Elsevier

Journal of Chromatography B

Volume 832, Issue 1, 17 February 2006, Pages 58-66
Journal of Chromatography B

Simultaneous determination of urinary dialkylphosphate metabolites of organophosphorus pesticides using gas chromatography–mass spectrometry

https://doi.org/10.1016/j.jchromb.2005.12.030Get rights and content

Abstract

In this study, we developed a safe and sensitive method for the simultaneous determination of urinary dialkylphosphates (DAPs), metabolites of organophosphorus insecticides (OPs), including dimethylphosphate (DMP), diethylphosphate (DEP), dimethylthiophosphate (DMTP), and diethylthiophosphate (DETP), using a pentafluorobenzylbromide (PFBBr) derivatization and gas chromatography–mass spectrometry (GC–MS). Several parameters were investigated: pH on evaporation, reaction temperature and time for the derivatization, the use of an antioxidant for preventing oxidation, and a clean-up step. The pH was set at 6, adjusted with K2CO3, and the reaction temperature and time of derivatization were 80 °C and 30 min, respectively. Sodium disulfite was chosen as an antioxidant. The clean-up step was performed with a Florisil/PSE mini-column to remove the unreacted PFBBr and sample matrix. This established procedure markedly shortened the sample preparation time to only about 3 h, and completely inhibited the unwanted oxidization of dialkylthiophosphates. The limits of determination (LOD) were approximately 0.3 μg/L for DMP, and 0.1 μg/L for DEP, DMTP, and DETP in 5 mL of human urine. Within-series and between-day imprecision for the present method using pooled urine spiked with DAPs was less than 20.6% in the calibration range of 1–300 μg/L, and the mean recovery was 56.7–60.5% for DMP, 78.5–82.7% for DEP, 88.3–103.9% for DMTP, and 84.2–92.4% for DETP. This method detected geometric mean values of the urinary DAPs in Japanese with and without occupational exposure to OPs, 16.6 or 27.4 for DMP, 1.0 or 0.7 for DEP, 1.3 or 2.3 for DMTP, and 1.0 or 1.1 μg/L for DETP, respectively. The present method, which does not require special equipment except for GC–MS, is quick, safe, and sensitive enough to be adopted in routine biological monitoring of non-occupational as well as occupational exposure to OPs.

Introduction

Organophosphorus compounds have been widely and effectively used as insecticides with applications in agriculture and pest control [1]. However, it is well known that severe exposure to organophosphorus insecticides (OPs) in human and animal causes an acute cholinergic syndrome such as miosis, salivation, seizures, paralysis, neuromuscular and cardiac conduction disorders [2], [3] through the inhibition of acethylcholinesterase [4], [5]. Moreover, it has been reported that occupational long-term exposure could cause a decrease in sensory nerve conduction velocity [6] and enduring extrapyramidal symptoms as well as transient motor and psychiatric symptoms [7].

Occupational human exposure to OPs is usually evaluated by determination of the decrease of cholinesterase activity in blood between pre- and post-exposure, since it has large inter-individual variability [8], [9]. However, this indicator is not sensitive enough to monitor the body burden of OPs in the general population, and blood collection from many people is not always feasible. Therefore, it is necessary to develop a biological monitoring method using urine that is sensitive enough to detect low exposure levels to OPs.

OPs are mainly metabolized into any of the following compounds and excreted in urine: dimethylphosphate (DMP), diethylphosphate (DEP), dimethylthiophosphate (DMTP), and diethylthiophosphate (DETP) (Table 1) [11]. The determination of these dialkylphosphates (DAPs) in human urine has been reported as a sensitive indicator for non-occupational background exposure levels [10], [11], [12], [13]. Consequently, many biological monitoring studies using DAPs in human urine have been reported [10], [12], [14], [15], [16]. However, it is difficult to analyze OP metabolites due to their low urinary concentration and the complicated procedure needed to extract and derivatize metabolites from urine.

Previously reported methods for the extraction of DAPs were liquid phase extraction [15], [17], column clean-up [14], [18], lyophilization [10], and azeotropic distillation [11], [19]. Unfortunately, urinary DAP extraction using the lyophilization or azeotropic distillation technique is extremely time consuming (over 16–24 h) and is labor intensive [10]. Pentafluorobenzylbromide (PFBBr), which is able to yield a single reaction product [20], is widely used for the determination of trace amounts of urinary DAPs in the general population [10], [14], [15], [17], [21]. However, the handling of PFBBr is troublesome due to its highly irritating nature.

In this paper, using gas chromatography–mass spectrometry equipped with electron ionization system (GC–MS-EI), we improved some conditions of evaporation, derivatization, and clean-up for removing PFBBr to facilitate the determination of urinary DAPs. The present method was then applied to measure DAP concentrations in 48 urine samples collected from a Japanese population in order to evaluate non-occupational as well as occupational OP exposure levels. We focused on four DAPs (DMP, DEP, DMTP, and DETP) since the pest control operators, our study population, seldom used pesticides metabolized to dimethyldithiophosphate (DMDTP) or diethyldithiophosphate (DEDTP), and the reported urinary levels of DMDTP and DEDTP are very low in the general population [10], [16].

Section snippets

Reagents

DMP tetramethylammonium salt (99.9% purity), DMTP ammonium salt (98.9%), DEP (98.2%), and DETP ammonium salt (95.2%) were purchased from Hayashi Pure Chemical Ind. (Osaka, Japan), and dibutylphosphate (DBP), used for an internal standard (I.S.), was from Tokyo Kasei Kogyo (Tokyo, Japan). Diethyl ether, acetonitrile, n-hexane, acetone, and toluene, which are pesticide residue grade, and sodium sulfate anhydrous, sodium chloride (NaCl), sodium disulfite (Na2S2O5), l-ascorbic acid, potassium

Optimum conditions for method

To optimize the conditions for the determination of DAPs, we examined the reaction time and temperature for derivatization, pH and stability on evaporation and derivatization, and separation of unreacted PFBBr. Five milliliters of human urine spiked with a known amount of DAPs (final concentration 10 mg/L) was used except for the optimization study of the derivatization condition. There was no interfering peak for internal standard DBP.

Conclusion

The present method allows us to determine four urinary DAPs simultaneously, quickly, safely, and with high sensitivity. It does not require special equipment except for GC–MS, needs only 3 h for the sample preparation, and completely inhibits the unwanted oxidization of dialkylthiophosphates. The PFB-DAPs in injection samples remain stable for over 36 h after preparation. A single operator will be able to determine about 100 urine samples within 3 or 4 working days. Thus, this method can be

Acknowledgements

This work was supported in part by Health and Labor Sciences Research Grants (Research on Risk of Chemical Substances) from the Ministry of Health, Labor and Welfare of Japan, and Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

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