215

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Current Pharmaceutical Analysis, 2017, 13, 215-223

RESEARCH ARTICLE
ISSN: 1573-4129 Volume 12, Number 4
eISSN: 1875-676X

GC-MS Method for the Analysis of Thirteen Opioids, Cocaine and

Cocaethylene in Whole Blood Based on a Modified Quechers Extraction Impact
Factor:
0.885

BENTHAM
SCIENCE

Emanuele Amorim Alves1,2,3*†, Ana Sofia Agonia2†, Sara Manuela Cravo4, Carlos Manuel Afonso4,5,
Annibal Duarte Pereira Netto6, Maria de Lourdes Bastos1, Félix Carvalho1 and Ricardo Jorge
Dinis-Oliveira1,2,7*

1
UCIBIO, REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University
of Porto, Porto, Portugal; 2Department of Public Health and Forensic Sciences, and Medical Education, Faculty of
Medicine, University of Porto, Porto, Portugal; 3EPSJV – Polythecnical School of Health Joaquim Venâncio, Oswaldo
Cruz Foundation, Rio de Janeiro, Brazil; 4Center of Medical Chemistry (CEQUIMED-UP), Faculty of Pharmacy,
University of Porto, Porto, Portugal; 5Interdisciplinary Center of Marine and Enviromental Investigation
(CIIMAR/CIMAR), Porto, Portugal; 6Department of Analytical Chemistry, Chemistry Institute, Fluminense Federal
Current Pharmaceutical Analysis

University, Niterói, Brazil; 7IINFACTS - Institute of Research and Advanced Training in Health Sciences and
Technologies, Department of Sciences, University Institute of Health Sciences (IUCS), CESPU, CRL, Gandra, Portugal

Abstract: Background: QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) is a methodology
previously developed to extract pesticides from vegetables and fruits and has been fully applied for dif-
ferent analytical approaches.
Objective: In the present study, a rapid and less laborious modified QuEChERS extraction method for the
quantification of 13 opioids [codeine, morphine, heroin, 6-acetylmorphine (6-AM), desomorphine, ethyl-
morphine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), 2-ethyl-5-methyl-3,3-
diphenyl-1-pyrroline (EMDP), papaverine, tramadol, O-desmetyltramadol (M1) and, tapentadol], cocaine
and cocaethylene in whole blood was developed and validated by Gas Chromatography-Mass Spectrometry.
ARTICLE HISTORY
Method: The modification of QuEChERS method consisted in the pretreatment of the whole blood sam-
Received: November 18, 2015 ples using ultrasonication, the use of ethyl acetate as extraction solvent and a previous step of sample
Revised: April 12, 2016
Accepted: April 29, 2016 alcalinization. The use of dispersive separation steps such as Dispersive Solid-Phase Extraction (dSPE) or
sorbents such as Primary Secondary Amine (PSA) was suppressed to minimize the errors and, to improve
DOI:
10.2174/15734129126661605021638 the velocity of the analysis.
46
Results: The method proved to be selective and the regression analysis for the analytes was linear in the
range of 31.2-2000 ng/mL with correlation coefficients > 0.98. The coefficients of variation did not ex-
ceed 15%. The lowest limit of detection and quantification for all the analytes were below the therapeutic
range of the drugs. The recoveries of the analytes ranged from 52.4 to 95.0%.
Conclusion: The developed method can provide a rapid, effective and “greener” process for the analysis
of a wide range of opioids drugs in whole blood samples and can be applied to clinical and forensic ante-
mortem and postmortem cases.
Keywords: Opioids, drugs of abuse, QuEChERS, whole blood, GC-EI/MS, clinical and forensic toxicology.

INTRODUCTION [1-2]. The use of such biological matrices demands an ex-

traction/purification pre-treatment before its chroma-
Clinical and Forensic Toxicology are strongly based in tographic analysis. Solid-phase extraction (SPE) and liquid-
Analytical Chemistry. Whole blood, urine and solid tissues
liquid extraction (LLE) are common techniques used for this
as well as alternative samples such as oral fluid, hair and
purpose. LLE is a classical and simple technique that uses
meconium are commonly used for toxicological analysis
solvents to selectively extract target compounds [3, 4]. How-
ever, LLE has a number of drawbacks that limit its use,
*Address correspondence to these authors at the Department of Biological namely reduced selectivity, large solvent consumption and
Sciences, Laboratory of Toxicology, Faculty of Pharmacy, University of difficulty of automation. Moreover, formation of emulsions
Porto, Rua José Viterbo Ferreira nº 228, 4050-313 Porto, Portugal; Tel: can interfere with the phase-separation process due to the
+351 220428597; E-mails: manuhpa@hotmail.com; ricardinis@med.up.pt
†The first two authors contributed equally to this work. difficulty to separate emulsified organic phases, and errors

