Characterization of the metabolites of rosmarinic acid in human liver microsomes using liquid chromatography combined with electrospray ionization tandem mass spectrometry

Rosmarinic acid (RA) is a phenolic acid originally isolated from the herb medicine Rosmarinus officinalis. The purpose of this study was to identify the metabolites of RA. RA was incubated with human liver microsomes in the presence of β-nicotinamide adenine dinucleotide phosphate tetrasodium salt and/or uridine diphosphate glucuronic acid using glutathione (GSH) as a trapping agent. After 60-min incubation, the samples were analyzed using high-resolution liquid chromatography tandem mass spectrometry. Under the current conditions, 14 metabolites were detected and identified. Our data revealed that RA was metabolized through the following pathways: the first pathway is the oxidation of catechol to form ortho-quinone intermediates, which react with GSH to form mono-GSH adducts (M1, M2, and M3) and bis-GSH adducts (M4 and M5); the second pathway is conjugation with glucuronide to yield acylglucuronide (M7), which further reacts with GSH to form RA-S-acyl-GSH adduct (M9); the third pathway is hydroxylation to form M10, M11, and M12, which further react with GSH to form mono-GSH adducts (M13 and M14); the fourth pathway is conjugation with GSH through Michael addition (M6); the fifth pathway is conjugation with glucuronidation, forming M8, which is the major metabolic pathway of RA.     

Characterization of the metabolites of H3B-6545 in vitro and in vivo by using ultra-high performance liquid chromatography combined with electrospray ionization linear ion trap-orbitrap tandem mass spectrometry

H3B-6545 is a selective ERα covalent antagonist, which has been demonstrated to be effective in anti-tumor. To fully understand its mechanism of action, it is necessary to investigate the in vitro and in vivo metabolic profiles. For in vitro metabolism, H3B-6545 (50 μM) was incubated with the hepatocytes of rat and human for 2 h. For in vivo metabolism H3B-6545 was orally administered to rats at a single dose of 10 mg/kg, and plasma, urine and fecal samples were then collected. All samples were analyzed by using ultra-high performance liquid chromatography combined with linear ion trap-orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MS) operated in positive ion mode. The structures of the metabolites were elucidated by comparing their MS and MS² spectra with those of parent drug. A total of 11 metabolites, including a GSH adduct, were detected and structurally identified. M2, M7 and M8 were further unambiguously identified by using reference standards. Among these metabolites, M1, M5, M7 and M10 were newly found and reported for the first time. The metabolic pathways of H3B-6545 included deamination (M8 and M9), dealkylation (M2, M3 and M10), N-hydroxylation (M6), hydroxylation (M1 and M4), formation of amide derivatives (M5 and M7) and GSH conjugation (G1).     

Characterization of in vitro metabolites of irisflorentin by rat liver microsomes using high‐performance liquid chromatography coupled with tandem mass spectrometry

Belamcanda chinensis has been extensively used as antibechic, expectorant and anti‐inflammatory agent in traditional medicine. Irisflorentin is one of the major active ingredients. However, little is known about the metabolism of irisflorentin so far. In this work, rat liver microsomes (RLMs) were used to investigate the metabolism of this compound for the first time. Seven metabolites were detected. Five of them were identified as 6,7‐dihydroxy‐5,3′,4′,5′‐tetramethoxy isoflavone (M1), irigenin (M2), 5,7,4′‐trihydroxy‐6,3′,5′‐trimethoxy isoflavone (M3), 6,7,4′‐trihydroxy‐5,3′,5′‐trimethoxy isoflavone (M4) and 6,7,5′‐trihydroxy‐5,3′,4′‐trimethoxy isoflavone (M5) by means of NMR and/or HPLC‐ESI‐MS. The structures of M6 and M7 were not elucidated because they produced no MS signals. The predominant metabolite M1 was noted to be a new compound. Interestingly, it was found to possess anticancer activity much higher than the parent compound. The enzymatic kinetic parameters of M1 revealed a sigmoidal profile, with V_max = 12.02 μm /mg protein/min, K_m = 37.24 μm , CL_int = 0.32 μL/mg protein/min and h = 1.48, indicating the positive cooperation. For the first time in this work, a new metabolite of irisflorentin was found to demonstrate a much higher biological activity than its parent compound, suggesting a new avenue for the development of drugs from B. chinensis, which was also applicable for other herbal plants. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization of the metabolite of AdipoRon in rat and human liver microsomes by ultra‐high‐performance liquid chromatography combined with Q‐Exactive Orbitrap tandem mass spectrometry

