High-performance liquid chromatography–tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites

Applications of tandem mass spectrometry (MS/MS) techniques coupled with high-performance liquid chromatography (HPLC) in the identification and determination of phase I and phase II drug metabolites are reviewed with an emphasis on recent papers published predominantly within the last 6 years (2002–2007) reporting the employment of atmospheric pressure ionization techniques as the most promising approach for a sensitive detection, positive identification and quantitation of metabolites in complex biological matrices. This review is devoted to in vitro and in vivo drug biotransformation in humans and animals. The first step preceding an HPLC-MS bioanalysis consists in the choice of suitable sample preparation procedures (biomatrix sampling, homogenization, internal standard addition, deproteination, centrifugation, extraction). The subsequent step is the right optimization of chromatographic conditions providing the required separation selectivity, analysis time and also good compatibility with the MS detection. This is usually not accessible without the employment of the parent drug and synthesized or isolated chemical standards of expected phase I and sometimes also phase II metabolites. The incorporation of additional detectors (photodiode-array UV, fluorescence, polarimetric and others) between the HPLC and MS instruments can result in valuable analytical information supplementing MS results. The relation among the structural changes caused by metabolic reactions and corresponding shifts in the retention behavior in reversed-phase systems is discussed as supporting information for identification of the metabolite. The first and basic step in the interpretation of mass spectra is always the molecular weight (MW) determination based on the presence of protonated molecules [M+H]⁺ and sometimes adducts with ammonium or alkali-metal ions, observed in the positive-ion full-scan mass spectra. The MW determination can be confirmed by the [M-H]^- ion for metabolites providing a signal in negative-ion mass spectra. MS/MS is a worthy tool for further structural characterization because of the occurrence of characteristic fragment ions, either MS ⁿ analysis for studying the fragmentation patterns using trap-based analyzers or high mass accuracy measurements for elemental composition determination using time of flight based or Fourier transform mass analyzers. The correlation between typical functional groups found in phase I and phase II drug metabolites and corresponding neutral losses is generalized and illustrated for selected examples. The choice of a suitable ionization technique and polarity mode in relation to the metabolite structure is discussed as well.     

Bioanalytical methods for determination of tamoxifen and its phase I metabolites: a review

The selective estrogen receptor modulator tamoxifen is used in the treatment of early and advanced breast cancer and in selected cases for breast cancer prevention in high-risk subjects. The cytochrome P450 enzyme system and flavin-containing monooxygenase are responsible for the extensive metabolism of tamoxifen into several phase I metabolites that vary in toxicity and potencies towards estrogen receptor (ER) alpha and ER beta.An extensive overview of publications on the determination of tamoxifen and its phase I metabolites in biological samples is presented. In these publications techniques were used such as capillary electrophoresis, liquid, gas and thin layer chromatography coupled with various detection techniques (mass spectrometry, ultraviolet or fluorescence detection, liquid scintillation counting and nuclear magnetic resonance spectroscopy). A trend is seen towards the use of liquid chromatography coupled to mass spectrometry (LC-MS). State-of-the-art LC-MS equipment allowed for identification of unknown metabolites and quantification of known metabolites reaching lower limit of quantification levels in the sub pg mL⁻¹ range. Although tamoxifen is also metabolized into phase II metabolites, the number of publications reporting on phase II metabolism of tamoxifen is scarce. Therefore the focus of this review is on phase I metabolites of tamoxifen.We conclude that in the past decades tamoxifen metabolism has been studied extensively and numerous metabolites have been identified. Assays have been developed for both the identification and quantification of tamoxifen and its metabolites in an array of biological samples. This review can be used as a resource for method transfer and development of analytical methods used to support pharmacokinetic and pharmacodynamic studies of tamoxifen and its phase I metabolites.     

New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal.     