17-;/17 $58.00+.00 © 2017 Bentham Science Publishers

216 Current Pharmaceutical Analysis, 2017, Vol. 13, No. 3 Alves et al.

can occur when extracting compounds of interest, conse- (internal standard, IS) was obtained from LGC Standards.
quently questioning the obtained quantitative values [5]. SPE The purity of all standards were higher than 98.5%. Helium
has high selectivity and has been used for many biological C-60 (99.99%) was ordered from Gasin (Portugal). Nitrogen
matrices [6-7]. Although some automation exists, SPE is a was supplied by AirLiquid (Algés, Portugal). All the rea-
complex, laborious, high cost and time-consuming method- gents used were from the highest available grade.
ology in an extensive multi-steps process [8].
Sample Collection
In 2003, Anastassiades et al. [9] developed a new extrac-
tion method named QuEChERS (Quick, Easy, Cheap, Effec- Blank whole blood samples for method validation were
tive, Rugged and Safe) to analyze pesticides in vegetables obtained from healthy volunteers who gave informed con-
and fruits, removing sugars, lipids, organic acids, steroids, sent, and collected in tubes containing EDTA. Any preserva-
proteins, pigments and water excess, all in one step. Its ad- tive such as sodium fluoride was added.
vantages are the quicker and easier handling, the use of low
solvent volumes and low cost when compared with other Preparation of Stock, Quality Control and Working So-
extraction methods. This process involves a sample extrac- lutions
tion using acetonitrile, followed by addition of anhydrous
Separate commercially available 1 mg/mL methanolic so-
magnesium sulfate (MgSO4) and sodium chloride (NaCl) to lutions for each analyte and IS were used as stock solutions.
decrease solubility of organic drugs in aqueous phase, and to
They were prepared and stored at -20ºC until use. The heroin
reduce the amount of water in the organic phase [9-10]. Only
solutions were prepared in chloroform due to their instability
few studies have applied QuEChERS in human samples.
when stored in methanolic solutions [9]. Working solutions
Some successful reports, included the extraction of pesti-
(2000 ng/mL) were prepared by dilution from each stock
cides [11] and drugs of abuse in human whole blood [10, 12]
solution in 1 mL of mL of blank whole blood. For calibration
and muscle samples [13-15]. Most studies applying QuECh- standards and quality control (QC) samples, the working
ERS to complex biological samples such as whole blood,
solutions were diluted in blank blood by serial dilutions. For
describes the use of a pre-treatment step with Primary Sec-
the calibration standards serial dilutions were made to origi-
ondary Amine (PSA) [8, 12]. PSA showed to be an effective
nate the 2000, 1000, 500, 250, 125, 62.5 and 31.2 ng/mL
sorbent for removal of various matrices and significantly
concentrations. For QC spiking solutions (low, 31.2; me-
reducing matrix-enhancement effect. The surface of PSA
dium, 250; high, 2000 ng/mL), serial dilutions from the work
contains many primary secondary amino groups, which solutions were also used. No dilution integrity was per-
could selectively adsorb fatty acids an important interference
formed as part of the method validation since selected stan-
in whole blood samples [16].
dard concentrations cover most concentration found in clini-
Due the large number of cases that every year reaches cal and forensic settings as previously demonstrated [10].
clinical and forensic laboratories, it is imperative to use fast
extraction, detection and quantitative analytical methods to Samples Extraction Using QuEChERS
analyze toxicological substances in biological specimens.
For extraction, 300
L of spiked whole blood with work
Opioids have a great role in fatal intoxications in Europe, as
concentrations of each analyte was alkalinized using one
reported in The European Report about Drugs. About 3.5%
drop of NaOH 0.1M, to obtain a pH around 10.0. Samples
of all deaths of Europeans 15-39 years old were drug over-
were vortexed and then centrifuged at 10,000g for 5 minutes.
doses, opioids were found in about three-quarters of fatal
The resulting supernatants were separated and, then soni-
overdoses [17]. cated for 5 min in an ultrasonic bath (Fig. 1A). 300
L of the
Therefore, the goal of our work was the development and supernatant was pipetted into a tube containing a previously
validation of a modified QuEChERS method for the simulta- pulverized mixture of 50mg of NaCl and 100mg of MgSO4
neous extraction of 13 opioids from whole blood samples (1:2), 500 μL of ethyl acetate and two metal spheres. The
and to analyze them by GC-MS. samples were vortexed for 10 seconds and then centrifuged
at 7,300g for 2 minutes. The organic phase was carefully
MATERIAL AND METHODS transferred to a dry and clean glass tube. This extraction
process was repeated to maximize extraction efficiency. All
Reagents and Standards
samples were evaporated to dryness using a nitrogen flow at
Ethyl acetate and sodium sulfate were purchased from room temperature (Fig. 1B). The time effort was approxi-
Carlo Erba (Milan, Italy), N-methyl-N-(trimethylsily) mately 30 minutes for fourteen different samples and a daily
trifluoroacetamide (MSTFA) was purchased from Sigma- calibration curve. 30
L of N-methyl-N-(trimethylsily)
Aldrich (St Louis, MO, USA). Magnesium sulfate and so- trifluoroacetamide (MSTFA) were added and samples heated
dium chloride in powder form and analytical grade were at 80ºC for 30 min to accomplish silylation. An aliquot of 1
purchased from Merck (Darmstadt, Germany). Codeine,
L of the derivatized extract was injected into the GC-IT/MS
morphine, heroin, 6-AM, desomorphine, ethylmorphine, system (Fig. 1C).
methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine
(EDDP), 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP), GC-MS Conditions
papaverine, tramadol, tapentadol, cocaine and cocaethylene
Quantitative and qualitative GC-MS analyses were per-
certified standards were purchased from Lipomed AG (Ar-
formed on a Trace GC 2000 Series ThermoQuest gas chro-
lesheim, Switzerland). O-desmetyltramadol (M1) was a gen-
matograph equipped with ion-trap GCQ Plus ThermoQuest
erous gift from Grünenthal (Amadora, Portugal). Phenacetin
Finnigan mass detector (Austin, Texas, USA). Chroma-
QuEChERS and Drugs of Abuse Current Pharmaceutical Analysis, 2017, Vol. 13, No. 3 217

