[The profiles of free organophosphorus poisons in the bile of rabbits poisoned with different organophosphates].

OBJECTIVE: To study the process and significance of the distribution of free organophosphorus poisons (FOPs) in the blood and bile of rabbits poisoned with organophosphates. METHODS: Seventy two livid blue rabbits, male, 2 - 2.5 kg in weight, were divided into 3 groups: trichlorfon (556.0 mg/kg), monocrotophos (11.12 mg/kg) and methyl parathion (37.05 mg/kg). Each group consisted of 24 rabbits. All organophosphates were administered by subcutaneous route. Blood and bile were collected at time intervals of 1, 6, 24, 96 hours after administration. Blood cells and plasma were separated. Acetylcholinesterase (AChE) activity was measured with dithiobisnitrobenzoic acid (DTNB) enzyme kinetic method. The levels of FOPs in plasma and bile were determined with enzyme inhibited method. RESULTS: From 1 h to 96 h after administration negative correlation was found between time and FOP concentration in plasma (trichlorfon, r = -0.74, P < 0.01; monocrotophos, r = -0.55, P < 0.01; methyl parathion, r = -0.69, P < 0.01), and it was also found in bile between time and FOP concentration of trichlorfon (r = -0.97, P < 0.01) and monocrotophos (r = -0.71, P < 0.01). There is no linear correlation between time and concentration of methyl parathion in bile (r = -0.14, P > 0.05). When FOPs in plasma were not detectable at 96 h after administration, high levels of FOPs could still be detected in bile [trichlorfon (300.3 +/- 174.44) IU/L; monocrotophos (362.8 +/- 136.62) IU/L; methyl parathion (101.0 +/- 75.85) IU/L]. CONCLUSION: The bile is the most important store for FOPs in animal. FOPs can exist in bile over 96 h. The process of poisoning is a changing process of absorption, distribution and redistribution and it is different owing to various physical and chemical properties of organophosphates.

Determination of metrifonate enantiomers in blood and brain samples using liquid chromatography on a chiral stationary phase coupled to tandem mass spectrometry.

A novel enantioselective assay is described for the simultaneous determination of the metrifonate enantiomers BAY z 7216 and BAY z 7217 in extracts of whole blood samples obtained from rats, mice, rabbits and Beagle dogs as well as in rat brain tissue using liquid chromatography tandem mass spectrometry (LC/MS/MS) with thermally and pneumatically assisted electrospray ionization (TurboIonSpray(R)). Chromatographic separation is achieved on a chiral normal phase column with a mobile phase containing 0.25% water only. The total run time per sample is 11.0 min giving chromatographic base line separation of the enantiomers. Compared with previous methods this assay offers a higher sample throughput, excellent ruggedness and higher sensitivity. The limits of quantification for each enantiomer are 5.00 microg/L from 0.5 mL whole blood and 7.50 ng/g (ppb) using 0.333 g brain tissue, respectively. Similar assay specifications have been derived for the two enantiomers. The method has been validated for the analysis of blood samples from low and high dosed preclinical pharmacokinetic and toxicokinetic studies, corresponding to two analytical working ranges like e.g. 5.00 to 1000 microg/L and 200 to 40000 microg/L (0. 200 to 40.0 mg/L). For rat brain tissue the validated concentration range is 7.50 to 750 ng/g (ppb).

Development, validation and application of assays to quantify metrifonate and 2,2-dichlorovinyl dimethylphosphate in human body fluids.

Gas chromatographic procedures [GC with electron-capture detection (ECD) and GC-MS] for the quantitative analysis of metrifonate and its active metabolite 2,2-dichlorovinyl dimethylphosphate (DDVP) in human blood and urine were developed, validated, and applied to the analysis of clinical study samples. Analysis of metrifonate involved extraction of acidified blood with ethyl acetate followed by solid-phase clean-up of the organic extract. Acidified urine was extracted with dichloromethane and the residue of evaporated organic phase was reconstituted in toluene. ECD and diethyl analogue of metrifonate internal standard (I.S.) were used for quantitation of metrifonate. The metrifonate lower limit of quantitation (LOQ) was 10.0 microg/l. The DDVP metabolite was chromatographed separately after cyclohexane extraction of acidified blood and urine using d6-DDVP I.S. and MS detection. The LOQ of DDVP was 1 microg/l. Stability studies have confirmed that the matrix should be acidified prior to storage at -20 degrees C or -80 degrees C to inhibit chemical and enzymatic degradation of the analytes and to avoid overestimation of DDVP concentrations. Metrifonate was found to be stable in acidified human blood after 20 months of storage at -20 degrees C and after 23 months of storage at -80 degrees C. Under these conditions DDVP was found to be stable after 12 months of storage. Both assay procedures were cross-validated by different world-wide laboratories and found to be accurate and robust during analyses of clinical study samples.