Characterization of metabolites of Celecoxib in rabbits by liquid chromatography/tandem mass spectrometry

The metabolism of the anti-inflammatory drug Celecoxib in rabbits was characterized using liquid chromatography (LC)/tandem mass spectrometry (MS/MS) with precursor ion and constant neutral loss scans followed by product ion scans. After separation by on-line liquid chromatography, the crude urine samples and plasma and fecal extracts were analyzed with turbo-ionspray ionization in negative ion mode using a precursor ion scan of m/z 69 (CF(3)) and a neutral loss scan of 176 (dehydroglucuronic acid). The subsequent product ion scans of the [M - H] ions of these metabolites yielded the identification of three phase I and four phase II metabolites. The phase I metabolites had hydroxylations at the methyl group or on the phenyl ring of Celecoxib, and the subsequent oxidation product of the hydroxymethyl metabolite formed the carboxylic acid metabolite. The phase II metabolites included four positional isomers of acyl glucuronide conjugates of the carboxylic acid metabolite. These positional isomers were caused by the alkaline pH of the rabbit urine and were not found in rabbit plasma. The chemical structures of the metabolites were characterized by interpretation of their product ion spectra and comparison of their LC retention times and the product ion spectra with those of the authentic synthesized standards.     

Determination of zolpidem, a new sleep-inducing agent, and its metabolites in biological fluids: pharmacokinetics, drug metabolism and overdosing investigations in humans.

For the determination of zolpidem, a new sleep inducer, and its metabolites in human plasma and urine, three methods were developed that are suitable for pharmacokinetics, drug metabolism and overdosing investigations. The methods used for pharmacokinetic and drug metabolism studies are based on column-switching high-performance liquid chromatography; they do not require any sample manipulation because the plasma or diluted urine is injected into a pre-column where clean-up and preconcentration take place. The analytes are transferred by valve-switching to the C18 analytical column for chromatography. To investigate overdose cases, urine samples only are used: the method is simple, because the diluted urine can be injected directly into the analytical column (phenyl type). This allows the identification and quantification of the principal urinary metabolite of zolpidem, the unchanged drug being practically undetectable. All the methods use fluorescence detection, which affords high sensitivity and selectivity. It is necessary to use a method capable of the determination of metabolites even if these are apparently pharmacologically inactive, because in different physiopathological populations the qualitative and quantitative metabolic profiles of zolpidem could be different. The method designed for the investigation of (accidental or deliberate) overdose cases is, as required on such occasions, simple and rapid, with good selectivity with respect to commonly prescribed psychotropic drugs.     

New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal. Received: 23 July 1999 / Revised: 9 September 1999 / Accepted: 16 September 1999     

Selective system of identification and determination of antidepressants and neuroleptics in serum or plasma by solid-phase extraction followed by high-performance liquid chromatography with photodiode-array detection in analytical toxicology.

A selective off-line solid-phase isolation of antidepressants, neuroleptics and other structurally related basic drugs from plasma or serum prior to high-performance liquid chromatography was tested and optimized for general use in toxicological analyses where concomitant drugs can be encountered. The sequential elution preseparated drug mixtures and simplified the subsequent analytical steps. High isolation efficiencies of cyano-bonded silica cartridges from Baker for fifteen amine drugs were determined. Isocratic chromatography on octadecylsilica proved to be very suitable for broad practical applications in complicated cases. The identification of an unknown peak was supported by photodiode-array detection in the range 200-400 nm with a resolution of 2 nm. The linearity of the assay from therapeutic to toxic concentrations was attained. Sufficient sensitivities covering low therapeutic levels of parent drugs and their demethylated metabolites were reached. The system is flexible and allows various methods of quantitative assay to be devised according to the conditions of a particular case in clinical or forensic toxicology.     

Direct analysis of alkylnaphthalenes in crude oils by two-dimensional capillary gas chromatography

Summary Diaromatics are geochemically significant constituents of crude oils. Their determination is usually achieved by elaborate prefractionation methods, such as medium pressure liquid chromatography and HPLC, prior to capillary gas chromatography. The present contribution describes the quantitative analysis of methylnaphthalenes, ethylnaphthalenes, and dimethylnaphthalenes in selected crude oils by two-dimensional capillary GC. Since the method does not comprise any work-up procedure the determination of geochemical parameters (alkylnaphthalene concentration ratios) is performed on the original, untreated crude oil samples. Accordingly, the analytical results reflect the original composition. The influence by evaporational losses in the laboratory is minimized.     

Quantitative screening and matrix effect studies of drug discovery compounds in monkey plasma using fast-gradient liquid chromatography/tandem mass spectrometry.

