High-performance liquid chromatographic separation and isolation of quinidine and quinine metabolites in rat urine

A procedure for the separation and isolation of the urinary metabolites of quinidine and quinine by reversed-phase high-performance liquid chromatography is described. Nine metabolites of quinidine and eight metabolites of quinine were detected in the urine of male Sprague-Dawley rats after a single dose of quinidine or quinine (50 mg kg^?1). Following extraction from urine, the metabolites were separated on either an analytical or a semi-preparative reversed-phase column by gradient elution. After isolation and derivatization, the metabolites were analyzed by gas chromatography and gas chromatography—mass spectrometry.     

Quantitative analysis of steroid profiles from urine by capillary gas chromatography : I. Accuracy and reproducibility of the sample preparation

A method is described for the determination of steroid profiles from urine by means of gas chromatography using high-efficiency glass capillary columns. The accuracy and reproducibility of essential steps in the sample preparation (extraction of steroids and steroid conjugates by means of XAD-2, enzymatic hydrolysis with Helix pomatia juice, solvolysis in acidified ethyl acetate and alkali wash) are established using different endogenously labelled urine samples, obtained from normal subjects to whom labelled steroids had been administered. Preliminary results are given on the reproducibility of the derivatization procedure (formation of methoxime-trimethylsilyl (MO-TMS) ethers), the gas chromatographic analysis and the whole method. Two procedures for the purification of MO-TMS steroid derivatives are compared. Application of the method to urine samples of patients with various endocrine disorders is included.     

Confirmation of cocaine exposure by gas chromatography-mass spectrometry of urine extracts after methylation of benzoylecgonine

Volatility and thermal stability are necessary physical-chemical properties for analysing a substance by gas chromatography. A derivatization step is required before gas chromatography of benzoylecgonine (the main metabolite of cocaine). In the literature, reactions such as silylation, perfluoroalkylation or alkylation are the most frequently used to derivatize benzoylecgonine. However, they allow the formation of products sensitive to moisture or require a purification step. So, a procedure to derivatize benzoylecgonine with diazomethane before gas chromatographic analysis was evaluated. A study was performed to evaluate the efficiency of conversion of benzoylecgonine in cocaine, the necessary time for reaction and the stability of ethereal solution of diazomethane. The reaction was shown to be very fast in mild conditions and there was no need for a further purification step. When benzoylecgonine was extracted from urine by solid-phase extraction and derivatized with diazomethane, concentrations as low as 25 ng/ml could be detected using GC-MS in the full scan mode.     

Identification of aminoethylcysteine ketimine decarboxylated dimer in human plasma

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) is a natural sulfur-containing tricyclic compound detected, until now, in human urine and bovine cerebellum. Recently, the antioxidant properties of this compound, and particularly its protective effect on the in vitro oxidation of low-density lipoproteins, have been demonstrated. In this paper, the identification of AECK-DD in human plasma by means of gas chromatography, high-performance liquid chromatography and gas chromatography–mass spectrometry, performed after a simple and fast purification procedure, is reported.     

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.     

Methodology for the identification of the urinary metabolites of the analgesic-narcotic antagonist, codorphone, by gas chromatography mass spectrometry

Methodology is presented for the identification of codorphone and its metabolites in urine samples using gas chromatography mass spectrometry. The procedure focuses on the clean-up of biological samples and a derivatization technique suitable for these samples. Sep-Pak C-18 cartridges were employed in the clean-up procedure permitting the biological sample to be derivatized in a relatively small volume of reagents. The derivatization procedure incorporated a one-step trimethylsilyloxime reaction to prevent enol formation while simultaneously derivatizing free hydroxyl groups with the excess trimethylsilylimidazole present in the reaction mixture. This was followed by the addition of BSTFA directly to this reaction mixture to complete derivatization of any metabolites possessing dealkylation of the nitrogen. Using this derivatization scheme, synthetic metabolites were analyzed by gas chromatography mass spectrometry, and their mass spectra were characterized emphasizing the diagnostic fragment ions observed in the spectra. To illustrate the usefulness of this methodology, a urine sample obtained from a dog that had been dosed with codorphone was analyzed by gas chromatography mass spectrometry, and the metabolites were identified by comparison to the mass spectra of the synthetic derivatives.     

The urinary metabolites of 17alpha-ethynylestradiol-9alpha,11xi-3H in women. Chromatographic profiling and indentification of ethynyl and non-ethynyl compounds.

