Determination of ethylene oxide-hemoglobin adduct by silylation and gas chromatography-electron impact-mass spectrometry

A gas chromatography-electron impact ionization-mass spectrometric (GC-EI-MS) assay was developed for the determination of ethylene oxide-hemoglobin adduct (N-2-hydroxyethylvaline, HEVal). HEVal and deuterated HEVal (d(4)-HEVal) were synthesized for identification and quality control. Globin samples were separated from red blood cells (RBCs) by acidic isopropanol and extracted with ethyl acetate. HEVal adduct in globin was transformed to HEVal-pentafluorophenylthiohydantoin derivative by modified Edman-degradation method, which was extracted from globin with diethylether. d(4)-HEVal was used as an internal reference standard. The dried extract was derivatized with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBDMSTFA)-NH(4)I (1000:4, w/w) containing 0.4 mg of dithioerthritol. The TBDMS derivative of HEVal had very good chromatographic property and offered sensitive response of GC-EI-MS. The recovery of HEVal was about 81.6% and the coefficient of variation was 5.0% at the concentration of 311 pmol/g. Low limit of detection (LOD) of the assay was 1.8 pmol/g in 0.1g hemoglobin. The experiments have demonstrated to detect background level of HEVal adduct in human blood. HEVal adduct in globin was detected between 12 and 6573 pmol/g.     

Development and validation of a gas chromatography/ion trap-mass spectrometry method for simultaneous quantification of cocaine and its metabolites benzoylecgonine and norcocaine: application to the study of cocaine metabolism in human primary cultured renal cells

Acute renal failure is a common finding in cocaine abusers. While cocaine metabolism may contribute to its nephrotoxic mechanisms, its pharmacokinetics in kidney cells is hitherto to be clarified. Primary cultures of human proximal tubular cells (HPTCs) provide a well-characterized in vitro model, phenotypically representative of HPTCs in vivo. Thus, the present work describes the first sensitive gas chromatography/ion trap-mass spectrometry (GC/IT-MS) method for measurement of cocaine and its metabolites benzoylecgonine (BE) and norcocaine (NCOC) using a primary culture of HPTCs as cellular matrix, following solid phase extraction (SPE) and derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). The application of this methodology also enables the identification of two other cocaine metabolites: ecgonine methyl ester (EME) and anhydroecgonine methyl ester (AEME). The validation of the method was performed through the evaluation of selectivity, linearity, precision and accuracy, limit of detection (LOD), and limit of quantification (LOQ). Its applicability was demonstrated through the quantification of cocaine, BE and NCOC in primary cultured HPTCs after incubation, at physiological conditions, with 1 mM cocaine for 72 h. The developed GC/IT-MS method was found to be linear (r2 > 0.99). The intra-day precision varied between 3.6% and 13.5% and the values of accuracy between 92.7% and 111.9%. The LOD values for cocaine, BE and NCOC were 0.97±0.09, 0.40±0.04 and 20.89±1.81 ng/mL, respectively, and 3.24±0.30, 1.34±0.14 and 69.62±6.05 ng/mL as LOQ values.     

Selection of the derivatization reagent--the case of human blood cholesterol, its precursors and phytosterols GC-MS analyses

Phytosterols (PS; β-sitosterol and campesterol) and cholesterol precursors (CP; desmosterol and lathosterol) have been suggested as important biochemical markers of cholesterol intestinal absorption and liver biosynthesis, respectively. Given that these compounds appear in human blood in low amounts, sensitive and accurate methodology is required, such as gas chromatography-mass spectrometry (GC-MS) the most frequently used. One of the most critical factors of the GC analytical determination is the step of derivatization. Thus, the main objective of the present study was compare and select the better (one out of three) silylation mixtures as follows: N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide/ammonium iodide (MTBSTFA:NH(4)I), N-O-bis-(trimethylsilyl) trifluoroacetamide/trimethylchlorosilane (BSTFA:TMCS), and N-methyl-N-(trimethylsilyl)-trifluoroacetamide/1,4-dithioerythritol/trimethyliodosilane (MSTFA:DTE:TMIS). The results of this study are discussed and accompanied by a brief review on the importance and principles of derivatization process, specifically in silylation reactions in GC-MS sterols analyses. Furthermore, a scrutiny of some published results is presented, along with additional information about mass spectral data of these potentially useful compounds. Interestingly, the results of the study showed that from the three validated methodologies, the selected one, based on the best relation specificity/sensibility, is MSTFA:DTE:TMIS. With this silylation procedure for simultaneous determination of PS and CP in human serum by GC-MS in selected ion monitoring (SIM) mode, good linearity (r(2)≥0.931), precision (repeatability ranging from 0.92 to 3.91 CV and intermediate precision ranging from 5.12 to 6.33) and recoveries (≥93.6%) were obtained. Thus, it proved to be a helpful methodology in the quantification of predominant serum cholesterol origin in each patient.     