Pharmacokinetics and pharmacodynamics of metrifonate in humans.

We studied the pharmacokinetics and pharmacodynamics [red blood cell (RBC) cholinesterase (ChE) inhibition] of metrifonate (MTF) and its active anti-ChE metabolite, dichlorvos (DDVP) in Alzheimer's disease (AD) patients and normal controls after oral MTF. In Study I conducted for 6 h, 3 patients with prior MTF exposure received oral MTF (7.5 mg/kg). Plasma ChE inhibition peaked to 78.5 +/- 12.3% at 15 min, while maximum RBC ChE inhibition seen at 1 h was 61.0 +/- 11.0%. Plasma ChE inhibition was unchanged at 6 h, whereas RBC ChE recovered with a t1/2 of 7.0 +/- 3.5 h. In Study II, 6 patients and 6 controls with no prior MTF exposure were given oral MTF. Mean plasma t1/2 of MTF was 2.3 +/- 0.3 h with ChE recovery t1/2 of 9.0 +/- 3.3 (plasma) and 26.6 +/- 15.2 days (RBC) after 7.5 mg/kg MTF. The short drug t1/2, long ChE recovery t1/2 and the achievement of high ChE inhibition levels with minimal side effects suggest the potential use of this drug for Alzheimer therapy.

High-performance liquid chromatographic method using ultraviolet detection for measuring metrifonate and dichlorvos levels in human plasma.

We report high-performance liquid chromatographic methods using ultraviolet detection, developed for the first time in our laboratory with sensitivity to detect clinically significant concentrations of metrifonate (MTF), an experimental drug for Alzheimer disease, and its active anticholinesterase metabolite, dichlorvos (DDVP). The determination limit of the method for MTF and DDVP was 1 microgram/ml and 40 ng/ml, respectively. Stability of MTF and DDVP at various temperatures in water, buffered solutions and in human plasma were also studied.

Pharmacokinetics of metrifonate and its rearrangement product dichlorvos in whole blood.

Six male healthy volunteers were given single oral doses of 7.5 mg/kg of metrifonate and concentrations of metrifonate and dichlorvos were determined in whole blood using a standardized sampling procedure. Blood was collected in test tubes containing equal volumes of 0.74 M phosphoric acid for the determinations of metrifonate and dichlorvos with gas chromatography and mass spectrometry at different time points for up to 24 hr. Cholinesterases were also determined in blood haemolyzed with water. Metrifonate was quickly absorbed with a Cmax of 50.5 +/- 18.9 mumol/l which was obtained between 0.17 to 1 hr after drug intake. Mean whole blood t1/2, Clo and AUC were 2.07 +/- 0.24 hr, 0.34 +/- 0.06 l/hr/kg and 89.2 +/- 16 mumol.hr/l respectively. The concentrations of dichlorvos closely followed those of metrifonate with a constant ratio of 0.01 to 0.02. The concentrations of metrifonate were detectable for up to 8 hr but those of dichlorvos had fallen below the level of determination by this time. Both plasma and red blood cell cholinesterases were readily inhibited and were still low after 24 hr. None of the volunteers complained of side effects.

Determination of metrifonate and dichlorvos in whole blood using gas chromatography and gas chromatography-mass spectrometry.

Analytical methods for determining metrifonate and dichlorvos in whole blood and a sampling procedure suitable for pharmacokinetic studies in man are described. Metrifonate concentrations were determined after chloroform extraction using gas chromatography-nitrogen-phosphorus detection. The within-assay coefficients of variation were 4 and 9% at 19.4 and 0.8 mumol/l (limits of determination), respectively. Dichlorvos was determined using gas chromatography-mass spectrometry of toluene extracts. The within-assay coefficients of variation were 2 and 5% at 225 and 50 nmol/l (limits of determination), respectively. Since both substances are chemically unstable, the blood was collected by dripping it directly from the vein into 0.74 M phosphoric acid.

