Enabling the inclusion of non-hydrolysed sulfated long term anabolic steroid metabolites in a screening for doping substances by means of gas chromatography quadrupole time-of-flight mass spectrometry.

The World Anti-Doping Agency (WADA) publishes yearly their prohibited list, and sets a minimum required performance limit for each substance. To comply with these stringent requirements, the anti-doping laboratories have at least two complementary methods for their initial testing procedure (ITP), one using gas chromatography - mass spectrometry (GC-MS) and the other using liquid chromatography-MS (LC-MS). Anabolic androgenic steroids (AAS) have in previous years consistently been listed as the most frequently detected class of compounds. Over the last decade, evidence has emerged where a longer detection time is attained by focusing on sulfated metabolites of AAS instead of the conventional gluco-conjugated metabolites. Despite a decade of research on sulphated AAS using LC-MS, no LC-MS ITP has been developed that combines this class of compounds with the other mandatory targets. Such combination is essential for economical purposes. Recently, it was demonstrated that the direct injection of non-hydrolysed sulfates is compatible with GC-MS. Using this approach and by taking full use of the open screening capabilities of the quadrupole time of flight MS (QTOF-MS), this work describes for the first time a validated ITP that allows the detection of non-hydrolysed sulfated metabolites of AAS while, simultaneously, remaining capable of detecting a vast range of other classes of compounds, as well as the quantification of endogenous steroids, as required for an ITP compliant with the applicable WADA regulations. The method contains 263 compounds from 9 categories, including stimulants, narcotics, anabolic androgenic steroids and beta-blockers. Additionally, the advantages of the new method were illustrated by analysing excretion samples of drostanolone, mesterolone and metenolone. No negative effects were observed for the conventional markers and the detection time for mesterolone and metenolone increased by up to 150% and 144%, respectively compared to conventional markers.

Comparison of Androgen Levels, Endocrine and Metabolic Indices, and Clinical Findings in Women with Polycystic Ovary Syndrome in Uygur and Han Ethnic Groups from Xinjiang Province in China.

BACKGROUND The aim of this study was to compare androgen levels, endocrine and metabolic indices, and clinical findings in women with polycystic ovary syndrome (PCOS) in Uygur and Han ethnic groups from Xinjiang Province, China. MATERIAL AND METHODS Between January 2016 to May 2017 clinical data were collected from Uygur (N=82) and Han (N=100) women diagnosed with PCOS, including age, body mass index (BMI), the Ferriman-Gallwey (mFG) hirsutism score, and waist-to-hip ratio (WHR). Blood samples obtained from all study participants were used to measure androgenic steroid levels, including androgen, androstenedione, dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), and the free androgen index (FAI). Endocrine indices measured included sex-hormone binding globulin (SHBG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), and prolactin (PL). Metabolic indices measured included insulin, glucose, total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), triglyceride (TG), and low-density lipoprotein (LDL). RESULTS The FAI in Uygur women with PCOS (4.89) was significantly increased compared with Han women with PCOS (2.78) (p<0.05); androgen levels were significantly correlated with the FAI, glucose, insulin, TC, HDL, and LDL (p<0.05); androstenedione levels were positively correlated with glucose and insulin levels (p<0.05). In Han women with PCOS, androgen levels were negatively correlated with TG levels and positively correlated with TC levels (p<0.05); the FAI was positively correlated with glucose and insulin levels (p<0.05). CONCLUSIONS There were significant differences in androgen levels, endocrine, and metabolic indices in women with PCOS between the Uygur and Han ethnic groups from Xinjiang Province in China.

Detection and metabolic investigations of a novel designer steroid: 3-chloro-17α-methyl-5α-androstan-17ß-ol.

In 2012, seized capsules containing white powder were analyzed to show the presence of unknown steroid-related compounds. Subsequent gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) investigations identified a mixture of 3α- and 3ß- isomers of the novel compound; 3-chloro-17α-methyl-5α-androstan-17ß-ol. Synthesis of authentic reference materials followed by comparison of NMR, GC-MS and gas chromatography-tandem mass spectrometry (GC-MS/MS) data confirmed the finding of a new 'designer' steroid. Furthermore, in vitro androgen bioassays showed potent activity highlighting the potential for doping using this steroid. Due to the potential toxicity of the halogenated steroid, in vitro metabolic investigations of 3α-chloro-17α-methyl-5α-androstan-17ß-ol using equine and human S9 liver fractions were performed. For equine, GC-MS/MS analysis identified the diagnostic 3α-chloro-17α-methyl-5α-androstane-16α,17ß-diol metabolite. For human, the 17α-methyl-5α-androstane-3α,17ß-diol metabolite was found. Results from these studies were used to verify the ability of GC-MS/MS precursor-ion scanning techniques to support untargeted detection strategies for designer steroids in anti-doping analyses. Copyright © 2015 John Wiley & Sons, Ltd.

