Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of sarin and soman-butyrylcholinesterase adducts in human plasma.

Organophosphorus nerve agent (OPNA) adducts formed with human butyrylcholinesterase (HuBuChE) can be used as biomarker of OPNA exposure. Indeed, intoxication by OPNAs can be confirmed by the LC/MS2 analysis of a specific HuBuChE nonapeptide on which OPNAs covalently bind. A fast, selective, and highly sensitive online method was developed to detect sarin and soman adducts in plasma, including immunoextraction by anti-HuBuChE antibodies, pepsin digestion on immobilized enzyme reactors (IMER), and microLC/MS2 analysis of the OPNA adducts. The potential of three different monoclonal antibodies, covalently grafted on sepharose, was compared for the extraction of HuBuChE. The online method developed with the most promising antibodies allowed the extraction of up to 100% of HuBuChE contained in plasma and the digestion of 45% of it in less than 40 min. Moreover, OPNA-HuBuChE adducts, aged OPNA adducts, and unadducted HuBuChE could be detected (with S/N > 2000), even in plasma spiked with a low concentration of OPNA (10 ng mL-1). Finally, the potential of this method was compared to approaches involving other affinity sorbents, already described for HuBuChE extraction. Graphical abstract Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of organophosphorous nerve agent adducts formed with human butyrylcholinesterase.

Simultaneous measurement of tabun, sarin, soman, cyclosarin, VR, VX, and VM adducts to tyrosine in blood products by isotope dilution UHPLC-MS/MS.

This work describes a new specific, sensitive, and rapid stable isotope dilution method for the simultaneous detection of the organophosphorus nerve agents (OPNAs) tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VR, VX, and VM adducts to tyrosine (Tyr). Serum, plasma, and lysed whole blood samples (50 µL) were prepared by protein precipitation followed by digestion with Pronase. Specific Tyr adducts were isolated from the digest by a single solid phase extraction (SPE) step, and the analytes were separated by reversed-phase ultra high performance liquid chromatography (UHPLC) gradient elution in less than 2 min. Detection was performed on a triple quadrupole tandem mass spectrometer using time-triggered selected reaction monitoring (SRM) in positive electrospray ionization (ESI) mode. The calibration range was characterized from 0.100-50.0 ng/mL for GB- and VR-Tyr and 0.250-50.0 ng/mL for GA-, GD-, GF-, and VX/VM-Tyr (R(2) ≥ 0.995). Inter- and intra-assay precision had coefficients of variation of ≤17 and ≤10%, respectively, and the measured concentration accuracies of spiked samples were within 15% of the targeted value for multiple spiking levels. The limit of detection was calculated to be 0.097, 0.027, 0.018, 0.074, 0.023, and 0.083 ng/mL for GA-, GB-, GD-, GF-, VR-, and VX/VM-Tyr, respectively. A convenience set of 96 serum samples with no known nerve agent exposure was screened and revealed no baseline values or potential interferences. This method provides a simple and highly specific diagnostic tool that may extend the time postevent that a confirmation of nerve agent exposure can be made with confidence.

Quantitation of five organophosphorus nerve agent metabolites in serum using hydrophilic interaction liquid chromatography and tandem mass spectrometry.

Although nerve agent use is prohibited, concerns remain for human exposure to nerve agents during decommissioning, research, and warfare. Exposure can be detected through the analysis of hydrolysis products in urine as well as blood. An analytical method to detect exposure to five nerve agents, including VX, VR (Russian VX), GB (sarin), GD (soman), and GF (cyclosarin), through the analysis of the hydrolysis products, which are the primary metabolites, in serum has been developed and characterized. This method uses solid-phase extraction coupled with high-performance liquid chromatography for separation and isotopic dilution tandem mass spectrometry for detection. An uncommon buffer of ammonium fluoride was used to enhance ionization and improve sensitivity when coupled with hydrophilic interaction liquid chromatography resulting in detection limits from 0.3 to 0.5 ng/mL. The assessment of two quality control samples demonstrated high accuracy (101-105%) and high precision (5-8%) for the detection of these five nerve agent hydrolysis products in serum.

Forensic analysis of soman exposure using characteristic fragment ions from protein adducts.