[The forensic medical aspects of the toxicokinetics of organophosphate insecticides]. / Sudebno-meditsinskie aspekty toksikokinetiki fosfororganicheskikh insektitsidov.

The article deals with analysis of toxicokinetics of organophosphorous compounds (malathion and trichlorfon) in blood and of its changes in critical situations (shock, coma). In these forms of viability disturbances provided for mass-transfer and accumulation of poisonous substances in gastrointestinal tract their kinetics was shown to decelerate substantially. Active detoxification therapy improves markedly the parameters of organophosphorous insecticide kinetics. The parameters of toxicokinetics were recommended for use as objective criteria in estimation of intoxication gravity and therapy efficacy.

A rapid and sensitive quantitation of Dipterex in serum by solid-phase extraction and gas chromatography with flame thermionic detection.

A simple, sensitive, and rapid quantitative method for Dipterex in serum is described. A SepPak C18 cartridge for the extraction and gas chromatography with flame thermionic detection for determination are used. The detection limit is 2.5 ng/mL, and linearity is obtained in the range 5-500 ng/mL.

Levels of metrifonate and dichlorvos in plasma and erythrocytes during treatment of schistosomiasis with Bilarcil.

A method for the simultaneous quantitation of metrifonate (0,0-dimethyl-(1-hydroxy-2,2,2-trichloroethyl)-phosphonate) and dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP) in human blood has been worked out. It is based upon multiple labelling of the compounds with deuterium and gas phase analysis using the mass spectrometer as a selective detector. The amount of DDVP in plasma is about 1% of the amount of metrifonate. In erythrocytes the corresponding amount of DDVP is 0.5% or less of metrifonate. Both compounds reach peak levels in blood within two hours after oral dosing and are detectable for at least eight hours. Cholinesterase activity in plasma reaches zero levels within 15 min. and remains inhibited for more than eight hours. Red blood cell cholinesterase is inhibited only 60-80%. According to kinetic calculations, clearance of metrifonate occurs primarily via dichlorvos. If dichlorvos is the only active component, which in all likelihood it is, it's slow release may be important in the schistosomicidal effect. Clinical data in seven metrifonate treated patients revealed that mild vertigo subsiding in a few hours was the most common side effect.

[Metabolism of 32P-butonate and formation of vinylbutonate metabolite in warm-blooded animals]. / Zum Metabolismus von 32P-Butonat und zur Bildung des Metaboliten Vinylbutonat im Warmblüter.

The metabolic fate of butonate, a 32P-labelled organophosphorus insecticide (0.0-dimethyl-l-n-butyryloxy-2.2.2-trichloro-ethyl phosphonate) was investigated in blood serum of cattle in vitro and in mice in vivo, following intraperitoneal and oral administration of 200 mg/kg. Metabolites were separates by thin-layer chromatography, using two solvent systems, acetonitrile-water = 85:15 and ethyl ether, for polar or non-polar metabolites. The Rf-values as well as the partition coefficients for chloroform and water and ethyl ether to ten per cent H2SO4 and for n-hexane and acetonitrile are given for butonate and the metabolites together with the amounts of metabolite, following in vivo metabolic degradation. The blood level as well as residues in milk are given by the following graduation: trichlorphone greater than vinylbutonate greater than butonate greater than dichlorvos. The formation of the new metabolite vinylbutonate is discussed.

Plasma levels of metrifonate and dichlorvos during treatment of schistosomiasis with bilarcil.

Metrifonate (0,0-dimethyl-[1-hydroxy-2,2,2-trichloroethyl]-phosphonate), and its rearrangement product dichlorvos (2,2-dichlorovinyl dimethyl phosphate) (DDVP), were studied in plasma from two patients with schistosomiasis who were treated with Bilarcil. A mass fragmentographic technique was used. Isotopic variants of the substances were used as internal standards and to compensate for DDVP formed during the workup procedure. The results were related to erythrocyte and plasmacholinesterase determinations. The method described makes it possible to study pharmacokinetics in man and to relate this information to therapeutic effects. It is proposed that metrifonate acts as a slow release formulation for DDVP.