Analysis of rocuronium in human whole blood and tissues using liquid chromatography-tandem mass spectrometry.

A rapid, accurate and sensitive liquid chromatography-tandem mass spectrometry method has been developed for the determination of a quaternary nitrogen muscle relaxant, rocuronium, in human blood. The procedure involves protein precipitation with chloroform and trichloroacetic acid, and purification using methanol. The chromatography was performed using a phenyl-hexyl column (150 × 2.0 mm i.d., 3 µm; Phenomenex) with a mobile phase consisting of 5 mM ammonium formate (pH 3.0) and acetonitrile. Multiple reaction monitoring was used for quantification. The assay was linear over a concentration range of 4-500 ng/mL for rocuronium with R(2) ≥ 0.998. The recoveries for this compound ranged from 96.0 to 109.1%. The intra-day and inter-day precision was less than 10.5% and the accuracy ranged from 106.6 to 114.9%. The validated method was applied to quantify the content of rocuronium in blood and a variety of tissues of a victim suspected of overdose. In conclusion, the method was successfully applied for the analysis of rocuronium in biological samples for forensic toxicology.

Use of hydrogen as a carrier gas for the analysis of steroids with anabolic activity by gas chromatography-mass spectrometry.

Due to the impact in the media and the requirements of sensitivity and robustness, the detection of the misuse of forbidden substances in sports is a really challenging area for analytical chemistry, where any study focused on enhancing the performance of the analytical methods will be of great interest. The aim of the present study was to evaluate the usefulness of using hydrogen instead of helium as a carrier gas for the analysis of anabolic steroids by gas chromatography-mass spectrometry with electron ionization. There are several drawbacks related with the use of helium as a carrier gas: it is expensive, is a non-renewable resource, and has limited availability in many parts of the world. In contrast, hydrogen is readily available using a hydrogen generator or high-pressure bottled gas, and allows a faster analysis without loss of efficiency; nevertheless it should not be forgotten that due to its explosiveness hydrogen must be handled with caution. Throughout the study the impact of the change of the carrier gas will be evaluated in terms of: performance of the chromatographic system, saving of time and money, impact on the high vacuum in the analyzer, changes in the fragmentation behaviour of the analytes, and finally consequences for the limits of detection achieved with the method.

Feasibility of microdialysis to determine interstitial rocuronium concentration in the muscle tissue of anesthetized neurosurgical patients.

BACKGROUND: Microdialysis is used to determine the concentrations of substances in the extracellular fluid of tissues. To date, it has not been used to measure rocuronium concentrations in human muscle. We determined the ability of microdialysis to recover rocuronium from muscle interstitial tissue for the purpose of assessing the effect of chronic phenytoin therapy on muscle concentrations of rocuronium. METHODS: In a first phase, an in vitro study was performed to establish the ability of the assay to recover rocuronium. In a second phase, 17 patients undergoing brain surgery were enrolled. Eight patients were on chronic phenytoin therapy and the remaining 9 patients were not taking any antiepileptic agent (controls). Rocuronium was administered intravenously and muscle tissue samples for microdialysis were collected. RESULTS: The recovery rate of the in vitro assay was 36% at a pump rate of 1 microL/min. Rocuronium muscle tissue concentrations could be measured in 25 microdialysate samples. Rocuronium concentrations were similar in patients treated with phenytoin and in controls, although the doses required to obtain a similar effect were significantly higher in patients on chronic phenytoin treatment. CONCLUSIONS: Quantification of drug concentrations in muscle by means of microdialysis is technically feasible in the clinical setting and it might help in studying pharmacologic mechanisms of drug action. Based on our results the decrease in the degree of effect of rocuronium in the presence of chronic phenytoin therapy might seem to be due mainly to a pharmacokinetic mechanism.

Transformation of some 3alpha-substituted steroids by Aspergillus tamarii KITA reveals stereochemical restriction of steroid binding orientation in the minor hydroxylation pathway.