Table 1. The designated m/z for the opioids analyzed.

Pre-treatment A
300 µL of blood Analyte m/z
+
1 drop of NaOH 0.1M Phenacetin-tms 162, 236, 251
↓
Centrifuge 10.000 g, 5 minutes, 4 ºC Desomorphine-tms 148, 271, 286
↓ Codeine 146, 234, 371
5 minutes in ultrasonic bath
Morphine-tms 234, 268, 429
QuEChERS extraction B 6-AM-tms 203, 268, 399
Two metal spheres
+ Heroin-tms 327, 369, 310
Pulverized mixture of 100 mg MgSO4 + 50 mg NaCl Methadone 72, 233, 294
↓
500 µL of ethyl acetate EDDP 232, 262, 277
↓
100 µL of pre-treated blood EMDP 115, 130, 193
↓
Tramadol 58, 264, 336
Vortex (2×)
↓ M1 58, 322, 394
Centrifuge 7.300g, 2 minutes, 4ºC
↓ Ethylmorphine-tms 192, 234, 385
Separate the organic phase into a glass tube
Papaverine 293, 324, 338
↓
Dry under nitrogen flow Tapentadol 58, 133, 221
Derivatization procedure C Cocaine 82, 182, 303

30 µL of MSTFA Cocaethylene 82, 196, 317

↓
80ºC, 30 minutes
↓ and cocaine in Portugal between 2001 and 2013; these cases
Cool to room temperature did not register positivity for phenacetin. Typical recom-
↓ mendations on sample preparation of biological specimens
Injection of 1 μL into GC-IT/MS for systematic toxicological analysis suggests that for any
systematic toxicological assay procedure, active drugs
Fig. (1). Sample preparation procedure. A – Pre-treatment of blood should only be used as internal standards when no alternative
samples. B - Extraction of analytes by modified QuEChERS is available and after proofing their absence in the sample,
method. C – Derivatization procedure. which corresponds to the present case [15].

