Liquid chromatography coupled with hybrid ion trap time‐of‐flight mass spectrometry method for the determination of caulophine in rat bio‐samples and its pharmacokinetic study after intragastric and intraperitoneal administration

A rapid and precise liquid chromatography coupled with hybrid ion trap/time‐of‐flight mass spectrometry method to detect and quantify caulophine and its possible active metabolites in rat plasma and urine was developed. Samples were prepared by plasma protein precipitation combined with a liquid‐liquid extraction method. The separation was carried out on an InertSustain® C18 column with a mobile phase comprising methanol and 0.1% aqueous formic acid solution. The analysis was complete in 20 min with a flow rate of 0.4 mL/min. Taspine was used as the internal standard. Mass spectrometric detection was conducted with hybrid ion trap/time‐of‐flight equipped with electrospray ionization in the positive ion mode. The calibration curves of caulophine were linear over the concentration ranges of 0.002–0.20 μg/mL for plasma and 0.005–0.50 μg/mL for urine with the correlation coefficients greater than 0.998 in both cases. The method was successfully used to investigate the pharmacokinetics and bioavailability in rat plasma and urine samples after intragastric and intraperitoneal administration of caulophine sodium salt.     

Metabolic profiling comparison of isovitexin in normal and kidney stone model rats by ultra‐high‐performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry

Isovitexin, a bioactive flavonoid constituent isolated from Desmodii Styracifolii, is considered an adjuvant for antiurolithiasis diseases. In this study, an ultra‐high‐performance liquid chromatography coupled with hybrid triple quadruple time‐of‐flight mass spectrometry method was developed to characterize and compare the metabolic profiling of isovitexin experimented on normal and kidney stone model rats. The comparative research indicated that 28 metabolites (18 phase I and 10 phase II) in normal rats and 33 metabolites (20 phase I and 13 phase II) in kidney stone model rats were initially identified. The results of relative quantitative determination reflected that the contents of metabolites produced by deglycosylation, reduction, and isomerization in kidney stone model rats were greater than those in healthy rats. Instead, the levels of oxidative and dehydrogenated metabolites in normal groups were higher than those in kidney stone model groups. The results of this study are valuable and important for understanding the metabolic process of isovitexin in clinical application, and especially the metabolism study in kidney stone model rats could provide a beneficial reference for the further search of effective substances associated with the treatment of kidney stones.     

Identification of metabolites in human and rat urine after oral administration of Xiao‐Qing‐Long‐Tang granule using ultra high performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry

Xiao‐Qing‐Long‐Tang is a traditional Chinese formula used for the treatment of cold syndrome, bronchitis, and nasal allergies for thousands of years. However, the in vivo integrated metabolism of its multiple components and the active chemical constituents of Xiao‐Qing‐Long‐Tang remain unknown. In this study, a method using ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry was established for the detection and identification of the metabolites in human and rat urine after oral administration of Xiao‐Qing‐Long‐Tang. A total of 19 compounds were detected or tentatively identified in human urine samples, including eight prototypes and 11 metabolites. Also, a total of 50 compounds were detected or tentatively identified in rat urine samples, including 15 prototypes and 35 metabolites detected with either a highly sensitive extracted ion chromatogram method or the MS^E determination using Mass Fragment software. Our results indicated that phase Ⅱ reactions (e.g. glucuronidation and sulfation) were the main metabolic pathways of flavones, while phase I reactions (e.g. demethylation and hydroxylation) were the major metabolic reaction for alkaloids, lignans, and ginger essential oil. This investigation provided important structural information on the metabolism of Xiao‐Qing‐Long‐Tang and provided evidence to obtain a more comprehensive metabolic profile.     

Metabolite profiling of l‐isocorypalmine in rat urine,plasma, and feces after oral administration using high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry

l ‐Isocorypalmine, an active alkaloid compound isolated from Rhizoma Corydalis yanhusuo, has been reported to possess biological activity for treating cocaine use disorder. A high‐performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry method was established for identification of the metabolites of l ‐isocorypalmine in urine, plasma and feces samples of rats after a single intragastric gavage of l ‐isocorypalmine at a dose of 15 mg/kg. As a result, a total of 21 metabolites (six phase ? metabolites and fifteen phase II metabolites) were detected and tentatively identified by mass spectrometry and fragment ions from tandem mass spectrometry spectra. All metabolites were present in the urine samples, nine metabolites were found in the plasma samples and three metabolites were found in the feces samples. Results indicated that metabolic pathways of l ‐isocorypalmine included oxidation, dehydrogenation, demethylation, sulfate conjugation, and glucuronide conjugation. In addition, glucuronidation was the major metabolic reaction. Results of this investigation could provide significant experimental basis for efficacy, safety and action mechanism of l ‐isocorypalmine, which will be advantageous to new drug development for treating cocaine addiction.     