A higher-throughput bioanalytical method based on fast-gradient (1 min run time) high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) was developed for screen-type analyses of plasma samples from early drug discovery studies in support of exploratory pharmacodynamic studies. The HPLC system equipped with minibore column was interfaced with either atmospheric pressure chemical ionization (APCI) or electrospray (ESI) ionization techniques. The matrix ion suppression effect of both quantitative HPLC/MS/MS analyses was compared using the post-column infusion system. The use of the described methods provided advantages such as a shorter chromatographic region of ion suppression, less solvent consumption and shorter run times in comparison with standard analytical column HPLC/MS/MS methods. The analytical results obtained by both HPLC/MS/MS methods were in good agreement (within 15% of error) and displayed a good correlation with the pharmacodynamic outcome.     

Zirconia-based column high-performance liquid chromatography/atmospheric pressure photoionization tandem mass spectrometric analyses of drug molecules in rat plasma

A method using zirconia-based column high-performance liquid chromatography (HPLC) interfaced with an atmospheric pressure photoionization (APPI) source and a tandem mass spectrometer (MS/MS) was developed for the quantitative determination of new chemical entities in rat plasma in support of pharmacokinetics studies. The ionization suppression resulting from endogenous components of the biological matrices on the quantitative zirconia-based column HPLC/APPI-MS/MS method was investigated using the post-column infusion technique. The analytical results for 'rapid rat pharmacokinetics' for 12 drug discovery compounds, obtained by both silica-based phase (S-phase) and zirconia-based phase (Z-phase) chromatographic separation, are in good agreement in terms of accuracy. The application of a Z-phase column for high-temperature fast HPLC/MS/MS methods was explored to reduce the analysis time from 3 min to 30 s for column temperatures of 25-110 degrees C, respectively. The chromatographic retention times and peak responses of all analytes were found to be reproducible under high-temperature conditions following 100 continuous injections, with %CV less than 0.4 and 5, respectively.     

Determination of a 'GW cocktail' of cytochrome P450 probe substrates and their metabolites in plasma and urine using automated solid phase extraction and fast gradient liquid chromatography tandem mass spectrometry

A mass spectrometry based method for the simultaneous determination of an in vivo Greenford-Ware or 'GW cocktail' of CYP450 probe substrates and their metabolites in both human plasma and urine is described. The probe substrates, caffeine, diclofenac, mephenytoin, debrisoquine, chlorzoxazone and midazolam, together with their respective metabolites and stable isotope labelled internal standards, are simultaneously extracted from the biological matrix using solid phase extraction in 96-well microtitre plate format, automated by means of a custom built Zymark robotic system. The extracts are analysed by fast gradient high performance liquid chromatography (HPLC) with detection by tandem mass spectrometry (MS/MS) using thermally and pneumatically assisted electrospray ionisation in both positive and negative ion modes and selected reaction monitoring. The methods are specific, accurate and precise with intra- and inter-assay precision (%CV) of less than 15% for all analytes.     

Development and validation of LC-HRMS and GC-NICI-MS methods for stereoselective determination of MDMA and its phase I and II metabolites in human urine

3,4-Methylenedioxymethamphetamine (MDMA) is a racemic drug of abuse and its R- and S-enantiomers are known to differ in their dose-response curve. The S-enantiomer was shown to be eliminated at a higher rate than the R-enantiomer most likely explained by stereoselective metabolism that was observed in various in vitro experiments. The aim of this work was the development and validation of methods for evaluating the stereoselective elimination of phase I and particularly phase II metabolites of MDMA in human urine. Urine samples were divided into three different methods. Method A allowed stereoselective determination of the 4-hydroxy-3-methoxymethamphetamine (HMMA) glucuronides and only achiral determination of the intact sulfate conjugates of HMMA and 3,4-dihydroxymethamphetamine (DHMA) after C18 solid-phase extraction by liquid chromatography-high-resolution mass spectrometry with electrospray ionization. Method B allowed the determination of the enantiomer ratios of DHMA and HMMA sulfate conjugates after selective enzymatic cleavage and chiral analysis of the corresponding deconjugated metabolites after chiral derivatization with S-heptafluorobutyrylprolyl chloride using gas chromatography-mass spectrometry with negative-ion chemical ionization. Method C allowed the chiral determination of MDMA and its unconjugated metabolites using method B without sulfate cleavage. The validation process including specificity, recovery, matrix effects, process efficiency, accuracy and precision, stabilities and limits of quantification and detection showed that all methods were selective, sensitive, accurate and precise for all tested analytes.     

