Enantiomeric screening of racemic citalopram and metabolites in human urine by entangled polymer solution capillary electrophoresis: an innovatory robustness/ruggedness study

Several CE methods have been developed to achieve the chiral separation of citalopram (CIT) and its metabolites demethylcitalopram (DCIT), didemethylcitalopram (DDCIT), and citalopram N-oxide (CIT-NO). All of these compounds were present as racemic mixtures. The best method, which led to the first ever chiral screening of CIT, DCIT, DDCIT, and CIT-NO, involved the use of carboxymethyl-gamma-CD (CM-gamma-CD) and the entangled polymer hydroxypropylmethylcellulose (HPMC) as chiral and selectivity additives, respectively, in the buffer system. In an effort to improve the selectivity and sensitivity of the method, the chemical and instrumental parameters were optimized. The best conditions were short-end anodic hydrodynamic injection (6 s, 0.7 psi); as BGE pH 5, 20 mM phosphate buffer, 0.2% w/v CM-gamma-CD, 0.05% w/v HPMC; voltage of 28 kV with a ramp applied (0.4 s); cartridge temperature of 20 degrees C; detection at 205 nm. In addition, a simple and rapid achiral CE method for the determination of citalopram propionic acid (CIT-PA, the only anionic metabolite of CIT) is also reported for the first time. Prior to the electrophoretic procedure it was necessary to apply an extraction and preconcentration step to obtain analytes from the human urine samples. This was achieved using an optimized SPE process. Moreover, an innovatory experimental and statistical design approach, which involves the simultaneous evaluation of the global robustness and ruggedness effects, was applied. Both of the proposed methods proved to be very useful in the chiral pharmacokinetic screening of CIT and related metabolites in clinical human urine samples.     

Determination of debrisoquine and metabolites in human urine by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric analysis has been developed for the determination of debrisoquine and its metabolites in the urine of healthy individuals (controls) and patients with chronic renal failure. The sensitive and specific assay comprises selected-ion monitoring of the drug and the metabolites 4-hydroxydebrisoquine and 8-hydroxydebrisoquine using guanoxan as the internal standard. The limit of detection is ca. 0.2 microgram/ml. The clinical study shows that the healthy individuals and patients with chronic renal failure can be divided in two groups of extensive metabolizers and poor metabolizers, respectively. The extensive metabolizers excreted large amounts of 4-hydroxydebrisoquine and minor amounts of 8-hydroxydebrisoquine. The poor metabolizers excreted small amounts of 4-hydroxy metabolite, and no 8-hydroxydebrisoquine was detected in the urine.     

Screening and confirmatory analysis of beta-agonists, beta-antagonists and their metabolites in horse urine by capillary gas chromatography-mass spectrometry

A method for the screening and confirmatory analysis of beta-agonists and -antagonists in equine urine is described. Following initial enzymic hydrolysis, the basic drugs and metabolites are extracted using Clean Screen DAU or Bond Elut Certify cartridges, and analysed as their trimethylsilyl ether or 2-(dimethyl) silamorpholine derivatives by capillary gas chromatography-mass spectrometry. The method proved to be very sensitive and selective for basic drugs. After administration of therapeutic doses of propranolol, metoprolol, timolol, isoxsuprine and clenbuterol to thoroughbred horses, the parent compound/metabolites could be detected in urine for upto 14-120 h depending on the drug.     

Determination of pyrethroid metabolites in human urine by capillary gas chromatography-mass spectrometry

Summary An analytical method for the simultaneous determination of the pyrethroid metabolites cis and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid, cis 3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid, 3-phenoxybenzoic acid and 4-fluoro-3-phenoxybenzoic acid in human urine samples is described. The urine is subjected to acid-induced hydrolysis followed by exhaustive solvent extraction, covering both conjugated and free acids, followed by a common derivatisation step yielding the corresponding methyl esters. Quantitation was by diastereomeric, capillary gas chromatography-mass spectrometry. It appears that 4-fluoro-3-phenoxybenzoic acid is a characteristic urinary marker for cyfluthrin exposure. The limits of determination are 0.5–1.0 g L^–1 urine depending on the metabolites concerned. The applicability of the method was tested on urine samples from pest control operators exposed occupationally to cypermethrin and cyfluthrin.     