AdipoRon is an orally active adiponectin receptor agonist. The aim of this study was to characterize the metabolites of AdipoRon in rat and human liver microsomes using ultra‐high performance liquid chromatography combined with Q‐Exactive Orbitrap tandem mass spectrometry (UPLC‐Q‐Exactive‐Orbitrap‐MS) together with data processing techniques including extracted ion chromatograms and a mass defect filter. AdipoRon (10 μm ) was incubated with liver microsomes in the presence of NADPH and this resulted in a total of 11 metabolites being detected. The identities of these metabolites were characterized by comparing their accurate masses and fragment ions as well as their retention times with those of AdipoRon using MetWorks software. Metabolites M1–M3, M6, and M8–M11 were identified for the first time. Metabolite M4, the major metabolite both in rat and human liver microsomes, was further confirmed using the reference standard. Our results revealed that the metabolic pathways of AdipoRon in liver microsomes were N‐dealkylation (M2), hydroxylation (M, M5–M9), carbonyl reduction (M4) and the formation of amide (M10 and M11). Our results provide valuable information about the in vitro metabolism of AdipoRon, which would be helpful for us to understand the mechanism of the elimination of AdipoRon and, in turn, its effectiveness and toxicity.     

Characterization of in vitro metabolites of deoxypodophyllotoxin in human and rat liver microsomes using liquid chromatography/tandem mass spectrometry

The in vitro metabolism of deoxypodophyllotoxin (DPT), a medicinal herbal product isolated from Anthriscus sylvestris (Apiaceae), was investigated in rats and human microsomes and human recombinant cDNA-expressed CYPs. The incubation of DPT with pooled human microsomes in the presence of NADPH generated five metabolites while its incubation with dexamethasone (Dex)-induced rat liver resulted in seven metabolites (M1-M7) with major metabolic reactions including mono-hydroxylation, O-demethylation and demethylenation. Reasonable structures of the seven metabolites of DPT could be proposed, based on the electrospray tandem mass spectra. Chemical inhibition by ketoconazole and metabolism studies with human recombinant cDNA-expressed CYPs indicated that CYP 3A4 and 2C19 are the major CYP isozymes in the metabolism of DPT in human liver microsomes.     

Characterization of in vitro metabolites of rutaecarpine in rat liver microsomes using liquid chromatography/tandem mass spectrometry

Following incubation of rutaecarpine, a new cyclooxygenase-2 inhibitor, with rat liver microsomes, the structures of the metabolites were characterized by liquid chromatography with tandem mass spectrometry. Nine metabolites corresponding to mono- or dihydroxylated rutaecarpine were formed. Characteristic product ions for the identification of rutaecarpine metabolites were observed at m/z 136, 158 and 286. The loss of water led to the fragment ion at m/z 286, indicating the hydroxylation of the aliphatic ring. The fragment ion at m/z 136 indicated the hydroxylated form of the phenyl group of the quinazolinone moiety, while that at m/z 158 indicated the hydroxylated form of the aromatic ring of the indole moiety.     

Metabolic profiles of ribociclib in rat and human liver microsomes using liquid chromatography combined with electrospray ionization high-resolution mass spectrometry

Ribociclib is a highly specific CDK4/6 inhibitor. Determination of the metabolism of ribociclib is required during the drug development stage. In this study, metabolic profiles of ribociclib were investigated using rat and human liver microsomes. Metabolites were structurally identified by liquid chromatography electrospray ionization high-resolution mass spectrometry operated in positive-ion mode. The metabolites were characterized by accurate masses, MS² spectra and retention times. With rat and human liver microsomes, a total of 10 metabolites were detected and further identified. No human-specific metabolites were detected. The metabolic pathways of ribociclib were oxygenation, demethylation and dealkylation. Most importantly, two glutathione (GSH) adducts were identified in human liver microsomes fortified with GSH. The formation of the GSH adducts was hypothesized to be through the oxidation of electron-rich 1,4-benzenediamine to a 1,4-diiminoquinone intermediate, which is highly reactive and can be trapped by GSH to form stable metabolites. The current study provides an overview of the metabolic profiles of ribociclib in vitro, which will be of great help in understanding the efficacy and toxicity of this drug.     

