Determination of 4-hydroxyandrost-4-ene-3,17-dione metabolism in breast cancer patients using high-performance liquid chromatography-mass spectrometry

A sensitive procedure for studying the metabolism of the steroidal aromatase inhibitor 4-hydroxy-androst-4-ene-3,17-dione (4OHA) was developed based on enzyme hydrolysis, liquid-liquid extraction and reversed-phase liquid chromatography coupled with a mass spectrometer (LC-MS) using a thermospray interface. Seven metabolites were identified from the hydrolysed urine samples together with the parent drug. The major routes of metabolism were via dehydrogenation, reduction of the ketone functional groups, reduction at the C-4-C-5 double bond and hydroxylation at the C-5 position. Confirmation of the identity of 4OHA and its metabolites isolated from female patients' urine samples was accomplished by comparison of the retention times of their corresponding synthetic standards on LC-MS. We have demonstrated that this technique is particularly suitable for studying the metabolism of steroidal drugs.     

Biotransformation of (+)-androst-4-ene-3,17-dione

Fermentation of (+)-androst-4-ene-3,17-dione (1) with Curvularia lunata for 10 days yielded five oxidative and reductive metabolites, androsta-1,4-diene-3,17-dione (2), 17beta-hydroxyandrosta-1,4-dien-3-one (3), 11alpha-hydroxyandrost-4-ene-3,17-dione (4), 11alpha,17beta-dihydroxyandrost-4-en-3-one (5) and 15alpha-hydroxyandrosta-1,4-dien-17-one (6). The structures of these metabolites were elucidated on the basis of spectroscopic techniques. These microbially transformed products were assayed against the clinically important enzymes, tyrosinase and prolyl endopeptidase.     

A short efficient synthesis of 19-norandrost-4-ene-3,17-dione

A three-step conversion of 3aα-H-4α-(3′-propionic acid)-7aβ-methylhexahydro-1,5-indanedione into 19-norandrost-4-ene- 3,17-dione is described.     

Identification of novel circulating coffee metabolites in human plasma by liquid chromatography-mass spectrometry

This study reports a liquid chromatography-mass spectrometry method for the detection of polyphenol-derived metabolites in human plasma without enzymatic treatment after coffee consumption. Separation of available standards was achieved by reversed-phase ultra performance liquid chromatography and detection was performed by high resolution mass spectrometry in negative electrospray ionization mode. This analytical method was then applied for the identification and relative quantification of circulating coffee metabolites. A total of 34 coffee metabolites (mainly reduced, sulfated and methylated forms of caffeic acid, coumaric acid, caffeoylquinic acid and caffeoylquinic acid lactone) were identified based on mass accuracy (<4 ppm for most metabolites), specific fragmentation pattern and co-chromatography (when standard available). Among them, 19 circulating coffee metabolites were identified for the first time in human plasma such as feruloylquinic acid lactone, sulfated and glucuronidated forms of feruloylquinic acid lactone and sulfated forms of coumaric acid. Phenolic acid derivatives such as dihydroferulic acid, dihydroferulic acid 4'-O-sulfate, caffeic acid 3'-O-sulfate, dimethoxycinnamic acid, dihydrocaffeic acid and coumaric acid O-sulfate appeared to be the main metabolites circulating in human plasma after coffee consumption. The described method is a sensitive and reliable approach for the identification of coffee metabolites in biological fluids. In future, this analytical method will give more confidence in compound identification to provide a more comprehensive assessment of coffee polyphenol bioavailability studies in humans.     

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.     

Identification of polymer additives by liquid chromatography-mass spectrometry

LC-MS at different experimental conditions was used to construct a library of MS spectra of polymer additives. Combination of retention time information derived from the chromatogram with molecular mass and fragment ion information derived from MS and MS/MS was used for the identification of 20 additives. Mixtures of different additives and extracts of LDPE films were prepared and analyzed as unknowns. All 20 additives could be identified, 15 with 100% certainty.     

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.     

Determination of dithiocarbamates and metabolites in plants by liquid chromatography-mass spectrometry

A quantitative matrix solid-phase dispersion and liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) method is outlined for the simultaneous analysis of dithiocarbamates (DTCs) and their degradation products in plants. Compounds analyzed are dazomet, disulfiram, thiram and the metabolites ethylenthiourea and propylenthiourea. The performance of two different sample preparation protocols, the proposed one and other based on solid-phase extraction, as well as, of both atmospheric pressure ionization sources, APCI and electrospray, were compared. The effect of several parameters on the extraction, separation and detection was studied. Dithiocarbamates and metabolites were dispersed with carbograph, eluted with a mixture of dichloromethane-methanol, and then, identified by monitoring the base peak of the spectra corresponding to [M + H]+. The method was validated for avocados, cherries, lemons, nuts, oat, oranges, peaches, rice and tomatoes. Average recoveries varied from 33 to 109%, and relative standard deviation were between 4 and 21% with limits of quantification ranged from 0.25 to 2.5 mg kg(-1), except for thiram and disulfiram, which were not recovered from fruits with high acid content. The procedure was applied to the determination of DTCs and their metabolites in fruits, vegetables and cereals taken from different markets of Valencia, Spain.     

Identification of new urinary metabolites of trimeprazine in rats by gas chromatography-mass spectrometry.

The metabolites of trimeprazine were identified in urine of rats by gas chromatography-mass spectrometry. After the oral administration of trimeprazine, the urinary metabolites were extracted with diethyl ether before or after hydrolysis with beta-glucuronidase. The identified metabolites were N-demethyltrimeprazine,3-hydroxytrimeprazine,N-demethyl-3-++ +hydroxytrimeprazine and trimeprazine sulphoxide.     