tographic separation was achieved using a capillary columm Method Validation

(30 m  0.25 mm  0.25
m, cross-linked 5% diphenyl and
95% dimethyl polysiloxane) from Restek® (Pennsylvania, The validation of the method was performed accordingly
USA) and high-purity helium C-60 as carrier gas. An initial to European Medicines Agency [16]. The evaluated parame-
temperature of 80ºC was maintained for 1 min, increased to ters were selectivity, limit of detection (LOD), lower limit of
300ºC at 10ºC/min rate, and held for 5 min giving a total run quantification (LLOQ), precision, accuracy, recovery, matrix
time of 28 min. The flow of the carrier gas was maintained at effect and linearity of the method. Calibration curves were
1.0 mL/min. The injector port was set at 280ºC. Quantifica- prepared by serial dilutions starting from the working solu-
tion was performed in selected ion monitoring mode (SIM) tions in blank whole blood prepared with standard solutions
with splitless injection. The most abundant ions with higher of codeine, morphine, heroin, 6-AM, desomorphine,
m/z were selected for quantification and, second and third ethylmorphine, methadone, 2-ethylidene-1,5-dimethyl-3,3-
more abundant m/z ions were used for qualification, consid- diphenylpyrrolidine (EDDP), 2-ethyl-5-methyl-3,3-diphenyl-
ering the mass spectra of the standards previously analyzed 1-pyrroline (EMDP), papaverine, tramadol, O-desmetylt-
and the retention times of each analyte [11-13]. Elucidation ramadol (M1), tapentadol, cocaine and cocaethylene. The
of their structures was not performed. The designated m/z for QC samples were also prepared from working solutions
the each analyte is present in Table 1. For qualitative analy- starting from the high concentration with serial dilutions for
sis all analytes were analyzed simultaneously. medium and low concentrations.
Phenacetin was chosen as IS since it proved to be effec- Proof of Applicability
tive for opioids analysis as described previously [14]. Al-
though phenacetin has been reported to be used to “cut” her- Whole blood samples were collected from volunteers un-
oin and cocaine samples, we performed a thorough retro- dergoing methadone administration as a replacement therapy
spective analysis of forensic intoxication involving heroin of heroin dependence and from rats exposed to desomor-
218 Current Pharmaceutical Analysis, 2017, Vol. 13, No. 3 Alves et al.

phine, tramadol and tapentadol. The human samples were environment should be ionized and consequently not soluble
obtained under the consent of the volunteers. in organic solvents. In our work, the use of an alkaline pH
during the extraction process was an important modification
The animal study was performed using adult male Wistar
to compensate the absence of the Primary Secondary Amine
rats obtained from Harlan (Udine, Italy), with mean weight
of 250±10g. Animals were kept under standard laboratory (PSA) step.
conditions (12h/12h light/darkness, 22±2ºC room tempera- Salting out steps: The “salting out” process follows the
ture, 50-60% humidity) for at least 1 week (quarantine) be- original method using MgSO4 and NaCl salts, being only
fore starting the experiments. Animals were allowed access miniaturized for adaptation to whole blood samples [23].
to tap water and rat chow ad libitum during the quarantine Anhydrous MgSO4 is an effective drying agent with an exo-
period. Animals’ experiments were licensed by the Portu- thermic process of hydration, which increases the extraction
guese General Directorate of Veterinary Medicine and ap- efficiency [22, 24] and NaCl decreases the solubility of or-
proved by the Ethical Committee of Faculty of Pharmacy of ganic drugs in their aqueous phase, increasing their concen-
the University of Porto (protocol number 0269/26/EA). tration in the organic solvent.
Housing and experimental treatment of animals were in ac-
cordance with the guidelines defined by the European Coun- Sample preservation: Fluoride preservation with a final
cil Directive (2010/63/EN). Six male Wistar rats were daily concentration of 1-5% sodium (or potassium) fluoride by
exposed to desomorphine (1 mg/Kg) during five consecutive weight is usually recommended for whole blood samples and
days. After the last day, animals were euthanized and whole has great role avoiding the enzymatic loss of esters [1]. The
blood was collected. For tapentadol and tramadol analysis whole blood sample collection was performed using only