A new,validated HPLC‐MS/MS method for the simultaneous determination of the anti‐cancer agent capecitabine and its metabolites: 5′‐deoxy‐5‐fluorocytidine, 5′‐deoxy‐5‐fluorouridine, 5‐fluorouracil and 5‐fluorodihydrouracil,in human plasma

A rapid and selective liquid chromatography/tandem mass spectrometric method was developed for the simultaneous determination of capecitabine and its metabolites 5′‐deoxy‐5‐fluorocytidine (5′‐DFCR), 5′‐deoxy‐5‐fluorouracil (5′‐DFUR), 5‐fluorouracil (5‐FU) and dihydro‐5‐fluorouracil (FUH₂) in human plasma. A 200 μL human plasma aliquot was spiked with a mixture of internal standards fludarabine and 5‐chlorouracil. A single‐step protein precipitation method was employed using 10% (v/v) trichloroacetic acid in water to separate analytes from bio‐matrices. Volumes of 20 μL of the supernatant were directly injected onto the HPLC system. Separation was achieved on a 30 × 2.1 mm Hypercarb (porous graphitic carbon) column using a gradient by mixing 10 mm ammonium acetate and acetonitrile–2‐propanol–tetrahydrofuran (1 : 3 : 2.25, v/v/v). The detection was performed using a Finnigan TSQ Quantum Ultra equipped with the electrospray ion source operated in positive and negative mode. The assay quantifies a range from 10 to 1000 ng/mL for capecitabine, from 10 to 5000 ng/mL for 5′‐DFCR and 5′‐DFUR, and from 50 to 5000 ng/mL for 5‐FU and FUH₂ using a plasma sample of 200 μL. Correlation coefficients (r²) of the calibration curves in human plasma were better than 0.99 for all compounds. At all concentration levels, deviations of measured concentrations from nominal concentration were between ?4.41 and 3.65% with CV values less than 12.0% for capecitabine, between ?7.00 and 6.59% with CV values less than 13.0 for 5′‐DFUR, between ?3.25 and 4.11% with CV values less than 9.34% for 5′‐DFCR, between ?5.54 and 5.91% with CV values less than 9.69% for 5‐FU and between ?4.26 and 6.86% with CV values less than 14.9% for FUH₂. The described method was successfully applied for the evaluation of the pharmacokinetic profile of capecitabine and its metabolites in plasma of treated cancer patients. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of bilobetin metabolites,in vivo and in vitro,based on an efficient ultra‐high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry strategy

Bilobetin, a natural compound extracted from Ginkgo biloba, has various pharmacological activities such as antioxidation, anticancer, antibacterial, antifungal, anti‐inflammatory, antiviral, and promoting osteoblast differentiation. However, few studies have been conducted and there are no reports on its metabolites owing to its low content in nature. In addition, it has been reported to have potential liver and kidney toxicity. Therefore, this study aimed to identify the metabolites of bilobetin in vitro and in vivo. Bilobetin was incubated with liver microsomes to determine metabolites in vitro, and faeces and urine were collected after oral administration to rats to determine metabolites in vivo. After the samples were processed, they were measured using ultra‐high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. As a result, a total of 21 and 9 metabolites were detected in vivo and in vitro, respectively. Demethylation, demethylation and loss of water, demethylation and hydrogenation, demethylation and glycine conjugation, oxidation, methylation, oxidation and methylation, and hydrogenation were the main metabolic pathways. This study is the first to identify the metabolites of bilobetin and provides a theoretical foundation for the safe use of bilobetin in clinical application and the development of new drugs.     

Comprehensive characterization of multiple components and metabolites of Xiaojin Capsule based on ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

Xiaojin Capsule, a classic traditional Chinese medicine formula, has been used to treat mammary cancer, thyroid nodules, and hyperplasia of the mammary glands. However, its systematic chemical information remained unclear, which hindered the interpretation of the pharmacology and the mechanism of action of this drug. In this research, an ultra high performance liquid chromatography coupled with a quadrupole time‐of‐flight mass spectrometry method was developed to identify the complicated components and metabolites of Xiaojin Capsule. Two acquisition modes, including the MS^Energy mode and fast data directed acquisition mode, were utilized for chemical profiling. As a result, 156 compounds were unambiguously or tentatively identified by comparing their retention times and mass spectrometry data with those of reference standards or literature. After the oral administration of Xiaojin Capsule, 53 constituents, including 24 prototype compounds and 29 metabolites, were detected in rat plasma. The obtained results were beneficial for a better understanding of the therapeutic basis of Xiaojin Capsule. A high‐resolution and efficient separation method was firstly established for systematically characterizing the compounds of Xiaojin Capsule and the associated metabolites in vivo, which could be helpful for quality control and pharmacokinetic studies of this medicine.     