Highly sensitive determination of estrone and estradiol in human serum by liquid chromatography-electrospray ionization tandem mass spectrometry

A highly sensitive and specific quantification method of estrone and estradiol in human serum was described based upon the use of picolinoyl derivatization and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) in a positive mode. Estrogens were treated with picolinoyl chloride hydrochloride or picolinic acid and 2-methyl-6-nitrobenzoic anhydride followed by a solid-phase extraction with ODS cartridge. Picolinoyl derivatization proceeded quantitatively even in a microscale, and the picolinoyl esters provided simple positive ESI-mass spectra showing [M+H](+) as base peaks for these estrogens. The picolinoyl derivatives of these estrogens showed 100-fold higher detection response compared to underivatized intact molecules by LC-ESI-MS (selected reaction monitoring). Using this derivatization, estrogens spiked in the charcoal treated human serum samples were analyzed with limit of quantification (LOQ), intra-day accuracy and precision of 1.0pg/ml, 96.0% and 9.9% for estrone, and 0.5pg/ml, 84.4% and 12.8% for estradiol, respectively. Estrone and estradiol added to the crude serum samples were recovered with comparable LOQ and accuracy obtained for the charcoal treated serum samples as well.     

Gas chromatography-mass spectrometry method for determination of phenylalanine and tyrosine in neonatal blood spots

In this paper we developed a simple, rapid and sensitive method for the quantitative analysis of phenylalanine (Phe) and tyrosine (Tyr) in dried blood spots of newborns by gas chromatography-mass spectrometry (GC-MS). Phe and Tyr in blood samples were reacted with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide at 120 degrees C for 30 min and their corresponding single derivatives were obtained. Phe and Tyr were determined by measurement of their derivatives by GC-MS in the selected ion monitoring mode. Contents of Phe and Tyr in blood spots were calculated by external standard method. The ratio of Phe to Tyr was used for neonatal screening for phenylketonuria. The present method only took a few minutes to perform and required minimal sample preparation. In addition it provided low detection limits of 1.2 micromol l(-1) for Phe and 1.6 micromol l(-1)for Tyr.     

Capillary gas chromatographic determination of proteins and biological amino acids as N(O)-tert.-butyldimethylsilyl derivatives

Forty-seven biological amino acids containing all 22 protein amino acids were derivatized to N(O)-tert.-Butyldimethylsilyl (tBDMSi) derivatives by a single-step reaction with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide and successfully separated on an HP-1 capillary column. The relative standard deviations of the relative molar responses of most amino acids were <5%. Cystine seems to be partially converted into cysteine during derivatization. An increase in carrier gas flow-rate towards the end of the analysis by inlet pressure programming with electron pressure control avoided the peak broadening and adsorption of the derivatives with high boiling points on the column and especially increased sensitivity of cystine to 5 pmol. Glutamine was converted almost completely into pyroglutamic acid during prolonged storage of a standard solution prepared in 0.01 M HCl but not during derivatization. These results compared with those for the phenylthiocarbamyl derivatives analysed by HPLC and the analytical results reported in the literature on soybean hydrolysate showed good agreement except for cysteine. The results for the amino acid composition of bovine serum albumin also showed good agreement with results in the literature except for cysteine. In human urine, seventeen free amino acids were detected as tBDMSi derivatives.     

Analytical derivatization-a tool for determination of orotic acid

Derivatization of orotic acid (OA) into various forms (trimethylsilylderivate, alkyl ester and per-methylated derivate) and their evaluation by GC/MS is described. The tested approach includes ion-exchange SPE clean-up, evaporation and chemical reaction with different types of derivatization agents (N,O-bis-(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane, butanol with acetylchloride and ethereal solution of diazomethane). Derivate originated in the reaction with diazomethane was used for determination of urinary orotic acid by GC/MS. Detection limit of 0.28 micromol l(-1) was reached using the ion 82 m/z in single ion monitoring (SIM) mode. Linearity of the method was tested within the range of 3.4-2503.4 micromol l(-1) covering physiological and pathological levels of orotic acid in urine sample. Recoveries were within the range 93.7-110.6%. Application of the method on the patient with defect of ornithine transcarbamylase (OTC) was demonstrated as well.     

