative standard deviation (RSD).
concentrations from 0.5 ng mL 1 to 100 ng mL 1. They were calcu- These results are also shown in the table, and they were lower than
lated by dividing the difference between the measured concentra- 10% in most cases. LODs (signal-to-noise ratio of 3) are also pre-
tions for spiked and non-spiked samples by the concentrations of sented, and all of them were below 1 ng mL 1 with the exception
the compounds added. The thus obtained values can be considered of anisyl alcohol.
satisfactory, generally between 70% and 110%, excluding 3 of the The aim of the present work is to develop a methodology suitable
25 target analytes, limonene, benzyl alcohol, and anisyl alcohol. for the analysis of the target compounds in different kinds of water
For these last compounds, recoveries were very low, with average samples and, consequently, it is necessary to evaluate possible
E. Becerril-Bravo et al. / Chemosphere 81 (2010) 1378–1385 1383
A/A IS
1.01 C2 H3 Cl3 25
A/A IS
0.02 2
10 25
0.91
CHCl3 50
10 0.01
0.81
20 C2 H3 Cl3 0
0.71 0
Time Solvent Temperature NaCl Time Solvent Temperature NaCl
(min) (° C) (%) (min) (° C) (%)
Citral Lyral
0.96 C2 H3 Cl3 0.25
CHCl3 20
0.23
20
0.86 25 0.21 10 50
2
A/A IS
A/A IS
2
50 0.19 25
10
0.76 0
0.17
0
CHCl3 C2 H3 Cl3
0.66 0.15
Time Solvent Temperature NaCl Time Solvent Temperature NaCl
(min) (° C) (%) (min) (° C) (%)
b Methyl-2-octynoate
0.24
Anisyl alcohol
0.93
0.89 0.20
20 % NaCl 0.16 20 % NaCl
0.85
A/A IS
A/A IS
0.81 0.12
0.77 0.08
0.69 0.00
CHCl3 C 2 H 3 Cl 3 CHCl3 C 2 H 3 Cl 3
Fig. 3. (a) Main effects and (b) interaction effect (BD, solvent – addition of salt) plots for some selected fragrance allergens.
Table 3
Linearity, limits of detection, recovery and precision of the method.
1
Compound Linearity LOD (ng mL ) Recovery (RSD)%
R2 0.5 ng mL 1a
2 ng mL 1a
10 ng mL 1a
100 ng mL 1b
BBWc (10 ng mL 1
) STPc (2 ng mL 1
)
Limonene 0.9986 0.30 25.0 (8.5) 19.0 (6.4) 16.1 (10) 18.2 9.0 (10) 19.9 (3.5)
Benzyl alcohol 0.9995 0.95 21.5 (15) 21.9 (8.3) 19.7 25 (23) 20.9 (4.3)
Linalol 0.9999 0.042 113 (8.1) 89.1 (6.0) 95.4 (2.3) 91.3 87.9 (8.3) 96.3 (1.6)
Methyl-2-octynoate 0.9998 0.055 79.2 (6.8) 81.6 (0.2) 88.7 (4.5) 81.7 81.7 (4.5) 81.8 (10)
Citronellol 0.9997 0.28 91.3 (11) 97.5 (4.3) 86.5 113 (5.3) 94.5 (8.0)
Citral 0.9994 0.064 88.0 (10) 95.6 (6.1) 96.9 (6.5) 85.8 108 (13) 96.4 (3.6)
Geraniol 0.9993 0.45 84.2 (1.9) 95.8 (2.9) 84.7 79.2 (2.9) 91.4 (4.7)
Cinnamaldehyde 0.9999 0.017 99.8 (6.9) 88.7 (5.9) 90.6 (3.7) 81.2 114 (3.1) 88.2 (8.0)
Hydroxycitronellal 0.9999 0.049 88.5 (1.1) 71.8 (2.7) 70.5 (6.9) 63.1 81.4 (3.7) 73.1 (7.0)
Anisyl alcohol 0.9981 1.15 29.4 (3.9) 27.5 (3.9) 26.3 29.1 (14) 22.4 (7.0)
Cinnamyl alcohol 0.9974 0.25 75.8 (8.6) 83.4 (7.5) 70.2 81.5 (6.9) 78.8 (5.8)
Eugenol 0.9998 0.006 105 (6.4) 96.5 (6.6) 96.4 (3.0) 85.1 109 (3.9) 98.6 (5.8)
Methyl eugenol 0.9999 0.0065 105 (8.5) 95.0 (5.1) 96.3 (1.9) 87.8 109 (7.5) 93.3 (3.0)
Isoeugenol 0.9987 0.037 78.0 (8.6) 83.5 (12) 92.6 (5.8) 83.5 82.6 (3.0) 85.5 (9.5)
Coumarin 0.9996 0.011 96.5 (3.6) 86.3 (6.4) 86.6 (4.8) 75.2 98.7 (11) 83.3 (2.1)
Ionone 0.9998 0.012 85.2 (6.7) 77.2 (1.2) 83.3 (7.0) 77.9 90.0 (5.6) 75.4 (0.9)