Metabolites of 17alpha-ethynylestradiol (EE2) were obtained from human urine following ingestion of tritium-labeled EE2. Over 95% of the recovered activity was found as conjugated steroids and these were separated into four groups by chromatography of the urine extract on Sephadex LH-20 with chloroform-methanol (1/1) + 0.01M NaCl. The two major conjugate fractions appeared to be almost exclusively glucosiduronates. Enzymatic hydrolysis liberated at least ten different EE2 metabolites as shown by chromatography on Sephadex LH-20 with benzene-methanol (85/15). After additional separation and purification of these metabolites, positive identification was obtained for nine radioactive compounds by either gas liquid chromatography-mass spectrometry or reverse-isotope recrystallization. Five were ethynyl compounds: EE2, 2-MeO EE2, 16beta-OH EE2, 2-OH EE2 and 6alpha-OH EE2. The other four were de-ethynylated estrogens: estrone, estradiol-17beta, estriol, and 2-Me-O-estradiol-17beta.     

Detection of new urinary exemestane metabolites by gas chromatography coupled to mass spectrometry

Exemestane is an aromatase enzyme complex inhibitor. Its metabolism in humans is not fully described and there is only one known metabolite: 17β-hydroxyexemestane. In this work, excretion studies were performed with four volunteers aiming at the detection of new exemestane metabolites in human urine by gas chromatography coupled to mass spectrometry (GC-MS) after enzymatic hydrolysis and liquid-liquid extraction. Urine samples collected from four volunteers were analyzed separately. The targets of the study were mainly the 6-exomethylene oxidized metabolites. Two unreported metabolites were identified in both free and glucuconjugated urine fractions from all four volunteers, both of them were the result of the 6-exomethylene moiety oxidation: 6ξ-hydroxy-6ξ-hydroxymethylandrosta-1,4-diene-3,17-dione (metabolite 1) and 6ξ-hydroxyandrosta-1,4-diene-3,17-dione (metabolite 2). Furthermore, only in glucoconjugated fractions from all volunteers, one metabolite arising from the A-ring reduction was identified as well, 3ξ-hydroxy-5ξ-androst-1-ene-6-methylene-17-one (metabolite 3). The molecular formulae of all these metabolites were ascertained by the determination of exact masses using gas chromatography coupled to high resolution mass spectrometry (GC-HRMS). Moreover, all metabolites were confirmed using an alternative derivatization with methoxyamine and MSTFA/TMS-imidazole.     

Sensitive gas chromatographic method for determination of mercapturic acids in human urine

A method was developed for sensitive determination of the specific benzene metabolite S-phenylmercapturic acid and the corresponding toluene metabolite S-benzylmercapturic acid in human urine for non-occupational and occupational exposure. The sample preparation procedure consists of liquid extraction of urine samples followed by precolumn derivatization and a clean-up by normal-phase HPLC. Determination of analytes occurs by gas chromatography with electron-capture detection. With this highly sensitive method (detection limits 60 and 65 ng/l, respectively) urinary S-phenylmercapturic and S-benzylmercapturic acid concentrations for non-occupationally exposed persons (e.g. non-smokers) can be measured precisely in one chromatographic run. Validation of the method occured by comparison with a HPLC method we have published recently.     

Application of solid-phase microextraction combined with derivatization to the determination of amphetamines by liquid chromatography

This work evaluates the utility of solid-phase microextraction (SPME) in the analysis of amphetamines by liquid chromatography (LC) after chemical derivatization of the analytes. Two approaches have been tested and compared, SPME followed by on-fiber derivatization of the extracted amphetamines, and solution derivatization followed by SPME of the derivatives formed. Both methods have been applied to measure amphetamine (AP), methamphetamine (MA), and 3,4-methylenedioxymethamphetamine (MDMA), using the fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC) and carbowax-templated resin (CW-TR)-coated fibers. Data on the application of the proposed methods for the analysis of different kind of samples are presented. When analyzing aqueous solutions of the analytes, both approaches gave similar analytical performance, but the sensitivity attainable with the solution derivatization/SPME method was better. The efficiencies observed when processing spiked urine samples by the SPME/on-fiber derivatization approach were very low. This was because the extraction of matrix components into the fiber coating prevented the extraction of the reagent. In contrast, the efficiencies obtained for spiked urine samples by the solution derivatization/SPME approach were similar to those obtained for aqueous samples. Therefore, the later method would be the method of choice for the quantification of amphetamines in urine.     

