Determination of 6-(2'-chlorophenyl)-4-hydroxy-4H-imidazo[1,5-a] [1,4]benzodiazepine-3-carboxamide, a mixed agonist-antagonist anxiolytic agent, in human plasma and urine by gas chromatography-negative-ion chemical-ionization mass spectrometry

A simple, sensitive and specific assay was developed for the determination in plasma and urine of 6-(2'-chlorophenyl)-4-hydroxy-4H-imidazo[1,5-alpha] [1,4]benzodiazepine- 3-carboxamide, compound I, a mixed agonist-antagonist anxiolytic agent. A hexadeuterated analogue of the compound was added to plasma or urine as the reference standard. The titled compound was extracted with benzene at pH 11. Following evaporation of the solvent, the residue was reacted with pentafluoropropionic anhydride in the presence of triethylamine. The derivatizing reagents were evaporated, and the carbonitrile derivative of the analyte was extracted into ethyl acetate at pH 11. The residue remaining after removal of the ethyl acetate was silylated with bis(trimethylsilyl)trifluoroacetamide, and a portion of this solution was analyzed by gas chromatography-negative-ion chemical-ionization mass spectrometry. The mass spectrometer was set to monitor, in the gas chromatographic effluent, the M-. ion of the titled compound and its hexadeuterated reference standard. The ratio of these two ions was calculated and converted to a concentration of analyte using a calibration curve that was generated from the analyses of control plasma fortified with various amounts of analyte and a fixed amount of the hexadeuterated reference standard. The limit of quantitation of the assay was 1 ng/ml for plasma and urine.     

Determination of trace levels of pyrethroid metabolites in human urine by capillary gas chromatography-high resolution mass spectrometry with negative chemical ionization

Summary Applications of high-resolution gas chromatography and high-resolution mass spectrometry (GC-MS) for identification and quantitation of trace amounts of pyrethroid metabolites in human urine samples are demonstrated. The method covers the pyrethroid metabolitescis- andtrans-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropane carboxylic acid (cis- andtrans-DCCA),cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DBCA), 4-fluoro-3-phenoxybenzoic acid (FPBA), and 3-phenoxybenzoic acid (3-PBA). After acid-induced hydrolysis of urine samples and exhaustive solvent extraction, a carbodiimide-coupled esterification of the free carboxylic acids with hexafluoroisopropanol (HFIP) is applied. Identification of the derivatives formed is achieved by low-resolution electron-impact mass spectrometry (EIMS) using an ion-trap detector. Quantitation was by capillary gas chromatography—high-resolution mass spectrometry using negative chemical ionization (GC-NCIMS). 2-Phenoxybenzoic acid (2-PBA) served as internal standard. The limits of detection forcis- andtrans-DCCA,cis-DBCA, FPBA and 3-PBA were 0.03 μg L⁻¹ or below. The applicability of the presented method was tested on urine samples of persons exposed to low levels of pyrethroids.     

Gas chromatographic-electron-impact mass fragmentometric determination of lysergic acid diethylamide in urine

A sensitive method for the detection and quantitation of lysergic acid diethylamide (LSD) in urine was developed. After initial solvent extraction, the compound was further purified by liquid-liquid extraction or by solid-phase extraction. The trimethylsilyl derivative of LSD was detected by gas chromatography-mass spectrometry (GC-MS) operated in the electron-impact mode with selected-ion monitoring. The presence of LSD was confirmed by comparing retention times and relative abundances of ions of unknowns with that of a standard. The recovery of this procedure was greater than 89%. The intra-run and inter-run coefficients of variation were less than 5% and less than 7%, respectively. This procedure allows detection of LSD concentrations as low as 29 pg/ml. Quantitation of LSD was linear over the concentration range 50-2000 pg/ml.     