Lilial 0.9997 0.010 112 (6.7) 89.9 (5.7) 90.2 (7.2) 86.6 88.8 (7.0) 79.5 (1.9)
Amyl cinnamal 0.9999 0.024 102 (7.4) 75.3 (14) 85.7 (3.9) 80.3 72.9 (7.3) 79.1 (4.2)
Lyral 0.9999 0.063 91.8 (8.9) 76.2 (3.1) 75.4 (11) 69.7 89.2 (5.9) 89.9 (0.5)
Amyl cinnamyl alcohol 0.9999 0.28 96.2 (15) 106 (3.4) 84.8 116 (4.8) 108 (6.4)
Farnesol 0.9999 0.55 81.5 (9.0) 81.8 (7.5) 81.7 113 (9.1) 86.7 (7.2)
Hexylcinnam-aldehyde 0.9999 0.026 79.8 (8.8) 72.0 (0.8) 76.6 (3.8) 77.0 70.0 (6.5) 69.7 (11)
Benzyl benzoate 0.9996 0.014 89.0 (10) 86.8 (4.5) 92.5 (6.8) 83.2 96.3 (3.9) 92.4 (4.5)
Benzyl salicylate 1.0000 0.013 95.0 (7.6) 97.5 (1.4) 95.2 (6.3) 85.8 95.3 (4.5) 106 (4.1)
Benzyl cinnamate 0.9993 0.023 70.3 (6.1) 91.0 (2.0) 85.8 (9.2) 78.1 89.4 (5.3) 90.4 (9.2)
a
n = 4.
b
n = 2.
c
n = 3.
1384 E. Becerril-Bravo et al. / Chemosphere 81 (2010) 1378–1385
0.121
0.092
baby bathwater (BBW), were spiked with the target compounds at
STP
0.995
4.98
3.75
ASP
satisfactory for all compounds, with the exception of the three same
compounds with low recovery from milliQ water. These low recov-
SPW4
0.165
0.058
>LOD
eries were consistent in all samples, and also consistent with the
ones obtained from ultrapure water. Therefore, the method can be
considered suitable for the determination of 22 of the target com-
SPW3
0.183
0.101
0.077
pounds in real samples. The concentration of the other three com-
pounds, limonene, benzyl alcohol, and anisyl alcohol, could be
SPW2
0.133
0.215
0.180
0.407
0.190
0.630
0.093
0.090
0.987
0.550
0.017
0.053
>LOD
>LOD
0.177
0.093
0.052
>LOD
0.721
0.040
levels found for some of the target analytes. The samples from pub-
1.02
lic washing places (PWP), also showed the presence of some aller-
gen fragrances. These samples were taken at evening hours, in the
BBW13
0.444
0.417
0.148
0.247
0.556
0.340
5.33
21.6
6.93
2.70
204
0.759
0.289
51.1
11.4
4.25
43.4
8.94
30.2
0.706
2.11
0.829
0.232
5.95
4.15
1.82
5.67
BBW9
4. Conclusions
3385
4.23
32.8
2.18
4.55
0.378
0.300
2.50
0.385
0.979
2.22
87.4
2.83
4.29
11.5
0.225
0.233
0.990
2.13
0.462
0.364
0.861
0.894
4.78
5.23
4.66
4.39
11.5
23.0
6.02
BBW4
0.046
0.089
0.040
3.95
Target compounds found in different types of water (ng mL
0.364
0.500
7.59
9.25
2.48
1.13
128
0.462
0.840
2.64
19.9
6.55
91.8
23.4
1.77
23.2
2.08
0.133
0.620
0.086
0.200
Acknowledgements
Benzyl cinnamate
Cinnamyl alcohol
Benzyl salicylate
Benzyl benzoate
Hexylcinnam-
Isoeugenol
Limonenea
Citronellol
Coumarin
Eugenol
Ionone
Linalol
Lyral
Lilial
Table 4
References Ma, J.J., Du, X., Zhang, J.W., Li, J.C., Wang, L.Z., 2009. Ultrasound-assisted
emulsification–microextraction combined with flame atomic absorption
spectrometry for determination of trace cadmium in water samples. Talanta
Bassereau, M., Chaintreau, A., Duperrex, S., Joulain, D., Leijs, H., Loesing, G., Owen, N.,
80, 980–984.
Sherlock, A., Schippa, C., Thorel, P.J., Vey, M., 2007. GC–MS quantification of
Niederer, M., Bollhalder, R., Hohl, C., 2006. Determination of fragrance allergens in
suspected volatile allergens in fragrances. 2. Data treatment strategies and
cosmetics by size-exclusion chromatography followed by gas chromatography–
method performances. J. Agric. Food Chem. 55, 25–31.
mass spectrometry. J. Chromatogr. A 1132, 109–116.
