JOURNAL OF MASS SPECTROMETRY, VOL.

32, 99È102 (1997)

Development of a Method to Identify Keto Acids in

Ozonated Fulvic Acid Solutions

Yuefeng Xie1* and David A. Reckhow2

1 Environmental Pollution Control Program, School of Science, Engineering and Technology, Penn State Harrisburg,
Middletown, Pennsylvania 17057-4898, USA
2 Department of Civil and Environmental Engineering, University of Massachusetts, Amherst, Massachusetts 01003, USA

A gas chromatographic-mass spectrometric method, combining an O-(2,3,4,5,6-pentaÑuorobenzyl)hydroxylamine–

diazomethane double derivatization, was developed to identify major keto acids in ozonated fulvic acid solutions.
Three keto acids, glyoxylic, pyruvic, and ketomalonic acid, four aldehydes and two analytical artifacts were identi-
Ðed. The detailed mass spectra, obtained in both electron impact and positive chemical ionization modes, and their
fragment assignments are presented. Owing to the similarity of their electron impact mass spectra, electron impact
mass spectra along with positive chemical ionization mass spectra are needed to identify these keto acids reliably.
The e†ects of the formation of keto acids on water quality are also discussed.
J. Mass Spectrom. 32, 99È102 (1997)
No. of Figures : 4 No. of Tables : 2 No. of Refs : 24
KEYWORDS : electrospray ; non-covalent complexes ; calmodulin, imidazopyrazine ; low affinity

INTRODUCTION EXPERIMENTAL

Since the 1970s, a number of disinfection byproducts

(DBPs) have been identiÐed in chlorinated drinking Ozonated waters
waters. These DBPs include trihalomethanes, haloacetic
acids, trihaloacetaldehydes, cyanogen halides, haloace- Solutions of aquatic fulvic acid were used as model
tones, haloacetonitriles and MX.1h7 Because of con- drinking waters. These were prepared at 4 mg l~1 dis-
cerns over the health eþects of these compounds, the solved organic carbon (DOC) from a concentrated
US Environmental Protection Agency has proposed fulvic acid aqueous stock solution which was isolated
new DBP regulations in the coming disinfectants– from Thousand Acre reservoir (Athol, MA, USA).
disinfection byproducts (D-DBP) rule.8 Ozonation has These solutions were buþered at pH 7 with 10 mM
been used in many water utilities in North America as a total phosphates. They were then ozonated in batch by
treatment technique for controlling DBPs.9 Like chlo- addition of a concentrated ozone stock solution. Ozone
rine, ozone also reacts with natural organic matter in was applied at a dose ratio of 1 mg O per mg DOC.
3 with ozone in
water to produce various organic byproducts. Com- Fulvic acid samples were allowed to react
pared with chlorinated DBPs, however, very little infor- the dark for 3 h before derivatization.
mation is available on the ozonation byproducts.10 This
has been partly attributed to the difficulty in quantify-
ing and identifying these compounds. Analytical diffi- Sample derivatization
culties stem from the high polarity, hydrophilicity and
instability of the ozonation byproducts. An analytical A derivatization method based on that reported by
method employing O-(2,3,4,5,6-pentaýuorobenzyl) Xiong et al.14 was used. In a glass bottle, 20 ml of
hydroxylamine (PFBHA) derivatization and gas chro- PFBHA (Aldrich Chemical, Milwaukee, WI, USA)
matography with electron capture detection (GC/ECD) aqueous solution (6 mg ml~1) was added to a 1000 ml
has been reported for low molecular mass aldehydes in unquenched water sample. This was incubated at 45 ¡C
drinking waters.11h13 In another development, Xiong et for 1.75 h, after which the sample was cooled to room
al.14 used a method employing PFBHA and diazo- temperature. Following the addition of 40 ml of con-
methane derivatization to quantify glyoxylic acid in centrated sulfuric acid, the sample was extracted with
solutions of ozonated fulvic acid. 2 ] 30 ml of methyl tert-butyl ether (MTBE) for 5 min
The objective of this study was to develop a gas chro- in a separatory funnel. The combined MTBE extracts
matographic mass spectrometric (GC/MS) method to were concentrated to 2 ml under a gentle ýow of nitro-
identify major keto acids in ozonated drinking water gen, then 2 ml of diazomethane MTBE solution, pre-
and fulvic acid samples. pared in accordance with the method of Fales et al.,15
was added to the concentrated extract. The extract was
replaced in a refrigerator at 4 ¡C for 15 min. After
* Correspondence to : Y. Xie. quenching the residual diazomethane with silica gel, the

CCC 1076È5174/97/010099È04 Received 2 July 1996

( 1997 by John Wiley & Sons, Ltd. Accepted 3 October 1996
100 Y. XIE AND D. A. RECKHOW

extract was concentrated again to about 100 ll under

nitrogen and submitted to GC/MS analysis.