In vivo biotransformation of 17 alpha-methyltestosterone in the horse revisited: identification of 17-hydroxymethyl metabolites in equine urine by capillary gas chromatography/mass spectrometry

The in vivo phase I biotransformation of 17 alpha-methyltestosterone in the horse leads to the formation of a complex mixture of regio- and stereoisomeric C(20)O(2), C(20)O(3) and C(20)O(4) metabolites, excreted in urine as glucuronide and sulphate phase II conjugates. The major pathways of in vivo metabolism are the reduction of the A-ring (di- and tetrahydro), epimerisation at C-17 and oxidations mainly at C-6 and C-16. Some phase I metabolites have been identified previously by positive ion electron ionisation capillary gas chromatography/mass spectrometry (GC/EI + MS) mainly from the characteristic fragmentation patterns of their methyloxime-trimethylsilyl ether (MO-TMS), enol-TMS or TMS ether derivatives. Following oral administration of 17 alpha-methyltestosterone to two castrated thoroughbred male horses, the glucuronic acid conjugates excreted in post-administration urine samples were selectively hydrolysed by E. coli beta-glucuronidase enzymes. Unconjugated metabolites and the steroid aglycones obtained after enzymatic deconjugation were isolated from urine by solid-phase extraction, derivatised as MO-TMS ethers and analysed by GC/EI + MS. In addition to some of the known metabolites previously identified from the characteristic mass spectral fragmentation patterns of 17 alpha-methyl steroids, some isobaric compounds exhibiting a diagnostic loss of 103 mass units from the molecular ions with subsequent losses of trimethylsilanol or methoxy groups and an absence of the classical D-ring fragment ion were detected. From an interpretation of their mass spectra, these compounds were identified as 17-hydroxymethyl metabolites, formed in vivo in the horse by oxidation of the 17-methyl moiety of 17 alpha-methyltestosterone. This study reports on the GC/EI + MS identification of these novel 17-hydroxymethyl C(20)O(3) and C(20)O(4) metabolites of 17 alpha-methyltestosterone excreted in thoroughbred horse urine.     

Early identification of isovaleric aciduria using photodiode array detection for liquid chromatographic profiling of urinary carboxylic acids

The 190 nm high performance liquid chromatographic photodiode array profiling of the urinary carboxylic acids of the first urine of a newborn affected with isovaleric aciduria afforded an abnormal peak at 27.8 min. This peak was greatly increased in the carboxylic acid profiling of the 14 h urine sample from the same infant. Isolation of this peak by fraction collecting; solvent extraction of the eluent; trimethylsilyl derivatization of the residue and gas chromatographic/mass spectrometric analysis identified the compound as isovalerylglycine. Correlation of the 190 nm absorbance of isovalerylglycine (y) with concentration (x) afforded a least squares curve: y = 476.4x-13.72 (r = 0.99); run-to-run variation 6.92%; day-to-day variation 8.88% with a minimum detectable concentration of 25 micrograms/ml.     

New designer drug 2,5-dimethoxy-4-ethylthio-beta-phenethylamine (2C-T-2): studies on its metabolism and toxicological detection in rat urine using gas chromatography/mass spectrometry

Studies are described on the metabolism and the toxicological analysis of the phenethylamine-derived designer drug 2,5-dimethoxy-4-ethylthio-beta-phenethylamine (2C-T-2) in rat urine using gas chromatography/mass spectrometry (GC/MS) after enzymatic cleavage of conjugates, liquid-liquid extraction and derivatization. The structures of 14 metabolites were assigned tentatively by detailed interpretation of their mass spectra. Identification of these metabolites indicated that 2C-T-2 was metabolized by sulfoxidation followed by N-acetylation and either hydroxylation of the S-ethyl side chain or demethylation of one methoxy group, O-demethylation of the parent compound followed by N-acetylation and sulfoxidation, deamination followed by reduction to the corresponding alcohol followed by partial glucuronidation and/or sulfation or by oxidation to the corresponding acid followed either by partial glucuronidation or by degradation to the corresponding benzoic acid derivative followed by partial glucuronidation. Furthermore, 2C-T-2 was metabolized by N-acetylation of the parent compound followed either by O-demethylation and sulfoxidation or by S-dealkylation, S-methylation and sulfoxidation. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction microwave-assisted acetylation allowed the detection of an intake of a dose of 2C-T-2 in rat urine, which corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-T-2 in human urine.     