Achiral and chiral high-performance liquid chromatographic determination of flubendazole and its metabolites in biomatrices using UV photodiode-array and mass spectrometric detection

Flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, belongs to the group of benzimidazole anthelmintics, which are widely used in veterinary and human medicine. The phase I flubendazole biotransformation includes a hydrolysis of the carbamoyl methyl moiety accompanied by a decarboxylation (hydrolysed flubendazole) and a carbonyl reduction of flubendazole (reduced flubendazole). Flubendazole is a prochiral drug, hence a racemic mixture is formed during non-stereoselective reductions at the carbonyl group. Two bioanalytical HPLC methods were developed and validated for the determination of flubendazole and its metabolites in pig and pheasant hepatic microsomal and cytosolic fractions. Analytes were extracted from biomatrices into tert-butylmethyl ether. The first, achiral method employed a 250 mm x 4 mm column with octylsilyl silica gel (5 microm) and an isocratic mobile phase acetonitrile-0.025 M KH(2)PO(4) buffer pH 3 (28:72, v/v). Albendazole was used as an internal standard. The whole analysis lasted 27 min at a flow rate of 1 ml/min. The second, chiral HPLC method, was performed on a Chiralcel OD-R 250 mm x 4.6 mm column with a mobile phase acetonitrile-1 M NaClO(4) (4:6, v/v). This method enabled the separation of both reduced flubendazole enantiomers. The enantiomer excess was evaluated. The column effluent was monitored using a photodiode-array detector (scan or single wavelength at lambda=246 nm). Each of the analytes under study had characteristic UV spectrum, in addition, their chemical structures were confirmed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) experiments. Stereospecificity in the enzymatic carbonyl reduction of flubendazole was observed. While synthetic racemic mixture of reduced flubendazole was separated to equimolar amounts of both enantiomers, practically only one enantiomer was detected in the extracts from all incubates.     

Determination of S-carboxymethyl-L-cysteine and some of its metabolites in urine and serum by high-performance liquid chromatography using fluorescent pre-column labelling.

Pre-column labelling techniques are described for the determination of S-carboxymethyl-L-cysteine (CMC) and its metabolites in urine and plasma samples by high-performance liquid chromatography (HPLC) without prior extraction. All substances containing an amino group were converted into fluorescent fluorenylmethyl derivatives with 9-fluorenylmethyloxycarbonyl chloride (FMOC). Deaminated or N-acetylated carbocysteine metabolites were coupled with 1-pyrenyldiazomethane (PDAM) to give fluorescent PDAM esters. Similar results were obtained with the two commercially available and stable diazomethane derivatives PDAM and 9-anthryldiazomethane (ADAM). Following double derivatization with PDAM and FMOC, in a single chromatographic run with two fluorescence detectors connected in series, amines and amino(carboxylic) acids could be detected by their FMOC residues and, simultaneously, carboxylic acids were detected as fluorescent PDAM esters. The (R) and (S) enantiomers of the sulphoxides of CMC, of methylcysteine and of N-acetyl CMC were separated, although the reversed-phase HPLC system did not contain a chiral additive or stationary phase designed for the separation of enantiomers. The methods do not include liquid extraction steps and can therefore be performed either manually or automatically using an HPLC autosampler. These methods were used for the investigation of a disputed pharmacogenetic polymorphism of S-oxidation of CMC in humans, which until now has most often been studied using paper chromatography. The described techniques were applied to the determination of CMC and its metabolites in human urine and plasma samples.     

High Performance Liquid Chromatographic Analysis of Tolmetin,Indomethacin and Sulindac in Plasma

Abstract

Isocratic and gradient reversed phase high-performance liquid chromatographic (HPLC) methods for the quantitation of tolmetin, indomethacin, and sulindac and their respective metabolites in plasma were developed. Only the determination of the parent drugs was possible using the isocratic technique. Specific, simultaneous determination of each drug and its respective metabolites was achieved using the gradient technique. The effect of pH and ionic concentration of the mobile phase on retention time was studied. Statistical analysis demonstrated excellent precision and linearity over the following ranges: 1–40, 0.1–3, and 0.1–3 ug/ml plasma for tolmetin, indomethacin, and sulindac respectively. Both methods have been applied to the analysis of patient samples.     