Bossio, J.P., Harry, J., Kinney, C.A., 2008. Application of ultrasonic assisted extraction
Ozcan, S., Tor, A., Aydin, M.E., 2009a. Application of ultrasound-assisted
of chemically diverse organic compounds from soils and sediments.
emulsification–micro-extraction for the analysis of organochlorine pesticides
Chemosphere 70, 858–864.
in waters. Water Res. 43, 4269–4277.
Capelo, J.L., Mota, A.M., 2005. Ultrasonication for analytical chemistry. Cur. Anal.
Ozcan, S., Tor, A., Aydin, M.E., 2009b. Determination of selected polychlorinated
Chem. 1, 193–201.
biphenyls in water samples by ultrasound-assisted emulsification–
Chunhong, J., Xiaodan, Z., Li, C., Min, H., Pingzhong, Y., Ercheng, Z., 2010. Extraction
microextraction and gas chromatography-mass-selective detection. Anal.
of organophosphorus pesticides in water and juice using ultrasound-assisted
Chim. Acta 647, 182–188.
emulsification–mixroextraction. J. Sep. Sci. 33, 244–250.
Pumure, I., Renton, J.J., Smart, R.B., 2010. Ultrasonic extraction of arsenic and
EU, 2009. Regulation (EC) No. 1223/2009 of the European Parliament and of the
selenium from rocks associated with mountaintop removal/valley fills coal
Council of 30 November 2009 on Cosmetic Products (recast). Official Journal of
mining: estimation of bioaccessible concentrations. Chemosphere 78, 1295–
European Union, p. 59.
1300.
Fontana, A.R., Altamirano, J.C., 2010. Sensitive determination of 2,4,6-
Rastogi, S.C., Johansen, J.D., Menné, T., Frosch, P., Bruze, M., Andersen, K.E.,
trichloroanisole in water samples by ultrasound assisted emulsification
Lepoittevin, J.P., Wakelin, S., White, I.R., 1999. Contents of fragrance allergens
microextraction prior to gas chromatography–tandem mass spectrometry
in children’s cosmetics and cosmetic-toys. Contact Dermatitis 41, 84–88.
analysis. Talanta 81, 1536–1541.
Regueiro, J., Llompart, M., Garcia-Jares, C., Garcia-Monteagudo, J.C., Cela, R., 2008.
Fontana, A.R., Wuilloud, R.G., Martínez, L.D., Altamirano, J.C., 2009. Simple approach
Ultrasound-assisted emulsification–microextraction of emergent contaminants
based on ultrasound-assisted emulsification–microextraction for determination
and pesticides in environmental waters. J. Chromatogr. A 1190, 27–38.
of polibrominated flame retardants in water samples by gas chromatography–
Regueiro, J., Llompart, M., Psillakis, E., Garcia-Monteagudo, J.C., Garcia-Jares, C.,
mass spectrometry. J. Chromatogr. A 1216, 147–153.
2009. Ultrasound-assisted emulsification–microextraction of phenolic
IFRA. International Fragrance Association. <http://www.ifraorg.org/Home/
preservatives in water. Talanta 79, 1387–1397.
page.aspx/3> (accessed June 2010).
SCCNFP/0017/98, 1999. Final Fragrance Allergy in Consumers Brussels.
Lamas, J.P., Sanchez-Prado, L., Garcia-Jares, C., Llompart, M., 2009. Solid-phase
Sgorbini, B., Ruosi, M.R., Cordero, C., Liberto, E., Rubiolo, P., Bicchi, C., 2010.
microextraction gas chromatography–mass spectrometry determination of
Quantitative determination of some volatile suspected allergens in cosmetic
fragrance allergens in baby bathwater. Anal. Bioanal. Chem. 394, 1399–1411.
creams spread on skin by direct contact sorptive tape extraction-gas
Lamas, J.P., Sanchez-Prado, L., Garcia-Jares, C., Llompart, M., 2010. Determination of
chromatography–mass spectrometry. J. Chromatogr. A 1217, 2599–2605.
fragrance allergens in indoor air by active sampling followed by ultrasound-
WHO. Guidelines for Safe Recreational Water Environments. vol. 2. Swimming Pools
assisted solvent extraction and gas chromatography–mass spectrometry. J.
and Similar Environments. World Health Organization. <http://www.who.int/
Chromatogr. A 1217, 1882–1890.
water_sanitation_health/bathing/bathing2/en/>. (accessed June 2010)
Leijs, H., Broekhans, J., van Pelt, L., Mussinan, C., 2005. Quantitative analysis of the
26 allergens for cosmetic labeling in fragrance raw materials and perfume oils. J.
Agric. Food Chem. 53, 5487–5491.