Identification of triacylglycerols by high-performance liquid chromatography-gas-liquid chromatography and liquid chromatography-mass spectrometry

Triacylglycerols from rat adipose tissue were chromatographed by high-performance liquid chromatography (HPLC), with a gradient of propan-2-ol in acetonitrile as the mobile phase. Fractions of the material eluting from the column were collected and analysed by automated gas - liquid chromatography of the fatty acid methyl esters obtained after transmethylation. Triacylglycerols were identified by using a combination of their fatty acid content and elution time from the HPLC column. Fractions corresponding to whole peaks or groups of peaks were also collected and re-chromatographed on a liquid chromatography - mass spectrometry system equipped with a belt interface. For most triacylglycerols, good agreement was obtained between the two methods, although mass spectrometric identification of the early eluting peaks was complicated by poor resolution of the triacylglycerols on the HPLC system.     

Structural elucidation of metabolites of ritonavir and indinavir by liquid chromatography-mass spectrometry

The structural elucidation of metabolites of ritonavir and indinavir, HIV-protease inhibitor drugs, by liquid chromatography-electrospray ionization mass spectrometry is described. Ritonavir and indinavir were biotransformed separately by incubation with transplant quality human liver microsomes. The incubation mixture was then analyzed by HPLC coupled to ion trap (ITMS) and triple quadrupole mass analyzers. The metabolites retained most of the structural features of the parent molecules. Baseline chromatographic resolution of isobaric species by gradient elution HPLC permitted rapid structural identification of these metabolites. Both drugs were biotransformed primarily by oxidative and hydrolytic pathways to numerous metabolites that retained many of the features of the parent molecules. Triple quadrupole and ion trap mass spectrometry were applied jointly to thoroughly detect and thoroughly characterize these metabolites. Furthermore, retention-time and data-dependent scanning assured acquisition of detailed MS-MS spectra for rapid detection of metabolic pathways of ritonavir and indinavir. Comparison of the ITMS and triple quadrupole data showed qualitative and quantitative differences in the mass spectral patterns, suggesting that these instruments should be used in parallel to ensure comprehensive metabolite detection and characterization by LC-MS.     

Tentative identification of new metabolites of epimedin C by liquid chromatography-mass spectrometry

Epimedin C is one of the major bioactive constituents of Herba Epimedii. The aim of this study is to characterize and elucidate the structure of metabolites in the rat after administration of epimedin C. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) scan in positive ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer. A total of 18 metabolites were characterized by the changes in their protonated molecular masses, their MS/MS spectrum and their retention times compared with those of the parent drug. The results reveal possible metabolite profiles of epimedin C in rats; the metabolic pathways including hydrolysis, hydroxylation, dehydrogenation, demethylation and conjugation with glucuronic acid and different sugars were observed. This study provides a practical approach for rapidly identifying complicated metabolites, a methodology that could be widely applied for the structural characterization of metabolites of other compounds.     

Determination of theophylline and its metabolites in biological samples by liquid chromatography-mass spectrometry

Liquid chromatography–mass spectrometry (LC–MS) is a powerful tool for analysis of drugs and their metabolites. We used a column-switching system in combination with atmospheric pressure chemical ionization LC–MS (LC–APCI–MS) for the determination of theophylline and its metabolites in biological samples. The separation was carried out on a reversed-phase column using methanol–20 mM ammonium acetate as a mobile phase at a flow-rate of 1 ml/min in 30 min. In the mass spectrum, the molecular ions of these drugs and metabolites were clearly observed as base peaks. This method is sufficiently sensitive and accurate for the pharmacokinetic studies of these drugs.     

Elucidation of urinary metabolites of fluoxymesterone by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry

The suitability of liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC-MS) for the elucidation of fluoxymesterone metabolism has been evaluated. Electrospray ionization (ESI) and collision induced dissociation (CID) fragmentation in LC-MS/MS and electron impact spectra (EI) in GC-MS have been studied for fluoxymesterone and two commercially available metabolites. MS(n) experiments and accurate mass measurements performed by an ion-trap analyser and a QTOF instrument respectively have been used for the elucidation of the fragmentation pathway. The neutral loss scan of 20 Da (loss of HF) in LC-MS/MS has been applied for the selective detection of fluoxymesterone metabolites. In a positive fluoxymesterone doping control sample, 9 different analytes have been detected including the parent compound. Seven of these metabolites were also confirmed by GC-MS including 5 previously unreported metabolites. On the basis of the ionization, the CID fragmentation, the accurate mass of the product ions and the EI spectra of these analytes, a tentative elucidation as well as a proposal for the metabolic pathway of fluoxymesterone has been suggested. The presence of these compounds has also been confirmed by the analysis of five other positive fluoxymesterone urine samples.     

Structure elucidation of drug metabolites using thermospray liquid chromatography-mass spectrometry

Thermospray liquid chromatography-mass spectrometry (LC-MS) has been used to provide structural information both from in vitro and in vivo experiments. This paper will describe the more salient aspects of the technique that have emerged. The ability of the interface to handle gradients was essential for its successful application to metabolism studies, owing to the wide range of compound polarity involved. The examples discussed in this paper include the use of LC-MS in the analysis of in vitro incubations of drugs with hepatocyte cell cultures and the direct analysis of plasma samples from in vivo studies in the dog.     

State-of-the-art in liquid chromatography-mass spectrometry.

Impressive progress has been made in the technology and application of combined liquid chromatography-mass spectrometry (LC-MS) in the past decennium. From a technique, that could only be used by a specialist, it has developed into a routinely applicable technique. LC-MS has become the method-of-choice of analytical support in many stages of drug development within pharmaceutical industries and has found its way into environmental, biochemical and other laboratories. This paper provides a perspective on the current technology, principles and applications of LC-MS.