Femtomole detection of amino acids and dipeptides by gas chromatography—negative-ion chemical ionization mass spectrometry following alkylation with pentafluorobenzyl bromide

Amino acids and di- and tripeptides were derivatized by extractive alkylation using pentafluorobenzyl bromide (PFBBr) followed by reaction with heptafluorobutyric anhydride (HFBA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). Good chromatographic separation and the formation of intense diagnostic ions were observed for the derivative when examined using gas chromatography—negative-ion chemical ionization mass spectrometry (GC—NICI-MS). Of the 20 amino acids investigated, only Arg and Glu could not be detected by this method. Also, dipeptides which included neutral amino acid residues were derivatized with more success than those containing either acidic or basic residues. Each of the amino acids or dipeptides formed one major derivative with the exception of Asn which formed two derivatives with either one or two HFB groups. This derivatization method was optimized with respect to the reaction temperature, reaction time, and choice of derivatizing reagents. Recoveries of derivatized [³H]-labeled Phe, Lys, and Thr were 76, 55, and 34%, respectively. Linearity was observed from 10 to 2000 pg of Ala per vial; selected-ion monitoring provided a detection limit of less than 150 fg with a signal-to-noise (S/N) ratio of 80 to 1. This method has proven to work well with urine samples and shows great promise for the detection of small peptides at low levels.     

Stable isotope dilution analysis of salicylic acid and hydroquinone in human skin samples by gas chromatography with mass spectrometric detection

A sensitive and accurate gas chromatographic-mass spectrometric (GC-MS) method has been developed for the quantitative determination of salicylic acid (SA) and hydroquinone (HQ) from human skin samples and cosmetic emulsions. Deuterium labeled SA-d(6) and HQ-d(6) were used as internal standards (IS). The samples were extracted with methanol, dried under nitrogen and derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)+1% trimethylchlorosilane (TMCS). Quantification was performed in SIM mode with a limit of quantification (LOQ) of 50 ng ml(-1) for SA and 10 ng ml(-1) for HQ. The inter-day variation (R.S.D.) was less than 5% and the accuracy was better than 13.3% for both compounds. The recoveries from the different matrices ranged between 93.1 and 103.3% for SA, and 97.3 and 100.8% for HQ.     

Screening for forensically relevant benzodiazepines in human hair by gas chromatography-negative ion chemical ionization-mass spectrometry

A procedure is presented for the detection in human hair of forensically relevant benzodiazepines, i.e. nordiazepam, oxazepem, bromazepam, diazepam, lorazepam, flunitrazepam, alprazolam and triazolam. The method involves decontamination of hair with methylene chloride, pulverization in a ball mill, incubation of 50 mg powdered hair in Soerensen buffer (pH 7.6) in the presence of prazepam-d₅ used as internal standard, liquid-liquid extraction with diethyl ether-chloroform (80:20, v/v) and gas chromatography-mass spectrometry using negative chemical ionization after derivatization with, N,O-bis(trimethylsilyl)trifluoroacetamide plus 1% trimethylchlorosilane. The limits of detection for all benzodiazepines ranged from 1 to 20 pg/mg using a 50-mg hair sample. Coefficients of variation and extraction recoveries, ranging from 7.4 to 25.4% and 47.6 to 90%, respectively, were found suitable for a screening procedure. One hundred and fifteen samples were submitted to this screening procedure, and specimens tested positive for nordiazepam (0.20-18.87 ng/mg, n = 42) and its major metabolite oxazepam (0.10-0.50 ng/mg, n = 14), flunitrazepam (19–148 pg/mg, n = 31), lorazepam (31–49 pg/mg, n = 4) and alprazolam (0.3-1.24 ng/mg, n = 2). Bromazepam, diazepam and triazolam were not detected.     

