Quantitation of metrifonate and dichlorvos in blood and tissues by gas chromatography—Mass spectrometry

FUNDAMENTALAND APPLIEDTOXICOLOGY 1:230-237 (1981)

Quantitation. of Metrifonate and Dichlorvos in Blood and Tissues by Gas Chromatography - Mass Spectrometry INGRID NORDGREN Department of Toxicology, Karolinska lnstitutet, Box 60 400, S-104 01 Stockholm, Sweden

ABSTRACT

Metrifonate (0,0-dimethyl-( 1-hydroxy-2,2,2-trichloroethyl)-phosphonate) is an organophosphorus compound where there are excellent possibilities to make studies in man. Metrifonate and its rearrangement product dichlorvos (2,2-dichlorovinyl dimethyl phosphate, D D V P ) were studied in human blood from schistosomiasis patients treated with Bilarcil ~. A m a s s fragmentographic technique was employed. Deuterium labelled variants of the substances were used, both as'internal standards and to compensate for D D V P formed during the workup procedure. The amount of D D V P in plasma was about I% ofthe amount of metrifonate. In erythrocytes the corresponding amount of D D V P in percent of metrifonate was half or less. Both c o m p o u n d s reached peak levels within two hours and were detectable for at least eight hours. The results were compared to erythrocyte and plasma Cholinesterase determinations. Levels of metrifonate and D D V P , together with cholinesterase activity, have also been studied in mouse brain, liver and kidney. It is proposed that metrifonate acts as a slow release formulation for D D V P . Clearance of metrifonate in man occurs primarily via D D V P . Mild vertigo subsiding in a few hours was the most common side-effect.

(Kharasch and Bengelsdorf, 1955; Bengelsdorf, 1956; Metcalf et aL, 1959; Sohr and Lohs, 1965; Dedek et aL, 1969) (Fig. 1A). A method was developed for the analysis of both metrifonate and DDVP formed in vivo in organs of experimental animals or in human blood (Nordgren et aL, 1978 and 1980). When developing this method, the problem was that we could expect a formation of DDVP both in vivo and during the workup procedure. By the use of a mass fragmentographic technique and several deuterium labelled isotopes of metrifonate and DDVP, it is possible to distinguish between the DDVP formed in vivo and the DDVP formed during the workup procedure. The isotopes used are shown in Fig. lB. 2H6-DDVP can only be formed from the internal standard 2H6-metrifonate. Since we can measure how much 2Hsmetrifonate has been rearranged to 2Hs-DDVP, and this must happen during the workup procedure (2H6-metrifonate is added after the blood samples have been withdrawn), we can also calculate how much unlabelled DDVP has been formed from unlabelled metrifonate during the same workup.The rest of the unlabelled DDVP, consequently, has been formed in vivo. Metrifonate- and DDVP-levels, as well as cholinesterase activity, have been studied in plasma and erythrocytes in patients treated with Bilarcil~ (metrifonate) against schistosomiasis (Nordgren et aL, 1981). Determinations have also been carried out in brain, liver and kidney from mice.

INTRODUCTION One of the organophosphorus compounds where there are excellent possibilities to make studies in man is metrifonate (0,O-dimethyl-(1 -hydroxy-2,2,2-trichloroethyl)-phosphonate). Metrifonate, by now, has been given in about 5 million doses to patients in the tropics in the treatment of schistosomiasis. It is estimated that there are 200 million people in Asia, Africa, South America and the Caribbean suffering from this disease. Metrifonate is transformed in both aqueous and organic solvents into the active cholinesterase inhibitor dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP). The mechanism for this transformation has been extensively studied

MATERIALS A N D METHODS Subjects Seven patients have been studied. The dosage of metrifonate varied between 7.5 - 10 mg/kg body weight in a single dose repeated after 2 weeks, Side effects were recorded on a standardized form. Electrocardiographic and electroencephalographic examinations were performed after each drug administration.

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230

Fundam. Appl. ToxicoL (!)