Identification of phenprocoumon metabolites in human urine by high-performance liquid chromatography and gas chromatography-mass spectrometry

The oral anticoagulant phenprocoumon is eliminated in urine mainly as the glucuronide conjugate to an extent of 20% of the dose. The urine from patients undergoing phenprocoumon therapy was investigated and the following metabolites were isolated and identified: 7-hydroxyphenprocoumon as the main component, and 4'-hydroxyphenprocoumon and 6-hydroxyphenprocoumon as conjugates. They were characterized by high-performance liquid chromatography and, after methylation, by gas chromatography-mass spectrometry.     

Characterization of prednisone, prednisolone and their metabolites by gas chromatography-mass spectrometry

Human urinary metabolites of the synthetic corticosteroids prednisone and prednisolone were detected in the course of gas chromatographic steroid profiling as methoxime-trimethylsilyl derivatives. Metabolites were provisionaly identified by combined gas chromatography-mass spectrometry. The major metabolites were 11-keto/11-hydroxy conversion products, 20-hydroxy and 4,5-dihydro analogues of the parent drugs. Cortisone, 6-hydroxy and fully saturated A-ring compounds were minor metabolites. Retention indices and mass spectral data are presented.     

Toxicological detection of ethylenediamine and piperazine antihistamines and their metabolites in urine by computerized gas chromatography-mass spectrometry

Summary A gas chromatographic-mass spectrometric procedure for the detection of the following ethylenediamine and piperazine antihistamines and their metabolites in urine after acid hydrolysis, extraction and acetylation is described: Adeptolon, antazoline, bamipine, buclizine, chlorcyclizine, chloropyramine, cinnarizine, clemizole, cyclizine, etodroxizine, histapyrrodine, hydroxyzine, meclozine, mepyramine, oxatomide and tripelenamine. The acetylated extract was analysed by computerized gas chromatography-mass spectrometry. Using ion chromatography with the selective ions m/z 58, 72, 85, 125, 165, 183, 198 and 201 the possible presence of ethylenediamine or piperazine antihistamines or their metabolites was indicated. The identity of positive signals in the reconstructed ion chromatograms was then confirmed by a visual or computerized comparison of the stored full mass spectra with the reference spectra. The ion chromatograms, reference mass spectra and gas chromatographic retention indices (OV-101) are documented. The procedure presented is integrated in a general screening procedure (general unknown analysis) for several groups of drugs.

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Toxikologischer Nachweis von Ethylendiamin- und Piperazin-Antihistaminica und ihren Metaboliten im Urin mittels computerunterstützter Gas-Chromatographie-Massenspektrometrie

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Part of these results was reported at the Symposium Klinisch-Toxikologische Analytik of the Austrian and German Societies of Clinical Chemistry, Salzburg, Austria, September 14–16, 1987 [20]     

Systematic toxicological analysis of drugs and their metabolites by gas chromatography-mass spectrometry.

Gas chromatographic-mass spectrometric (GC-MS) procedures for the systematic toxicological analysis of several categories of drugs relevant to clinical toxicology, forensic toxicology and doping control are reviewed. Papers from 1981 to 1991 are taken into consideration. They describe the detection of acute or chronic intoxication and the detection of drug abuse. Screening procedures are included for the following categories: barbiturates and other sedative-hypnotics, anticonvulsants, benzodiazepines, antidepressants, phenothiazine and butyrophenone neuroleptics, central stimulants (amphetamines, cocaine), hallucinogens (LSD, phencyclidine, tetrahydrocannabinol), opioid (narcotic) and other potent analgesics, non-opioid analgesics, antihistamines (histamine H1-receptor blockers), antiparkinsonian drugs, beta-blockers (beta-adrenoceptor blockers), antiarrhythmics (class I and IV), diuretics, laxatives and their metabolites. Methods for confirmation of results obtained by screening procedures using immunoassay or chromatographic techniques are also included. GC-MS procedures for the simultaneous detection of several categories of drugs, the so-called "general unknown analysis", are reviewed. The toxicological question to be answered and the consequence for the choice of an adequate method, the sample preparation and the chromatography itself are discussed. The basic information about the biosample assayed, work-up, GC column, mass spectral detection mode, reference data and sensitivity of each procedure are summarized in tables, arranged according to the category of drug. Examples of typical GC-MS applications are presented. Fragment ions that are suitable for mass spectral screening for particular categories of drugs and for general unknown are tabulated.     