Ultrasound-assisted emulsification–microextraction for the analysis
of several high production volume chemicals in environmental
waters
Abstract
A method based on in-situ derivatization-ultrasound-assisted emulsification–
microextraction (USAEME) and gas chromatography-mass spectrometry
(GC-MS) has been developed for the analysis of benzothiazole (BTZ), 4-tert-
butylphenol (TBP), 2-phenylphenol (PP) and piperonyl butoxide (PBO) in
waters. These chemicals are characterized by its high production volume and
worldwide spread use as part of the composition of many goods, insecticides,
as well as intermediates in chemical processes. The extraction conditions
including the in-situ acetylation of the phenols, were optimized using an
experimental design strategy. In the proposed procedure, compounds were
extracted during 5 min in an acoustically emulsified media formed by 200 ȝL
chloroform and 10 mL sample. The derivatization-USAEME process
provided an efficient and exhaustive extraction with the advantage that after
centrifugation, the extract was ready for GC-MS and GC-MS/MS analysis.
Validation of the method was performed using environmental water samples.
Recoveries between 68 and 111% were obtained, and precision was
characterized by RSD values d10%. The limits of quantification (LOQs) were
at the sub-ng per millilitre level (0.004-0.072 ng mL-1). The procedure was
applied to the determination of the target compounds in different waters
including influent and effluent waters of sewage treatment plants. The
presence of the target compounds was confirmed in the samples.
In addition, we report for the first time that the presence of chlorine as
disinfection agent in the water did not affect BTZ and PBO, which remained
stable even at 4 mg L-1 of chlorine concentration, whereas the phenolic
compounds (TBP and PP) required lower chlorination levels to be
transformed into several mono- and di-chlorinated derivatives.
1
Introduction
2
Analytical methods developed for these chemicals are generally scarce. Some
of these compounds have been included in waters studies usually associated to
the analysis of pesticides (PBO and PP) or to the analysis of corrosion
problems (BTZ). Most methods are based on liquid-liquid extraction
techniques [13] and solid-phase extraction (SPE) [14] using large volumes of
sample. The use of solid-phase microextraction has also been reported [15].
To allow investigating the occurrence of these pollutants in waters, a simple
methodology based on liquid-liquid microextraction assisted by ultrasounds is
proposed for the first time. The ultrasound-assisted emulsification–
microextraction (USAEME) technique has been proposed by Regueiro et al
[16], and is based on the emulsification of a microvolume of organic
extractant in an aqueous sample by ultrasound radiation and further separation
of both liquid phases by centrifugation. The application of ultrasonic radiation
accelerates the mass-transfer process between two immiscible phases, which
together with the large surface of contact between both phases leads to an
increment in the extraction efficiency in a minimum time. After its
introduction in 2008, USAEME has found successful application worldwide
[17-21].
The proposed procedure includes in-situ acetylation of the phenolic
compounds (TBP and PP). The optimization of the derivatization-extraction
conditions is conducted using experimental design tools.
Several environmental water samples including river water as well as samples
taken from different sewage treatment plants, have been analyzed, showing
the presence of the compounds in the samples. In addition, since chlorination
is very common as water disinfection method, experiments have been
performed to establish the behaviour of the studied compounds in front of
chlorination. To the best of our knowledge, this is the first time that the
formation of chlorinated derivatives of TBP and PP is reported.
3
heptahydrate and sodium thiosulphate were purchased from Alfa Aesar
(Karlsruhe, Germany).
Individual stock solutions of each compound were prepared in acetone and a
standard mixture solution was prepared in acetone at a final concentration of
about 10 μg mL-1. Different standard working solutions were obtained by
appropriate dilution and stored in amber colored vials at -20 ºC. Water
samples were filtrated through a 0.45 μm Millipore HA membrane filter
(Billerica, MA, USA).
Chlorination
Experiments of chlorination were performed using chlorine water solutions
prepared by proper dilution of the analytical reagent (sodium hypochlorite).
These solutions were spiked with the standard mixture of analytes to obtain a
10 ng mL-1 concentration. After the selected reaction time, the excess of free
chlorine was removed with sodium tiosulphate (0.1 mg mL-1), and USAEME
was carried out as described before. In these experiments, the derivatizing
agents were not added to avoid for possible side-reactions.
4
PTV injector. This injector was equipped with a 12 cm×2mm i.d. Silcoseeve
liner (Thermofinnigan). Two μL of sample were injected onto the PTV
injector in the constant flow mode set at 1 mL min-1. The temperature of the
injector was initially set at 40ºC, then increased to 220ºC at a rate of 14.5ºC/s
where it was maintained for 9 min. The PTV split/splitless valve was operated
in the splitless mode until the temperature of 220ºC was achieved. Once the
temperature stabilised, it was maintained for 1.3 min, then changed to the split
mode. Compounds were separated on a 30m×0.25mm i.d. column, coated
with 0.25 m of 95% dimethyl–5% phenyl polysiloxane phase (Thermo TR-
5ms, San Jose, CA, USA). The temperature of the column was initially set at
80ºC for 1 min, and then increased at a rate of 10ºC/min to its final
temperature of 280ºC, which was maintained for 5 min. Helium was used as
the carrier gas at a constant flow of 1 mL min-1. The transfer line was set at
280ºC with the external ion source at 230ºC. Electron impact ionization (EI)
positive mode (+70eV) was used in the mass spectrometer and the damping
gas flow in the ion trap was set to 0.9 ml min-1 .The precursor ions were
selected among the most intense characteristic ions of the MS spectrum,
giving rise to the most efficient MS/MS transitions in the ion trap. Whenever
possible, two or three product ions were monitored in the full MS/MS spectra
for unambiguous identification of the analytes.
