Determination of Dimethyl Phosphate (DMP) As a Stable Metabolite in A
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Journal of Analytical Toxicology, Vol. 28, April 2004 [ TechnicalNote Acute Poisoning with Phosphamidon: Determination of Dimethyl Phosphate (DMP) as a Stable Metabolite in a Case of Organophosphate Insecticide Intoxication Downloaded from https://academic.oup.com/jat/article/28/3/198/701920 by guest on 01 October 2021 F.A. Tarbah, B. Kardel, S. Pier, O. Temme, and T. Daldrup* Institute of Legal Medicine, Heinrich-Heine-University, P.O. Box 10 10 07, D-40001 Duesseldoff, Germany Abstract rhea, urination, miosis, muscular twitching like bron- chiospasm, emesis, lacrimation, and salivation (mnemomic DUMBELS) (10). Many organophosphate pesticides (OP) such as phosphamidon are The relationship between OP concentrationsin body fluid and unstable in aqueous solutions and especially in blood in the presence of esterases. In a case of intoxication, the phosphamidon the severity of patients' reactions has not been thoroughly in- concentration in serum decreased from 10 mg/L to 4.4 mg/L after vestigated to date. This type of data is required to obtain a storage at -20~ for six months; nearly complete degradation was more accurate diagnoses,thus enabling the prediction of the ef- observed after three years. Dimethyl phosphate (DMP) is a ficacy of a clinical treatment (I i). metaboUte of phosphamidon, mevinphos, dicrotophos, Many OPs are unstable in aqueous solution and especially in monocrotophos, dichlorvos, and trichlorfon. A gas blood becauseof the presence of esterases.Complete degrada- chromatographic-mass spectrometric method with deuterated tion of the OP during storage was frequently observed. OPs DMP-d6 as internal standard for the determination of DMP in are degraded more rapidly by esteraseactivities than by other biological material was validated. DMP was found in all of the chemical mechanisms (4,12-15). The organophosphorothioates patient's samples (3.9 and 4.9 mg/L in blood, 33.5 and 50.4 mg/L like ch]orpyrifos methy] are unstable in aqueous solution but in urine, and 8.1 mg/L in gastric fluid), even after storage at -20~ stable in blood for at least 72 h. A possible explanation is that for up to 3 years. No hints for a degradation of DMP when spiked in fresh blood and stored at 4~ for 1 week and stored in water proteins and lipids in biological specimens may effectively sta- over a time period of 10 months. Looking for the stable bilize chlorpyrifos methyl (16). metabolites like DMP in cases of suspected OP intoxication is A typical degradation product is para-nitrophenol, which can recommended. be found in blood and urine at high concentrations in the case of parathion intoxication (17). Stability of OPs in serum sam- pies during storage was measured in 23 different OPs, which were mixed with serum containing 10 m~mL EDTA and stored Introduction up to 10 days at 4 and -20~ The recovery rates of the 23 OPs ranged between 50% (dimethoate) and 133% (dialifos) (18). The Organophosphate pesticides (OP) are responsible for nu- general population can be exposedto organophosphorus com- merous acute and even fatal poisonings (1-5), and therefore pounds because of the application of pesticides in household must always be considered when solving forensic and clinical and private garden. In general, the measurement of urinary cases (6-8). The mechanism of toxicity is the inhibition of alkyl phosphate metabolites is a more sensitive index of expo- acetylcholine esterase due to direct enzyme binding, resulting sure to OPs than cho]inesterase inhibition. The degradation in the accumulation of acetylcholine at the nerve synapses. In products of many OPs are the alkyl phosphates such as O,O- fatal cases, this leads to the disruption of nerve functions (cen- dimethyl phosphate (DMP), O,O-diethyl phosphate (DEP), O,O- trally and peripherally), tonic convulsion (limbs stretched and diethylthio phosphate (DETP), O,O-dimethyldithio phosphate rigid) or more likely clonic convulsion (rapid repetitive move- (DMDTP), O,O-diethyldithio phosphate (DEDTP), and O,O- ment), respiratory failure, and, when untreated, to asphyxiation dimethy]thio phosphate (DMTP). These phosphates are well (9). The muscarine and nicotine-like symptoms observed after known as markers of occupational OP exposure (19-25). Aprea poisoning with such anticholinesterase agents include diar- et al. (19) published a study in which 124 persons without oc- cupational exposure were examined in Italy. DMP was found to be of 10 ]J~JLurine. Hardt and Angerer (20) had investigated 54 "Author to whom correspondence should be addressed. E-mail: [email protected]. spot urine samples from non-occupational subjects in Ger- 198 Reproduction (photocopying)of editorialcontent of this journal is prohibitedwithout publisher'spermission. Journal of Analytical Toxicology,Vol. 28, April 2004 many. Their analyses revealed a mean concentration of DMP in A