3larch~April, 1981

SYMPOSIUM ON I~ROPHYLAXIS AND TREATMENT OF ORGANOPttOSPttATE POISONING

ISOTOPES USED CH30,,

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(1) 3-4/81

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Sampling Blood samples, 10 mL whole blood, were collected in Venoject| evacuated blood collection tubes. Blood levels in all patients were followed upon two successive treatments at a two weeks interval. Chemicals All chemical reagents were of analytical grade. The metrifonate was a gift from Bayer Chemical Co. The deuterated metrifonate was synthesized by Lars Fagerlind at the National Defence Research Institute, according to Schrader (1963). DDVP and 2H6-DDVP were gifts from the Shell Chemical Co. 2H4-DDVP was synthesized by Dr. Kurt Leander. "Astra 1397" (10-(e-diethylaminopropionyl)-pheno-thiazine HCI) is commercially available from the Astra Research Development Laboratory (S-151 85 S6dert~lje, Sweden). Ouantitation o f metrifonate and dichlorvos The plasma samples were analyzed according to Nordgren et al. (1978) and (1980). One mL of plasma was diluted with 3 mL of saline. After addition of the internal standards 2HGmetrifonate and 2H4-DDVP, the sample was extracted w i t h 4 mL of hexane. Metrifonate was found in the aqueous phase 232

and DDVP in the hexane phase. The aqueous phase was extracted with 4 mL chloroform, which was then dried with sodium sulphate, filtered, evaporated to dryness and the metrifonate was silanized with 50 pL BSTFA (N,O-bis(trimethylsilyl)-trifluoroacetamide) and analyzed mass fragmentographically. The hexane phase was washed twice with a saturated sodium chloride solutioh, dried with sodium sulfate, filtered, evaporated to dryness, dissolved in 50 pL chloroform and analyzed for DDVP mass fragmentographically. The erythrocytes were washed once with saline. The blood sample was centrifuged, the plasma was removed and the erythrocytes diluted to the original volume with saline, mixed and centrifuged again. The saline was discarded and the erythrocytes diluted to the original volume with distilled water and shaken. One mL of this was then diluted with 3 mL of saline and analyzed for metrifonate and DDVP according to the plasma analysis described above. Metrifonate was analyzed on an LKB 9000 mass spectrometer equipped with multiple ion detection. Standard curves with known amounts of ~H- and 2H6-metrifonate were run in the same way. Metrifonate was analyzed at m / z 317 (IHmetrifonate, Fig. 2) and m / z 323 (2H6-metrifonate). The gas Fundam. Appl. Tox/col. (!)

31arch~April, 1981

SYMPOSIUM ON PROPHYLAXIS AND TREATMENT OF ORGANOPHOSPHATE POISONING

chromatographic column used was a glass column 1.5 m in length and with an i.d. of 2 mm packed with 3%OV-17 on Gas Chrom Q. The flash heater was kept at 200 ~ the column at 160 ~ and the separator and ion source at 250 ~ and 270 ~ respectively. The helium flow was 20 mL/min. A typical mass fragmentogram is seen in Fig. 3. DDVP was analyzed on an LKB 2091 mass spectrometer with a computer controlled MID-system (Fig. 4). DDVP was analyzed at m / z 113 (2H4-DDVP) and m / z 115 (2He-DDVP). Standard curves with known amounts of 1H-DDVP, 2H~-DDVP and ~H6-DDVP were run in the same way. The gas chromatographic column used was a capillary column 25 m in length and with an i.d. of 0.25 mm coated w i t h OV-101. The flash heater was kept at 170 ~ the column at 130 ~ the separator at 250 ~ and the ion source at 270 ~ The helium flow was 1 mL/min. A typical mass fragmentogram is seen in Fig. 5.

are about the same as in plasma. Five of the patients exhibited lower peak levels of metrifonate and DDVP after the second dose, and the peak maximum appeared somewhat later. The plasma cholinesterase is inhibited almost completely and recovers slowly after one day. The erythrocyte cholinesterase activity dropped to about 20-40% of the pre-exposure value and recovered more slowly than the activity in plasma. At the second treatment, the plasma and erythrocyte cholinesterase activity have not recovered completely. The activity varies between 47-78% of the enzyme values before the first treatment for both types of esterases. Erythrocyte cholinesterase activity was found to reach a lower level after the second dose than after the first one and it also seemed to recover more slowly after the second dose. As after the first dose, the activity in plasma was next to zero. Among the patients studied, we have noticed only mild sideeffects. Three patients complained of mild vertigo subsiding in a few hours, and tw O complained of nausea. Electroencephalograms, liver and kidney function tests and routine hematology examir~ations were all normal. In one patient, abnormal T-wave flattening of the electrocardiogram was observed after the first dose of metrifonate, however, not after the second treatment. The levels of metrifonate and DDVP, together with cholinesterase activity, have also been studied in brain, liver and kidney from mice after i.p. injection of metrifonate 125 mg/kg. Table 1 shows the metrifonate and DDVP levels in these organs. The metrifonate levels in liver and kidney are approximately the same, but the amount of DDVP found in kidney is lower than that in liver despite the fact that the cholinesterase activity is much higher in liver than in kidney.