Specific MS/MS conditions and retention times for each target compound are
listed in Table 1.
An Agilent 7890A gas chromatograph provided with a 5975C inert MSD with
triple axis detector and a 7693 autosampler from Agilent Technologies (Palo
Alto, CA, USA), was also employed for several extraction and chlorination
experiments. The temperatures of the transfer line, the quadrupole and the ion
source were set at 290, 150 and 230 °C, respectively. The system was
5
operated by Agilent MSD ChemStation E.02.00.493 software. Separation was
carried out on a HP5 capillary column (30 m × 0.25 mm i.d., 0.25 ȝm film
thickness) from Agilent Technologies (Palo Alto, CA, USA). Helium (purity
99.999%) was employed as carrier gas at a constant column flow of 1.0 mL
miní1. The GC oven temperature was programmed from 80 °C (held 2 min) to
280 °C at 10 °C miní1 (held 3 min) (total analysis time = 25 min). After 1 min
the split was opened at a flow of 75 mL miní1 and the injector temperature
was kept at 220 °C. The injection volume was 1 ȝL. The mass spectra detector
(MSD) was operated in the scan mode and the mass range was varied from 40
to 500 m/z, starting at 4 min and ending at 25 min. The electron multiplier
was set at a nominal value of 1200 V.
6
considered was the salting-out effect. Sodium chloride addition was evaluated
at three concentration levels in the aqueous solution, 0% (no addition), 15%
(w/v) and 30% (w/v).
The selected optimization strategy consisted on a mixed levels fraction
screening design consisting of a fraction 3*23-1 design, involving a total of 12
experiments. Factor levels and the corresponding identification keys are
summarized in Table 2. The selected design allows to determine which factors
have a statistically significant effect, as well as which are the significant
interactions between factors. Experiments have been performed using 10-mL
aliquots of milliQ water spiked with the analytes at a concentration of 5 ng
mLí1.
Table 2. Factors and levels selected for the experimental design optimization
Factor level
Factor Key
Low Intermediate High
NaCl (%) A 0 15 30
Time (min) B 5 10
Chloroform Trichloroethane
Solvent type C
(CLF) (TCE)
Derivatizing
D No Yes
reagents
Numerical analysis of data resulting from the experimental design was made
using the Statgraphics XV Centurion statistical software package
(Manugistics, Rockville, MD, USA). ANOVA results are shown in Table 3.
For the sake of simplicity, only the interactions between factors that were
significant in any case have been included. ANOVA information can be
clearly shown in the Pareto charts (Figure 1a).
7
In these graphics the length of each bar is proportional to the absolute value of
its associated standardized effect. Vertical line in the graphs represents the
statistically significant bound at the 95% confidence level.
Main effects plots are displayed in Figure 1b. This kind of plots shows the
main effects with a line drawn between the low and the high level of the
corresponding factors. The length of the lines is proportional to the effect
magnitude of each factor in the extraction process, and the sign of the slope
indicates the level of the factor that produces the highest response.
Figure 1. (a) Pareto charts showing the significant factors (95%) for the target
analytes (see factor codes in Table 2). (b) Main effects plots (CLF:
chloroform; TCE: trichloroethane).
a
BTZ TBP
A AD
BC D
C BC
A
CD
AC
AC C
B CD
D B
0 2 4 6 8 10 0 2 4 6 8 10
Standardized effects Standardized effects
PP PBO
D D
AD AA
A BC
AC
BC
B
BD CD
C A
B C
0 4 8 12 16 0 2 4 6 8 10
Standardized effects Standardized effects
b
BTZ TBP
4100 6000 Yes
30%
3800 5500 0%
TCE
10 3 *A/AIS
TCE
10 3 *A/AIS
3500 10 5000 10
Yes
5
3200 No 4500
5 CLF
CLF 30%
2900 4000
0% No
2600 3500
NaCl Time (min) Solvent Derivatizing NaCl Time (min) Solvent Derivatizing
reagents reagents
PP PBO
2400 2700
Yes Yes
2000 0% 2400 10
10 3 *A/AIS
TCE
10 3 *A/AIS
15%
1600 5 CLF TCE 2100 5
1200 10 CLF
1800
800 30%
No
400 1500
No 30%
0%
0 1200
NaCl Time (min) Solvent Derivatizing NaCl Time (min) Solvent Derivatizing
reagents reagents
8
(Figure 1b). For two of the studied compounds (TBP and PP), higher
efficiency was observed when there is no addition of sodium chloride, while
for BTZ better responses are obtained when 30% of sodium chloride is added.
