Improved Cleanup and Determination of Dialkyl Phosphates in the Urine of Children Exposed to Organophosphorus Insecticides

Abstract

Analysis of dialkylphosphate urinary metabolites of organophosphorus insecticides has been used to estimate dose in nonoccupationally exposed populations, including children. Analytical methods must continue to be improved in order to accurately and reproducibly measure less than 10 ng/mL of these metabolites. Dialkyl phosphates are commonly determined as their pentafluorobenzyl bromide derivatives via gas chromatography (GC) with flame photometric detection. Presented here is an improved method for precleanup of urine using solid-phase extraction, followed by derivatization and GC analysis. The method includes the quantitative determination of the following dialkyl phosphate metabolites: dimethylphosphate, diethylphosphate, dimethylthiophosphate, diethylthiophosphate, and dimethyldithiophosphate. Additional cleanup of urine samples allows for increasing sample size and improving sensitivity while minimizing interferences and variability associated with derivatization. Sample aliquot size was 5 mL with limits of quantitation of 10 ng/mL of urine for dimethylphosphate and diethylphosphate and 2 ng/mL of urine for dimethylthiophosphate, diethylthiophosphate, and dimethyldithiophosphate. This level of method sensitivity allows for quantitative determination of trace dialkyl phosphates in approximately 75% of individuals in nonoccupationally exposed populations. This streamlined method increases sample throughput, provides a clean extract for analysis, and requires no custom glassware.

Measurement of dialkyl phosphate metabolites of organophosphorus pesticides in human urine using lyophilization with gas chromatography-tandem mass spectrometry and isotope dilution quantification

“…For example, a gummy syrup is produced after azeotropic distillation. Moate et al (1999) speculated that the alkylation of OP metabolites could be hindered by encapsulation of DAP metabolites in this gummy syrup. They found that a mixture of acetonitrile and dimethylformamide could dissolve the gummy residue and yield a better derivatization of DMP and DEP.…”

“…Because the alkylation of DAP metabolites is a thermosensitive reaction and the sulfur-containing DAPs degrade at high temperatures (Fenske and Leffingwell, 1989;Moate et al, 1999;Oglobline et al, 2001 a, b), optimization of the reaction temperature and time is critical. Typically, the sulfur-containing molecules (DMTP, DMDTP, DETP, and DEDTP) react at room temperature while DMP and DEP often require high temperatures to react.…”

“…Typically, the sulfur-containing molecules (DMTP, DMDTP, DETP, and DEDTP) react at room temperature while DMP and DEP often require high temperatures to react. Several methods use a two-step derivatization process to accommodate both groups of DAPs (Reid and Watts, 1981;Aprea et al, 1996a, b;Moate et al, 1999). Because performing two separate derivatization reactions is cumbersome and tedious, we sought to achieve an efficient single-step reaction that was effective in derivatizing DMP and DEP but did not degrade the other DAPs.…”

“…Since the 1970s, urinary dialkylphosphate (DAP) metabolites of OP pesticides have been quantified in human urine as a dosimeter for exposure and bodily adsorption of OP pesticides (Bradway and Shafik, 1977;Reid and Watts, 1981;Bardarov and Mitewa, 1989;Fenske and Leffingwell, 1989;Weisskopf and Seiber, 1989;Drevenkar et al, 1991;Aprea et al, 1994Aprea et al, , 1996aAprea et al, , b, 1999Aprea et al, , 2000Davies and Peterson, 1997;Loewenherz et al, 1997;Moate et al, 1999;Simcox et al, 1999. Fenske et al, 2000Hardt and Angerer, 2000;Heudorf and Angerer, 2001;Lu et al, 2001;Oglobline et al, 2001a, b;Bravo et al, 2002;CDC, 2002;Cocker et al, 2002;Garfitt et al, 2002a, b).…”

“…Typical isolation or extraction techniques reported in the literature include liquid-liquid extraction using polar solvents (Hardt and Angerer, 2000), solid-phase extraction (SPE) (Lores and Bradway, 1976;Weisskopf and Seiber, 1989;Driskell et al, 1999), azeotropic distillation using acetonitrile:water azeotropes (7:1) (Reid and Watts, 1981;Fenske and Leffingwell, 1989;Moate et al, 1999;Bravo et al, 2002), and lyophilization (Davies and Peterson, 1997;Oglobline et al, 2001 a, b). Because the DAP metabolites are inherently polar with low pK a 's, the liquid-liquid extraction of these metabolites into an organic phase is difficult, resulting in poor recoveries.…”