GC/MS analysis

GC/MS analysis was carried out on an HP-5988 quad-

rupole mass spectrometer (Hewlett-Packard, San Fern-
ando, CA, USA). The split–splitless injector was
operated in the splitless mode, and a 30 m ] 0.32 mm
i.d. (0.25 lm ülm thickness) PTE-5 capillary column
(Supelco, Bellefonte, PA, USA) was used. The column
oven was kept at 50 ¡C for 2 min, then ramped to
250 ¡C at a rate of 4 ¡C min~1 and held at 250 ¡C for 8 Figure 1. Total ion chromatogram of a derivatized ozonated fulvic
min. The injector temperature was 200 ¡C and the acid sample.
transfer line was kept at 280 ¡C. In the electron impact
(EI) mode, the ion source temperature was 200 ¡C, the in the double-derivatized extract of the ozonated fulvic
electron energy was 70 eV and the mass scan range was acid solution are shown in Fig. 2. The m/z values of 59,
30–400 u. In positive chemical ionization (PCI) mode, 181 and 211 in these mass spectra indicate
the ion source temperature was 100 ¡C, the electron [COOCH ]`, [C F CH ]` and [C F CH ON]` frag-
3 6 5 2 6 5 2
energy was 240 eV and mass scan range was 100–400 u.
Methane was used as the reagent gas for PCI. Data
were acquired and stored for subsequent analysis using
an HP 59970 MS ChemStation. Other GC/MS oper-
ating conditions were similar to those reported pre-
viously.16

RESULTS AND DISCUSSION

A number of peaks were found in the total ion chro-

matogram of the derivatized extract from the ozonated
fulvic acid sample, as shown in Fig. 1. Four mono-
aldehydes, with a predominant fragment at m/z 181,
were identiüed, as shown in Table 1. The double peaks
indicated the existence of isomers of derivatized acetal-
dehyde. There is no isomer for derivatized formalde-
hyde. No double peaks were observed for isomers of
the derivatized propanal and butanal. This might be
due to inadequate separation of aldehydes under the
operating conditions, which were optimized for keto
acids. All of these aldehydes have been reported by
other researchers.11h13
Three other peaks, with fragments at m/z 59 and 181,
were found in the methylated, PFBHA-derivatized
extract. Without methylation none of these three peaks Figure 2. EI mass spectra of derivatized keto acids in ozonated
was observed. The EI mass spectra of these three peaks fulvic acid solutions (base peaks 181 are 100%).

Table 1. Compounds identiÐed in ozonated fulvic acid solutions

Proposed
molecular Proposed chemical structures
Peak No. mass Derivatives Ozonation byproducts

1 225 C F —CH —O—NxCH H—CO—H

6 5 2 2
2 239 C F —CH —O—NxCH—CH H—CO—CH
6 5 2 3 3
3 253 C F —CH —O—NxCH—C H H—CO—C H
6 5 2 2 5 2 5
4 267 C F —CH —O—NxCH—C H H—CO—C H
6 5 2 3 7 3 7
5 283 C F —CH —O—NxCH—COOCH H—CO—COOH
6 5 2 3
6 297 C F —CH —O—NxC(CH )—COOCH CH —CO—COOH
6 5 2 3 3 3
7 391 C F —CH —O—NxCH—C F Analytical artifact
6 5 2 6 5
8 391 C F —CH —O—NxCH—C F Analytical artifact
6 5 2 6 5
9 341 C F —CH —O—NxC(COOCH ) HOOC-CO-COOH
6 5 2 3 2
 IDENTIFICATION OF KETO ACIDS IN OZONATED WATERS 101

Figure 4. Mass spectra of an analytical artifact in ozonated fulvic

acid solutions.