Aspergillus tamarii contains an endogenous lactonization pathway which can transform progesterone to testololactone in high yield through a sequential four step enzymatic pathway. In this pathway testosterone is formed which primarily undergoes oxidation of the C-17beta-alcohol to a C-17 ketone but, can also enter a minor hydroxylation pathway where 11beta-hydroxytestosterone is produced. It was recently demonstrated that this hydroxylase could monohydroxylate 3beta-hydroxy substituted saturated steroidal lactones in all four possible binding orientations (normal, reverse, inverted normal, inverted reverse) on rings B and C of the steroid nucleus. It was therefore of interest to determine the fate of a series of 3alpha-substituted steroidal analogues to determine stereochemical effect on transformation. Hydroxylation on the central rings was found to be restricted to the 11beta-position (normal binding), indicating that the 3alpha-stereochemistry removes freedom of binding orientation within the hydroxylase. The only other hydroxylation observed was at the 1beta-position. Interestingly the presence of this functional group did not prevent lactonization of the C-17 ketone. In contrast the presence of the 11beta-hydroxyl completely inhibited Baeyer-Villiger oxidation, a result which again demonstrates that single functional groups can exert significant control over metabolic handling of steroids in this organism. This may also explain why lactonization of 11beta-hydroxytestosterone does not occur. Lactonization of the C-17 ketone was not significantly affected by the 3alpha-alcohol with significant yields achieved (53%). Interestingly a time course experiment demonstrated that the presence of the 3alpha-acetate inhibited the Baeyer-Villiger monooxygenase with its activity being observed 24h later than non-acetate containing analogues. Apart from oxidative transformations observed a minor reductive pathway was revealed with the C-17 ketone being reduced to a C-17beta-alcohol for the first time in this organism.

Analysis of non-ketoic steroids 17alpha-methylepithiostanol and desoxymethyl- testosterone in dietary supplements.

Dietary supplements containing 17alpha-methyl-2,3-epithio-5alpha-androstane-17beta-ol (17alpha-methylepithiostanol), which is a 17-methylated analogue of epithiostanol or a prodrug of desoxymethyltestosterone (17alpha-methyl-5alpha-androst-2-en-17beta-ol), have recently appeared on the Internet. 17alpha-Methylepithiostanol and desoxymethyltestosterone are classified as prohibited substances on the World Anti-Doping Agency (WADA) list. Two preparations, EPISTANE and P-PLEX, were obtained from the Internet so that their contents could be investigated. This study involved gas chromatography/mass spectrometry (GC/MS) analysis after trimethylsilyl (TMS) derivatization, liquid chromatography/mass spectrometry (LC/MS) in atmospheric pressure photoionization (APPI) mode and nuclear magnetic resonance (NMR) spectroscopy. Analysis using LC/MS in APPI mode would be a useful tool for detecting heat-labile and non-polar steroids.Although the labelling of EPISTANE indicates that it contains 17alpha-methyl-2alpha, 3alpha-epithio-5alpha-androstane-17beta-ol only, 17alpha-methyl-2beta,3beta-epithio-5alpha-androstane-17beta-ol and desoxymethyltestosterone were identified in the supplement. The results showed that P-PLEX contained desoxymethyltestosterone and its isomer 17alpha-methyl-5alpha-androst-3-en-17beta-ol. Urine samples can be screened after EPISTANE or P-PLEX administration using the normal screening procedure for anabolic steroids with GC/MS.

Studies on neurosteroids XX. Liquid chromatography-tandem mass spectrometric method for simultaneous determination of testosterone and 5alpha-dihydrotestosterone in rat brain and serum.

Testosterone (T) and 5alpha-dihydrotestosterone (DHT) are now referred to not only as androgenic steroid hormones, but also as neuroactive steroids, because they elicit anesthetic and anxiolytic effects. Methods using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) coupled with derivatization are developed and validated to examine rat brain and serum levels of T and DHT and their stress-induced changes. The steroids are extracted with methanol-acetic acid from the brain tissue or serum, purified using solid-phase extraction cartridges, derivatized with a permanently charged reagent, 2-hydrazino-1-methylpyridine, and subjected to LC-MS-MS. [19,19,19-(2)H3]-T is used as the internal standard. The intra- and inter-assay coefficients of variation are below 10.0%, and the analytical recoveries are 98.1-103.0%. The developed methods are applied to the animal study and it was found that a fair amount of DHT is continuously and locally synthesized in the brain, and its level is not changed by the immobilization stress and depends on the brain T level.

Simultaneous determination of rocuronium and its eight impurities in pharmaceutical preparation using high-performance liquid chromatography with amperometric detection.