Rapid preparation of human urine and plasma samples for analysis of F2-isoprostanes by gas chromatography-mass spectrometry

Reliable MS-based methods have been developed for the measurement of free and esterified F2-isoprostanes. However, prior to sample analysis several steps of purification, including solid-phase extraction followed by TLC or HPLC, are usually required, making it tedious to analyze large sample numbers, e.g., for population studies. We report a quick sample purification method using anion exchange solid phase extraction (SPE), which is highly selective for acidic compounds. Urine and hydrolyzed plasma of healthy individuals were acidified before SPE extraction, washed with 4 different solvent mixtures and finally eluted with ethyl acetate. The eluted samples were first derivatized with pentafluorobenzyl bromide followed by a second derivatization with bis-(trimethylsilyl)trifluoroacetamide. F2-isoprostanes were analyzed by GC-MS-NCI. The method was highly sensitive; the limit of detection at 5:1 signal-to-noise ratio was 0.037 ng/ml and 0.007 ng/mg creatinine for plasma and urine, respectively. Anion exchange SPE extraction for F2-isoprostane showed recovery of 55-65% and high linearity for concentration 0-1.0 ng/ml for urine (CV=4.08%, r2=0.990) and 0-0.5 ng/ml for plasma (CV=4.07%, r2=0.998). Fasting for 6h significantly increased plasma F2-isoprostanes levels, which has implications for the design of intervention studies using this biomarker.     

Detection of cholesteryl ester hydroperoxide isomers using gas chromatography-mass spectrometry combined with thin-layer chromatography blotting

Oxidative modification of low-density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. During the oxidation of LDL, cholesteryl esters, the major lipid components in LDL, are oxidized to cholesteryl ester hydroperoxides (CEOOH). The isomers of CEOOH may reflect the reactive species that initiate the peroxidation reaction. In the current study, a novel analytical method for the determination of CEOOH isomers, especially cholesteryl linoleate hydroperoxide isomers, was developed using the combination of two chromatographic techniques: (i) thin-layer chromatography blotting with diphenyl-1-pyrenylphosphine (DPPP) fluorescent detection (DPPP-TLC blotting) and (ii) gas chromatography-electron ionization-mass spectrometry (GC-EI-MS). CEOOH was applied to DPPP-TLC blotting, the obtained DPPP-derived fluorescent spots containing cholesteryl ester hydroxides were extracted and derivatized (hydrogenation, transmethylation, and trimethylsilylation), and the formed methyl ester/trimethylsilylether derivatives of hydroxyoctadecenoic acid were then analyzed by GC-EI-MS. The CEOOH isomers were determined by selected ion monitoring of isomer-specific fragment ions originated from the alpha-cleavage of the trimethylsilyloxyl group. Using these two chromatographic techniques, we were able to detect isomeric CEOOH in the oxidized human LDL. Our results indicated that GC-EI-MS analysis combined with DPPP-TLC blot is a specific method for analyzing cholesteryl ester hydroperoxide isomers in biological samples such as oxidized LDL.     

Identification and determination of the enantiomers of moprolol and their metabolites in human urine by high-performance liquid chromatography and gas chromatography—mass spectrometry

A simple and sensitive high-performance liquid chromatographic (HPLC) method using chiral derivatization was developed to screen and determine the enantiomers of moprolol and their metabolites in human urine. The recovery of (+)- and (−)-moprolol from urine was 70.8–81.1% at different concentrations. The coefficients of variation (C.V.) were less than 3.2 and 6.5% for intra- and inter-assays, respectively. Moprolol could be detected in urine up to 24 h after oral administration of a 50-mg dose of moprolol. Unconjugated and conjugated enantiomers of moprolol and their metabolites were analyzed by gas chromatography (GC). A gas chromatographic—mass spectrometric (GC—MS) confirmatory method was established to identify the metabolites of moprolol. The double derivatization procedure for moprolol and their metabolites with S-(−)-menthyl chloroformate [(−)-MCF] and N-methyl(trimethylsilyl)trifluoroacetamide (MSTFA) gave very good GC—MS properties of the derivatized compounds and provided reliable structural information for their confirmation analysis. This is the first published report on the use of a GC—MS method for the detection of the enantiomers of moprolol and their metabolites in human urine.     

Discriminative determination of alkyl methylphosphonates and methylphosphonate in blood plasma and urine by gas chromatography-mass spectrometry after tert.-butyldimethylsilylation

