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A novel and rapid procedure based on liquid chromatography/tandem mass spectrometry (LC/
MS/MS) for the determination of dialkyl phosphates (metabolites of organophosphorus pesticides)
in human urine has been developed. After addition of 40 mM tetrabutylammonium acetate, 10 mL of
urine sample were directly injected into the LC/MS/MS system. The method was validated yielding
calibration curves with correlation coefficients greater than 0.997 and repeatability coefficient of
variation (CV) lower than 9%. The accuracy and precision were evaluated by direct injection of
spiked samples at 10 and 100 mg/L obtaining recoveries between 78 and 119% with coefficients of
variation below 12%. Limits of detection of 1 mg/L for diethyl phosphate (DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP) and 2 mg/L for dimethyldithiophosphate
(DMDTP) were achieved, all the analytes being detected in negative ion mode. The fragmentation
pathway of dialkyl phosphates allowed us the use of an additional transition for confirmation in
order to improve their identification in real-world samples. The applicability of the LC/MS/MS
method was demonstrated by applying it to the analysis of urine samples of farmers exposed to the
organophosphorus pesticide chlorpyrifos. Good correlation between application of the product in
the field (citrus orchards), concentration levels of dialkyl phosphates and levels of the chlorpyrifosspecific metabolite (1,3,5-trichloro-2-pyridinol) was obtained. Copyright # 2002 John Wiley & Sons,
Ltd.
is frequently followed by a solvent exchange step appropriate to GC determinations. Detection limits between 2 and
100 mg/L are usually achieved in these methods.616 Mass
spectrometric detection is sometimes used for analyte
confirmation.14,16 Recently, GC/MS17 and GC/MS/MS18
methods have been used for the quantitative determination
of dialkyl phosphates in urine.
Obviously, the traditional LC detectors, UV and fluorescence, are not appropriate for the direct determination of
dialkyl phosphates due to the lack of chromophore or
fluorophore moieties in the analyte molecules. The recent
introduction of atmospheric pressure interfaces, mainly
electrospray ionization (ESI), for coupling LC to MS has
popularized the use of LC/ESI-MS for the determination of
many analytes unsuitable for conventional liquid chromatography.19,20 In addition, the use of tandem mass spectrometry (MS/MS), due to its inherent high selectivity,21,22
allows the direct injection of complex matrices into the LC
system.
Nowadays, the potential of LC/MS and LC/MS/MS
techniques in the direct determination of pesticide metabolites in urine has been demonstrated2,23 and some compounds similar to organophosphorus metabolites such as
phosphonates,24 inositol phosphate25 or dibutyl phosphate26
have been determined by LC/MS methods.
In this paper, a new analytical method based on direct
injection into an LC/MS/MS system is developed for the
sensitive determination of dialkyl phosphates in urine
samples. To our knowledge, this paper describes the first
application of LC/MS/MS in the analysis of alkyl phosphates in urine samples. The potential of the developed
procedure for the biomonitoring of chlorpyrifos exposure in
pesticide applicators has been confirmed.
EXPERIMENTAL
Reagents and chemicals
Diethylthiophosphate (DETP) potasium salt, diethyldithiophosphate (DEDTP) potasium salt, dimethyl chlorophosphate (DMClP) and dimethyl chlorothiophosphate
(DMClTP) were purchased from Aldrich (Milwaukee, WI,
USA); diethyl phosphate (DEP) was purchased from Supelco
(Bellfonte, PA, USA). HPLC-grade acetonitrile was purchased from ScharLab (Barcelona, Spain). LC-grade water
was obtained by purifying demineralized water in a
Nanopure II system (Barnstead Newton, MA, USA). Analytical-grade formic acid (HCOOH, content >98%) was
purchased from Fluka (Buchs, Switzerland) and tetrabutylammonium acetate (TBA) was obtained from Sigma (St
Louis, MI, USA).
