Metabonomics study of atherosclerosis rats by ultra fast liquid chromatography coupled with ion trap-time of flight mass spectrometry

An ultra fast liquid chromatography coupled with IT-TOF mass spectrometry (UFLC/MS-IT-TOF) metabonomic approach was employed to study the plasma and urine metabolic profiling of atherosclerosis rats. Acquired data were subjected to principal component analysis (PCA) for differentiating the atherosclerosis and the control groups. Potential biomarkers were screened by using S-plot and were identified by the accurate mass and MSⁿ fragments information obtained from UFLC/MS-IT-TOF analysis. 12 metabolites in rat plasma and 8 metabolites in urine were identified as potential biomarkers. Concentrations of leucine, phenylalanine, tryptophan, acetylcarnitine, butyrylcarnitine, propionylcarnitine and spermine in plasma and 3-O-methyl-dopa, ethyl N2-acetyl-l-argininate, leucylproline, glucuronate, t6A N(6)-(N-threonylcarbonyl)-adenosine and methyl-hippuric acid in urine decreased in atherosclerosis rats. Ursodeoxycholic acid, chenodeoxycholic acid, LPC (C16:0), LPC (C18:0) and LPC (C18:1) in plasma and hippuric acid in urine were in higher levels in atherosclerosis rats. The alterated metabolites demonstrated abnormal metabolism of phenylalanine, tryptophan, bile acids and amino acids. This research proved that metabonomics is a promising tool for disease research.     

Impurity analysis of gentamicin bulk samples by improved liquid chromatography-ion trap mass spectrometry

Several gentamicin bulk samples from different origins were investigated using an LC/MS method.LC equipped with ion trap MS with positive ionization was performed on a Capcell Pak C18(AQ) column with the mobile phase containing 50 mM trifluoroacetic(TFA) and methanol.Impurities present in batches of gentamicin bulk samples were elucidated and compared according to their fragmentation behavior.In total seventeen impurities present in samples,five impurities were not elucidated and two compounds were identifi...     

A sensitive and specific liquid chromatography/tandem mass spectrometry method for determination of echinacoside and its pharmacokinetic application in rats

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the determination of echinacoside in rat plasma was established and fully validated. A single step of liquid–liquid extraction with n‐butanol was utilized. Chromatographic separation of the analyte and the internal standard (IS), chlorogenic acid, from the sample matrix was performed using a Capcell‐MG C₁₈ analytical column (100 2.0 mm × 5 µm), with a gradient of acetonitrile and water containing 0.1% acetic acid as the mobile phase. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization source operated in negative ion selected reaction monitoring mode. The method was linear in the concentration range 10–2500 ng/mL. The deviations of both intra‐ and inter‐day precisions (RSD) were 7.1% and the assay accuracies were within 99.2–106.5%. Echinacoside proved to be stable during sample storage, preparation and analysis when an antioxidant solution was used. The method was successfully applied to a pharmacokinetic study in rats after an intragastric administration of echinacoside (100 mg/kg). With the lower limit of quantification at 10 ng/mL, this method proved to have sufficient selectivity, sensitivity and reproducibility for the pharmacokinetic study of echinacoside. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of metabolites of tanshinone IIA in rats by liquid chromatography/tandem mass spectrometry

The metabolism of tanshinone IIA was studied in rats after a single-dose intravenous administration. In the present study, 12 metabolites of tanshinone IIA were identified in rat bile, urine and feces with two LC gradients using LC-MS/MS. Seven phase I metabolites and five phase II metabolites of tanshinone IIA were characterized and their molecular structures proposed on the basis of the characteristics of their precursor ions, product ions and chromatographic retention time. The seven phase I metabolites were formed, through two main metabolic routes, which were hydroxylation and dehydrogenation metabolism. M1, M4, M5 and M6 were supposedly tanshinone IIB, hydroxytanshinone IIA, przewaquinone A and dehydrotanshinone IIA, respectively, by comparing their HPLC retention times and mass spectral patterns with those of the standard compounds. The five phase II metabolites identified in this research were all glucuronide conjugates, all of which showed a neutral loss of 176 Da. M9 and M12 were more abundant than other identified metabolites in the bile, which was the main excretion path of tanshinone IIA and the metabolites. M12 was the main metabolite of tanshinone IIA. M9 and M12 were proposed to be the glucuronide conjugates of two different semiquinones and these semiquinones were the hydrogenation products of dehydrotanshinone IIA and tanshinone IIA, respectively. This hydrogenized reaction may be catalyzed by the NAD(P)H: quinone acceptor oxidoreductase (NQO). The biotransformation pathways of tanshinone IIA were proposed on the basis of this research.     