Sensitive liquid chromatographic/tandem mass spectrometric method for the determination of beclomethasone dipropionate and its metabolites in equine plasma and urine

Beclomethasone dipropionate (BDP) is a potent pro-drug to beclomethasone (BOH) and is used in the treatment of chronic and acute respiratory disorders in the horse. The therapeutic dose of BDP (325 microg per horse) by inhalation results in very low plasma and urinary concentrations of BDP and its metabolites that pose a challenge to detection and confirmation by equine forensic laboratories. To solve this problem, a method involving the use of a liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed for the detection, confirmation and quantification of the analytes in equine samples. Ammonium formate or acetate buffer added to LC mobile phase favored the formation of [M + H](+) ions from BDP and its metabolites, whereas formic acid led to the formation of sodium and potassium adduct ions ([M + Na](+), [M + K](+)) together with [M + H](+) ions. Acetonitrile, on the other hand, favored the formation of abundant solvent adduct ions [M + H + CH(3)CN](+) with the analytes under electrospray ionization (ESI) and atmospheric pressure chemical ionization conditions. In contrast, methanol formed much less solvent adduct ions than acetonitrile. The solvent adduct ions were thermally stable and could not be completely desolvated under the experimental conditions, but they were very fragile to collision-induced dissociation (CID). Interestingly, these solvent adduct ions were observed on a triple-quadrupole mass spectrometry but not on an ion trap instrument where helium used as a damping gas in the ion trap might cause the solvent adduct ions desolvated by collision. By CID studies on the [M + H](+) ions of BDP and its metabolites, their fragmentation paths were proposed. In equine plasma at ambient temperature over 2 h, BDP and B21P were hydrolyzed in part to B17P and BOH, respectively, but B17P was not hydrolyzed. Sodium fluoride added to equine plasma inhibited the hydrolysis of BDP and B21P. The matrix effect in ESI was evaluated in equine plasma and urine samples. The method involved the extraction of BDP and its metabolites from equine plasma and urine samples by methyl tert-butyl ether, resolution on a C(8) column with a mobile phase gradient consisting of methanol and ammonium formate (2 mmol l(-1), pH 3.4) and multiple reaction monitoring for the analytes on a triple-quadrupole mass spectrometer. The detection limit was 13 pg ml(-1) for BDP and B17P, 25 pg ml(-1) for BOH and 50 pg ml(-1) for B21P in plasma and 25 pg ml(-1) for BOH in urine. The method was successfully applied to the analysis of equine plasma and urine samples for the analytes following administration of BDP to horses by inhalation. B17P, the major and active metabolite of BDP, was detected and quantified in equine plasma up to 4 h post-administration by inhalation of a very low therapeutic dose (325 microg per horse) of BDP.     

Characterization and quantification of fluoxymesterone metabolite in horse urine by gas chromatography/mass spectrometry

Fluoxymesterone, an anabolic steroid with the 17alpha-methyl,17beta-hydroxy group, has been developed as an oral formulation for therapeutic purposes. However, it is also used illegally in racehorses to enhance racing performance. In this study, we detected 9alpha-fluoro-17,17-dimethyl-18-norandrostane-4,13-dien-11beta-ol-3-one by gas chromatography/mass spectrometry (GC/MS), which has not been reported as a fluoxymesterone metabolite so far in horse. It was synthesized for use as a reference standard, and characterized on the basis of (1)H NMR and (13)C NMR spectra, as well as GC/MS EI mass spectra of TMS derivatives. It was excreted as the main metabolite in horse urine, and its reference standard could be synthesized easily. Therefore, this metabolite could be a useful target for a doping test of fluoxymesterone in racehorses.     