The verification of exposure to nerve agents is a serious challenge, especially in cases of soman (GD) poisoning. Protein adducts are reliable biomarkers, that provide forensic information and evidence during incidents of terrorism or sporadic poisoning. Mass spectrometry, coupled with a proteomics approach, was established for the forensic analysis of GD-based protein adducts. The fragmentation pathways of GD-based protein adducts were investigated for the first time using electrospray ionization tandem mass spectrometry. Three abundant natural loss product ions, [M+2H-54]2+ (loss of two carbon cations), [M+2H-72]2+ (loss of tert-butyl and methyl moieties), and [M+2H-84]2+ (loss of the pinacolyl moieties), were observed in each of the GD-labeled adducts, and the product ions were independent of protein structure and exposure route. A unique mechanism for the formation of product ions involving GD-protein adducts is proposed here. These findings support the development of a simple and precise forensic analysis technique to rapidly verify GD poisoning using these three GD-related product ions.

Biomarkers of low-level exposure to soman vapor: comparison of fluoride regeneration to acetylcholinesterase inhibition.

The nerve agent O-pinacolyl methylphosphonofluoridate, also known as soman or by its military designation GD, is a highly toxic organophosphorous compound that exerts its effects through inhibition of the enzyme acetylcholinesterase (AChE). In the present study, a fluoride ion based regeneration assay was developed to quantify the level of soman present in the blood of rats following a low-level whole-body inhalation exposure. It was hypothesized that the amount of regenerated nerve agent in the blood would be dose dependent in rats subjected to a whole-body inhalation exposure to a low-level dose of soman vapor, and that the fluoride ion-based regeneration method would be more sensitive for the detection of a low-level exposure to soman vapor than the measurement of whole blood AChE activity. Regenerated soman was dose-dependently detected in both the red blood cells (RBCs) and plasma of exposed rats at all concentrations tested (0.033-0.280 mg/m(3) for a 240-min exposure). Significant inhibition of whole blood AChE activity did not occur below a concentration of 0.101 mg/m(3), and was only depressed by approximately 10-25% at concentrations ranging from 0.101 mg/m(3) to 0.280 mg/m(3). This study is the first to utilize a fluoride ion-based regeneration assay to demonstrate the dose-dependent increases in soman in the blood following whole-body inhalation exposure to low levels of vapor. Additionally, the results of the present study demonstrate that the fluoride ion based regeneration assay was approximately threefold more sensitive than the measurement of AChE activity in the blood for the detection of exposure to soman, and also that miosis is a more sensitive marker of soman exposure than inhibition of AChE activity.

Validation and application of a GC-MS method for determining soman concentration in rat plasma following low-level vapor exposure.

A method for determining the chemical warfare agent soman (GD) in rat plasma has been validated and applied to low-level inhalation exposure studies currently being conducted. This method utilizes a fluoride ion-based regeneration assay with isotope dilution followed by large volume injection gas chromatography with ammonia chemical ionization mass spectrometric detection. Following sample preparation by solid phase extraction, chromatographic separation was achieved using a 14% cyanopropylphenyl/86% dimethyl polysiloxane capillary column with a total run time of 18.16 min. Soman and the deuterated isotope ((2)H(4)-soman) internal standard were detected using the selected ion monitoring mode and quantitated using the ammonia adduction ratio of m/z ions 200/204. A reproducible linear relationship was obtained for the quantitative concentration range of 10 pg on-column to 1000 pg on-column (r(2) = 0.9995) for standards in ethyl acetate with a detection limit of 5.65 pg on-column, and an average recovery of 93% in plasma. This sensitive method was successfully applied to the analysis of soman in rat plasma immediately post-exposure, resulting in the construction of dose-response plots.

Modifications to the organophosphorus nerve agent-protein adduct refluoridation method for retrospective analysis of nerve agent exposures.

Organophosphorus nerve agents (OPNAs) continue to pose a threat to military personnel and the general public because of their toxicity and their potential use as weapons of mass destruction. An effective method for the detection of human exposure to OPNAs involves the refluoridation of nerve agents adducted to the serum protein butyrylcholinesterase. The regenerated agents are then enriched by solid-phase extraction and quantified by isotope-dilution gas chromatography-mass spectrometry. We have previously reported improvements that resulted in a 10-fold increase in sensitivity. We have now made further changes to the method that include the addition of confirmation ions, the addition of soman (GD) to the assay, the expansion of the linear range, and the elimination of high-volume injection to decrease background noise and run time while improving sensitivity. This report includes the standard operating procedures for this method for tabun, sarin, soman, cyclohexylsarin, and VX and validation studies. The method's limits of detection ranged from 5.5 to 16.5 pg/mL for the G analogue of VX and GD, respectively. Characterization of quality control (QC) materials resulted in an average coefficient of variation of 15.1% for the five analytes in low QC pools and 11.7% in high QC pools.