The addition of acetylating agents showed a significant higher extraction
efficiency for TBP, PP and PBO. Although PBO is not acetylated, it seems
that the presence of the derivatizing reagents increases the tendency of PBO to
dissolve into the extracting phase. Regarding the extraction solvent, this factor
was significant for BTZ, and in this case, 1,1,1-trichloroethane gave responses
of only about 10% higher than those obtained using chloroform. Since the
significantly lower prize of chloroform, this last solvent was finally selected.
The extraction time was not significant in any case.
The interaction between the two most important main factors, salt and
derivatizing reagents addition (AD), was significant for TBP and PP, even
being the most important factor for the extraction of TBP (see Figure 1a). The
presence of NaCl has an adverse effect on the extraction of the compounds
from the aqueous solution containing the acetylating reagents.
In view of the results of the experimental design, the selected general
conditions for the simultaneous in-situ derivatization-USAEME of the target
compounds from water samples were as follows: addition of the acetylation
reagents to the sample, and extraction with chloroform employing a
sonication time of 5 min.
9
samples by the added concentrations. As can be seen in Table 4, recoveries
were satisfactory for all compounds, with values ranging from 68.3 to 111%.
The precision of the method was evaluated by calculating the relative standard
deviation (RSD) at the same concentration levels. Results are also shown in
Table 4, and they were lower than 10% in all cases.
Linearity was tested using acetylated standards prepared in chloroform at
concentrations ranging between 2 and 1000 ng mL-1. Correlation coefficients
(R) between 0.9999 and 1.0000 were obtained for all compounds (Table 4).
Instrumental detection limits (IDL) were estimated for a signal-to-noise ratio
of 3 (S/N=3), and values ranged from 0.001 to 0.006 ng mL-1. Since the
presence of BTZ was observed in some of the procedural blanks, the LOQ for
this compound was estimated as that corresponding to the average amount of
analyte giving a response that is the blank signal plus 10 times the standard
deviation (LOQ = blank signal + 10SD). The source of BTZ in blanks might
be due to their presence at low levels in the ultrapure water or to
contamination through the analytical process. As shown in Table 4, LOQ
values ranged from 0.004 ng mL-1 to 0.072 ng mL-1.
Finally, the proposed method was applied to the analysis of several non-
spiked water samples, including river water, and the effluent and influent
waters from different STPs. The presence of the targets was confirmed in the
samples. BTZ was found above the quantification limit in all the samples, at
concentrations in a range of 0.10-0.37 ng mL-1 (see Table 5). TBP and PP
were found in some of the effluents, whereas PBO was found in three of the
seven samples at concentrations over the LOQ.
Table 5. Target compounds found in river and wastewaters samples (ng mL-1)
Comp. River1 River2 Ef. STP1 Ef. STP2 Ef. STP4 Inf. STP3 Inf. STP4
BTZ 0.137 0.222 0.204 0.370 0.164 0.123 0.101
TBP - - - 0.113 - - -
PP - - 0.0886 0.123 - - -
PBO - - 0.024 - - 0.025 0.031
Chlorination experiments
The target compounds have been found in waters that may be treated for
drinking and other uses such as recreational. Chlorination is a widespread
procedure of water disinfection that can decompose and thus be useful to
remove the target chemicals, but it also may generate undesirable chlorination
by-products.
Sodium hypochlorite exists in aquatic solution mainly as hypochlorous acid
and hypochlorite anion, which can react with organic compounds by addition,
substitution or oxidation. In disinfection processes, many factors would have
influence on chlorination disinfection by-products formation and distribution,
e.g. chlorine dosage and chlorination time.
10
Some experiments were carried out in order to check if the studied
compounds were degraded in the presence of chlorine. In this case, sodium
hypochlorite was added to MilliQ water (pH= 5.6) to reach a final
concentration of 4 mg L-1 expressed as chlorine, which is the maximum
residual disinfectant level (MRDL) regulated by EPA [28], and about 10 times
over the usually recommended minimum breakpoint chlorine concentration
[29]. The resulting mixture was extracted and analyzed after 1 hour and 5
hours of reaction. These experiments were performed without adding
derivatization reagents to avoid for possible interferences. Complete
degradation of TBP and PP was observed after 1 hour of reaction, whilst the
responses of BTZ and PBO were not affected (Figure 2a).
Figure 2. (a) Relative responses obtained after 1 and 5 hours of reaction with
chlorine (4 mg L-1) (Rt : Response at time t; R0 : response at time 0) (b) Effect
of the chlorine concentration on the degradation of TBP and PP (Rc :
Response at concentration C; R0 : response at concentration 0).
a
0h 1h 5h
120
100
80
100xRt /R0
60
40
20
0
BTZ TBP PP PBO
120
100
Chlorine concentration:
80 0 mg/L
100xRC/R 0
0.2 mg/L
60
1.5 mg/L
40 4 mg/L
20
0
TBP PP
11
The influence of the initial concentration of chlorine on the degradation of
TBP and PP was evaluated at 1.5 h of reaction. As can be seen in Figure 2b,
the concentration of TBP and PP decreased with increasing concentrations of
chlorine. The exposition to low concentrations of chlorine (0.2 mgL-1) did
only decrease the TBP and PP concentrations in 3 and 13%, respectively,
whereas the residual concentrations of both compounds at 1.5 mgL-1 chlorine
level in water were 16 and 5%, respectively.