Simultaneous analysis of substrates,products, and inhibitors of xanthine oxidase by high-pressure liquid chromatography and gas chromatography

Procedures are presented for the simultaneous analysis of hypoxanthine, xanthine, allopurinol, oxipurinol, and uric acid in standard mixtures and physiological fluids using gas chromatography (gc) or high-pressure liquid chromatography (hplc). Excellent correlation was obtained between the two methods for hypoxanthine, xanthine, oxipurinol, and uric acid. There are advantages and disadvantages to both methods. hplc requires no prior derivatization, uses isocratic elution with a buffer containing no organic solvent, and has 50- to 100-fold greater sensitivity than gc. Simpler methods of prepurification, readily adapted to clinical laboratories, can be used for hplc analysis. Although substances that are found in some urine samples from cancer patients interfere with hplc, separations by gc are not affected by these substances.     

Simultaneous determination of isbufylline and its major metabolites in rabbit blood and urine by reversed-phase high-performance liquid chromatography

A sensitive high-performance liquid chromatographic assay for isbufylline and its major metabolites in rabbit blood and urine is described. After extraction, samples were eluted by a linear reversed-phase gradient. Specimens obtained after intravenous administration of isbufylline to rabbits were analysed to identify and subsequently quantify the potential metabolites. Using the ultraviolet absorption trace on the recorder as a reference, elution fractions were collected and analysed by mass spectrometry with the direct inlet system and gas chromatography—mass spectrometry after derivatization. Seven metabolites were identified and another five quantified. The method is specific, accurate, reproducible and recommended for pharmacokinetic studies.     

Gas—liquid chromatography of isobutyl ester,N(O)-heptafluorobutyrate derivatives of amino acids on a glass capillary column for quantitative separation in clinical biology

A gas chromatographic method adapted to routine analysis has been developed for quantitative separation on glass capillary columns for free proteic and other known amino acids normally or abnormally found in physiological fluids. The procedure involves ion-exchange chromatography and isobutyl ester, N(O)-heptafluorobutyrate derivatization of free plasma and urine amino acid samples. Derivatized components were ascertained by combined gas chromatography—mass spectrometry. The use of glass for the capillary column is mandatory to achieve qualitative and quantitative analysis of the known occurring amino acids in urine and small plasma samples. Quantitative analysis of several types of human amino acid disorders are presented.     

Direct determination of the ratio of tetrahydrocortisol+allo-tetrahydrocortisol to tetrahydrocortisone in urine by LC-MS-MS

The 11beta-hydroxysteroid dehydrogenase (11beta-HSD) is responsible for the interconversion of both the hormonally inactive cortisone and the active cortisol. This enzyme activity, which has implications in the pathogenesis of numerous diseases, is reflected in the ratio of tetrahydrometabolites of cortisol (allo-tetrahydrocortisol and tetrahydrocortisol) to those of cortisone (tetrahydrocortisone). Several methods have been proposed in the literature to determine such a ratio in urine. Most of them require tedious and extensive extraction and derivatization steps and make use of gas-chromatographic techniques, including gas chromatography coupled to mass spectrometry (GC-MS). We present here an alternative approach for the direct determination of such a ratio in urine by using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS), based on a minimal sample treatment. Actually, the limit of detections (LODs) for pure standards in water permitted a simple dilution of the urine samples prior to the analysis, hence, an accurate optimization of the high performance liquid chromatography (HPLC) separation was needed in order to get rid of the severe influence of the urine matrix on the ionization efficiency. Besides, the nature of some interfering species was deeply investigated, as well as the suitability of some commercial deuterated steroids as internal standards. All these led to the final method, which was based on a HPLC separation on a C8 column and a ternary gradient water/methanol/acetonitrile. In parallel, an appropriate sample preparation was set up, which consisted of an enzymatic hydrolysis of the conjugated species and a followed 1:20 dilution. Preliminary measurements on real urine samples were performed as well.     

Mass spectral characterization of doxylamine and its rhesus monkey urinary metabolites

This study describes the use of mass spectrometry (MS), high-performance liquid chromatography (HPLC) and chemical derivatization techniques for the identification of doxylamine and five rhesus monkey urinary metabolites. The analyses were performed using chemical ionization mass spectrometry with either methane or ammonia as the reagent gas. The confirmation of the structures of two of these urinary metabolites was aided by the synthesis of doxylamine N-oxide and desmethyldoxylamine and by the use of methylation and acetylation derivatization techniques. Doxylamine N-oxide, desmethyldoxylamine, didesmethyldoxylamine, and two metabolites which resulted from the cleavage of the aliphatic tertiary nitrogen side chain to the subsequent 2-[1-phenyl-1-(2-pyridinyl)ethoxy]acetic acid or 2-[1-phenyl-1-(2-pyridinyl)ethoxy]methanol compounds were isolated and identified from rhesus monkey urine. Additional data concerning the mass spectral analysis of derivatization or reaction products from the three chloroformate reactions with doxylamine, and the synthesis and separation techniques which afforded mass spectral identification of the urinary metabolites are also presented.     