In vitro metabolite identification using fast gradient high performance liquid chromatography combined with tandem mass spectrometry.

Metabolite identification can provide critical information for further synthesis during the lead optimization stage of drug discovery. Fast chromatographic gradient methodology was explored to reduce the analysis time for metabolite identification by liquid chromatography/tandem mass spectrometry while maintaining chromatographic separation of all components of interest. Short HPLC columns (2 cm) containing 3 microm particles were used and the chromatographic conditions involved elevated flow rates (1.0-2.5 mL/min) and fast, full range linear gradients (<2 min). Detection was accomplished by fast scanning triple quadrupole mass spectrometers or a quadrupole time-of-flight mass spectrometer. Analysis of liver microsomal incubations of several compounds indicates that separation of isomeric metabolites as well as the parent compound is feasible with analysis times not exceeding 2 minutes.     

Simultaneous determination of prochlorperazine and its metabolites in human plasma using isocratic liquid chromatography tandem mass spectrometry

Oral prochlorperazine (PCZ), an antiemetic, undergoes extensive first-pass metabolism. The study developed a simultaneous analytical method for PCZ and its major metabolites, prochlorperazine sulfoxide (PCZSO), N-demethylprochlorperazine (NDPCZ) and 7-hydroxyprochlorperazine (PCZOH), in human plasma using an isocratic liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Deproteinized plasma specimens were separated using a 3 μm particle size octadecylsilyl column, and the run time was 10 min. The calibration curves were linear over the concentration ranges of 0.01-40 μg/L for PCZ, NDPCZ and PCZOH, and 0.05-80 μg/L for PCZSO. The intra- and inter-assay precisions and accuracies were within 7.0 and 99-104% and within 9.0 and 99-105%, respectively. The lower limits of quantification in human plasma were 10 ng/L for PCZ, NDPCZ and PCZOH, and 50 ng/L for PCZSO. The validated method was applied to the determination of plasma samples in 37 cancer patients receiving PCZ. Large interindividual variations were observed in plasma concentrations of PCZ, PCZSO, NDPCZ and PCZOH (relative standard deviation, 89.4, 88.7, 86.4 and 78.2%, respectively). In conclusion, this simultaneous LC-MS/MS method with acceptable analytical performance can be helpful for evaluating the pharmacokinetics of PCZ, including the determination of its metabolites in cancer patients and in clinical research.     

Screening and characterization of reactive metabolites using glutathione ethyl ester in combination with Q-trap mass spectrometry

The present study describes a new analytical approach for the detection and characterization of chemically reactive metabolites using glutathione ethyl ester (GSH-EE) as the trapping agent in combination with hybrid triple quadrupole linear ion trap mass spectrometry. Polarity switching was applied between a negative precursor ion (PI) survey scan and the positive enhanced product ion (EPI) scan. The negative PI scan step was carried out monitoring the anion at m/z 300, corresponding to deprotonated gamma-glutamyl-dehydroalanyl-glycine ethyl ester originating from the GSH-EE moiety. Samples resulting from incubations in the presence of GSH-EE were cleaned and concentrated by solid-phase extraction, followed by the PI-EPI analysis. Unambiguous identification of GSH-EE-trapped reactive metabolites was greatly facilitated by the unique survey scan of the anion at m/z 300, which achieved less background interference, in particular, from endogenous glutathione adducts present in human liver microsomes. Further structural characterization was achieved by analyzing positive MS(2) spectra that featured rich fragments without mass cutoff and were acquired in the same liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The effectiveness and reliability of this approach was evaluated using a number of model compounds in human liver microsomal incubations, including acetaminophen, amodiaquine, carbamazepine, 4-ethylphenol, imipramine and ticlopidine. In addition, iminoquinone reactive metabolites of mianserin were trapped and characterized for the first time using this method. Compared to neutral loss (NL) scanning assays using GSH as the trapping agent, the results have demonstrated superior selectivity, sensitivity, and reliability of this current approach.     