高效液相色谱-离子阱-飞行时间质谱法鉴定碱性嫩黄中的杂质及高效液相色谱法制备碱性嫩黄标准物质

该文建立了一种高效液相色谱-离子阱-飞行时间质谱（HPLC-IT-TOF-MS）分析非法食品添加剂碱性嫩黄样品中杂质成分的方法。对碱性嫩黄样品中的杂质进行多级质谱分析,根据各碎片离子的精确质量数推测出该杂质的具体组成,确定这个杂质为4-（亚氨基（4-（甲基氨基）苯基）甲基）-N,N-二甲基苯胺盐酸盐,从而推断出碱性嫩黄的合成方法及杂质的可能来源。同时建立了制备碱性嫩黄标准物质的方法,分别选用了粒径为10μm和5μm的制备液相色谱柱进行两次制备高效液相色谱分离纯化,进样量分别为1 mL和500μL,最终采用分析型高效液相色谱峰面积归一化法得到纯度为99.52%的碱性嫩黄标准物质。扣除0.34%水分含量,0.13%灰分含量,采用质量平衡法确定制备的物质最终纯度为99.05%,并通过UV、IR、LC-MS和NMR四大谱图进行化学结构的确认。该方法简单、高效,可拓展应用于其他非法食品添加剂标准物质的制备。     

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.     

Rapid identification of efavirenz metabolites in rats and humans by ultra high performance liquid chromatography combined with quadrupole time‐of‐flight tandem mass spectrometry

An in vivo study of efavirenz metabolites in rats and human patients with ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry combined with MetabolitePilot^MT software is reported for the first time. Considering the polarity differences between the metabolites, solid‐phase extraction and protein precipitation were both applied as a part of the sample preparation method. The structures of the metabolites and their fragment ions were identified or tentatively characterized based on the accurate mass and MS² data. As a result, a total of 15 metabolites, including 11 from rat samples and 13 from human samples, were identified or tentatively characterized. Two metabolites and several new metabolism pathways are reported for the first time. This study provides a practical approach for identifying complicated metabolites through the rapid and reliable ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry technique, which could be widely used for the investigation of drug metabolites.     

Quantitative analysis of acetaminophen and its six metabolites in rat plasma using liquid chromatography/tandem mass spectrometry

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. It is mainly metabolized by phase 1 and 2 reactions in the liver, and thus it could be involved in many drug–drug interactions. Therefore, the study of APAP metabolism is important in toxicological and pharmacokinetic studies. The objective of this study was to develop a rapid and sensitive method for the determination of APAP and its six metabolites in rat plasma for the pharmacokinetic studies. APAP and its metabolites were separated through a Capcell Pak MGII C₁₈ column and quantitated with a 16 min run in a triple‐quadruple mass spectrometer. The mobile phases were composed of 0.1% formic acid in either 95% water or 95% acetonitrile and analysis was performed twice in positive and negative modes. Validations such as accuracy, precision, recovery, matrix effect and stability were found to be within acceptance criteria of validation guidelines, indicating that the assay was applicable to the determination of the plasma concentrations of drug and its six metabolites. In conclusion, we developed an LC‐MS/MS method for the quantitative analysis of APAP and its six metabolites in rat plasma, and this method appears to be useful for pharmacokinetic/toxicokinetic studies of APAP and its metabolites in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Application of liquid chromatography-ion trap mass spectrometry to the characterization of the 16-membered ring macrolide josamycin propionate

Coupled liquid chromatography and ion trap mass spectrometry (LC/MS) was used for the characterization of the semi-synthetic 16-membered ring macrolide josamycin propionate. On-line identification of impurities in this antibiotic complex was performed with an ion trap mass spectrometer without recourse to time-consuming isolation and purification procedures. Ion trap mass spectrometry is ideally suited to identification of impurities because it provides MSn capability, enabling multiple stages of mass spectrometry to obtain the maximum amount of structural information for a given molecule. The ion trap was used with an electrospray ionization source operated in the positive ion mode or with an atmospheric pressure chemical ionization source operated in the negative ion mode. The identity of the unknown compounds was deduced using the MS/MS and MSn collision-induced dissociation spectra of reference substances or structural analogs as interpretative templates, combined with knowledge about the nature of functional group fragmentation behavior. Given the importance attached to the identification of impurities of unknown identity in pharmaceutical substances, this study is useful for companies producing josamycin propionate. The knowledge of the fragmentation behavior is also of importance in further research on other 16-membered macrolides.     