Figure 3. PCI mass spectra of derivatized keto acids in ozonated

fulvic acid solutions.
PCI was used to gain the molecular mass information
on these peaks. The PCI mass spectra of a derivatized
sample are shown in Fig. 3. The three ions in each PCI
ments, respectively. For the fragment [C F CH ]`, the mass spectrum were assigned the structures [M ] H]`,
6 for
5 the
2 three
(A ] 1) isotopic contributions for carbon [M ] C H ]` and [M ] C H ]`. Therefore, from
derivatives are 6.1, 7.6 and 6.5% of (A) ; respectively. 2 mass
5 3 5
these PCI spectra, the molecular masses of these
These data match the theoretical isotopic contribution three compounds were determined to be 283, 297 and
(7.7%) fairly well. These fragments suggest that the 341, respectively. By logical structural assignments for
compounds contain carboxyl and carbonyl groups. both the EI and PCI mass spectra (Table 2), these three
Several other fragments higher than m/z 211 were also peaks were identiüed as glyoxylic, pyruvic and ketoma-
present at very low abundances. However, owing to the lonic acid derivatives. This result was also conürmed by
similarity of the EI mass spectra of these three peaks, it matching the mass spectra of PFBHA–diazomethane-
is difficult to assign deünite structures to these mass derivatized standards. These standards were prepared
spectra. from the pure commercially available compounds, gly-
oxylic, pyruvic, and ketomalonic acid.
By using a similar derivatization procedure, Xiong et
al.14 identiüed glyoxylic acid in an ozonated fulvic acid
Table 2. Proposed fragment ion assignments for keto acid solution. Mass spectra that resemble those of deriv-
derivativesa atives of pyruvic and ketomalonic acid were also pre-
Proposed m /z of mass fragments (abundance, % of base peak)b sented by Xiong et al., but, the authors were unable to
fragment ion Glyoxylic acid Pyruvic acid Ketomalonic acid assign deünite structure to these mass spectra. Other
ÍM É OH˽ 266 (ND) 280 (1.0) 324 (ND) studies suggest that the keto acids may be general ozon-
ÍM É NO˽ 253 (0.4) 267 (0.7) 311 (ND) ation products of a wide range of organic compounds.
ÍM É OCH ˽ 252 (0.7) 266 (1.1) 310 (0.7) Using diþerent derivatization techniques, glyoxylic acid
3
ÍC F CH ON˽ 211 (1.4) 211 (1.1) 211 (2.6) has been found in ozonated solutions of acetic, maleic
6 5 2
ÍC F CH N˽
6 5 2
195 (1.7) 195 (1.6) 195 (1.9) and oxalacetic acid and glyoxal.17h19 Pyruvic acid was
ÍC F CH ˽ c 182 (6.1) 182 (7.6) 182 (6.5) reported in an ozonated solution of 2-methyl-4-
6 5 2
ÍC F CH ˽ 181 (100) 181 (100) 181 (100) chlolophenoxyacetic acid.20 Ketomalonic acid was
6 5 2
ÍC F ˽ 167 (1.6) 167 (1.7) 167 (1.7) reported in ozonated solutions of malonic acid, oxa-
6 5
ÍC F CH˽ 161 (2.9) 161 (2.0) 161 (1.8)
6 4 lacetic acid, tartronic acid, phenol, aniline, pheoxyacetic
ÍC F ˽ 117 (2.0) 117 (1.8) 117 (1.4)
5 3
ÍCOOCH ˽ 59 (2.4) 59 (6.1) 59 (6.0)
acid and p-cresol.18, 20
3 By comparison with an ozonated Super-Q water
a Base peak is underlined ; ND, not detected. blank, two other major peaks were identiüed as analyt-
b The abundance is given in parentheses.
c (A ½ 1) isotopic contribution for carbon.
ical artifacts. These were apparently formed by the
reaction between the residual ozone and PFBHA. Since
102 Y. XIE AND D. A. RECKHOW

these two peaks were present in the unmethylated pyruvate clearly promoted the growth of strain NOX
extract, this indicated that no carboxylic function in tap water.
groups were present in these compounds. By logical
structural assignments of the EI and PCI mass spectra,
as shown in Fig. 4, these two artifacts were identiüed as CONCLUSIONS
those shown in Table 1. The similar mass spectra and
the same molecular masses indicated the existence of
isomers. These two peaks were also reported as bypro- A GC/MS method combined with PFBHA–
ducts of residual chlorine and PFBHA.12 diazomethane double derivatization was developed to
As reported here, these three keto acids have been identify keto acids in ozonated waters. Three keto
found in ozonated drinking water at a level which is acids, glyoxylic, pyruvic and ketomalonic acid, were
about 5–10 times higher than that of the alde- identiüed in ozonated fulvic acid solutions. Two ana-
hydes.21h23 Since these keto acids are readily biode- lytical artifacts were also identiüed in the derivatized
gradable, they can be removed through a biological extract. Owing to the similarity of the EI mass spectra
ülter after ozonation.22 Owing to the insigniücant level of keto acid derivatives, EI mass spectra together with
of keto acids in the bioülter effluent, they are unlikely PCI mass spectra are needed to identify these keto acids
to be of human health concern. The correlation reliably.
between keto acid and assimilable organic carbon
(AOC) has been reported previously.22,23 The total
keto acid concentration correlated well with AOC Acknowledgements
(NOX) in a variety of waters. This indicates that keto
acid concentration is a potential chemical surrogate for This project was supported by the US National Science Foundation
under grant number BCS-8818468. Dr Edward H. Bryan was the
AOC or biodegradable dissolved organic carbon grant officer. The authors also acknowledge the generous ünancial
(BDOC). Corroborating research was conducted by van assistance from Hewlett-Packard and technical assistant from Dr R.
der Kooij et al.,24 who showed that glyoxylate and V. Rajan.

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