The kinetics of the degradation of TBP and PP in the presence of 1.5 mgL-1
chlorine were followed and the results are depicted in Figure 3. As can be
seen, in less than one hour of reaction (c.a. 45 min) the concentrations of both
compounds were reduced to half, and the degradation of both compounds was
complete (>94%) after 5 h reaction.
60
PP 0.989 1.93 0.36
50
40
30
20
10
0
0 5 10 15 20
Time (h)
The degradation of PP was slightly faster than the one of TBP. These
reactions followed pseudo-first order kinetics and the first-order rate constants
(kap) can be determined as the slopes of the straight-line equations obtained
from linear regression: lnC vs. time plot. The corresponding half-lives are
calculated using the following expression: t1/2 = ln 2/kap. These date are also
showed in Figure 3.
The degradation reactions of TBP and PP in the presence of chlorine led to the
formation of several by-products, five of which were detected in these
experiments, as can be seen in the chromatogram of a chlorinated water
sample (1.5 mgL-1) depicted in Figure 4. These by-products were tentatively
identified on the basis of their mass spectra and the results are shown in
Figure 4. Two compounds are formed by chlorination of TBP (monochloro-
TBP and dichloro-TBP), and the other three are generated when PP combined
12
with one and two chlorine atoms (two isomers have been detected for the
monochlorinated derivative). Although the formation of chlorination
disinfection by-products of phenolic compounds [30, 31], and endocrine
disruptors [32, 33] has been previously reported, to the best of our knowledge
this is the first time that the formation of chlorinated by-products of TBP and
PP is described.
Figure 5 shows the formation and degradation kinetics of TBP and PP
detected by-products. It can be appreciated that monochloro-TBP reaches it
maximum at 40 min (Figure 5a), when the degradation of the parent TBP
reaches the 50% (see Figure 3). At this reaction time, the formation of the
dichloro-TBP is 20% to rapidly grow to its maximum at 1.5 h. For both
compounds, kinetics curves are very similar showing that at 5 h the residual
concentrations of both derivatives are in the range of 20-35% and remain
stable in the frame of the experiments (24 h). Figure 5b shows the kinetic
behaviour of PP by-products. In this case, monochloro-derivatives are rapidly
formed, reaching their 100% at 40 min. After this time, the first
monochlorinated by-product transforms in dichloro-PP, which quickly reaches
its maximum at 1.5 h. Degradation-formation curves of monochloro-PP2 and
dichloro-PP are similar from 1.5 h to the end of the kinetics experiment (24h),
showing that both derivatives are very stable, with remaining water
concentrations of 60-80%.
13
Figure 4. Ion chromatograms of the chlorination obtained TBP and PP by-
products (chlorine concentration: 1.5 mg L-1), showing the experimental and
NIST mass spectra of the by-products.
Relative abundance
Counts m/z: 169.00
50
2000
141 184
Monochloro-TBP 51 63 77 89 96 105 114 129 156
1600 0
169
100 NIST MS Search 2.0 spectrum:
Relative abundance
Phenol, 2-chloro-4-(1,1-dimethylethyl)-
1200 (Match: 88%)
800 50
141 184
51 77 129
400 0
63 89 97 105 114 153
50 70 90 110 130 150 170 190
0
100 Experimental spectrum 203
Relative abundance
Counts m/z: 203.00
50
8000 Dichloro-TBP 175 218
51 63 77 89 103 123 139 150 163 191
6000 0
NIST MS Search 2.0 spectrum: 203
100
Relative abundance
Phenol, 2,6-dichloro-4-(1,1-dimethylethyl)-
(Match: 97%)
4000
50
175 218
2000 77 89 103
50 63 123 139 152 163 187
0
60 80 100 120 140 160 180 200 220
0
100 Experimental spectrum 204
Relative abundance
0
100 Experimental spectrum 238
Relative abundance
168 202
8000 50
69 87 101 149 183 223
0
238
100 NIST MS Search 2.0 spectrum:
6000
Relative abundance
(1,1’-Biphenyl)-2-ol, 3,5-dichloro
(Match: 89%)
4000 50 139
168
14
Figure 5. (a) Formation and degradation kinetics of TBP by-products. (b)
Formation and degradation kinetics of PP by-products
a
MonochloroTBP
DichloroTBP
100
80
100xRt /Rmax
60
40
20
0
0 5 10 15 20
Time (h)
b
MonochloroPP1
MonochloroPP2
100 DichloroPP
80
100xRt /Rmax
60
40
20
0
0 5 10 15 20
Time (h)
Conclusions
In the present work, a method based on ultrasound-assisted emulsification–
microextraction (USAEME) has been developed for the analysis in water of
four high production volume chemicals (benzothiazole, 4-tert-butylphenol, 2-
phenylphenol and piperonyl butoxide). USAEME is proposed as an efficient
and non-expensive extraction technique. Under the optimized conditions
15
established after a multivariate study of the USAEME process, good
recoveries were obtained for all compounds, even in complex water samples
(sewage water). Method precision was satisfactory and quantification limits at
the sub-nanogram per millilitre level were obtained. The proposed method
was applied to the analysis of several real water samples including rivers and
STP influent and effluent waters. Since no matrix effects were observed,
quantification could be carried out by means of external calibration using
standards in chloroform, allowing high throughput of the analysis and
procedural simplicity. The presence of the studied compounds was confirmed
in the water samples. In addition, we found that in chlorinated waters the
phenolic compounds (TBP and PP) were transformed into mono- and di-
chlorinated derivatives, some of them quite persistent, while BZT and PBO
remained unchanged during at least 24 h, even at chlorine concentrations of 4
mgL-1.
