High-throughput cytochrome P450 (CYP) inhibition screening via a cassette probe-dosing strategy. VI. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2A6, 3A4, 2C9, 2D6 and 2E1

The inhibition potential of drugs towards five major human hepatic cytochrome P450 (CYP) isozymes (CYP2A6, 3A4, 2C9, 2D6, and 2E1) was investigated via cassette dosing of the five probe substrates (coumarin, midazolam, tolbutamide, dextromethorphan, and chlorzoxazone) in human liver microsomes using a 96-well plate format. After microsomal incubations had been terminated with formic acid, the five marker metabolites (7-hydroxycoumarin, 1'-hydroxymidazolam, 4-hydroxytolbutamide, dextrorphan, and 6-hydroxychlorzoxazone) were simultaneously quantified using direct injection/online guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS). Several advantages resulted from the use of a short C(18) guard cartridge (4 mm in length) for DI-GCE/MS/MS, including minimal sample preparation, fast online extraction, short analysis time (2.5 min), and minimal source contamination. In addition, this method demonstrated an inter-day accuracy range from -8.7 - 7.4% with a precision less than 8.3% for the quantification of all the marker metabolites. The inhibition assay for the five CYP isozymes was evaluated using their known selective inhibitors via individual and cassette dosing of the probe substrates. The IC(50) values measured via cassette dosing were consistent with those observed via individual dosing, which were all in agreement with the reported values. In addition, the validated assay was used to evaluate the inhibitory potential of 23 generic drugs (randomly selected) towards the five CYP isozymes. The results suggest the integration of the cassette dosing strategy and the DI-GCE/MS/MS method can provide a reliable in vitro approach to screening the inhibitory potential of new chemical entities, with maximal throughput and cost-effectiveness, in support of drug discovery and development.     

Application of polarity switching in the identification of the metabolites of RO9237

Polarity switching mass spectrometry is an efficient way to collect structural data on drug metabolites. The value of this approach is illustrated with the in vitro metabolism of RO9237. Metabolites are identified by positive and negative electrospray ionization (ESI) full scan mass spectrometry, MS/MS and MS(3) using unlabelled and (14)C-radiolabelled versions of the drug. Comparison of the relative detectability of these metabolites by +ESI and -ESI shows that neither ESI mode is universal. It is advantageous to screen for metabolites using both positive and negative ionization modes. This is especially true for phase II metabolism which tends to make molecules more polar and often more acidic. Identification of phase II metabolites also benefits greatly from MS(3) experiments because the conjugating groups typically are cleaved in MS/MS and information on the core structure is only obtained in MS(3). A special case of phase II metabolism is the generation of glutathione (GSH) conjugates from reactive metabolites. The detection of GSH conjugates also benefits from generating both positive and negative ESI mass spectral data.     

Imaging with mass spectrometry: Which ionization technique is best?

The use of mass spectrometry (MS) to acquire molecular images of biological tissues and other substrates has developed into an indispensable analytical tool over the past 25 years. Imaging mass spectrometry technologies are widely used today to study the in situ spatial distributions for a variety of analytes. Early MS images were acquired using secondary ion mass spectrometry and matrix-assisted laser desorption/ionization. Researchers have also designed and developed other ionization techniques in recent years to probe surfaces and generate MS images, including desorption electrospray ionization (DESI), nanoDESI, laser ablation electrospray ionization, and infrared matrix-assisted laser desorption electrospray ionization. Investigators now have a plethora of ionization techniques to select from when performing imaging mass spectrometry experiments. This brief perspective will highlight the utility and relative figures of merit of these techniques within the context of their use in imaging mass spectrometry.     

High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. I. Development of direct injection/on-line guard cartridge extraction/tandem mass spectrometry for the simultaneous detection of CYP probe substrates and their metabolites

A highly efficient direct injection/on-line guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS) method utilizing electrospray polarity switching was developed for the simultaneous detection of probe substrates and marker metabolites of seven human hepatic cytochrome P450 (CYP) isozymes: CYP1A2, 2A6, 3A4, 2C9, 2C19, 2D6 and 2E1. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for analysis by DI-GCE/MS/MS. This method employed an extremely short C(18) cartridge (4 mm in length) which allowed rapid cleanup of sample matrices while retaining the analytes an appropriate time (2. 0-2.2 min). From 1.5 to 2.7 min the effluent was directed to the mass spectrometer for detection otherwise diverted to waste. As a result of the efficient on-line extraction, matrix (e.g., salts and proteins) suppression was minimized. In addition, no visible source contamination was observed and system performance (chromatographic and mass spectrometric) did not significantly deteriorate after 500 consecutive injections. Electrospray polarity switching was strategically executed on a Micromass Quattro II mass spectrometer by establishing dummy ion transitions to protect the analytes from the interference of the overwhelming noise which was unavoidable for the first transition scanned following each polarity switch. This unique strategy led to the simultaneous detection of seven CYP probe substrates and seven corresponding marker metabolites (12 by positive mode and 2 by negative mode).     