Analysis of cypermethrin residues and its main degradation products in soil and formulation samples by gas chromatography-electron impact-mass spectrometry in the selective ion monitoring mode

A fast, simple and inexpensive sample preparation method based on the matrix solid-phase extraction (SPE) technique is proposed for the isolation of cypermethrin and its metabolite residues from soils. Both the extraction and clean-up procedures were carried out in two steps and target compounds were determined by gas chromatography coupled with electron-impact mass spectrometry (GC-EI/MS). The characteristic ions and fragmentation mechanism of cypermethrin were evaluated by electron impact ionization mass spectrometry (EI/MS). After the optimization of different parameters, such as the extraction solvent, the pesticide was extracted from the matrix with methanol/acetone in a Soxhlet extractor, cleaned up on a Florisil column by elution with a mixture of 30% ethyl acetate in n-hexane and analyzed by gas chromatography-electron impact ionization mass spectrometry (GC-EI/MS) in the selected ion-monitoring mode (SIM) with permethrin as internal standard. Recovery was in the range 77–118% with relative standard deviations (RSD) between 2.5% and 10.2%. The limit of detection (LOD) was 6.5?µg/kg for cypermethrin. The developed method was linear in the injection range 6–30?ng, with correlation coefficients greater than 0.9957.     

Metabolic studies with phenobarbitone, primidone and their N-alkyl derivatives: quantification of substrates and metabolites using chemical ionization gas chromatography-mass spectrometry

Metabolic studies with phenobarbitone, primidone and some of their N-alkyl derivatives required the concurrent assay of any mixture of these substrates (twelve compounds) and their major metabolites (an additional twenty-two compounds) in urine. The method described in the present report met this requirement by incorporating two complementary derivatization techniques into a gas chromatographic-mass spectrometric (GC-MS) assay procedure. Following hydrolysis of conjugates with beta-glucuronidase, urine samples were extracted with ethyl acetate (3 X 5 ml). The combined extracts were dried over sodium sulphate, divided into two equal portions, and the solvent was removed. One residue was derivatized by propylation using 1-iodopropane with base catalysis. The other residue was silylated using methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide. The derivatives in each case were analysed by GC-MS, using temperature-programmed packed-column GC and chemical ionization MS. Mass spectra were acquired over an appropriate mass range, and peak areas for the compounds of interest were determined from specific mass chromatograms. Satisfactory precision, accuracy, specificity and sensitivity were obtained for all analytes. All compounds produced satisfactory derivatives by at least one procedure; twelve compounds could be analysed by both techniques. The method illustrates the utility of chemical ionization GC-MS for the simultaneous quantitative analysis of multiple related analytes in complex biological samples.     

Detection of Anti-Ulcer Drugs and Their Metabolites in Horse Urine by Liquid Chromatography – Mass Spectrometry

This paper describes a convenient method for the extraction and detection of eight anti-ulcer drugs simultaneously in horse urine, a relatively complex and viscous matrix, using a single-step liquid-liquid extraction followed by high-performance liquid chromatography – mass spectrometry (LC-MS). Anti-ulcer drugs were isolated from horse urine by salting out and liquid-liquid extraction. Detection of these drugs at concentrations below 1 ng mL^?1 could be achieved using LC-MS-MS in the positive atmospheric pressure chemical ionisation (APCI) mode. The above analysis was also extended to elimination studies of horses administered with ranitidine and omeprazole. The urinary elimination data of these two drugs suggest that ranitidine was eliminated from horse urine at a much slower rate than omeprazole. In addition, three ranitidine metabolites and six omeprazole metabolites were identified for the first time in equine urine samples.     

Determination on the microscale of plasmatic lactic acid as its t-butyldimethylsilyl derivative by stable-isotope dilution using capillary gas chromatography/mass spectrometry.

A new sensitive and precise method for the determination of lactic acid in plasmatic microsamples (50 microL) has been developed. Lactic acid was directly extracted from plasma by ethyl acetate in acidic conditions, and analysed as its di-t-butyldimethylsilyl derivative by capillary gas chromatography/electron-impact mass spectrometry (GC/MS). The internal standard was a previously synthesized deuterated compound, 3-[2H]-(2R)-lactic acid. The method gives good reproductibility and precision, the overall standard deviation being better than 3%. The GC/MS assay was in good agreement with the enzymatic determination.     