高效液相色谱-质谱/质谱法测定蛋黄粉中硝基呋喃代谢产物

采用固相基质分散技术, 液-液分配净化, 同位素内标定量, 建立了蛋黄粉中呋喃它酮、呋喃西林、呋喃妥因和呋喃唑酮等硝基呋喃类药物代谢产物残留的高效液相色谱-质谱/质谱测定方法. 方法测定低限为0.5 μg/kg;线性范围为0.5～6.0 μg/kg;室内验证回收率范围为90.06%～109.8%;相对标准偏差2.0%～7.7%. 该方法适用于残留检测实验室对蛋黄粉类基质中硝基呋喃代谢产物的监控检测.     

超高效液相色谱-电喷雾串联质谱法测定生活饮用水中49种药物及5种代谢物

建立了超高效液相色谱-电喷雾串联质谱(UPLC-ESI MS/MS)分析生活饮用水中54种药物的方法。采用HLB固相萃取柱对水样中的目标化合物进行富集净化,以5 mL甲醇洗脱;洗脱液用氮气吹至近干,用0.4 mL 0.1%甲酸水溶液定容,上机分析;ACQUITY UPLC^TMBEH C₁₈柱用作色谱分离,以0.1%甲酸水溶液-甲醇为流动相进行梯度洗脱;多反应监测(MRM)模式进行检测;目标药物使用基质外标法定量。54种药物在自备井水、市政末梢水和地表水中的加标回收率分别为58.7%~104.4%、53.1%~109.5%和50.7%~118.8%,相对标准偏差(n=6)分别为0.3%~12.8%、1.0%~15.5%和0.4%~19.3%;方法定量限为0.002~5.000 ng/L。将建立的方法应用于北京部分自备井水、市政末梢水和地表水样品的分析,结果在自备井水样中检出26种药物。     

Structure elucidation of aplidine metabolites formed in vitro by human liver microsomes using triple quadrupole mass spectrometry

The cyclic depsipeptide aplidine is a new anti-cancer drug of marine origin. Four metabolites of this compound were found after incubation with pooled human microsomes using gradient high-performance liquid chromatography with ultraviolet detection. After chromatographic isolation, the metabolites have been identified using nano-electrospray triple quadrupole mass spectrometry. A highly specific sodium-ion interaction with the cyclic structure opens the depsipeptide ring, and cleavage of the amino acid residues gives sequence information when activated by collision-induced dissociation in the second quadrupole. The aplidine molecule could undergo the following metabolic reactions: hydroxylation at the isopropyl group (metabolites apli-h 1 and apli-h 2); C-dealkylation at the N(Me)-leucine group (metabolite apli-da); hydroxylation at the isopropyl group and C-dealkylation at the N(Me)-leucine group (metabolite apli-da/h), and C-demethylation at the threonine group (metabolite apli-dm). The identification of these metabolites formed in vitro may greatly aid the elucidation of the metabolic pathways of aplidine in humans.     

In vitro metabolic fate of alizapride: evidence for the formation of reactive metabolites based on liquid chromatography‐tandem mass spectrometry

The study of the formation of reactive metabolites during drug metabolism is one of the major areas of research in drug development since the link between reactive metabolites and drug adverse effects was well recognized. In particular, it has been shown that acrolein, a reactive carbonyl species sharing carbonylating and alkylating properties, binds covalently to nucleophilic sites in proteins, causing cellular damage. Alizapride, (±)‐6‐methoxy‐N‐{[1‐(prop‐2‐en‐1‐yl)‐pyrrolidin‐2‐yl]methyl}‐1H‐benzotriazole‐5‐carboxamide, is a N‐allyl containing dopamine antagonist with antiemetic properties for which no data concerning its metabolic fate are so far reported. The study of the in vitro metabolism of alizapride showed the formation of acrolein during the oxidative N‐deallylation. Moreover, the formation of an epoxide metabolite has been also described suggesting its role as a putative structural alert. The reactivity of the acrolein and the epoxide generated in alizapride metabolism was demonstrated by the formation of the corresponding adducts with nucleophilic thiols. Overall, ten metabolites have been identified and characterized by electrospray ionization tandem mass spectrometry analysis allowing to propose an in vitro metabolic scheme for alizapride. At the best of our knowledge, this is the second case of a drug involved in the generation of acrolein during its metabolism being the first represented by cyclophosphamide. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of seven metabolites of oxyresveratrol in rat urine and bile using liquid chromatography/tandem mass spectrometry