A method for determining two nerve gas hydrolysis products, alkyl (ethyl, isopropyl and pinacolyl) methylphosphonates (RMPAs) and methylphosphonate (MPA), separately, in human plasma and urine samples was developed, using two different deproteinization procedures. In the first method, the plasma sample was deproteinized by adding a fourfold volume of acetonitrile, followed by passing the supernatant through a Bond Elut strong anion-exchange (SAX) cartridge [fluoride (F(-)) form]. After washing the cartridge with water and methanol, the RMPAs were eluted with a 3% (v/v) solution of methanolic ammonia, and analyzed by gas chromatography-mass spectrometry (GC-MS) after tert.-butyldimethylsilyl (TBDMS) derivatization. The detection yields of TBDMS derivatives of RMPAs were in the range of 69 to 99%, in contrast to the poor yields obtained when only acetonitrile deproteinization pretreatment was used (yield: 13-26%). The yield of the TBDMS derivative of MPA was very low (8%), however. In a the second method, a plasma sample was deproteinized by adding a half volume of 10% (w/v) trichloroacetic acid (TCA), and the resulting supernatant was extracted with diethyl ether to remove TCA, the aqueous fraction was then passed through a Bond Elut SAX cartridge. After washing the cartridge with 0.5% (v/v) methanolic ammonia, MPA was eluted with 3% (v/v) methanolic ammonia. The detection yield of the TBDMS derivative of MPA was nearly quantitative. A pretreatment method using SAX solid-phase extraction was also developed for the cleanup of a urine sample, in which the sample was directly applied to a Bond Elut SAX cartridge, followed by elution of the RMPAs and MPA with 3% (v/v) methanolic ammonia, which were then derivatized and analyzed by GC-MS. The detection yields of TBDMS derivatives of RMPAs and MPA were in the range of 61 to 97%.     

Changes in the sugarcane metabolome with stem development. Are they related to sucrose accumulation?

Sucrose content increases with internode development down the stem of sugarcane. In an attempt to determine which other changes in metabolites may be linked to sucrose accumulation gas chromatography-mass spectrometry was used to obtain metabolic profiles from methanol/water extracts of four samples of different age down the stem of cultivar Q117. Extracts were derivatized with either N-methyl-N-(trimethylsilyl) trifluoracetamide (TMS) or N-methyl N-(tert-butyldimethylsilyl) trifluoroacetamide (TBS) separately in order to increase the number of metabolites that could be detected. This resulted in the measurement of 121 and 71 metabolites from the TMS and TBS derivatization, respectively. Fifty-five metabolites were identified using commercial and publicly available libraries. Statistical analysis of the metabolite profiles resulted in clustering of tissue types. Particular metabolites were correlated with the level of sucrose accumulation, which as expected increased down the stem. Metabolites, such as tricarboxylic acid cycle intermediates and amino acids, were more abundant in the M2 sample (meristem to internode 2) that was actively growing and decreased in an apparently coordinated developmentally programmed manner in more mature internodes down the stem. However, other metabolites such as trehalose and raffinose showed positive correlations with sucrose concentration. Here we discuss the technique used to measure metabolites in sugarcane and the changes in metabolite abundance down the sugarcane stem.     

Determination of lincomycin residues in salmon tissues by gas chromatography with nitrogen-phosphorus detection

A sensitive method for the determination of lincomycin residues in fish tissues is described. Lincomycin was extracted from fish tissues with phosphate buffer (pH 4.5). The extract was concentrated with a C₁₈ solid-phase extraction cartridge and further cleaned up by solvent extraction. Lincomycin was derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide to form a trimethylsilyl derivative before being analyzed by gas chromatography with nitrogen-phosphorus detection. Coumaphos was used as the internal standard. Assays showed good linearity in the range 25–250 ppb (ng/g) (r = 0.9994). Recoveries of fortified lincomycin at 50, 100 and 200 ppb were>80% with relative standard deviation <6%. The limit of detection of the method was 1.7 ppb and the limit of quantitation was 3.8 ppb.     

Preparation of completely 6-O-desulfated heparin and its ability to enhance activity of basic fibroblast growth factor

Although regioselective removal of 6-O-sulfate groups of heparin has been undertaken by several researchers, complete 6-O-desulfation with little side reaction has not been attained successfully. In this work, a modified method with a certain silylating reagent, N-methyl-N-(trimethylsilyl)trifluoroacetamide, has been established to produce completely 6-O-desulfated heparin with few other chemical changes. The degrees of 6-O-desulfation were estimated by means of chemical disaccharide analyses and/or (13)C NMR spectra. Although the completely 6-O-desulfated heparin lost about 20% of 2-O-sulfate groups, any other chemical changes and depolymerization were not detected. The completely 6-O-desulfated heparin displayed strong inhibition of COS-1 cell adhesion to basic fibroblast growth factor (bFGF)-coated well in a dose-dependent manner, as was clarified by the competitive cell-adhesion assay. Furthermore, the completely 6-O-desulfated heparin was shown to promote in vitro A31 fibroblast proliferation in a dose-dependent manner in the presence of bFGF. These results suggest that signal transduction through bFGF/bFGF receptor in A31 cells occurs in the absence of 6-O-sulfate groups in heparin. The involvement of 6-O-sulfate group(s) of heparin/heparan sulfate in the promotion of bFGF mitogenic activity was reported by several groups. This discrepancy between our results and those of other groups would be due to the differences in molecular size of heparin/heparan sulfate derivatives and/or cell species used for the assay.     