Radiolabelled soman binding to sera from Rats, Guinea Pigs and Monkeys.

Soman is a highly toxic organophosphorus chemical warfare compound that binds rapidly and irreversibility to a variety of serine active enzymes, i.e., butyryl- and acetyl-cholinesterases and carboxylesterase. The in vivo toxicity of soman has been reported to vary significantly in different animal species, such as rats and guinea pigs or non-human primates. This species variation makes it difficult to identify appropriate animal models for therapeutic drug development under the US Food and Drug Administration (FDA) Animal Rule. Since species variation in soman toxicity has been correlated with species variation in serum carboxylesterase, we undertook to determine if serum from guinea pigs, rats and non-human primates bound different levels of soman in vitro in the presence of equimolar concentrations of soman. Our results demonstrated that the amount of soman bound in the serum of rats was 4 uM, but essentially null in guinea pigs or non-human primates. The results strongly correlate with the presence or absence of carboxylesterase in the serum of animals and the difference in the toxic dose of soman in various species. Our results support prior suggestions that guinea pigs and non-human primates may be better animal models for the development of antidotes under the FDA Animal Rule.

Investigation of dried blood sampling with liquid chromatography tandem mass spectrometry to confirm human exposure to nerve agents.

A method was developed to detect and quantify organophosphate nerve agent (OPNA) metabolites in dried blood samples. Dried blood spots (DBS) and microsampling devices are alternatives to traditional blood draws, allowing for safe handling, extended stability, reduced shipping costs, and potential self-sampling. DBS and microsamplers were evaluated for precision, accuracy, sensitivity, matrix effects, and extraction recovery following collection of whole blood containing five OPNA metabolites. The metabolites of VX, Sarin (GB), Soman (GD), Cyclosarin (GF), and Russian VX (VR) were quantitated from 5.0 to 500 ng mL-1 with precision of ≤16% and accuracy between 93 and 108% for QC samples with controlled volumes. For unknown spot volumes, OPNA metabolite concentrations were normalized to total blood protein to improve interpretation of nerve agent exposures. This study provides data to support the use of DBS and microsamplers to collect critical exposure samples quickly, safely, and efficiently following large-scale chemical exposure events.

Simultaneous Time-concentration Analysis of Soman and VX Adducts to Butyrylcholinesterase and Albumin by LC-MS-MS.

A sensitive method for the purification and determination of two protein adducts, organophosphorus (OP)-BChE and OP-albumin adducts, in a single sample using a simultaneous sample preparation method was developed and validated using liquid chromatography-tandem mass spectrometry. First, we isolated O-ethyl S-2-diisopropylaminoethyl methyl phosphonothiolate (VX) and O-pinacolyl methylphosphonofluoridate (soman, GD)-BChE adducts using an immunomagnetic separation (IMS) method and the HiTrap™ Blue affinity column was subsequently used to isolate and purify VX and GD-albumin adducts from the plasma of rhesus monkeys exposed to nerve agents. Additionally, we examined the time-concentration profiles of two biomarkers, VX and GD-nonapeptides and VX and GD-tyrosines, derived from OP-BChE and OP-albumin adducts up to 8 weeks after exposure. Based on the results, we determined that VX and GD-tyrosine is more suitable than VX and GD-nonapeptide as a biomarker owing to its longevity. This integrated approach is expected to be applicable for the quantification of other OP-BChE and OP-albumin adducts in human plasma, thus serving as a potential generic assay for exposure to nerve agents.

Distortion product otoacoustic emissions as non-invasive biomarkers and predictors of soman-induced central neurotoxicity: a preliminary study.

The organophosphorus nerve agent soman is an irreversible cholinesterase (ChE) inhibitor that can produce long-lasting seizures and brain damage in which the neurotransmitters acetylcholine and glutamate are involved. These same neurotransmitters play key-roles in the auditory function. It was then assumed that exploring the hearing function may provide markers of the central events triggered by soman intoxication. In the present study, distortion product otoacoustic emissions (DPOAEs), a non-invasive audiometric method, were used to monitor cochlear functionality in rats administered with a moderate dose of soman (45 microg/kg). DPOAEs were investigated either 4h or 24h post-challenge. In parallel, the effects of soman on whole blood and brain ChE activity and on brain histology were also studied. The first main result is that DPOAE intensities were significantly decreased 4h post-soman and returned to baseline at 24h. The amplitude changes were well related to the severity of symptoms, with the greatest change being recorded in the rats that survived long-lasting convulsions. The second main result is that baseline DPOAEs recorded 8 days before soman appear to predict the severity of symptoms produced by the intoxication. Indeed, the lowest baseline DPOAEs corresponded to the occurrence of long-lasting convulsions and brain damage and to the greatest inhibition in central ChE. These results thus suggest that DPOAEs represent a promising non-invasive tool to assess and predict the central consequences of nerve agent poisoning. Further investigations will be carried out to assess the potential applications and the limits of this non-invasive method.