Application of optical isomer analysis by diastereomer derivatization GC/MS to determine the condition of patients with short bowel syndrome

To establish a method for separating the optical isomers of lactic acid, we modified the derivatization steps in our procedure for urinary mass-screening for inborn errors of metabolism. For chiral recognition, we chose O-trifluoroacetyl-(-)-menthylation derivatization instead of our previous method, trimethylsilyl derivatization, and the samples were then analyzed under GC/MS by capillary gas chromatography on a DB-5MS column. This method can be used to follow-up the condition of a patient with short bowel syndrome and to prevent onset and/or seizure. d-Lactic acid was also isolated from the urine of healthy controls as one of the main peaks in the chromatogram.     

Validation of a reversed-phase HPLC method for quantitative amino acid analysis.

A semi-automated method for amino acid derivatization and analysis has been validated for use in analysis of protein biopharmaceuticals. The method includes protein hydrolysis, o-phthalaldehyde derivatization, and reversed-phase high-performance liquid chromatography analysis in a general-purpose UV-visible high-performance liquid chromatography system. Amino-acid derivatization is performed automatically by the high-performance liquid chromatography autosampler right before injection. The required validation parameters, i.e., specificity, linearity, accuracy, precision, limit of detection, and limit of quantification, were studied for bovine serum albumin and for a recombinant human Fab fragment. The method can be employed as an absolute quantification method for determination of extinction coefficients of recombinant proteins.     

Endogenous alkaloids in man XXVI. Determination of the dopaminergic neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) in biological samples using gas chromatography with selected ion monitoring

Highly chlorinated β-carboline have a potential in vivo relevance to Parkinson's disease. In this paper, a gas chromatographic method for the determination of the neurotoxic 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo), the condensation product of tryptamine and chloral hydrate, is described. The specific and sensitive assay involves purification of the biological samples by solid-phase extraction with C₁₈ cartridges, derivatization with heptafluorobutyric anhydride, and chromatography on a non-polar fused-silica capillary column. Detection of TaClo was achieved by the registration of characteristic mass fragments of the TaClo heptafluorobutyric amide derivative using selected ion monitoring. The method was utilized to detect and quantify TaClo in blood, urine, bile, faeces, and brain tissue of rats treated with this alkaloid-type heterocycle. Four-fold deuterium-labelled TaClo was used as an internal standard.     

Application of FRIT fast atom bombardment liquid chromatography/mass spectrometry for the determination of acetylcholine levels in rat brain regions

This report describes the use of FRIT fast atom bombardment (FAB) liquid chromatography/mass spectrometry for the analysis of acetylcholine in rat brain regions. Direct assessment of acetylcholine levels is possible without the need for either derivatization or extensive sample preparation. Quantification is accomplished by monitoring intact molecular cations of acetylcholine and a deuterated internal standard. The results are compared with those obtained by conventional pyrolysis gas chromatography/mass spectrometry and by liquid chromatography with electrochemical detection.     

Purification and separation of plant gibberellins from their precursors and glucosyl conjugates

A procedure using two small preparative columns (in sequence) of C₁₈ reverse phase Bondapak B material with methanolic extracts of plant tissue (Pisum sativum L., Malus domestica Borkh., Pimpinella anisum L.) yields two fractions: (i) gibberellin (GA) precursors, and (ii) free GA/GA methyl esters (GA-Me)/GA glucosyl conjugates. The discrete separation of (iii) free GA/GA-Me from (iv) GA glucosyl conjugates is then accomplished by a combination of differential solvent solubility and SiO₂ partition chromatography. All fractions are almost pigment free, and appreciable dry weight purification was accomplished for the GA precursor and free GA/GA-Me fractions. Solvent volumes can be kept low, no buffer salts are introduced, and each fraction (i, iii, iv) can be subjected directly to preparative or analytical reverse phase C₁₈ high performance liquid chromatography without recourse to solvent partitioning, and often without further purification.