Oxyresveratrol (trans‐2,4,3′,5′‐tetrahydroxystilbene) is a major compound isolated from Smilax china, a Chinese herbal medicine. The rat urine and bile samples were pretreated by solid‐phase extraction method after oral administration at a dose of 100 mg/kg of oxyresveratrol. Seven metabolites were identified by LC‐MS/MS method with electrospray ionization in negative ion mode. The results indicated that main metabolites of oxyresveratrol were monoglucuronided and monosulfated oxyresveratrol. Based on the results, the metabolic pathway of oxyresveratrol in rat urine and bile was proposed. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of benzene,toluene, ethylbenzene,and xylene metabolites in human urine using electromembrane extraction combined with liquid chromatography and tandem mass spectrometry

For the first time, electromembrane extraction combined with liquid chromatography and tandem mass spectrometry was applied for the determination of urinary benzene, toluene, ethylbenzene, and xylene metabolites. S‐Phenylmercapturic acid, hippuric acid, phenylglyoxylic acid, and methylhippuric acid isomers were extracted from human urine through a supported liquid membrane consisting of 1‐octanol into an alkaline acceptor solution filling the inside of a hollow fiber by application of an electric field. Various extraction factors were investigated and optimized using response surface methodology, the statistical method. The optimum conditions were established to be 300 V applied voltage, 15 min extraction time, 1500 rpm stirring speed, and 5 mM ammonium acetate (pH 10.2) acceptor solution. The method was validated with respect to selectivity, linearity, accuracy, precision, limit of detection, limit of quantification, recovery, and reproducibility. The results showed good linearity (r² > 0.995), precision, and accuracy. The extract recoveries were 52.8–79.0%. Finally, we applied this method to real samples and successfully measured benzene, toluene, ethylbenzene, and xylene metabolites.     

Simultaneous determination of pharmaceuticals and some of their metabolites in wastewaters by high performance liquid chromatography with tandem mass spectrometry

The present work describes the development of a sensitive and reliable analytical method based on solid‐phase extraction followed by analysis using liquid chromatography with tandem mass spectrometry for the simultaneous determination of pharmaceuticals from antibiotics (fluoroquinolones, sulfonamides, and their N⁴‐acetyl metabolites, and trimethoprim as sulfonamides synergist) and anthelmintics groups. SPE was optimized using different cartridges (Strata‐X, Oasis HLB, Strata C₁₈‐E, Isolute C₁₈, SampliQ C₈/Si‐SCX). The highest recovery was achieved using Strata X cartridge (>80%) with good reproducibility (RSDs < 5%) despite various physicochemical properties of the compounds. Investigated analytes were identified and quantitatively determined by liquid chromatography with tandem mass spectrometry using multiple reaction monitoring. The method was shown to be linear over the concentration range of 0.05–30 μg/L for febantel and albendazole, and 0.10–60 μg/L for all other pharmaceuticals. Correlation coefficients were >0.99 for all compounds except for sulfamethazine (0.98). In order to demonstrate the applicability of the developed method, wastewater from the veterinary industry was analyzed. Results evidenced the presence of febantel, praziquantel, albendazole, enrofloxacin, sulfamethazine, and sulfadiazine.     