sixteen male Wistar rats were divided in two groups. At the EDTA once the samples were not stored and all the analysis
first group, tramadol was administered in two doses (thera- were performed on the same day after the collection.
peutic - 10 or toxic - 25 mg/Kg) in four animals per dose. Additional drugs: Besides opioids, in this method valida-
The second group had the same procedure but tapentadol tion we also included cocaine and cocaethylene. The pres-
was used. Both group animals were euthanized 24 h after the ence of these compounds relies on the fact that many poly-
injections to collect whole blood. For both substances ex- drug users self-administer combinations of heroin with co-
periments, blood was obtained by exsanguination using a caine (i.e., “speedball”) and ethanol [25]. Benzoylecgonine is
hypodermic heparinized needle. Blood samples were centri- the major cocaine metabolite in plasma by all routes of ad-
fuged (3000g, 4ºC, 10 min) and plasma was aliquoted and ministration [26-28]. Although benzoylecgonine is consid-
stored (-80ºC) for further analysis. ered pharmacologically inactive, it has a longer elimination
plasma half-life (0.5-1.5h versus 4-7h) [32-35] and, there-
RESULTS AND DISCUSSION
fore, it is the most commonly monitored metabolite for the
Several authors have applied QuEChERS to clean up bio- determination of cocaine abuse. Although previously consid-
logical samples for subsequent detection of drugs [8, 17-21]. ered for validation purposes, benzoilecgonine co-eluted with
Occasionally, sorbents were used after QuEChERS to im- other analytes and presented low recoveries even after ex-
prove removal of proteins and lipids, which are the most traction and pH optimization and therefore our QuEChERS
common interferents in biological samples [17-19]. To de- methodology proved not be effective for this analyte.
velop a fast and reliable method for clinical and forensic
applications, we aimed to reduce sample preparation steps in Method Validation
order to minimize errors and cost.
The analytical figures of merit of the developed method
Modified QuEChERS method are discussed in the following topics.
Selectivity. Selectivity is “the ability of an analytical
Sample clean-up: Sample clean-up is a very important
method to differentiate and quantify the analyte in the pres-
step in toxicological analysis aiming to isolate target sub-
stances from tissue interfering components such as proteins ence of other endogenous components in the sample” [16].
and lipids, and to concentrate analytes present in the speci- Six blank matrix samples were analyzed to evaluate chroma-
men. This method adds a rapid pre-treatment step using cen- tographic interferences. No interference peaks were detected,
trifugation and sonication to release the compounds linked to either in the retention times of all the analytes or in the phen-
blood proteins, especially albumin, followed by ethyl acetate acetin (IS) retention time (Fig. 2A; Fig. 2B). Therefore, the
as the extraction solvent replacing the acetonitrile of the analytical method is capable to differentiate our 15 analytes
classic QuEChERS methodology. These adaptations were and IS from endogenous components present in the sample.
shown to be suitable for the detection and quantification of Carry-over. During the validation process, injections of
the tested drugs of abuse in whole blood samples (Fig. 2A). calibration standards containing 20
g/mL of each analyte
Choice of solvent: The drug analytes tested are more (10 times the concentration of the higher curve calibration
soluble in ethyl acetate, it is easier and safer to handle, and is point) were followed by six blank sample injections of ethyl
considered a “solvent of choice” among a wide variety of acetate, to ensure that there was no carry-over from one in-
multiresidue tests available for different kinds of analytes jection to the next one. The obtained carry-over results were
[22]. Previously, basified acetonitrile was tested as extraction less than 20% of the LLOQ and less than 5% for the IS,
solvent but recoveries lower than 15% were obtained. Rais- which are within the proposed acceptance limits for this pa-
ing the ethyl acetate extraction pH to 10 takes into account rameter [16].
the chemical nature of all compounds which in an acidic
QuEChERS and Drugs of Abuse Current Pharmaceutical Analysis, 2017, Vol. 13, No. 3 219