Analyses of benzodiazepines and their metabolites in various biological matrices by LC-MS(/MS)

Benzodiazepines are among the most frequently prescribed drugs due to their sedative, hypnotic, anxiolytic, muscle relaxant and antiepileptic properties. Because of the high consumption of benzodiazepines worldwide, this class of drugs and their metabolites are frequently present in both clinical and forensic cases. For these reasons, the analysis of benzodiazepines and their metabolites in biological fluids is of great interest to clinicians and forensic toxicologists. This paper reviews procedures for multi-analyte single-stage (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) using different mass analyzers for the screening, identification and/or quantification of drugs, poisons and/or their metabolites in blood, plasma, serum or urine published since 2001. Basic information about the biosamples assayed, work-up, LC column, mobile phase, ionization type, mass spectral detection mode, matrix effects and validation data for each procedure is summarized. The feasibility of using LC-MS(/MS) techniques to identify and quantify benzodiazepines and their metabolites is also discussed.     

Identification of phase I and phase II metabolites of benfluron and dimefluron in rat urine using high-performance liquid chromatography/tandem mass spectrometry

Biotransformation products of two potential antineoplastic agents, benfluron and dimefluron, are characterized using our integrated approach based on the combination of high-performance liquid chromatography (HPLC) separation of phase I and phase II metabolites followed by photodiode-array UV detection and electrospray ionization tandem mass spectrometry (MS/MS). High mass accuracy measurement allows confirmation of an elemental composition and metabolic reactions according to exact mass defects. The combination of different HPLC/MS/MS scans, such as reconstructed ion current chromatograms, constant neutral loss chromatograms or exact mass filtration, helps the unambiguous detection of low abundance metabolites. The arene oxidation, N-oxidation, N-demethylation, O-demethylation, carbonyl reduction, glucuronidation and sulfation are typical mechanisms of the metabolite formation. The interpretation of their tandem mass spectra enables the distinction of demethylation position (N- vs. O-) as well as to differentiate N-oxidation from arene oxidation for both phase I and phase II metabolites. Two metabolic pathways are rather unusual for rat samples, i.e., glucosylation and double glucuronidation. The formation of metabolites that lead to a significant change in the chromophoric system of studied compounds, such as the reduction of carbonyl group in 7H-benzo[c]fluorene-7-one chromophore, is reflected in their UV spectra, which provides valuable complementary information to MS/MS data.     

Metabolite identification of artemether by data-dependent accurate mass spectrometric analysis using an LTQ-Orbitrap hybrid mass spectrometer in combination with the online hydrogen/deuterium exchange technique

Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is a first-line antimalarial drug used in areas of multi-drug resistance. Artemisinin drugs can be metabolized extensively in vivo and this seems related to their autoinduction pharmacokinetics. In the present study, the metabolite identification of ARM was performed by the generic data-dependent accurate mass spectrometric analysis, using high-resolution (HR) liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) and tandem mass spectrometry (MS/MS) LTQ-Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange for rapid structural characterization. The LC separation was improved allowing the separation of ARM parent drugs and their metabolites from their diastereomers. A total of 77 phase I metabolites of ARM were identified in rat liver microsomal incubates and rat urine, including dihydroartemisinin and artemisinin. In rat bile, 12 phase II metabolites were found. Accurate mass data were obtained in both full scan and HR-MS/MS mode to support assignments of metabolite structures. Online H/D exchange LC/HR-ESI-MS experiments provided additional evidence in differentiating dihydroxylated deoxy-ARM from mono-hydroxylated ARM. The results showed the main phase I metabolites of artemether are hydroxylated, dehydro, demethylated and deoxy products, and they will undergo subsequent phase II glucuronidation processes. Most metabolites were reported for the first time. This study also demonstrated the effectiveness of high-resolution mass spectrometry in combination with an online H/D exchange LC/HR-MS(n) technique in rapid identification of drug metabolites.     