Metabolism profiles of nuciferine in rats using ultrafast liquid chromatography with tandem mass spectrometry

Nuciferine (NF) is one of the main aporphine alkaloids existing in the traditional Chinese medicine Folium Nelumbinis (lotus leaves). Modern pharmacological studies have demonstrated that NF has a broad spectrum of bioactivities, such as anti‐HIV and anti‐hyperlipidemic effects, and has been recommended as a leading compound for new drug development. However, the metabolites and biotransformation pathway of NF in vivo have not yet been comprehensively investigated. The present study was performed to identify the metabolites of NF for exploring in vivo fates. Rat plasma and urine samples were collected after oral administration and prepared by liquid–liquid extraction with ethyl acetate. A method based on ultrafast liquid chromatography with tandem mass spectrometry was applied to identify the metabolites. Q1 (first quadrupole) full scan combined with a multiple reaction monitoring (MRM) survey scan were used for the detection of metabolites. MRM‐information‐dependent acquisition of enhanced product ions was used for the structural identification of detected metabolites. A total of 10 metabolites were identified, including phase I (demethylation, oxidation and dehydrogenation) and phase II (glucuronidation, sulfation and glutathione) biotransformation products. Demethylation is the main metabolic pathway of NF in the body. These results can help in improving understanding of the disposition and pharmacological mechanism of NF in the body. Copyright © 2016 John Wiley & Sons, Ltd.     

Metabolic profiles of neratinib in rat by using ultra‐high‐performance liquid chromatography coupled with diode array detector and Q‐Exactive Orbitrap tandem mass spectrometry

Neratinib is a tyrosine kinase inhibitor that has been approved by the US Food and Drug Administration for the treatment of breast cancer. However, its metabolism remains unknown. This study was carried out to investigate the in vitro and in vivo metabolism of neratinib using an UHPLC‐DAD‐Q Exactive Orbitrap‐MS instrument with dd‐MS² on‐line data acquisition mode. The post‐acquisition data was processed using MetWorks software. Under the current conditions, a total of 12 metabolites were detected and structurally identified based on their accurate masses, fragment ions and chromatographic retention times. Among these metabolites, M3, M10 and M12 were unambiguously identified using chemically synthesized reference standards. M6 and M7 (GSH conjugates) were the major metabolites. The metabolic pathways of neratinib were proposed accordingly. Our findings suggested that neratinib was mainly metabolized via O‐dealkylation (M3), oxygenation (M8), N‐demethylation (M10), N‐oxygenation (M12), GSH conjugation (M1, M2, M4, M5, M6 and M7) and N‐acetylcysteine conjugation (M9 and M11). The α,β‐unsaturated ketone was the major metabolic site and GSH conjugation was the predominant metabolic pathway. In conclusion, this study provided valuable metabolic data and would benefit the assessment of the contributions to the overall activity or toxicity from the key metabolites.     

Structural identification of SAR-943 metabolites generated by human liver microsomes in vitro using mass spectrometry in combination with analysis of fragmentation patterns

SAR-943 (32-deoxo rapamycin) is a proliferation signal inhibitor via interaction with the mammalian target of rapamycin (mTOR). Most importantly, SAR-943 has improved chemical stability compared to rapamycin (sirolimus) and is currently under investigation as a drug coated on coronary stents. It was the goal of this study to identify the SAR-943 metabolites generated after incubation with human liver microsomes using high-resolution mass spectrometry (MS) and MS/iontrap (MS(n)) and comparison of fragmentation patterns of the metabolites with those of SAR-943 and other known rapamycin derivatives. Our study showed that SAR-943 is mainly hydroxylated and/or demethylated by human liver microsomes. The structures of the following metabolites were identified: O-demethylated metabolites: 39-O-desmethyl, 16-O-desmethyl and 27-O-desmethyl SAR-943; hydroxylated metabolites: hydroxy piperidine SAR-943, 11-hydroxy, 12-hydroxy, 14-hydroxy, 23-hydroxy, 24-hydroxy, 25-hydroxy, 46-hydroxy and 49-hydroxy SAR-943; didemethylated metabolites: 16,39-O-didesmethyl and 27,39-O-didesmethyl SAR-943; demethylated-hydroxylated metabolites: 39-O-desmethyl, 23- or 24-hydroxy and 39-O-desmethyl, hydroxy piperidine SAR-943 and dihydroxylated metabolites: 12-,23- or 24-dihydroxy SAR-943. In addition, several other demethylated-hydroxylated and dihydroxylated metabolites were detected. However, their exact structures could not be identified.