100
M1
17.56
A
50

45

Relative Abundance
40

35

30
EDDP Desomorphine
17.80 19.99 Ethylmorphine
25 Tramadol
Cocaine Codeine 21.75
16.98
19.38 21.48
20 Morphine
Methadone
21.85
IS 18.81

Cocaethylene
15 6-AM
12.80 EMDP 22.41

19.81
Tapentadol 16.85
10
13.84 Heroin
22.32 Papaverine
5 24.67

100
B

50
Relative Abundance

25

12 14 16 18 20 22 24
Time (min)

Fig. (2) A- Chromatogram of analytes extracted by modified QuEChERS method at concentrations of 2
g/mL (1 –IS; 2 – Tapentadol; 3 –
EMDP; 4 – Tramadol; 5 – M1; 6 – EDDP; 7 – Methadone; 8 – Cocaine; 9 – Cocaethylene; 10 – Desomorphine; 11 – Codeine; 12 – Ethylmo-
prhine; 13 – Morphine; 14 – 6-AM; 15 – Papaverine; 16 - Heroine. B - Chromatogram of a blank sample. M1 – O-desmethyltramadol; 6-AM –
6-acethylmorphine; EDDP - 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EMDP - 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline; IS – Inter-
nal Standard.

Matrix Effect. For the matrix effect evaluation six cali- source [31], ionization mode [34], and flow rate [35] have
bration curves were performed. Three curves were prepared been found to influence the extent of the matrix effect.
using neat standard solutions diluted in pure solvent in a There are different approaches to avoid matrix effect in GC
range of 31.2-2000 ng/mL and, three curves were prepared analysis such as adding masking agents or analyte protec-
using spiked blank whole blood samples on the same con- tants to standards and samples but an efficient clean-up
centration range. The matrix effect factor was calculated process is the best strategy especially for complex matrices
considering the response of the analytes with matrix and in [36].
the absence of matrix. The values for all analytes were be-
Linearity. Peak-area ratios of analytes of interest to the
tween 80-120%. For the method tested the %CV obtained
internal standard were calculated. Calibration curves were
were less than 15%. Matrix-induced signal enhancement obtained by plotting peak-area ratios against concentration.
occurs in GC when active surfaces in the system (i.e., injec-
The determination coefficient (r2) was obtained for each lin-
tor, column and detector) cause retention and/or degradation
ear regression of three different curves of each analyte ob-
of analytes. In standard solutions without sample matrix,
tained from independent working solutions. The results ob-
only the analytes fill the active sites, which reduce the per-
tained are showed in Table 2. The r2 were >0.98 over the
centage of injected molecules eventually detected. In com-
concentration range, showing good linearity for all analytes.
plex injected extracts, the active sites are filled predomi-
nantly by matrix components, thereby increasing efficiency Limit of Detection and Limit of Quantification. LOD and
of analyte transfer through the GC system to the detector [29]. LLOQ were determined based on signal-to-noise ratio, by
The ionic suppression or enhancement at the interface affects comparing measured noise signals from samples with known
accuracy because standards in pure solvent do not undergo standard concentrations and establishing the minimum con-
this process [30]. The exact mechanism of ion suppression is centration level at which analyte can be detected and quanti-
not known; it seems that it may be caused by non-volatile fied. A signal-to-noise ratio of 3:1 and 10:1 was considered
material 31 or by compounds of high surface activity [32]. acceptable for LOD and LLOQ, respectively. The values of
The chemical nature of the analyte plays an important role LOD and LLOQ are listed on Table 2. Although LLOQ val-
too. Taylor [33] observed that matrix effects of polar com- ues obtained were higher than most forensic data in litera-
pounds are more relevant than those of less polar com- ture, we found them acceptable considering chromatographic
pounds. Some instrumental parameters such as the ionization system limitations, the time consuming analysis and the cost.
220 Current Pharmaceutical Analysis, 2017, Vol. 13, No. 3 Alves et al.

Table 2. A - Parameters of the analytical curves of thirteen opioids, cocaethylene and cocaine solutions (31.2 – 2000 ng/mL) ob-
tained by the least squares method in three different days. B - Precision, accuracy and recovery (%) for desomorphine
and codeine evaluation at 3 different spiked concentrations. LOD, limit of detection; LLOQ, limit of quantification.

A B

Concentration Quality control Intra-day Inter-day Accuracy

LOD LLOQ Recovery
Xenobiotic n=3 y = mx + b range r
concentration precision precision
(ng/mL) (ng/mL) (%, n=3) (%)
(ng/mL) (ng/mL) (%, n=3) (%, n=3)

day1 y = 0.00006x – 0.0059 31.2 - 2000 0.9851 31.2 11.6 5.6 104.0 78.8

Codeine day2 y = 0.00003x + 0.0028 31.2 - 2000 0.9842 63.6 73.4 250 13.5 15.1 112.7 89.7

day3 y = 0.0001x – 0.0189 31.2 - 2000 0.9817 2000 5.3 14.6 111.0 85.5

day1 y = 0.00002x – 0.0025 31.2 - 2000 0.9966 31.2 11.5 5.1 110.0 58.6

Morphine day2 y = 0.00001x + 0.0028 31.2 - 2000 0.9951 31.8 46.3 250 6.9 5.3 106.0 72.4

day3 y = 0.00001x + 0.0030 31.2 - 2000 0.9844 2000 9.4 8.8 102.4 71.4

day1 y = 0.000004x + 0.0042 31.2 - 2000 0.9958 31.2 12.3 11.5 110.1 79.3

Heroin day2 y = 0.000004x + 0.0063 31.2 - 2000 0.9969 32.7 65.2 250 13.7 8.8 107.3 82.1

day3 y = 0.00005x + 0.0002 31.2 - 2000 0.9975 2000 14.2 13.1 102.8 78.3

day1 y = 0.00003x – 0.0042 31.2 - 2000 0.9815 31.2 11.8 11.7 88.23 59.8

6-acethylmorphine day2 y = 0.00002x + 0.0033 31.2 - 2000 0.9961 81.7 123.4 250 6.4 9.3 89.0 52.4

day3 y = 0.00002x + 0.0053 31.2 - 2000 0.9830 2000 13.9 10.1 84.5 53.3

day1 y = 0.0001x – 0.0057 31.2 - 2000 0.9873 31.2 11.0 3.7 102.4 82.5

Desomorphine day2 y = 0.00005x + 0.0081 31.2 - 2000 0.9981 30.8 102.6 250 12 13.9 96.4 76.3

day3 y = 0.00006x + 0.0091 31.2 - 2000 0.9946 2000 15.1 14.8 112.3 83.7

day1 y = 0.00006x + 0.0002 31.2 - 2000 0.9808 31.2 8.5 11.6 81.6 87.8

Ethylmorphine day2 y = 0.00005x + 0.0070 31.2 - 2000 0.9982 16.2 54.1 250 6.4 11.0 90.8 63.2

day3 y = 0.00006x + 0.0075 31.2 - 2000 0.9978 2000 2.5 15.8 92.5 67.0

day1 y = 0.000004x + 0.0041 31.2 - 2000 0.9805 31.2 14.7 16.8 114.5 78.9

Methadone day2 y = 0.000007x + 0.0005 31.2 - 2000 0.9977 21.2 70.7 250 14.7 6.4 94.3 77.0