Database-assisted global metabolomics profiling of pleural effusion induced by tuberculosis and malignancy

Database-assisted global metabolomics has received growing attention due to its capability for unbiased identification of metabolites in various biological samples. Herein, we established a mass spectrometry (MS)-based database-assisted global metabolomics method and investigated metabolic distance between pleural effusion induced by tuberculosis and malignancy, which are difficult to be distinguished due to their similar clinical symptoms. The present method utilized a liquid chromatography (LC) system coupled with high resolution mass spectrometry (MS) working on full scan and data dependent mode for data acquisition. Unbiased identification of metabolites was performed through mass spectral searching and then confirmed by using authentic standards. As a result, a total of 194 endogenous metabolites were identified and 33 metabolites were found to be differentiated between tuberculous and malignant pleural effusions. These metabolites involved in tryptophan catabolism, bile acid biosynthesis, and β-oxidation of fatty acids, provided non-invasive biomarkers for differentiation of the pleural effusion samples with high sensitivity and specificity.     

Simultaneous quantitative cassette analysis of drugs and detection of their metabolites by high performance liquid chromatography/ion trap mass spectrometry

A method using high performance liquid chromatography (HPLC) coupled with ion trap mass spectrometry (MS) for simultaneous quantification of multiple drugs and detection of their metabolites is described. The new approach offers a significant increase in analytical throughput and is illustrated with analysis of the in vitro metabolism of 19 alpha-1a receptor antagonists. The compounds were separated into four cassette groups by using a computer program as well as by manual examination. The samples from incubation with dog liver microsomes were pooled into the designed cassette groups and analyzed by HPLC/electrospray (ESI) ion trap MS in full-scan mode. The metabolic stability of the drugs was determined by comparing their signals after incubation for 0 and 60 min, respectively. The quantitative results from the cassette analysis procedure agreed well with those obtained from conventional discrete analysis. In addition, the technique allowed simultaneous detection of metabolites formed during the same incubation without having to reanalyze the samples. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent multi-stage tandem mass spectrometry (MS(n)) on the ion trap instrument allowed confirmation of the detected metabolites.     

Recently developed GC/MS and LC/MS methods for determining NSAIDs in water samples

Pharmaceuticals have become major targets in environmental chemistry due to their presence in aquatic environments (following incomplete removal in wastewater treatment or point-source contaminations), threat to drinking water sources and concern about their possible effects to wildlife and humans. Recently several methods have been developed for the determination of drugs and their metabolites in the lower nanogram per litre range, most of them using solid-phase extraction (SPE) or solid-phase microextraction (SPME), derivatisation and finally gas chromatography mass spectrometry (GC-MS), gas chromatography tandem mass spectrometry (GC-MS/MS) and liquid chromatography electrospray tandem mass spectrometry (LC-ES/MS/MS). Due to the elevated polarity of non-steroidal anti-inflamatory drugs (NSAIDs), analytical techniques based on either liquid chromatography coupled to mass spectrometry (LC-MS) and gas chromatography coupled to mass spectrometry (GC-MS) after a previous derivatisation step are essential. The most advanced aspects of current GC-MS, GC-MS/MS and LC-MS/MS methodologies for NSAID analysis are presented.     

Strategy of using microsome-based metabolite production to facilitate the identification of endogenous metabolites by liquid chromatography mass spectrometry

One of the challenges in metabolomic profiling of complex biological samples is to identify new and unknown compounds. Typically, standards are used to help identify metabolites, yet standards cannot be purchased or readily synthesized for many unknowns. In this work we present a strategy of using human liver microsomes (HLM) to metabolize known endogenous human metabolites (substrates), producing potentially new metabolites that have yet to be documented. The metabolites produced by HLM can be tentatively identified based on the associated substrate structure, known metabolic processes, tandem mass spectrometry (MS/MS) fragmentation patterns and, if necessary, accurate mass measurements. Once identified, these metabolites can be used as references for identification of the same compounds in complex biological samples. As a proof of principle, a total of 9 metabolites have been identified from individual HLM incubations using 5 different substrates. Each metabolite was used as a standard. In the analysis of human urine sample by liquid chromatography MS/MS, four spectral matches were found from the 9 microsome-produced metabolite standards. Two of them have previously been documented as endogenous human metabolites, the third is an isomer of a microsome-metabolite and the fourth compound has not been previously reported and is also an isomer of a microsome-metabolite. This work illustrates the feasibility of using microsome-based metabolism to produce metabolites of endogenous human metabolites that can be used to facilitate the identification of unknowns in biological samples. Future work on improving the performance of this strategy is also discussed.     