Liquid chromatography–mass spectrometry for analysis of a novel β2‐adrenoceptor agonist trantinterol and its metabolites in beagle dog urine

A liquid chromatography–tandem mass spectrometry method was developed for the identification of metabolites of trantinterol, a novel β₂‐adrenoceptor agonist, in beagle dog urine. The separation of metabolites was performed on a reversed‐phase C₈ column using 0.1% formic acid in water and methanol (70 : 30, v/v) as the mobile phase. The structural information and elemental information of metabolites were acquired by an electrospray ionization tandem mass spectrometer and a quadrupole time‐of‐flight mass spectrometer, respectively. A total of 13 metabolites were detected and characterized on the basis of their tandem MS/MS fragmentation patterns. The accurate masses of nine metabolites were determined and two metabolites were further confirmed by comparing with reference standards. The metabolic pathways of trantinterol in beagle dog are proposed. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of the metabolites of biologically active xanthones isolated from Halenia elliptica D.Don by high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry

Metabolism study has been carried out on 1-hydroxy-2,3,5-trimethoxyxanthone(HM-1) and 1-hydroxy-2,3,4,7- tetramethoxyxanthone(HM-2),which are two biologically active ingredients isolated from the Tibetan herb,Halenia elliptica D.Don.,in rat liver microsomes in vitro.A method of high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry(LCMS~n-ESI-IT-TOF) was applied to analyze metabolites of HM-1 and HM-2 on line,and five metabolites were identified containing 1,5-dihydroxy-2,3-dimethoxyxanthone(HM-5),1,7-dihydroxy-2,3,4-trimethoxyxanthone(HM-9),1,4,7- trihydroxy-2,3-dimethoxyxanthone(HM-10),1,4-dihydroxy-2,3,7-trimethoxyxanthone(HM-11) and 1,2-dihydroxy-3,4,7- trimethoxyxanthone(HM-12).Among these metabolites,HM-9,HM-11,and HM-12 were isomers mutually.The results indicated that HM-1 and HM-2 occurred Phase I metabolic reaction of demethylation in rat microsomes in vitro.     

Development of a solid phase extraction protocol coupled with liquid chromatography mass spectrometry to analyze central carbon metabolites in lake sediment microcosms

An ion exchange solid phase extraction (SPE) strategy is developed for application with liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) for characterization of central carbon metabolites involved in methane assimilation and adjacent pathways in natural mixtures. For this purpose, short-time microcosm samples were obtained from lake sediment known to consume methane. Three SPE procedures were developed for the recovery of 51 targeted metabolites from five compound classes (amino acids, carboxylic acids, sugar phosphates, nucleotides and acyl-CoAs). The three SPE procedures employed were mixed mode (i) strong cation exchange, (ii) strong anion exchange and (iii) weak anion exchange. By spiking stable isotopic labeled standards, validation of the SPE procedures for the sediment extracts demonstrated that a 3 cm(3), 60 mg SPE sorbent bed provided effective loading capacity for targeted metabolites with an analytical variation of 16% RSD. We readily analyzed 32 of the targeted 51 metabolites using LC-MS/MS after sediment sample extraction, cleanup and pre-concentration. The remaining 19 targeted metabolites were either at, or below, the limit of detection. The current approach provides a good workflow for absolute quantification of intermediates in C(1)-carbon metabolism in natural microbial communities.     

基于液相色谱-质谱联用技术的代谢组学方法在细胞种属分类中的应用

细胞内的代谢产物可以反映细胞的生理状态。为了考察基于胞内代谢物的指纹图谱对不同种属细胞进行区分的可行性,利用基于超高效液相色谱-高分辨飞行时间质谱(UPLC-TOF MS)技术的代谢组学方法对5种不同来源的细胞进行分类,获得了小分子代谢产物的差异表达谱,并采用主成分分析(PCA)数据处理方法对各类细胞进行模式识别。研究结果表明,不同的细胞种属之间均能呈现显著性差异。该研究可从分子水平对细胞种属进行分类,为细胞种属的鉴定与评价提供了一种新的技术方法,为细胞组学的深入研究提供了一种潜在的、非常具有应用前景的技术手段。     

Determination of clopidogrel in human plasma by liquid chromatography/tandem mass spectrometry: application to a clinical pharmacokinetic study

A rapid and sensitive LC/MS/MS assay was developed and validated for the determination of clopidogrel in human plasma. Clopidogrel was extracted by single liquid-liquid extraction with pentane, and chromatographic separations were achieved on a C(18) column. The method was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), stability, accuracy and precision. The multiple reaction monitoring was based on m/z transition of 322.2 --> 211.9 for clopidogrel and 264.1 --> 125.1 for ticlopidine (internal standard). The total analytical run time was relatively short (3 min), and the LLOQ was 10 pg/mL using 0.5 mL of human plasma. The assay was linear over a concentration range from 10 to 10,000 pg/mL (r > 0.999). The intra- and inter-day accuracies were 101.3-108.8 and 98.4-103.5%, respectively, and the intra- and inter-day assay precisions were 1.9-5.5 and 4.4-8.1%, respectively. The developed assay method was applied to a pharmacokinetic study in human volunteers after oral administration of clopidogrel at a dose of 150 mg.     