Evaluation of dispersive liquid-liquid microextraction for the simultaneous determination of chlorophenols and haloanisoles in wines and cork stoppers using gas chromatography-mass spectrometry

Dispersive liquid-liquid microextraction (DLLME) coupled with gas chromatography-mass spectrometry (GC-MS) was evaluated for the simultaneous determination of five chlorophenols and seven haloanisoles in wines and cork stoppers. Parameters, such as the nature and volume of the extracting and disperser solvents, extraction time, salt addition, centrifugation time and sample volume or mass, affecting the DLLME were carefully optimized to extract and preconcentrate chlorophenols, in the form of their acetylated derivatives, and haloanisoles. In this extraction method, 1mL of acetone (disperser solvent) containing 30μL of carbon tetrachloride (extraction solvent) was rapidly injected by a syringe into 5mL of sample solution containing 200μL of acetic anhydride (derivatizing reagent) and 0.5mL of phosphate buffer solution, thereby forming a cloudy solution. After extraction, phase separation was performed by centrifugation, and a volume of 4μL of the sedimented phase was analyzed by GC-MS. The wine samples were directly used for the DLLME extraction (red wines required a 1:1 dilution with water). For cork samples, the target analytes were first extracted with pentane, the solvent was evaporated and the residue reconstituted with acetone before DLLME. The use of an internal standard (2,4-dibromoanisole) notably improved the repeatability of the procedure. Under the optimized conditions, detection limits ranged from 0.004 to 0.108ngmL(-1) in wine samples (24-220pgg(-1) in corks), depending on the compound and the sample analyzed. The enrichment factors for haloanisoles were in the 380-700-fold range.     

Determination of Salmeterol in Equine Urine and Serum

Salmeterol is a β₂-adrenergic agonist and an Association of Racing Commissioners International (ARCI) class 3 drug. Trade names of its xinafoate salt are Arial (Dompé), Salmetedur (Menarini), and Serevent (Glaxo). Salmeterol is routinely used to increase ease of breathing in race horses during their training. Due to its bronchodilating and central nervous system stimulant properties, its administration to a horse just prior to race time has the potential to affect the horse’s performance, therefore a reliable method of analysis for this compound is necessary. This paper describes a method for the identification and quantitation of salmeterol in equine urine using liquid-liquid extraction followed by liquid chromatography and tandem mass spectrometry (LC-MS-MS). Urine salmeterol concentrations peaked at about 2 h post-dose following administration of 500 ug both intravenously and intratracheally at concentrations of 14 ng mL^?1 and 4 ng mL^?1, respectively. Serum concentrations at 30 min were below the minimum level of quantitation.     

Positive- and negative-ion chemical ionization gas chromatography/mass spectrometry of polynuclear aromatic hydrocarbons

Four groups of isomeric polynuclear aromatic hydrocarbons (PAH) were examined by gas chromatography/mass spectrometry (GC/MS) using positive-ion chemical ionization and negative-ion chemical ionization with a variety of reagent gases in order to evaluate the utility of each; differentiation of isomers was the ultimate goal. Hydrogen positive-ion chemical ionization (PICI) yielded different spectra for all but one isomer pair while retaining sensitivity comparable to electron-impact mass spectrometry. Several experimental conditions in the negative-ion mode afforded distinctly different spectra for isomeric PAH, but often sensitivities were reduced. The thirteen model compounds divided approximately into three classes according to the types and extent of reactions of the molecular anion. Class 1 gave as good sensitivity as hydrogen PICI; class 2 gave isomer-dependent spectra, but reduced sensitivity; class 3 gave no isomer differentiation, but greatly enhanced sensitivity.     