A UPLC-MS/MS assay of the "Pittsburgh cocktail": six CYP probe-drug/metabolites from human plasma and urine using stable isotope dilution

The efficiency of drug metabolism by a single enzyme can be measured as the fractional metabolic clearance which can be used as a measure of whole body activity for that enzyme. Measurement of activity of multiple enzymes simultaneously is feasible using a cocktail approach, however, analytical approach using different assays for drug probes can be cumbersome. A quantitative ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based method for the rapid measurement of six cytochrome P450 (CYP) probe drugs and their relevant metabolites is described. The six specific probe substrates/metabolites are caffeine/paraxanthine (CYP1A2), flurbiprofen/4'-hydroxyflurbiprofen (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), debrisoquine/4-hydroxydebrisoquine (CYP2D6), chlorzoxazone/6'-hydroxychlorzoxazone (CYP2E1) and dapsone/N-monoacetyldapsone (NAT2). These probes were quantified by stable isotope dilution from plasma and urine. The present workflow provides a robust, fast and sensitive assay for the "Pittsburgh cocktail", and has been successfully applied to a clinical phenotyping study of liver disease. A representative group of 17 controls and patients with chronic liver disease were administered orally caffeine (100 mg), chlorzoxazone (250 mg), debrisoquine (10 mg), mephenytoin (100 mg), flurbiprofen (50 mg) and dapsone (100 mg). Urine (0 through 8 h) and plasma (4 and 8 h) samples were analyzed for drug/metabolite amounts by stable isotope dilution UPLC-MS/MS. The phenotypic activity of drug metabolizing enzymes was investigated with 17 patient samples. Selected reaction monitoring (SRM) was optimized for each drug and metabolite. In the method developed, analytes were resolved by reversed-phase by development of a gradient using a water/methanol solvent system. SRM of each analyte was performed in duplicate on a triple quadrupole mass spectrometer utilizing an 8 min analytical method each, one with the source operating in the positive mode and one in the negative mode, using the same solvent system. This method enabled quantification of each drug (caffeine, chlorzoxazone, debrisoquine, mephenytoin, flurbiprofen, and dapsone) and its resulting primary metabolite in urine or plasma in patient samples. The method developed and the data herein demonstrate a robust quantitative assay to examine changes in CYP enzymes both independently or as part of a cocktail. The clinical use of a combination of probe drugs with UPLC-MS/MS is a highly efficient tool for the assessment of CYP enzyme activity in liver disease.     

Mass spectrometric analysis of cyclofenil and its metabolites in human urine

Using gas chromatography/electron impact-mass spectrometry (GC/EI-MS) and high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), the structures of cyclofenil metabolites in human urine have been assigned. The hydroxyl metabolites liberated from the glucuronide conjugates after acid hydrolysis were characterized as the trimethylsilyl (O-TMS) derivatives using GC/MS. The conjugate glucuronide forms were detected without hydrolysis by HPLC/MS. Cyclofenil was not observed in urine. Tentative structures for the two metabolites are proposed.     

Application of a generic fast gradient liquid chromatography tandem mass spectrometry method for the analysis of cytochrome P450 probe substrates

A liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the fast routine analysis of selected CYP450 probe substrate metabolites in microsomal incubations, with no sample pretreatment. This has allowed fast and simple assessment of the potential effects which drug candidates may or may not have on the metabolism of specific CYP450 probe substrates, providing information which can then be used to rationalize in vivo interaction studies required in the clinic. This methodology takes advantage of fast gradient chromatography as a generic means of sample separation and analysis. It provides high throughput analysis compared to conventional gradient HPLC, with no significant loss in chromatographic performance. © 1998 John Wiley & Sons, Ltd.     

Metabolic profile of glyburide in human liver microsomes using LC‐DAD‐Q‐TRAP‐MS/MS

The sulfonylurea urea drug glyburide (glibenclamide) is widely used for the treatment of diabetes milletus and gestational diabetes. In previous studies monohydroxylated metabolites were identified and characterized for glyburide in different species, but the metabolite owing to the loss of cyclohexyl ring was identified only in mouse. Glyburide upon incubation with hepatic microsomes resulted in 10 metabolites for human. The current study identifies new metabolites of glyburide along with the hydroxylated metabolites that were reported earlier. The newly identified drug metabolites are dihydroxylated metabolites, a metabolite owing to the loss of cyclohexyl ring and one owing to hydroxylation with dehydrogenation. Among the 10 identified metabolites, there were six monohydroxylated metabolites, one dihydroxylated metabolite, two metabolites owing to hydroxylation and dehydrogenation, and one metabolite owing to the loss of cyclohexyl ring. New metabolites of glyburide were identified and characterized using liquid chromatography–diode array detector–quadruple‐ion trap–mass spectrometry/mass spectrometry (LC‐DAD‐Q‐TRAP‐MS/MS). An enhanced mass scan–enhanced product ion scan with information‐dependent acquisition mode in a Q‐TRAP‐MS/MS system was used to characterize the metabolites. Liquid chromatography with diode array detection was used as a complimentary technique to confirm and identify the metabolites. Metabolites formed in higher amounts were detected in both diode array detection and mass spectrometry detection. Copyright © 2012 John Wiley & Sons, Ltd.     