D e t e r m i n a t i o n o f cholinesterase a c t i v i t y Cholinesterase activity was determined according to the method of Augustinsson et al. (1978). RESULTS Seven patients have been studied in all. Fig. 6 and 7 show the results from one of these patients. The relationship of DDVP to metrifonate in plasma was around 1% in all cases, both after the first and second treatment (0.5 - 2.0%). In erythrocytes the amount of DDVP compared to metrifonate was half or less (0.2 - 0.6%). Peak levels of both compounds occur w i t h i n two hours, and the compounds are detectable during at least eight hours. If the plasma results are recalculated to whole blood volume, the levels of metrifonate in erythrocytes

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DISCUSSION

Metrifonate has been given routinely empirically in two doses of 7.5 mg/kg body weight at two weeks intervals (Lebrun and Cerf, 1960; Plestina et aL, 1972). Other therapeutically used doses have been tabulated by Holmstedt et aL (1978). The in vivo formation of DDVP from metrifonate has been chemically proven tp occur in man. The shape of the blood curves is su'ch that metrifonate can be said to act as a naturally occurring "slow-release formulation". If the direct acting anticholinesterase DDVP is the active component, the prolonged effect may be of essential importance for the schistosomicidal effect. Further studies on levels and enzyme inhibition in organs will have to be carried out. Pharmacokinetic calculations indicate that clearance of metrifonate in man occurs primarily via DDVP (Nordgren et al., 1981 ). In the tropics, other anticholinesterases may be used simultaneously as insecticides, and a double exposure is conceivable. Potentiation of action between organophosphates is known to result both from other organophosphates and from impurities occurring in the formulation (Aldridge et aL, 1979; Murphy, 1967).

234

In areas.where temephos (diphenphos, abate (O,O,O'O'tetramethyl O,O'-thiodi-paraphenylene phosphorthioate)) is used as s i m u l i u m farvicide, a combined exposure to the two a nticholinesterase compounds may frequently occur. Plestina (1978) studied, therefore, the effect of the toxicity of either compound upon simultaneous administration of temephos and metrifonate to rats. In rats fed diets containing temephos, he found no changes in the oral or intravenous toxicity of metrifonate. Thus, it seems unlikely that exposure to temephos would enhance metrifonate toxicity during schistosomiasis treatment. However, other combinations have not been tested. In the treatment of onchocerciasis, doses as high as 10 mg/kg • day for six consecutive days have been given. Vertigo and fall in blood pressure during the end of the treatment period was reported. 10 mg/kg body weight during three days, repeated several times at two weeks intervals, caused no complaints from the patients (Thylefors, personal communication). Metrifonate, by now, has been given to millions of patients in the tropics in the treatment of schistosomiasis. Accurate observations may be difficult under field conditions, but acute Fundam. ,,Ippl. Toxicol. (!)

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SYMPOSIUM ON PROPtIYLAXIS AND TREATMENT

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Fundam. Appl. Toxicol. (!)

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SYMPOSIUM ON PROPttYLAXIS AND TREATMENT OF ORGANOPHOSPttATE l'OISO~l~(-: TABLE 1 Levels of Metrifonate and DDVP in Brain, Liver and Kidney from Mice after i.p. Injection of Metrifonate 125 mg/kg Organ

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DDVP taglg 4- S.E.