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.     

Automated high-performance liquid chromatographic assay of enzymatic activities

High-performance liquid chromatography (HPLC) is a powerful technique which enables a reliable and quantitative determination of enzyme activities. The purpose of the work reported here was to develop an automatic assay of enzymatic activity. Using an automatic sample processor and injector, a program was developed which allows the complete automation of each step of analysis (calibration, enzymatic reaction, HPLC determination). This program can be adapted to different experimental requirements as each step can be performed independently and each input (time, volume, number of standards) is made by answering questions asked by instrument. Using this approach both kinetic and single-point determinations can be carried out, and in the latter case different samples can be analysed sequentially. This paper reports the automated analysis of trypsin.     

Simultaneous determination of a drug candidate and its metabolite in rat plasma samples using ultrafast monolithic column high-performance liquid chromatography/tandem mass spectrometry

An ultrafast bioanalytical method using monolithic column high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) was evaluated for the simultaneous determination of a drug discovery compound and its metabolite in plasma. Baseline separation of the two compounds was achieved with run times of 24 or 30 s under isocratic or gradient conditions, respectively. The monolithic column HPLC/MS/MS system offers shorter chromatographic run times by increasing flow rate without sacrificing separation power for the drug candidate and its biotransformation product (metabolite). In this work, the necessity for adequate chromatographic resolution was demonstrated because the quantitative determination of the drug-related metabolism product was otherwise hampered by interference from the dosed drug compound. The chromatographic performance of a monolithic silica rod column as a function of HPLC flow rates was investigated with a mixture of the drug component and its synthetic metabolite. The assay reliability of the monolithic column HPLC/MS/MS system was checked for matrix ionization suppression using the post-column infusion technique. The proposed methods were successfully applied to the analysis of study rat plasma samples for the simultaneous quantitation of both the dosed drug and its metabolite. The analytical results obtained by the proposed monolithic column methods and the 'standard' silica particle-packed HPLC column method were in good agreement, within 10% error.     

Pre-column derivatization with fluorescamine and high-performance liquid chromatographic analysis of drugs. Application to tocainide

The quantitative determination of tocainide, a new antiarrhythmic agent, by high-performance liquid chromatography (HPLC) is reported. The drug and a chemically similar internal standard were extracted from blood plasma with acetonitrile under salting-out conditions obtained by saturation of the aqueous medium with sodium chloride-sodium carbonate. The organic extract, without evaporation, was treated with borate buffer (pH 8.2) and fluorescamine. The resulting derivatives were chromatographed on an ODS reversed-phase column using a methanol-phosphate buffer (pH 7.0) mixture as mobile phase and were detected fluorometrically by monitoring the emission at 485 nm, with excitation at 395 nm. The intra-assay coefficients of variation were 3.0 and 4.3% for ten replicate 0.25 and 1.00 microgram/ml samples, respectively, and the inter-assay coefficient of variation was 3.6% for ten replicate 1.00 microgram/ml samples. The procedure is simple, rapid, sensitive, and specific. Several other drugs and drug metabolites also were derivatized with fluorescamine and chromatographed successfully. Pre-column derivatization with fluorescamine followed by HPLC with fluorometric detection may have significant advantages in drug analysis.     

Identification of new minor metabolites of penicillin G in human serum by multiple-stage tandem mass spectrometry

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were applied to characterize drug metabolites. Although these two methods have overcome the identification and structural characterization of metabolites analysis, they remain time‐consuming processes. In this study, a novel multiple‐stage tandem mass spectrometric method (MSⁿ) was evaluated for identification and characterization of new minor metabolism profiling of penicillin G, one of the β‐lactam antibiotics, in human serum. Seven minor metabolites including five phase I metabolites and two phase II metabolites of penicillin G were identified by using data‐dependent LC/MSⁿ screening in one chromatographic run. The accuracy masses of seven identified metabolites of penicillin G were also confirmed by mass spectral calibration software (MassWorks?). The proposed data‐dependent LC/MSⁿ method is a powerful tool to provide large amounts of the necessary structural information to characterize minor metabolite in metabolism profiling. Copyright © 2010 John Wiley & Sons, Ltd.