A sensitive and selective HPLC method with amperometric detection (HPLC-ED) for the determination of rocuronium bromide and its eight impurities has been developed. The analysis was performed on Hypersil 100 Silica column 5 microm (250 mm x 4.6 mm; Thermo Electron). The mobile phase consisting of 4.53 g l(-1) solution of tetramethylammonium hydroxide adjusted to pH 7.4 with 85% phosphoric acid:acetonitrile (1:9), was found the best for the separation and determination of the studied compounds. The chromatograms were recorded over 10 min using the amperometric detection at a potential +0.9 V of the glassy carbon electrode versus the reference electrode Ag/AgCl. The limit of quantitation was 45 ng ml(-1) for rocuronium and from 25 to 750 ng ml(-1) for the examined impurities. The proposed HPLC-ED method was successfully applied to the analysis of rocuronium and its impurities in Esmeron solution for injection.

Determination of interstitial rocuronium concentrations in the muscle tissue of anesthetized dogs by microdialysis.

INTRODUCTION: The objective was to establish and validate a microdialysis technique for the quantification of interstitial concentrations of the neuromuscular blocker, rocuronium, in the muscle tissue of dogs under steady-state conditions. METHODS: The standard and combined retrodialysis approaches were used for in vivo microdialysis probe calibration. After induction of anesthesia with pentobarbital (30 mg/kg), the left femoral vein was cannulated and blood drawn for protein binding determination. Microdialysis probes were inserted in the muscle and calibrated in vivo, using vecuronium as the calibrator. Each dog received a short 2-min infusion followed by a 120-min infusion of rocuronium via the right jugular vein and three microdialysis samples were collected at steady-state during a 2-h period. Samples were stored at -70 degrees C until HPLC analysis. RESULTS: Using combined retrodialysis, rocuronium unbound interstitial (C(ISFu)) and venous plasma (C(pssuv)) concentrations are in good agreement; with a ratio C(ISFu)/C(pssuv) of 100+/-11%. Using standard retrodialysis, this ratio was 47+/-7%. CONCLUSIONS: Combined retrodialysis is a more reliable and accurate technique for quantitative assessment of rocuronium interstitial concentrations especially for lengthy anesthetic procedures. These findings have potential implications, as drug concentrations in the site of action would be more relevant for concentration-effect relation of muscle relaxants.

Microbial transformation of hydrocortisone by Acremonium strictum PTCC 5282.

The ability of a genus of cephalosporium-like fungus isolated from soil, Acremonium strictum PTCC 5282, for hydrocortisone biotransformation has been investigated. This potential had not been previously examined. The fermentation yielded 11beta,17beta-dihydroxyandrost-4-en-3-one, 11beta,17alpha,20beta,21-tetrahydroxypregn-4-en-3-one and 21-acetoxy-11beta,17alpha,20-trihydroxypregn-4-en-3-one. Each microbial metabolite was purified and characterized using spectroscopic methods.

The detection and identification of quaternary nitrogen muscle relaxants in biological fluids and tissues by ion-trap LC-ESI-MS.

Quaternary nitrogen muscle relaxants pancuronium, rocuronium, vecuronium, gallamine, suxamethonium, mivacurium, and atracurium and its metabolites were extracted from whole blood and other biological fluids and tissues by using a solid-phase extraction procedure. The extracts were examined by using high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). The drugs were separated on a ODS column in a gradient of ammonium acetate buffer (pH 5.0) and acetonitrile. Full-scan mass spectra of the compounds showed molecular ions, and MS-MS spectra showed fragments typical of the particular compounds. LC-ESI-MS allowed an unequivocal differentiation of all muscle relaxants involved. The method was applied in a case of rocuronium and suxamethonium administration in a Caesarian section and in a case of intoxication by pancuronium injection. In both cases, the administered drugs could be detected and identified in the supplied samples.