[Studying chronic effects caused by alkaline products of from bituminous-salt masses obtained through destruction of sarin, soman and RVX].

The authors summarized study results on chronic effects caused by products of leaching from bituminous-salt masses obtained through destruction of sarin, soman and RVX. State of experimental rats was evaluated with integral informative tests (physiologic, biochemical, hematologic and morphologic) presenting changes in objective health parameters and revealing every disorder in organs and systems functioning.

Simultaneous quantification of soman and VX adducts to butyrylcholinesterase, their aged methylphosphonic acid adduct and butyrylcholinesterase in plasma using an off-column procainamide-gel separation method combined with UHPLC-MS/MS.

This work describes a novel and sensitive non-isotope dilution method for simultaneous quantification of organophosphorus nerve agents (OPNAs) soman (GD) and VX adducts to butyrylcholinesterase (BChE), their aged methylphosphonic acid (MeP) adduct and unadducted BChE in plasma exposed to OPNA. OPNA-BChE adducts were isolated with an off-column procainamide-gel separation (PGS) from plasma, and then digested with pepsin into specific adducted FGES*AGAAS nonapeptide (NP) biomarkers. The resulting NPs were detected by UHPLC-MS/MS MRM. The off-column PGS method can capture over 90% of BChE, MeP-BChE, VX-BChE and GD-BChE from their respective plasma materials. One newly designed and easily synthesized phosphorylated BChE nonapeptide with one Gly-to-Ala mutation was successfully reported to serve as internal standard instead of traditional isotopically labeled BChE nonapeptide. The linear range of calibration curves were from 1.00-200ngmL-1 for VX-NP, 2.00-200ngmL-1 for GD-NP and MeP-NP (R2≥0.995), and 3.00-200ngmL-1 for BChE NP (R2≥0.990). The inter-day precision had relative standard deviation (%RSD) of <8.89%, and the accuracy ranged between 88.9-120%. The limit of detection was calculated to be 0.411, 0.750, 0.800 and 1.43ngmL-1 for VX-NP, GD-NP, MeP-NP and BChE NP, respectively. OPNA-exposed quality control plasma samples were characterized as part of method validation. Investigation of plasma samples unexposed to OPNA revealed no baseline values or interferences. Using the off-column PGS method combined with UHPLC-MS/MS, VX-NP and GD-NP adducts can be unambiguously detected with high confidence in 0.10ngmL-1 and 0.50ngmL-1 of exposed human plasma respectively, only requiring 0.1mL of plasma sample and taking about four hours without special sample preparation equipment. These improvements make it a simple, sensitive and robust PGS-UHPLC-MS/MS method, and this method will become an attractive alternative to immunomagnetic separation (IMS) method and a useful diagnostic tool for retrospective detection of OPNA exposure with high confidence. Furthermore, using the developed method, the adducted BChE levels from VX and GD-exposed (0.10-100ngmL-1) plasma samples were completely characterized, and the fact that VX being more active and specific to BChE than GD was re-confirmed.

In vitro degradation of the four isomers of soman in human serum.

Starting from racemic soman (1,2,2-trimethylpropyl methylphosphonofluoridate), the degradation of its four stereoisomers in human serum (25 degrees, pH 8.8), has been studied at the nM level. Phosphylation of serum proteins is eliminated by preincubation of the serum with soman. A capillary gas chromatographic method with nitrogen-phosphorous detection allows the separation of the diastereoisomers. The total hydrolysis (enzymatic and non-enzymatic) rate constants of the isomers can then be resolved indirectly on the basis of the important rate difference between P(+) and P(-) isomers. The enzymatic hydrolysis rate constants are obtained by subtracting, for each isomer, the spontaneous (non-enzymatic) rate constant from the total hydrolysis rate constant. The non-enzymatic part of the hydrolysis is obtained from experiments in serum ultrafiltrate (30,000 NMWL). Enzymatic hydrolysis of C(+) P(+) soman occurs so rapidly that only a lower limit of the rate constant can be given. The other enzymatic rate constants are 0.016 min-1 for C(+)P(-), 0.74 min-1 for C(-)P(+) and 0.028 min-1 for C(-)P(-).