Standard stock solutions of DEP, DETP and DEDTP were
prepared by dissolving 25 mg powder, accurately weighed,
in 50 mL of HPLC-grade acetonitrile obtaining a final
concentration of 500 mg/mL. Stock solutions of synthesized
DMP and DMTP were diluted with HPLC-grade water to
obtain a concentration of 500 mg/mL. A standard stock
mixture of five dialkyl phosphates was prepared taking 5mL aliquots of each individual solution and diluting to
50 mL with HPLC-grade acetonitrile. For the LC/MS/MS
Copyright # 2002 John Wiley & Sons, Ltd.
1767
The preparation of dimethyl phosphate (DMP) and dimethylthiophosphate (DMTP) was carried out very efficiently by a simple hydrolysis of DMClP and DMClTP, as
shown in Fig. 1. A solution of triethylamine (3 mL) in 40 mL
LC-grade water/HPLC-grade acetonitrile (10:30) was prepared and stirred at 0 C. Then, DMClP (5 g) or DMClTP (5 g)
was carefully and quantitatively dissolved into this solution
and HPLC-grade acetonitrile was added in order to obtain
an exact volume of 200 mL, achieving concentrations of
21,800 and 22,118 mg/L for DMP and DMTP, respectively.
Liquid chromatography
Mass spectrometry
125a
125b
141a
141b
153a
153b
169a
169b
185
30
30
20
20
20
20
30
30
30
17
25
17
25
12
20
20
20
20
63
79
126
95
125
79
95
141
111
Quantification transition.
Confirmation transition.
Analytical procedure
Validation study
1769
Figure 2. Negative ion electrospray full-scan mass spectra (bottom) and product ion spectra (top) from (a) DEP, (b) DETP, (c) DEDTP,
(d) DMP, and (e) DMTP.
LC optimization
Validation study
1771
Figure 4. LC/ESI-MS/MS chromatograms for DMTP, DEP, DETP and DEDTP: (a) 10 mg/L standards, (b)
urine sample blank and (c) urine sample spiked at 10 mg/L.
DMTP
DEP
DETP
DEDTP
Repeatability (n = 7, CV%)
Range (mg/L)
10 mg/L
100 mg/L
51000
51000
51000
51000
0.999
0.997
0.999
0.999
9
4
3
7
8
7
2
4
DEDTP
a
b
10 mg/L
Sample
Sample
Sample
Sample
Sample
Sample
Sample
Sample
1
2
1
2
1
2
1
2
110
119
90
82
78
86
83
82
(9 )
(11)
(10)
(6)
(9)
(5)
(10)
(7)
100 mg/L
119
101
95
92
79
87
90
90
(12)
(12)
(9)
(6)
(6)
(4)
(5)
(5)
Recovery (%).
Coefficient of variation in parentheses (%).
specific metabolite of this pesticide, 1,3,5-trichloro-2-pyridinol (TCP), was also determined in all urine samples. The
method used was based on the direct injection of hydrolyzed
urine samples into an LC/LC/MS/MS system.2 Figure 5
shows good correlation between the behavior of the diethyl
phosphates and that of TCP, although the concentration
levels of TCP were much higher than those of DEP and
DETP. This was possibly due to the higher excretion rates of
very polar compounds like phosphates as they do not have
to suffer a phase II metabolism process in order to be
excreted. However, TCP has to be conjugated before the
excretion and, therefore, its metabolism will probably be
slower. As urine samples were collected early in the morning
after the day of exposure, dialkyl phosphates could be
largely excreted during the previous day, while TCP could
attain a maximum excretion level after some hours.
Figure 6(a) shows chromatograms of alkyl phosphates and
TCP for an exposed worker (sample a, see Fig. 5).
Confirmation as well as quantification transitions are also
shown in Fig. 6(b) for DEP and DETP in order to authenticate
the presence of these analytes.
Rapid Commun. Mass Spectrom. 2002; 16: 17661773
Figure 6. Chromatograms from urine sample of a farmer exposed to chlorpyrifos (sample a, collected on Thursday): (a) LC/ESIMS/MS chromatograms for alkyl phosphates and LC/LC/ESI-MS/MS chromatogram for TCP; (b) confirmation of the two dialkyl
phosphates derivatives found, (Q) quantitation transition; (q) confirmation transition.
Copyright # 2002 John Wiley & Sons, Ltd.
1773
CONCLUSIONS
REFERENCES
Acknowledgements