GC/MS characterization of urinary metabolites of doxylamine succinate: Identification of the aglycones formed from intestinal microflora metabolism of the polar glucuronide metabolites

This study describes the use of high performance liquid chromatography (HPLC) and capillary gas chromatography/mass spectrometry (GC/MS) in the characterization of polar glucuronide conjugates of doxylamine and their subsequent aglycones following deconjugation. Rat urinary extracts which contained doxylamine and both nonconjugated and conjugated doxylamine metabolites, were examined by HPLC before and after incubation with rat intestinal microflora. The subsequent deconjugated urinary metabolites and the nonconjugated products remaining in the urinary extracts were then isolated, acetylated, and assayed by GC/MS. Incubation with the intestinal microflora indicated that anaerobic bacteria were capable of effecting hydrolytic cleavage of these polar O-glucuronide metabolites of doxylamine and its demethylated products to their subsequent aglycones. GC/MS analysis was performed using a fused silica DB-5 GC column and was utilized for the identification of these deconjugated metabolites.     

Development and validation of a LC-MS/MS method based on a new 96-well Hybrid-SPE<Superscript>™</Superscript>-precipitation technique for quantification of CYP450 substrates/metabolites in rat plasma

A rapid and selective high-throughput HESI-LC-MS/MS method for determining eight cytochrome P450 probe drugs in one-step extraction and single run was developed and validated. The four specific probe substrates midazolam, dextromethorphan, tolbutamide, theophylline and their metabolites 1-hydroxymidazolam, dextrorphan, hydroxyl(methyl)tolbutamide, 1,3-dimethyluric acid, together with the deuterated internal standards, were extracted from rat plasma using a novel 96-well Hybrid-SPE™-precipitation technique. The bioanalytical assay was based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using selected reaction monitoring for drug (-metabolite) quantification. All analytes were separated simultaneously in a single run that lasted less than 11 min. The intra- and inter-day precisions for all eight substrates/metabolites were 1.62–12.81% and 2.09–13.02%, respectively, and the relative errors (accuracy) for the eight compounds ranged from −9.62% to 7.48% and −13.84% to 8.82%. Hence, the present method provides a robust, fast and reproducible analytical tool for the evaluation of four major drug metabolising cytochrome P450 (3A4, 2C9, 1A2 and 2D6) activities with a cocktail approach in rats to clarify herb–drug interactions. The method can be used as a basic common validated high-throughput analytical assay for in vivo interaction studies.     

Mass spectrometry applied to the analysis of estrogens in the environment

Environmental analytical chemistry has recently changed focus from analysis of non-polar, persistent contaminants (e.g. polychlorinated biphenyls and dioxins) to more polar and labile compounds that interfere with biological processes. For example, natural and synthetic estrogens and their metabolites have been detected in sewage treatment plant effluents at nanogram/liter concentrations that are similar to those at which both total sex reversal and intersex (containing both testes and ova) is induced in fish exposed to these compounds in laboratory experiments. The development of techniques for the analysis of natural and synthetic estrogens in biological fluids (i.e. serum and urine) has been a priority in the biomedical field. However, the recent recognition that estrogen hormones are contaminants in the environment that may contribute to endocrine disruption has focused attention on the need for highly sensitive and specific techniques that are applicable for trace analysis in complex environmental matrices. Three optimized mass spectrometric protocols have been developed for the determination and quantitation of steroid hormones in environmental matrices using gas chromatography/tandem mass spectrometry (GC/MS/MS), liquid chromatography/mass spectrometry selected ion monitoring, (LC/MS - SIM) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The advantages and disadvantages of each method are presented.     

Hyphenated mass spectrometric techniques-indispensable tools in clinical and forensic toxicology and in doping control

Hyphenated mass spectrometric techniques, particularly gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), are indispensable tools in clinical and forensic toxicology and in doping control owing to their high sensitivity and specificity. They are used for screening, library-assisted identification and quantification of drugs, poisons and their metabolites, prerequisites for competent expertise in these fields. In addition, they allow the study of metabolism of new drugs or poisons as a basis for developing screening procedures in biological matrices, most notably in urine, or toxicological risk assessment. Concepts and procedures using GC/MS and LC/MS techniques in the areas of analytical toxicology and the role of mass spectral libraries are presented and discussed in this feature article. Finally, perspectives of their future position are discussed.     

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。