Stereoselective hydrolysis of soman in human plasma and serum.

The contribution of various human serum and plasma fractions to the total hydrolysis rate constants of the four isomers of soman is studied. Spontaneous hydrolysis (as measured in buffer) occurs at a faster rate for the C(+)P(+)- and C(-)P(-)-isomers. A stereoselectively catalyzed hydrolysis of soman occurs in serum fractions IV and V (albumin). In fraction V the C(+)P(+)- and C(-)P(-)-isomers are hydrolyzed at a faster rate than their respective epimers, while in fraction IV-1 a stereoselective effect towards C(+)P(+)-soman is found. All the forementioned contributions, however, are negligible in comparison with the stereoselective enzymatic hydrolysis of the P(+)-isomers. The latter reaction is characterized by a significant lowering of the activation energy as compared with the spontaneous hydrolysis of the P(+)-isomers. Such a lowering in activation energy is not found for the hydrolysis of the P(-)-isomers in whole serum or plasma; hence it can be concluded that a phosphorylphosphatase hydrolyzes the P(+)-isomers in a stereoselective way, the P(-)-isomers either not being affected by this (these) enzyme(s) or the mechanism of catalysis being fundamentally different. This conclusion is in agreement with the observations on the influence of Hg2+ on the hydrolysis of soman in serum; the hydrolysis of the P(+)-isomers is significantly inhibited by 1 mM of Hg2+ while the P(-)-hydrolysis is unaffected by this concentration of Hg2+. The action of some potential inhibitors on this phosphorylphosphatase activity was studied. Iodoacetate did not inhibit nor did Ba2+, Sr2+, Co2+ or Mn2+ show a significant effect on the hydrolysis of the P(+)-isomers. On the other hand the hydrolytic activity in serum was nearly completely inhibited by EDTA but restored upon addition of Ca2+. These findings suggest that this enzymatic activity can be classified as an arylesterase (paraoxonase). Finally, the influence of pH on the hydrolytic activity shows a different pattern for C(+)P(+)- and C(-)P(+)-soman, which may suggest that more than one enzyme is involved in the degradation of soman.

Hydrolysis and binding of a toxic stereoisomer of soman in plasma and tissue homogenates from rat, guinea pig and marmoset, and in human plasma.

The fallen concentration of one of the two isomers of soman (1,2,2-trimethylpropyl methylphosphonofluoridate), i.e., C(+)P(-)-soman, was investigated in plasma and in homogenates of brain, lung, liver, kidney, diaphragm, skeletal muscle and mucosa of small intestines from rat, guinea pig and marmoset, and in human plasma (pH 7.5, 37 degrees). The decrease of the isomer concentration was followed by gas chromatographic determination of the residual concentration and proceeded in two phases due to a very rapid saturation of covalent binding sites for the isomer followed by catalysed hydrolysis. Estimates for the concentrations of covalent binding sites were obtained, which were relatively high in liver and kidney. Time periods for the hydrolysis of the isomer from a concentration of 40 ng/mL to 20 ng/mL were evaluated from the second reaction phase. It is concluded that the spontaneous and enzyme-catalyzed hydrolytic activities found for degradation of C(+)P(-)-soman in organs participating in central elimination are sufficiently high to account for the terminal half-life times of the isomer found in our toxicokinetic studies for the blood concentration after intoxication with 2-6 LD50 C(+/-)P(+/-)-soman. The hydrolytic activities are lower in the target organs for toxic action of soman, e.g., diaphragm and brain, especially for guinea pigs and marmosets.

Formation of soman (1,2,2-trimethylpropyl methylphosphonofluoridate) via fluoride-induced reactivation of soman-inhibited aliesterase in rat plasma.