Identification of metabolites of rupestonic acid in rat urine by liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Rupestonic acid, a potential anti‐influenza agent, is an important and characteristic compound in Artemisia rupestris L., a well‐known traditional Uighur medicine for the treatment of colds. In the present study, high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry was used to detect and identify the metabolites in rat urine after oral administration of rupestonic acid. A total of 10 metabolites were identified or partially characterized. The structure elucidations of the metabolites were performed by comparing the changes in accurate molecular masses and fragment ions with those of the parent compound. The results showed that the main metabolites of rupestonic acid in rat urine were formed by oxidation, hydrogenation and glucuronidation. A metabolism pathway was proposed for the first time based on the characterized structures. This metabolism study can provide essential information for drug discovery, design and clinical application of rupestonic acid. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitative determination of uridine in rabbit plasma and urine by liquid chromatography coupled to a tandem mass spectrometry

Recently a pyrimidine nucleoside, uridine, has been show to have a protective effect on cultured human corneal epithelial cells, and on dry eye animal model and patients. In this study, we introduce a sensitive liquid chromatography/tandem mass spectrometry method for the determination of uridine in rabbit plasma and urine. After protein precipitation with methanol including methaqualone (internal standard), the analyte was chromatographed on a reversed-phase column with a mobile phase of 0.1% formic acid aqueous solution and methanol (1:4, v/v). The accuracy and precision of the assay were in accordance with Food and Drug Administration regulations for the validation of bioanalytical methods. This method was used to measure the concentrations of uridine in plasma and urine after a single oral administration of 450 mg/kg uridine in rabbits.     

Simultaneous determination of zolazepam and tiletamine in dog plasma by liquid chromatography coupled to a tandem mass spectrometry

A mixture of tiletamine, a dissociative anesthetic, and zolazepam, a minor tranquilizer, has been widely used as an anesthetic or an immobilizing agent in a variety of animal species. However, interestingly, their pharmacokinetic behaviors have been published only in polar bears and pigs. In this study, we introduce a sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for determining the two drugs in dog plasma. After simple protein precipitation with acetonitrile including midazolam (internal standard), the analytes were chromatographed on a reversed‐phase column with a mobile phase of 10 m m ammonium acetate aqueous solution and acetonitrile (1:4, v/v). The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This method was used to measure the concentrations of zolazepam and tiletamine in plasma after a single intramuscular 10 mg dose of each in beagle dogs. Copyright © 2012 John Wiley & Sons, Ltd.     

Structural identification of the metabolites of ganoderic acid B from Ganoderma lucidum in rats based on liquid chromatography coupled with electrospray ionization hybrid ion trap and time‐of‐flight mass spectrometry

Ganoderic acid B (GAB), a representative triterpenoid in Ganoderma lucidum, possesses various pharmaceutical effects and has been used as a chemical marker in quality control of G. lucidum and related products. The metabolites of GAB in vivo after its oral administration to rats were investigated by liquid chromatography coupled with electrospray ionization hybrid ion trap and time‐of‐flight mass spectrometry. A total of 14 metabolites of GAB in rat plasma, bile and various organs were detected and identified by direct comparison with the authentic compounds and their characteristic mass fragmentation patterns. The results showed that oxidization and hydroxylation were the common metabolic pathways for GAB in rats. Moreover, some reduction metabolites of GAB were detected in rat kidney and stomach and glucuronidation only appeared in rat bile. This is the first report on the metabolites of GAB in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of praziquantel,pyrantel embonate,febantel and its active metabolites,oxfendazole and fenbendazole,in dog plasma by liquid chromatography/mass spectrometry

A liquid chromatography–electrospray–mass spectrometry method (LC/MS) has been developed and validated for determination of praziquantel (PZQ), pyrantel (PYR), febantel (FBT), and the active metabolites fenbendazole (FEN) and oxfendazole (OXF), in dog plasma, using mebendazole as internal standard (IS). The method consists of solid‐phase extractions on Strata‐X polymeric cartridges. Chromatographic separation was carried out on a Phenomenex Gemini C₆‐Phenyl column using binary gradient elution containing methanol and 50 mm ammonium–formate (pH 3). The method was linear (r² ≥ 0.990) over concentration ranges of 3–250 ng/mL for PYR andFEB, 5–250 ng/mL for OXF and FEN, and 24–1000 ng/mL for PZQ. The mean precisions were 1.3–10.6% (within‐run) and 2.5–9.1% (between‐run), and mean accuracies were 90.7–109.4% (within‐run) and 91.6–108.2% (between‐run). The relative standard deviations (RSD) were <9.1%. The mean recoveries of five targeted compounds from dog plasma ranged from 77 to 94%.The new LC/MS method described herein was fully validated and successfully applied to the bioequivalence studies of different anthelmintic formulations such as tablets containing PZQ, PYR embonate and FBT in dogs after oral administration. Copyright © 2015 John Wiley & Sons, Ltd.