Bioquímica del líquido quístico mamario, III: niveles del factor de crecimiento epidérmico, gonadotrofina coriónica y andrógenos conjugados / Biochemistry of breast cyst fluids, III: levels of NA, K, androgens, epidermal growth factor and human chorionic gonadotropin

En la última década se ha implementado una serie de análisis bioquímicos que permiten identificar varios tipos de líquidos quísticos (LQs). En el presente trabajo se confirma la presencia de polipéptidos y esteroides conjugados -como el factor de crecimiento epidérmico (FCE), el sulfato de dehidroepiandrosterona (S-DHEA) y el androstano-3Ó, 17ß-diol glucurónido (3Ó-Adiol G)- a veces en concentraciones muy elevadas con respecto a los niveles encontrados simultáneamente en el plasma circulante. Como contraste, la concentración del cortisol apenas alcanza a un 20 por ciento del normalmente hallado en el plasma. Se demuestra además que la concentración intraquística del 3Ó-Adiol G se correlaciona positiva y significativamente con la del S-DHEA (r = 0,8744, p < 0,0001) y con el FCE (r = 0,8949, p < 0,0001), con amplia variabilidad en los resultados. Se establece también una correlación negativa entre el 3Ó-Adiol G y el cociente Na/K (r = - 0,6592, p = 0,0001). Por último, se determinan los niveles de la gonadotrofina coriónica (hCG), utilizando un sistema automatizado de quimioluminiscencia, demostrándose que esta glicoproteína se encuentra en cantidades determinables (> 1,1 mUI/ml) en el 73,8 por ciento de los LQs analizados. En el 57,4 por ciento los niveles superan a los encontrados normalmente en el plasma que oscilan entre < 1,1 mUl/ml y 5,5 mUl/ml. En un 4,9 por ciento las concentraciones resultan significativamente elevadas, alcanzando hasta las 1.000 mUl/ml. Se demuestra una correlación negativa con alta significación estadística entre los valores normalizados de la hCG con los niveles del S-DHEA, del 3Ó-Adiol G y del FCE y una correlación positiva con el cociente NA/K. Se discute la posibilidad de que el FCE, los esteroides conjugados y la hCG puedan ser sintetizados de novo en el tejido epitelial que recubre las paredes del quiste

Bioquímica del líquido quístico mamario, III: niveles del factor de crecimiento epidérmico, gonadotrofina coriónica y andrógenos conjugados / Biochemistry of breast cyst fluids, III: levels of NA, K, androgens, epidermal growth factor and human chorionic gonadotropin

En la última década se ha implementado una serie de análisis bioquímicos que permiten identificar varios tipos de líquidos quísticos (LQs). En el presente trabajo se confirma la presencia de polipéptidos y esteroides conjugados -como el factor de crecimiento epidérmico (FCE), el sulfato de dehidroepiandrosterona (S-DHEA) y el androstano-3O, 17ß-diol glucurónido (3O-Adiol G)- a veces en concentraciones muy elevadas con respecto a los niveles encontrados simultáneamente en el plasma circulante. Como contraste, la concentración del cortisol apenas alcanza a un 20 por ciento del normalmente hallado en el plasma. Se demuestra además que la concentración intraquística del 3O-Adiol G se correlaciona positiva y significativamente con la del S-DHEA (r = 0,8744, p < 0,0001) y con el FCE (r = 0,8949, p < 0,0001), con amplia variabilidad en los resultados. Se establece también una correlación negativa entre el 3O-Adiol G y el cociente Na/K (r = - 0,6592, p = 0,0001). Por último, se determinan los niveles de la gonadotrofina coriónica (hCG), utilizando un sistema automatizado de quimioluminiscencia, demostrándose que esta glicoproteína se encuentra en cantidades determinables (> 1,1 mUI/ml) en el 73,8 por ciento de los LQs analizados. En el 57,4 por ciento los niveles superan a los encontrados normalmente en el plasma que oscilan entre < 1,1 mUl/ml y 5,5 mUl/ml. En un 4,9 por ciento las concentraciones resultan significativamente elevadas, alcanzando hasta las 1.000 mUl/ml. Se demuestra una correlación negativa con alta significación estadística entre los valores normalizados de la hCG con los niveles del S-DHEA, del 3O-Adiol G y del FCE y una correlación positiva con el cociente NA/K. Se discute la posibilidad de que el FCE, los esteroides conjugados y la hCG puedan ser sintetizados de novo en el tejido epitelial que recubre las paredes del quiste (AU)

Determination of rocuronium and its putative metabolites in body fluids and tissue homogenates.