After incubation (37 degrees) of rat blood or plasma with the nerve agent soman, (CH3)3C(CH3)C(H)O(CH3)P(O)F (7.7 microM), for 10 min, only a small amount of this organophosphate (7 or 1%, respectively) is left, as determined enzymatically (acetylcholinesterase) and gas chromatographically. Comparison of the results obtained with both analyses shows that this residual soman consists only of its P(-)-isomers. Incubation (25 degrees) at pH 4.8-6.1 of such soman-treated rat blood or plasma with sodium fluoride (2.5 mM) for 0.5 min leads to (i) a substantial increase of the P(-)-soman concentration, and (ii) a (partial) reactivation of the soman-inhibited aliesterase, proportional to the amount of generated P(-)-soman. These results indicate strongly that added fluoride ions regenerate soman by a reversal of the inhibition reaction. From the relationship between percentage of reactivation and increase of soman concentration the aliesterase concentration in rat plasma is calculated as 2.6 microM. Sodium fluoride has a similar effect in blood taken from rats to which soman was administered intravenously. The increase of the P(-)-soman concentration is higher with higher sodium fluoride concentrations and at lower pH values. In accordance with the absence of aliesterase, addition of sodium fluoride does not induce an increase of the P(-)-soman concentration in soman-treated human plasma.

On-line solid-phase extraction liquid chromatography-continuous flow frit fast atom bombardment mass spectrometric and tandem mass spectrometric determination of hydrolysis products of nerve agents alkyl methylphosphonic acids by p-bromophenacyl derivatization.

For proof of the presence of chemical warfare agents sarin, soman and VX, a rapid, accurate and sensitive method which allows us to determine their hydrolysis products ethyl methylphosphonic acid, isopropyl methylphosphonic acid and pinacolyl methyl phosphonic acid was explored by using continuous flow frit fast atom bombardment (FAB) LC-MS and LC-MS-MS. After derivatization of analytes with p-bromophenacyl bromide, LC-MS-MS analyses for screening were performed by a flow injection method. The three alkyl methylphosphonic acids (AMPAs) were eluted within 5 min, and the detection limits for the three AMPAs ranged from 1 to 5 ng/ml. For confirmation of the screening results, LC-MS-MS analysis with chromatographic separation was conducted by using a narrow bore column. The three AMPAs were all eluted with excellent separation within 25 min, and the detection limits ranged from 1 to 20 ng/ml. Quantitative measurement was performed by LC-MS in selected ion monitoring (SIM) mode with chromatographic separation. Linear calibration curves were obtained for the three AMPAs and the detection limits ranged from 0.5 to 3 ng/ml. The relative standard deviation for peak area ranged from 3.4 to 6.0% at 50 ng/ml for the three AMPAs.

Influence of portal vein and liver artery ligation on the toxicokinetics of soman in rabbits.

The portal vein, liver artery ligation treatment and the portal vein ligation treatment could increase the concentration of P(-) soman in rabbit blood 3.6-19.3 times as compared with soman control group at each time points after soman injection (43.2 microgkg(-1), i.v.). Toxicokinetics parameters showed that portal vein, liver artery ligation treatment and portal vein ligation treatment could reduce the clearance (CL) and distribution volume (V(d)). Meanwhile, they could significantly increase the AUC of soman in rabbits from 2.08+/-0.154 to 18.2+/-2.96 and 22.9+/-3.73 mg s l(-1), respectively. All these data showed that the liver and intestine play a very important role on elimination the free soman in rabbit's blood at high dosing of soman.

Effects of pretreatment with verapamil on the toxicokinetics of soman in rabbits and distribution in mouse brain and diaphragm.

The effects of verapamil on the elimination of soman in rabbit blood and distribution in mouse brain and diaphragm by determining the concentration of P(-)soman using the chirasil capillary gas chromatographic analysis method were studied in order to study the effects of verapamil on the metabolic detoxification of soman. Verapamil (10 mg kg(-1), im, 30 min before soman administration) could significantly reduce the concentration of P(-)soman in rabbit blood at 15, 60, 90, 120, 180 and 240 s after soman injection (43.2 microg kg(-1), iv) as compared to soman-treated control animal respectively. Toxicokinetics parameters showed verapamil could increase clearance rate from 20.8+/-1.51 to 44.3+/-7.0 ml kg(-1)s(-1) and reduce AUC of P(-)soman from 2.08+/-0.151 to 0.996+/-0.172 mg s l(-1). For experiments in mice, verapamil could reduce the concentration P(-)soman in diaphragm from 74.7, 70.5, 88.7 to 41.1, 39.0, 49.3 ng g(-1) at the time of 30, 90, 120 s after intoxication of soman subcutaneously vs. soman control respectively, but it had no influence on the concentration of free P(-)soman in brain. Verapamil accelerated the elimination of P(-)soman in the rabbits blood and reduced the distribution of P(-)soman in the mouse diaphragm.