Number of Animals

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side-effects would most likely have been reported. Mild sideeffects like nausea, colic, sweating (Lebrun and Cerf, 1960) and lassitude (Taalat et al., 1963) are on record. Davis and Bailey (1969) and Plestina et aL (1972) reported side-effects at a dose of 10 mg/kg but only a few complaints of a trivial nature were reported in schoolchildren given 7.5 mg/kg. This is in accordance with our own results. Mild vertigo was reported in three cases and nausea in two. The electrocardiographic abnormality observed in one patient did not seem to have any connection with the drug treatment, since it did not appear on the second treatment. I

The mild side-effects observed upon treatment with this organophosphate imetrifonate) readily respond to atropine treatment. The use of reactivators has been suggested but, to our knowledge, has not been found necessary. However, treatment of humans w i t h metrifonate presents a unique possibility to study both side-effects and the action of various doses of antidotes.: A C K N O WL ED G E M E N TS

This work has been supported by grants from the UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical Diseases (SCH) T 1 6 / 1 8 1 / B 2 / 3 1 and V 2 / 1 8 1 / 1 5 8 ; the Swedish Medical Research Council B81-25X-04041-09 and by funds from the Wallenberg Foundation. REFERENCES

Aldridge, W.N., Miles, J.W., Mount, D.L. and Verschoyle, R.D. (1979). The toxicological properties of impurities in malathion. Arch. Toxicoil 42:95-106. Augustinsson, K.-B., Eriksson, H. and Faijersson, Y. (1978). A new approach to determining cholinesterase activities in samples of whole blood. Clin. Chim. Acta 89:239-252.

Fundamental and Applied Toxicology

(!) 3-4/81

Bengelsdorf, I.S. (1956). Dialky1,8-chlorovinyl phosphates. A proposed scheme to predict the products of the action of base with dialkyl n-hydroxyphosphonic esters. J. Org. Chem. 21:475-476. Davis, A. and Bailey, R. (1969). Metrifonate in urinary schistosomiasis, lhdil II'hil Ilhh. Org.-t1:209-224. Dedek, W., Koch, H., Uhlenhut, G. and Br6se, F. (1969). Zur Kenntnis der Umsetzung ,.,on 3H-Trichlorphon zu DDVP. Z. Naturforsch. 2.tb:663-664. ltolmstedt, B., Nordgren, I., Sandoz, M. and Sundwall, A. (1978). Metrifonate: St.mmary of toxicological and pharmacological information available. Arch. To.vicoil .11:3-29. Kharash, M.S. and Bengelsdorf, l.S. (1955). The reaction of triethyl phosphite with a-trichloromethyl carbonyl compounds. J. Org. Chem. 20:1356-1362. Lebrun, A. and Cerf, C. (1960). Notb preliminare sur la toxicitb pour I'homme d'un insecticide organophosphor~., (Dipterex). Btdil I|:ll.O. 22:579-582. Metcalf, R.L., Fukuto, R.B. and March, R.B. (1959). Toxic action of Dipterex and DDVP to the house fly. J. Econ. A)ltomoL 52:44-49. Murphy, S.D. (1967). Malathion inhibition of esterases as a determinant of malathion toxicity. J. PharmacoL 156:352-365. Nordgren, i., Bengtsson, E., Holmstedt, B. and Pettersson, B.-M. (1981). Levels of metrifonate and dichlorvos in plasma and erythrocytes during treatment of schistosomiasis with Bilarcil| To be published in A e t a P h a r m a c o l o g i c a et Toxicologica.

Nordgren, I., Bergstriim, M., Holmstedt, B. and Sandoz, M.: Transformation and action of metrifonate. Arch. ToxiCOIl 41:31-41. Nordgren, I., Holmstedtl B., Bengtsson; E. and Finkel, Y. (1980). Plasma levels of metrifonate and dichlorvos during treatment of schistosomiasis with Bilarcil~. tim. J. Trop. Med. tl.vg. 29(3):426-430. Plestina, R. (1978). Temephoslmetrifonate, combined exposure. 9 Wld. Hlth. Org., Geneve, October 3-9, PDSIEC25/78.32. Piestina, R., Davis, A. and Bailey, D.R. (1972). Effect of metrifonate on blood cholinesterases in children during the treatment of schistosomiasis. Bull. I|'hil llhh. Org. 46:747-759. Schrader, G. (1963). Die Entwickhmg net[er insektizir Phosphors~iure-Ester. Weinheim/Bergstr.: Veiiag Chemie GmbH. Sohr, H. and Lohs, Kh. (1965). Zum Verhahen von Dipterex in W~ssriger L6sung. Mber. Dtsch. dkad. IViss. Berlbt 7:708-711. Talaat, S.M., Amin, N. and El Massry, B. (1963). The treatment of bilharziasis and other intestinal parasites with Dipterex. A preliminary report on one hundred cases. J. Egypt. Med. Assoc. 46:827-832. Thylefors, B. Personal communication.

237