Screening for diuretics in human urine by gas chromatography-mass spectrometry with derivatisation by direct extractive alkylation

A rapid, sensitive and reliable gas chromatographic-mass spectrometric (GC-MS) screening procedure for diuretics in human urine has been developed. The procedure uses derivatisation by extractive methylation directly from the urine. The suitability of a number of phase transfer reagents and solvents were studied for the detection of sixteen diuretics. The results obtained indicate that the screening procedure employing tetrahexylammonium hydrogensulphate at pH 12 with methyl iodide in toluene at room temperature was the most effective. This method gives selectivity and sensitivities down to 0.03-0.1 microgram/ml and provides a substrate suitable for GC-MS confirmation without further manipulation. The application of the method is demonstrated by the screening of urine for bumetanide, ethacrynic acid, acetazolamide, chlorothiazide and hydrochlorothiazide.     

Detection of methandienone (methandrostenolone) and metabolites in horse urine by gas chromatography-mass spectrometry.

The metabolic transformation of methandienone (I) in the horse was investigated. After administration of a commercial drug preparation to a female horse (0.5 mg/kg), urine samples were collected up to 96 h and processed without enzymic hydrolysis. Extraction was performed by a series of solid-liquid and liquid-liquid extractions, thus avoiding laborious purification techniques. For analysis by gas chromatography-mass spectrometry, the extracts were trimethylsilylated. Besides the parent compound I and its C-17 epimer II, three monohydroxylated metabolites were identified: 6 beta-hydroxymethandienone (III), its C-17 epimer (IV) and 16 beta-hydroxymethandienone (V). In addition, three isomers of 6 beta,16-dihydroxymethandienone (VIa-c) were discovered. Apparently, reduction of the delta 4 double bond of 16 beta-hydroxymethandienone (V) in the horse yields 16 beta,17 beta-dihydroxy-17 alpha-methyl-5 beta-androst-1-en-3-one (VII). Reduction of the isomers VIa-c results in the corresponding 6 beta,16,17-trihydroxy-17-methyl-5 beta-androst-1-en-3-ones (VIIIa-c). The data presented here suggest that screening for the isomers of VI and VIII, applying the selected-ion monitoring technique, will be the most successful way of proving methandienone administration to a horse.     

Quantitation of 17beta-nandrolone metabolites in boar and horse urine by gas chromatography-mass spectrometry

A method to quantify metabolites of 17beta-nandrolone (17betaN) in boar and horse urine has been optimized and validated. Metabolites excreted in free form were extracted at pH 9.5 with tert-butylmethylether. The aqueous phases were applied to Sep Pak C18 cartridges and conjugated steroids were eluted with methanol. After evaporation to dryness, either enzymatic hydrolysis with beta-glucuronidase from Escherichia coli or solvolysis with a mixture of ethylacetate:methanol:concentrated sulphuric acid were applied to the extract. Deconjugated steroids were then extracted at alkaline pH with tert-butylmethylether. The dried organic extracts were derivatized with MSTFA:NH4I:2-mercaptoethanol to obtain the TMS derivatives, and were subjected to analysis by gas chromatography mass spectrometry (GC/MS). The procedure was validated in boar and horse urine for the following metabolites: norandrosterone, noretiocholanolone, norepiandrosterone, 5beta-estran-3alpha, 17beta-diol, 5alpha-estran-3beta, 17beta-diol, 5alpha-estran-3beta, 17alpha-diol, 17alpha-nandrolone, 17betaN, 5(10)-estrene-3alpha, 17alpha-diol, 17alpha-estradiol and 17beta-estradiol in the different metabolic fractions. Extraction recoveries were higher than 90% for all analytes in the free fraction, and better than 80% in the glucuronide and sulphate fractions, except for 17alpha-estradiol in the glucuronide fraction (74%), and 5alpha-estran-3beta, 17alpha-diol and 17betaN in the sulphate fraction (close to 70%). Limits of quantitation ranged from 0.05 to 2.1 ng mL(-1) in the free fraction, from 0.3 to 1.7 ng mL(-1) in the glucuronide fraction, and from 0.2 to 2.6 ng mL(-1) in the sulphate fraction. Intra- and inter-assay values for precision, measured as relative standard deviation, and accuracy, measured as relative standard error, were below 15% for most of the analytes and below 25%, for the rest of analytes. The method was applied to the analysis of urine samples collected after administration of 17betaN laureate to boars and horses, and its suitability for the quantitation of the metabolites in the three fractions has been demonstrated.     