Determination of 7-aminoflunitrazepam in urine by dispersive liquid-liquid microextraction with liquid chromatography-electrospray-tandem mass spectrometry

Dispersive liquid-liquid microextraction (DLLME) and liquid chromatography-electrospray-tandem mass spectrometry (LC-ES-MS/MS) procedure was presented for the extraction and determination of 7-aminoflunitrazepam (7-aminoFM2), a biomarker of the hypnotic flunitrazepam (FM2) in urine sample. The method was based on the formation of tiny droplets of an organic extractant in the sample solution using water-immiscible organic solvent [dichloromethane (DCM), an extractant] dissolved in water-miscible organic dispersive solvent [isopropyl alcohol (IPA)]. First, 7-aminoFM2 from basified urine sample was extracted into the dispersed DCM droplets. The extracting organic phase was separated by centrifuging and the sedimented phase was transferred into a 300 μl vial insert and evaporated to dryness. The residue was reconstituted in 30 μl mobile phase (20:80, acetonitrile:water). An aliquot of 20 μl as injected into LC-ES-MS/MS. Various parameters affecting the extraction efficiency (type and volume of extraction and dispersive solvent, effect of alkali and salt) were evaluated. Under optimum conditions, precision, linearity (correlation coefficient, r² = 0.988 over the concentration range of 0.05-2.5 ng/ml), detection limit (0.025 ng/ml) and enrichment factor (20) had been obtained. To our knowledge, DLLME was applied to urine sample for the first time.     

Determination of urinary succinylacetone by capillary gas chromatography

The average analytical recovery of succinylacetone added to urine and separated by capillary gas chromatography was 69% for solvent extraction and 72% for anion exchange separation. Treating succinylacetone with hydroxylamine hydrochloride at a pH of less than 5 caused formation of a derivative separated by capillary gas chromatography into two isomers: 3-methyl-5- isoxazole propionate and 5-methyl-3- isoxazole propionate as their trimethylsilyl derivatives (molecular weight 227). In a pH greater than or equal to 5, succinylacetone dioxime was formed and separated into 3 isomers as their trimethylsilyl derivatives (molecular weight 404). Succinylacetone dioxime was converted to 3(5)-methyl-(3)5- isoxazole propionate whenever the pH of the solution was dropped to less than 5. Mass spectra of both derivatives are shown. This study demonstrates that capillary gas chromatography is suitable for use in urinary succinylacetone determination.     

Gas chromatographic determination of m- and p-hydroxyphenytoin in the urine of epileptic patients

An analytical method for determining phenytoin and its metabolites in the urine of epileptic patients is described. The analysis was performed for the m- and p-isomers of hydroxyphenytoin, the oxidative products of phenytoin, using gas chromatography. As an internal standard, 5-(4-methylphenyl)-5-phenylhydantoin was chosen; the extraction solvent from human urine was ether-chloroform (3:7). Phenytoin and its hydroxy isomers were satisfactorily determined by the modified on-column methylation technique on a 2% OV-17 column using temperature programming from 180 degrees C to 240 degrees C at 5 degrees C/min.     

新型抗炎镇痛剂SFZ-47及其代谢物的电喷雾离子阱质谱研究

用电喷雾离子阱质谱对警犬尿样中SFZ－47[3H-1,2-二氢－2－（4－甲基苯胺基）甲基－1－吡咯里嗪酮）及其4种代谢物进行了结构鉴定，利用质谱解析软件分析其裂解方式发现，它们在（＋）ESI－MS^2或（ )ESI－MS^3质谱中分别生成m/z122和脱吡咯里嗪酮母核的碎片，并发现葡萄苷酸型代谢物易于生成脱水（18u)和脱葡萄醛酸（176u)的碎片离子，这些特征可用于SFZ－47及结构类似物的体内生物转化研究。     

Gas chromatographic analysis of flunixin in equine urine after extractive methylation

A quantitative method for the analysis of flunixin, 2-(2-methyl-3-trifluoromethylanilino) nicotinic acid, in equine urine by gas chromatography with nitrogen-phosphorus detection has been developed. Flunixin and the internal standard, mefenamic acid, N-(2,3-xylyl) anthranilic acid, were analysed after extractive methylation of the carboxylic acid group using methyl iodide. The extraction and alkylation conditions of flunixin and mefenamic acid have been studied. The detection limit of the method was 0.25 mumol/l flunixin in urine (74 ng/ml). Flunixin was found to be conjugated to 96.5% in equine urine, and the conjugate was spontaneously hydrolysed to free flunixin. This approach can also be used to confirm the presence of flunixin or mefenamic acid in horse urine in the doping control of racehorses.