day3 y = 0.00001x + 0.0006 31.2 - 2000 0.9838 2000 5.7 16.2 104.3 84.4

day1 y = 0.00007x + 0.0003 31.2 - 2000 0.9985 31.2 2.2 1.7 99.9 93.9

EDDP day2 y = 0.00009x + 0.0015 31.2 - 2000 0.9999 7.1 33.6 250 2.7 15.8 108.2 95.0

day3 y = 0.00008x + 0.0010 31.2 - 2000 0.9985 2000 3.0 11.5 104.2 83.6

day1 y = 0.0001x – 0.0015 31.2 - 2000 0.9980 31.2 3.2 3.8 120.8 88.2

EMDP day2 y = 0.0001x – 0.0001 31.2 - 2000 0.9960 6.4 32.3 250 2.6 7.4 106.7 86.2

day3 y = 0.0001x + 0.0001 31.2 - 2000 0.9998 2000 1.5 1.4 106.1 90.6

day1 y = 0.00007x – 0.0081 31.2 - 2000 0.9825 31.2 13.0 16.5 136.0 65.4

Papaverine day2 y = 0.00004x + 0.0026 31.2 - 2000 0.9849 32.7 109.1 250 7.8 14.5 94.6 67.3

day3 y = 0.00003x – 0.0016 31.2 - 2000 0.9849 2000 9.3 0.6 99.1 72.9

day1 y = 0.00005x + 0.0030 31.2 - 2000 0.9894 31.2 11.1 13.4 114.0 89.7
Tramadol 7.4 31.2
day2 y = 0.00004x + 0.0062 31.2 - 2000 0.9940 250 6.8 11.1 101.5 76.0
QuEChERS and Drugs of Abuse Current Pharmaceutical Analysis, 2017, Vol. 13, No. 3 221

Table (2) contd….

A B

Concentration Quality control Intra-day Inter-day

LOD LLOQ Accuracy Recovery
Xenobiotic n=3 y = mx + b range r
concentration precision precision
(ng/mL) (ng/mL) (%, n=3) (%)
(ng/mL) (ng/mL) (%, n=3) (%, n=3)

day3 y = 0.00004x + 0.0059 31.2 - 2000 0.9952 2000 6.6 11.9 105.4 74.7

day1 y = 0.00005x + 0.0014 31.2 - 2000 0.9958 31.2 17.5 8.4 104.0 87.2

M1 day2 y = 0.00004x + 0.0032 31.2 - 2000 0.9969 12.4 49.2 250 5.9 15.3 96.5 59.7

day3 y = 0.00004x + 0.0002 31.2 - 2000 0.9923 2000 8.7 12.3 92.8 72.5

day1 y = 0.00200x + 0.0027 31.2 - 2000 0.9999 31.2 2.4 8.9 110.9 85.0

Tapentadol day2 y = 0.00200x + 0.0058 31.2 - 2000 0.9987 32.1 31.6 250 2.8 6.2 113.7 81.9

day3 y = 0.00200x + 0.0099 31.2 - 2000 0.9986 2000 6.9 10.7 111.0 84.3

day1 y = 0.00007x – 0.0024 31.2 - 2000 0.9880 31.2 12.7 13.9 94.4 73.1

Cocaine day2 y = 0.00005x + 0.0069 31.2 - 2000 0.9989 15 62.5 250 11.9 13.9 92.8 79.4

day3 y = 0.00008x + 0.0048 31.2 - 2000 0.9972 2000 5.5 11.1 95.6 74.7

day1 y = 0.00004x – 0.0022 31.2 - 2000 0.9873 31.2 15.1 10.4 104.0 68.5

Cocaethylene day2 y = 0.00002x + 0.0033 31.2 - 2000 0.9845 62 125 250 14.7 7.3 97.0 54.1