Determination of atenolol in human urine by gas chromatography-mass spectrometry method

A sensitive and efficient method was developed for the determination of atenolol in human urine by gas chromatography-mass spectrometry (GC-MS). Atenolol and metoprolol (internal standard, IS) were extracted from human urine with a mixture of chloroform and butanol at basic pH with liquid-liquid extraction. The extracts were derivatized with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and analyzed by GC-MS using a capillary column. The standard curve was linear (r = 0.99) over the concentration range of 50-750 ng/mL. Intra- and inter-day precision, expressed as the relative standard deviation were less than 5.0%, and accuracy (relative error) was better than 7.0%. The analytical recovery of atenolol from human urine has averaged 91%. The limit of quantification was 50 ng/mL. Also, the method was successfully applied to a patient with hypertension who had been given an oral tablet of 50 mg atenolol.     

Determination of inositol isomers and arabitol in human urine by gas chromatography-mass spectrometry

Summary A capillary gas chromatography method for the analysis of inositol isomers and arabitol (extendable to threitol and adonitol) is described and applied to urine after derivatization. The single ion monitoring technique allows a notable improvement in sensitivity and selectivity.     

Rapid assay of cocaine, opiates and metabolites by gas chromatography-mass spectrometry.

The simultaneous assay of cocaine, opiates and metabolites in small biological samples continues to be a difficult task. This report focuses upon tabulation of important techniques (extraction, derivatization, chromatographic conditions, detection mode, data acquisition) reported over the last decade that were used in the development of assays for these analytes. The most prevalent procedures for extraction of cocaine, opiates and metabolites were liquid-liquid and solid-phase extraction isolation methods. Following extraction analytes were derivatized and analyzed by gas chromatography-mass spectrometry. The technique most often used for chromatographic separation was fused-silica capillary column gas chromatography. Detection generally was performed by selected ion monitoring in the positive-ion electron-impact ionization mode, although full-scan acquisition and positive- and negative-ion chemical ionization methods have been used. It was apparent from the review that there is a continuing need for greater sensitivity and selectivity in the assay of highly potent opiates and for cocaine and metabolites.     

Identification of some important metabolites of boldenone in urine and feces of cattle by gas chromatography-mass spectrometry.

17 alpha-Boldenone (17 alpha-BOL) and/or 17 beta-boldenone (17 beta-BOL) appear occasionally in fecal matter of cattle. In addition to 17 alpha-BOL, a whole array of boldenone related substances can be found in the same samples. In vitro experiments with microsomal liver preparations and isolated hepatocytes combined with the excretion profiles found in urine and feces samples of in vivo experiments made it possible to identify several metabolites of 17 beta-BOL in 17 beta-BOL positive feces samples. In one animal treated with 17 beta-BOL, no 17 beta-BOL or its metabolites were present before treatment and most of these compounds disappeared gradually in time after the treatment was stopped. It is not clear what the origin is of 17 alpha-BOL and boldenone metabolites in samples screened routinely for the abuse of anabolic steroids and considered to be 'negative' because of the absence of 17 beta-BOL since other workers showed some evidence that 17 alpha-BOL can be of endogenous origin. However, in our hands, most of these 17 alpha-BOL positive samples, obtained during routinely performed screenings of cattle, contained large amounts of delta 4-androstene-3,17-dione (AED), which normally is absent from routinely screened negative samples. Furthermore, AED was absent in all samples obtained from the animals treated with 17 beta-BOL. We have no direct evidence that 17 alpha-BOL or 17 beta-BOL is of endogenous origin.     

Determination of ketamine and metabolites in urine by liquid chromatography-mass spectrometry

We have developed an analytical method by using liquid chromatography-mass spectrometry (LC-MS) to determine ketamine and its metabolites in urine. The ionization efficiency between two ionization modes (ESI and APCI) of LC-MS was compared to each other. An easy and simple sample preparation of urine samples was made by passing samples through a 0.22 μm PVDF syringe filter. The results indicated that the ionization efficiency of positive APCI mode is better than positive ESI mode for determination of trace ketamines. A wide linearity range of the research is from 5 to 250 ng mL⁻¹ and the detection limits for ketamine, norketamine and dehydronorketamine were 0.95, 0.48 and 0.33 ng mL⁻¹, respectively. The proposed method was tested by analyzing ketamine and metabolites in the urines of volunteers. The concentrations of ketamine, norketamine and dehydronorketamine are ranged of 5.4-131.0, 12.5-74.1 and 22.8-278.9 ng mL⁻¹, respectively and the ketamines concentration profiles in human urine were also determined. The results demonstrate the suitability of liquid chromatography-mass spectrometry approach to analyze trace amount of ketamine and its metabolites in urine.