Development and validation of a new gas flame ionization detector method for the determination of finasteride in tablets

A simple, rapid, and sensitive method based on gas chromatography with flame ionization detection is described for the determination of finasteride in tablets. The method is based on the derivatization of finasteride with N,O-bis(trimethylsilyl)trifluoroacetamide-1% trimethylchlorosilane at 60 degrees C for 30 min. The method was validated for specificity, linearity, precision, accuracy, robustness, and limit of quantification. The degree of linearity of the calibration curves, the percentage recoveries of finasteride, and the limit of detection (LOD) and limit of quantification (LOQ) for the gas chromatographic method were determined. The assay was linear over the concentration range of 10 to 50 microg ml(-1) (R approximately 0.999). LOQ and LOD (signal/noise ratio = 10) were found to be 10 and 2 microg ml(-1), respectively. The method was found to be simple, specific, precise, accurate, and reproducible. All of the validation parameters were within the acceptance range. The developed method was applied successfully to estimate the amount of finasteride in tablets. The results were compared statistically with those obtained by the official method using t and F tests. There was no significant difference between the two methods with respect to mean values and standard deviations at the 95% confidence level.     

Development and validation of a gas chromatography-mass spectrometry method for the simultaneous determination of buprenorphine, flunitrazepam and their metabolites in rat plasma: application to the pharmacokinetic study

Buprenorphine (BUP), a synthetic opioid analgesic, is frequently abused alone, and in association with benzodiazepines. Fatalities involving buprenorphine alone seem very unusual while its association with benzodiazepines, such as flunitrazepam (FNZ), has been reported to result in severe respiratory depression and death. The quantitative relationship between these drugs remain, however, uncertain. Our objective was to develop an analytical method that could be used as a means to study and explore, in animals, the toxicity and pharmacological interaction mechanisms between buprenorphine, flunitrazepam and their active metabolites. A procedure based on gas chromatography-mass spectrometry (GC-MS) is described for the simultaneous analysis of buprenorphine, norbuprenorphine (NBUP), flunitrazepam, N-desmethylflunitrazepam (N-DMFNZ) and 7-aminoflunitrazepam (7-AFNZ) in rat plasma. The method was set up and adapted for the analysis of small plasma samples taken from rats. Plasma samples were extracted by liquid-liquid extraction using Toxi-tubes A. Extracted compounds were derivatized with N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), using trimethylchlorosilane (TMCS) as a catalyst. They were then separated by GC on a crosslinked 5% phenyl-methylpolysiloxane analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode. Excellent linearity was found between 0.125 and 25 ng/microl plasma for BUP, 0.125 and 12.5 ng/microl for NBUP and N-DMFNZ, 0.125 and 5 ng/microl for FNZ, and between 0.025 and 50 ng/microl for 7-AFNZ. The limit of quantification was 0.025 ng/microl plasma for 7-AFNZ and 0.125 ng/microl for the four other compounds. A good reproducibility (intra-assay CV=0.32-11.69%; inter-assay CV=0.63-9.55%) and accuracy (intra-assay error=2.58-12.73%; inter-assay error=0.83-11.07%) were attained. Recoveries were 71, 67 and 81%, for BUP, FNZ and N-DMFNZ, respectively, and 51% for NBUP and 7-AFNZ, with CV ranging from 5.4 to 13.9%, and were concentration-independent. The GC-MS method was successfully applied to the pharmacokinetic study of BUP, NBUP, FNZ, DMFNZ and 7-AFNZ in rats, after administration of BUP and FNZ.     

Quantitative determination of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, in human plasma, using isotope dilution mass spectrometry

An original method is described for the determination in human plasma of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, by isotope dilution mass-spectrometry using 7,7-[2H2]-4-OHA as internal standard. This compound was synthesized starting from 7,7-[2H2]-4-androstene-3,17-dione. The procedure includes an extraction step using an Extrelut 1 column and a derivatization with N,o-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The minimum detection level of the method is 0.650 pg and the coefficients of variation for the 0.5 ng/ml (plasma) and 5 ng/ml (plasma) concentrations are 3.2% (within assay) and 6.7% (between assay) and 1.86% (within assay) and 2.3% (between assay) respectively.