A sensitive and selective HPLC method was developed for the quantification of the neuromuscular blocking agent rocuronium and its putative metabolites (the 17-desacetyl derivative and the N-desallyl derivative of rocuronium) in plasma, urine, bile, tissue homogenates and stoma fluid. Samples were prepared by extraction of the biological matrix with dichloromethane, after mixing with a KI-glycine buffer. After evaporation of the organic solvent the samples were chromatographed on a reversed-phase HPLC column, using an aqueous buffer-dioxane (84:16, v/v) as the mobile phase. The aqueous buffer consisting of 0.1 M sodium dihydrogen phosphate, 0.11 mM 9,10-dimethoxyanthracene-2-sulphonate (DAS), 0.11 mM 1-heptane-sulfonic acid, was adjusted to pH 3 with orthophosphoric acid. After separation, the eluent was extracted with dichloroethane, and the organic phase was led to a fluorimetric detector, operating at 385 nm (excitation) and 452 nm (emission). The method was validated for the assay in plasma, urine, bile, tissue homogenates and stoma fluid, by determination of the repeatability, reproducibility, accuracy, lower limit of quantification, lower limit of detection, extraction recovery, effect of sample volume, and stability in the biological matrix. The method was found to be sensitive (lower limit of quantification for rocuronium in plasma is 10 ng/ml) and accurate. The interference of concomitant drugs with the assay of rocuronium and its putative metabolites has been studied extensively. In order to confirm the identity of rocuronium and its putative metabolites, a TLC method was developed. The method has been applied successfully in several pharmacokinetic studies with rocuronium.

Effect of oily vehicles on absorption of mepitiostane by the lymphatic system in rats.

[14C]Mepitiostane in various vehicles was administered to the small intestine of anaesthetized rats with cannulated thoracic ducts, and the effect of lipids on lymphatic absorption was examined. The extent of lymphatic absorption was greatest when administered in triolein and sesame oil, which are triglycerides of long-chain fatty acids. Absorption in the presence of other vehicles was in the order of 10% Tween 80 aqueous solution greater than monolein greater than oleic acid approximately oleic acid/monolein (2:1 mol/mol) greater than aqueous suspension. Differences between the extents of lymphatic absorption of mepitiostane in the various formulations were not due to variation in the lymph flow but to the increased secretion of chylomicron and very low density lipoproteins. During absorption of mepitiostane from the small intestine, oil affected not only the penetration into epithelium cells and the metabolism in them, but also the partition between blood and lymph.

Determination of Arduan and its desacetyl metabolites in biological fluids.

The authors elaborated a method for the determination of Arduan and its desacetyl metabolites in biological fluids. The method is based on the use of labelled Arduan and on the determination of radioactivity of the parent drug and metabolites separated by ion-pair TLC in the development system of chloroform-dichloromethane-methanol 6 : 2 : 2 (v/v) 3% NaI (w/v).

Quantitation of pancuronium, 3-desacetylpancuronium, vecuronium, 3-desacetylvecuronium, pipecuronium and 3-desacetylpipecuronium in biological fluids by capillary gas chromatography using nitrogen-sensitive detection.

A sensitive and specific capillary gas chromatographic (GC) assay was developed for the quantitation of the quaternary ammonium steroidal neuromuscular blocking drugs pancuronium (PANC), vecuronium (VEC) and pipecuronium (PIP), as well as the metabolites 3-desacetylpancuronium (3-desPANC) and 3-desacetylvecuronium (3-des VEC) in plasma, bile and urine; the putative metabolite 3-desacetylpipecuronium (3-des PIP) was extracted and quantitated only in urine. The procedure employed a single dichloromethane extraction of the iodide ion-pairs of the monoquaternary or bisquaternary ammonium compounds (including internal and external standards) from acidified, ether-washed biological fluid followed by the formation of stable O-tert.-butyldimethylsilyl derivatives at the 3-hydroxy steroidal position of the metabolites. An automated capillary GC system fitted with a nitrogen-sensitive detector and an integrator was then used to analyze and quantitate both parent compounds and their derivatized metabolites. Optimal extraction, derivatization and GC conditions, as well as short-term stability and recoveries of these drugs and metabolites in plasma, are reported. Electron ionization mass spectrometry combined with GC was used to confirm the identities of compounds eluted from the column. The assay demonstrated a 10(3)-fold linear range up to 5000 ng/ml for PANC, VEC, 3-des VEC and PIP, and lower limits of detection with adequate precision of 2 ng/ml for PANC, VEC and PIP, and 4 ng/ml for 3-des VEC; 3-des PANC was linear from 8 to 500 ng/ml while 3-des PIP was linear from 25 to 1000 ng/ml. The precision (coefficient of variation) of the calibration curves for underivatized drugs and their derivatized metabolites over the linear ranges was 2-20% and the reproducibility of the assay over a range of clinical concentrations of these drugs found in human plasma was 5-16% for PANC, 2-4% for VEC and 6-11% for PIP. No interferences were detected in the assay of plasma samples from 106 surgical patients.