quantitative, rapid, and sensitive gas chromatographic- urine of 30 IJg/L. mass spectrometric (GC-MS) method with deuterated DMP-d6 Stability studies carried out over a two-week period have as internal standard after derivatization with pentafluorobenzyl shown that DMP is stable in urine under storage at room tem- bromide PFB-Br is developed (Figure 2). perature, 4~ or -20~ (26). Further investigations carried out in our laboratory gave no hint of a degradation of DMP when stored in blood at 4~ for I week and stored in water for 10 months, whereas mevinphos was nearly completely de- Case History graded to DMP after storage for about 72 h in blood at 4~ (un- published data). A 19-year-old previously healthy female was admitted to Suspected criminal intoxication with OP was the reason a the emergency unit. She had attempted suicide by ingesting method to determine DMP as stable alkylphosphate in biolog- an unknown insecticide. The insecticide, blood (without addi- ical samples, especially in postmortem material, was devel- tion of any preservatives), urine, and gastric fluid were sent oped. DMP is a stable metabolite and the degradation product to our laboratory. The medical investigation of the patient Downloaded from https://academic.oup.com/jat/article/28/3/198/701920 by guest on 01 October 2021 of mevinphos, dicmtophos, monocmtophos, dichiorphos, phos- revealed aspiration pneumonia, acute pancreatitis, tachycardia, phamidon, and trichiorfon (Figure 1). and irritability. Skin and mucous membrane were normal. Trade name Chemical name Chemical structure 1. Mevinphos O, O-dimethyI-O-(2-methoxycarbonyl- H3CO\ ~O 0 1-methylvinyl)-phosphate II Hsco/P~o_ C - CH-C- OCH5 I CHa 2. Dicrotophos O,O-dimethyI-O-(1-methyl-2-N,N- H3CO\ ~O dimethylcarbamoylvinyl)-phosphate CH;I CH5 3. Monocrotophos O,O-dimethyI-O-(1-methyl-2- H=CO _ 0 \-// o N- methylcarbamoylvinyl)-phosphate H=CO/I"\O _ C = CH'C-II NH-CH~ I CH= 4. Dichlorvos O-(2,2-Dichlorovinyl)-O,O-dimethylphosphate H3CO\_// _ 0 H3CO \0 - CH = CCIz 5. Phosphamidon O,O-dimethyI-O-(2-chloro-2-N,N- H3CO . O \_6 CJ 0 /CzH= diethylcarbamoyl-l-methylvlnyl)-phosphate H3CO/I"\O _ I II C-C-C-N, \ CH3 C21"~ 6. Trichlorfon O, O-dimethyl-(2,2,2-tdchloro-1 -hydroxyethyl)- H=CO . O phosphonate H~CO/~\CH-CCI3 I OH Figure I. Examplesof organophosphatepesticides metabolizing to produce DMP as stable metabolite. 199 Journal of Analytical Toxicology, Vol. 28, April 2004 Cholinesterase activity was reduced (100 U/L). The patient program: initial temperature 90~ for 6 min, 15~ to was conscious. The analysis of the unknown insecticide proved 250~ maintained for 1 rain; and carrier gas: nitrogen, 10 that it contained phosphamidon. The blood concentration mL/min constant flow. of phosphamidon determined by gas chromatography- GC--MS. The analyses were performed with a Hewlett-Packard nitrogen-phosphorus detection (GC-NPD) was 10 mg/L. The GC 5890 and MSD 5970 equipped with a Hewlett-Packard samples were stored at -20~ for 20 and 36 months before automatic liquid sampler HP 7673; column, HP-5 MS (30-m the DMP analyses. length, 0.25-ram i.d., 0.25-pro film thickness); carrier gas, helium (pressure 70 kPa); split/purge off time, 2 rain; injector temperature, 270~ transfer line temperature, 280~ and temperature program, initial temperature 60~ for Experimental 2 min, 40~ to 110~ 12~ to 170~ 40~ Materials to 300~ maintained for 3.5 rain (total run time: 17 min). All solvents used were of analytical grade with a purity greater The MS was in electron impact mode at 70 eV. using the selected ion monitoring mode, m/z 110, 116, 194, 306, and than 98%. Acetonitrile, acetone, ethanol, heptane, toluene, Downloaded from https://academic.oup.com/jat/article/28/3/198/701920 by guest on 01 October 2021 water (HPLC grade), and potassium carbonate were obtained 312 for DMP and DMP-d6 were measured (quantitation ions from E. Merck (Darmstadt, Germany). Dimethyl phosphate are underlined). originated from Ultra Scientific (North Kingston, RI). Deuterated dimethyl phosphate (DMP-d6) was prepared by Extraction procedure for OP general screening Prof. Dr. G. Machbert (Institute of Legal Medicine, Erlangen, In an Eppendorf tube, an aliquot of the samples (0.7 mL Germany). PFB-Br was obtained from Sigma Chemical Co. (St. blood, serum, urine and/or gastric juice) was mixed with 1 mL Louis, MO), phosphamidon was from Dr. S. U. I. Ehrenstoffer toluene for 2 min. (vortex) and centrifuged at 14,000 rpm (Augsburg, Germany), and mevinphos was from Riedel-De (17,500 xg) for 5 min at 4~ to avoid the problems of emulsion Hahn (Hannover, Germany). formation. A second aliquot was extracted in the same way, Biological materials (case of acute phosphamidon intoxica- but the internal standard (7 pL of 0.01% mevinphos in toluene) tion) include serum, urine, blood, gastric fluid, and the un- was added to the sample before extraction. The toluene layers known insecticide derived were from Remscheid Hospital, were transferred to glass vials.