day3 y = 0.00002x + 0.0049 31.2- 2000 0.9913 2000 8.8 9.3 103.5 70.6

Most literature, presents LLOQ data obtained by High Pres- The accuracy of an analytical method describes the
sure Liquid Chromatography (HPLC) coupled with tandem closeness of the determined value obtained by the method to
mass spectrometry (MS) techniques [36-39] which is much the nominal concentration of the analyte in percentage [16].
more sensitive for drug analysis. Nevertheless, GC-MS is a Accuracy percentage should be within ±20% of actual value
useful technique for forensic and clinical analysis due its for LLOQ and ±15% for other concentrations along the line-
reproducibility. Lerch and coworkers [40] described a GC- arity range. The accuracy was evaluated by spiking blank
MS technique for detection of opioids and cocaine obtaining whole blood samples with three different analytes concentra-
slightly lower LLOQ using SPE as the extraction methodol- tions (low, 31.2; medium, 250; high, 2000 ng/mL) and
ogy. Even with limitations, our technique showed to be com- through the calculation of the deviation of the percentage
parable with data described in literature. Moreover, the ob- between the calculated and the nominal value obtained from
tained LLOQ and LOD were derived from a multiresidual de QC samples concentrations (low, 31.2; medium, 250;
operation for simultaneous analysis of thirteen analytes. Im- high, 2000 ng/mL) [accuracy (%) = (experimental concentra-
provement of LOD and LLOQ can be made case by case, by tion/ QC concentration)
100].
reducing the numbers of ions included in the SIM data col-
The extraction recovery was calculated comparing the
lection process when a substance is suspected. Indeed, as
concentration of the analyte extracted from the matrix and
previously suggested the suspicion is an extremely important
the concentration of the analyte in a curve not extracted. It
pre-analytical step to vectorize which xenobiotics must be
was evaluated by spiking blank whole blood samples with
included in the toxicological analysis [41-44].
three different analytes concentrations (low, 31.2; medium,
Precision, Accuracy and Recovery. The results obtained 250; high, 2000 ng/mL) and through the calculation of the
are showed in Table 2. Precision was evaluated considering deviation of the percentage between the extracted and the
the threshold of 15% for %CV and 20% for the concentra- non-extracted value [Recovery (%) = (extracted [stan-
tion closer to the LLOQ. The precision of the analytical dard]/non-extracted [standard])
100]. Usually recovery of
method describes the closeness of repeated individual meas- extraction methods should be between 80-120%. According
ures of the analyte. Intra-day precision data was quantified to Anzillotti and colleagues [8], for QuEChERS it is accept-
by analyzing peak-area ratios of analytes of interest to the able to achieve an overall recovery value of 60–70% for
internal standard of three replicates of three different concen- non-, medium- and polar compounds. Therefore, the method
trations (low, 31.2; medium, 250; high, 2000 ng/mL) and showed good recovery values considering there was no dis-
calculating %CV. Peak-areas ratios of the same three con- persive solid-phase extraction (dSPE) step. Moreover, basic
centrations, injected on three consecutive days, were used to pH of extraction step proved to be important to extract basic
calculate inter-day precision. For this analysis all the samples drugs and may be used as an exchange to the addition of
sorbents like primary secondary amine (PSA).
were freshly fortified and extracted each day of analysis.
222 Current Pharmaceutical Analysis, 2017, Vol. 13, No. 3 Alves et al.

We also compared our validated modified QuEChERS CONFLICT OF INTEREST

methodology with classic solid-phase extraction (SPE). Con-
Authors declare no conflict of interest, particularly no fi-
sidering the basic nature of analytes, Oasis MCX SPE Column
nancial and personal relationships with other people or orga-
was used. Although slightly higher recoveries were obtained
nizations that could inappropriately influence (bias) this
with SPE (data not shown), the cost and the time consuming
were significantly greater. Moreover, QuEChERS methodol- work.
ogy proved to be effective in the proof of applicability step. ACKNOWLEDGEMENTS
Stability. Stability studies were performed concurrently Emanuele Alves and Annibal D. Pereira Netto acknowl-
with precision and accuracy analysis. The stock solutions of edges Brazilian agency Conselho Nacional de Desen-
the analytes did not show deviations (i.e., acceptable %CV) volvimento Científico e Tecnológico (CNPq) (process
during the 85 days that took the optimization and validation 245844/2012-0) for research grants and scholarship. Ricardo
process. Dinis-Oliveira acknowledges Fundação para a Ciência
e a Tecnologia (FCT) for his Investigator Grant
PROOF OF APPLICABILITY
(IF/01147/2013). Sara Cravo and Carlos Afonso acknowl-
From all 34 human samples, 23 were positive for opioids edges FCT through the strategic project CEQUIMED-UP
(Table S1), namely methadone and its main metabolites, (Pest-OE/SAU/UI4040/2014). This work was supported by
EDDP and EMDP. Methadone therapy is the most exten- FEDER under Program PT2020 (project 007265 -
sively evaluated and most used treatment for opioids addic- UID/QUI/50006/2013). The contribution of MSc. Pedro
tion [45]. It occurs due to its pharmacological characteristics Brandão is gratefully acknowledged.
such as high oral bioavailability, long half-live and the avail-
ability of a specific antagonist, needed for overdose cases SUPPLEMENTARY MATERIAL
[46]. In Portugal, the therapy consist in a weekly dose of 40 Supplementary material is available on the publisher's
mg of methadone chloride, which may be progressively in- website along with the published article.
creased until 200 mg, lasting 1 to 3 years. The variability of
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