Acknowledgements
This research was supported by FEDER funds and project CTQ2010-19831
(Ministerio de Ciencia e Innovacion, Spain). L. S.-P. and J.P.L. acknowledge
Xunta de Galicia for a postdoctoral Angeles Alvariño, and Isabel Barreto
contracts, respectively. E. B.-B thanks the Universidad Nacional Autonoma
de Mexico, Secretaría General, Dirección General de Asuntos del Personal
Academico, for the award of a research scholarship.
References
16
[12] H. Tovalin-Ahumada, L. Whitehead, Science of the Total
Environment, 376 (2007) 60.
[13] M.J. Gomez, M.M. Gomez-Ramos, A. Aguera, M. Mezcua, S.
Herrera, A.R. Fernandez-Alba, Journal of Chromatography A, 1216 (2009)
4071.
[14] R. Rodil, J.B. Quintana, E. Concha-Grana, P. Lopez-Mahia, S.
Muniategui-Lorenzo, D. Prada-Rodriguez, Chemosphere, 86 (2011) 1040.
[15] V. Casas, M. Llompart, C. Garcia-Jares, R. Cela, T. Dagnac,
Analytical and Bioanalytical Chemistry, 387 (2007) 1841.
[16] J. Regueiro, M. Llompart, C. Garcia-Jares, J.C. Garcia-Monteagudo,
R. Cela, J. Chromatogr. A, 1190 (2008) 27.
[17] J. Regueiro, M. Llompart, E. Psillakis, J.C. Garcia-Monteagudo, C.
Garcia-Jares, Talanta, 79 (2009) 1387.
[18] A.R. Fontana, R.G. Wuilloud, L.D. Martínez, J.C. Altamirano, J.
Chromatogr. A, 1216 (2009) 147.
[19] S. Ozcan, A. Tor, M.E. Aydin, Wat. Res., 43 (2009) 4269.
[20] J. Chunhong, Z. Xiaodan, C. Li, H. Min, Y. Pingzhong, Z. Ercheng, J.
Sep. Sci., 33 (2010) 244.
[21] N. Cabaleiro, F. Pena-Pereira, l.C.I. de, C. Bendicho, I. Lavilla,
Microchem. J., 99 (2011) 246.
[22] L. Sanchez-Prado, J.P. Lamas, M. Lores, C. Garcia-Jares, M.
Llompart, Anal. Chem., 82 (2010) 9384.
[23] E. Becerril-Bravo, J. Pablo Lamas, L. Sanchez-Prado, M. Lores, C.
Garcia-Jares, B. Jimenez, M. Llompart, Chemosphere, 81 (2010) 1378.
[24] I. Rodríguez, M.P. Llompart, R. Cela, J. Chromatogr. A, 885 (2000)
291.
[25] M. Llompart, M. Lourido, P. Landín, C. García-Jares, R. Cela, J.
Chromatogr. A, 963 (2002) 137.
[26] M. Polo, M. Llompart, C. Garcia-Jares, G. Gomez-Noya, M.-H.
Bollain, R. Cela, J. Chromatogr. A, 1124 (2006) 11.
[27] T.R. Croley, B.C. Lynn, Rapid Commun. Mass Spectrom., 12 (1998)
171.
[28] H.-J. Su, C.-J. Chao, H.-Y. Chang, P.-C. Wu, Atmospheric
Environment, 41 (2007) 1230.
[29] P. Mocho, V. Larroque, V. Desauziers, Analytical and Bioanalytical
Chemistry, 388 (2007) 147.
[30] F. Ge, L. Zhu, J. Wang, Desalination, 225 (2008) 156.
[31] H. Gallard, A. Leclercq, J.-P. Croué, Chemosphere, 56 (2004) 465.
[32] M. Deborde, S. Rabouan, H. Gallard, B. Legube, Environ. Sci.
Technol., 38 (2004) 5577.
[33] J. Hu, S. Cheng, T. Aizawa, Y. Terao, S. Kunikane, Environ. Sci.
Technol., 37 (2003) 5665.
17