High‐throughput LC–MS method for the rapid characterization of multiple chemical constituents and metabolites of Da‐Bu‐Yin‐Wan

Traditional Chinese medicine is the clinical experience accumulated by Chinese people against diseases. Da‐Bu‐Yin‐Wan is a famous traditional Chinese medicine formula consisting of Phellodendri amurensis Rupr., Anemarrhenae asphodeloides Bge., Radix Rehmanniae Preparata and Chinemys reevesii . In this study, ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight high‐definition mass spectrometry with the control software of Masslynx (V4.1) was established for comprehensive screening and identification of the chemical constituents and serum metabolites of Da‐Bu‐Yin‐Wan in vivo and in vitro. Consequently, 70 peaks in the methanol extract from Da‐Bu‐Yin‐Wan and 38 peaks absorbed into rat blood were characterized. The 70 constituents in vitro included alkaloids, flavonoids, polysaccharide, limonoids, flavonoid, etc. And the 38 constituents consist of 22 absorbed prototypes and 16 metabolites of Da‐Bu‐Yin‐Wan absorbed in vivo. We fully clarified the chemical constituents of Da‐Bu‐Yin‐Wan and provided a scientific strategy for the screening and characterization of the chemical constituents and metabolites of traditional Chinese medicine in vitro and in vivo.     

Characterization of chemical constituents and identification of absorbed components and metabolites in rat plasma of Fu‐Ke‐Zai‐Zao pills by ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Fu‐Ke‐Zai‐Zao pills, the famous traditional Chinese medicine formula, composed of 42 medicinal herbs, have been widely used to treat various gynecological diseases. However, the chemical constituents and metabolic profiling of Fu‐Ke‐Zai‐Zao pills remain largely unknown, which hampers improvement of the quality control and pharmacological elucidation of this formula. In the present study, a sensitive and selective ultra high performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry method was developed to separate and identify the comprehensive chemical constituents of Fu‐Ke‐Zai‐Zao pills. According to the results, a total of 83 compounds were identified, including phenylpropionic acids, flavonoids, terpenoids, triterpene saponins, and phthalides, and 81 compounds were first reported in Fu‐Ke‐Zai‐Zao pills. Moreover, the absorbed components and metabolites in rat plasma after intragastric administration of Fu‐Ke‐Zai‐Zao pills were also detected by the same analytical method. A total of 36 compounds were identified, including 21 prototypes and 15 metabolites. The latter were generated through the metabolic pathways of methylation and glucuronidation, and glucuronidated metabolites were the main constituents in the plasma. This is the first systematic study on the chemical constituents and metabolic profiling of Fu‐Ke‐Zai‐Zao pills, and the results provide valuable chemical information for further elucidating pharmacological effects and mechanism of action of Fu‐Ke‐Zai‐Zao pills.     

Evaluating novel silica hydride‐based stationary phases for the analysis of phytocannabinoids and other psychoactive drugs

Three silica hydride based novel chromatographic phases chemically‐bonded with allyloxy‐DL‐alpha‐tocopherol, allylpentafluorophenyl, and 1‐eicosene moieties were evaluated as separation media for selected phytocannabinoids and other substances of abuse. In order to assess column selectivity, a series of reference standards was analyzed and detected by using liquid chromatography with mass spectrometry. Further, quantitative detections of cannabidiol and tetrahydrocannabinol were attempted for the extracts of cannabis plants and cannabidiol gummy formulation. For potential bioanalytical applications, the columns were evaluated for substance screening in a human urine matrix. In summary, the newly developed columns are functional and effective for the analysis of phytocannabinoids and various psychoactive drugs with or without the presence of biological matrices.     

Cluster TOF‐SIMS imaging of human skin remains: analysis of a South‐Andean mummy sample

A skin sample from a South‐Andean mummy dating back from the XI^th century was analyzed using time‐of‐flight secondary ion mass spectrometry imaging using cluster primary ion beams (cluster‐TOF‐SIMS). For the first time on a mummy, skin dermis and epidermis could be chemically differentiated using mass spectrometry imaging. Differences in amino‐acid composition between keratin and collagen, the two major proteins of skin tissue, could indeed be exploited. A surprising lipid composition of hypodermis was also revealed and seems to result from fatty acids damage by bacteria. Using cluster‐TOF‐SIMS imaging skills, traces of bio‐mineralization could be identified at the micrometer scale, especially formation of calcium phosphate at the skin surface. Mineral deposits at the surface were characterized using both scanning electron microscopy (SEM) in combination with energy‐dispersive X‐ray spectroscopy and mass spectrometry imaging. The stratigraphy of such a sample was revealed for the first time using this technique. More precise molecular maps were also recorded at higher spatial resolution, below 1 µm. This was achieved using a non‐bunched mode of the primary ion source, while keeping intact the mass resolution thanks to a delayed extraction of the secondary ions. Details from biological structure as can be seen on SEM images are observable on chemical maps at this sub‐micrometer scale. Thus, this work illustrates the interesting possibilities of chemical imaging by cluster‐TOF‐SIMS concerning ancient biological tissues. Copyright © 2012 John Wiley & Sons, Ltd.     

基于非靶向代谢组学的茄梨和红茄梨成熟期果皮代谢产物的差异分析

为了探究西洋梨品种茄梨及其红色芽变红茄梨成熟期果皮代谢产物差异,采用超高效液相色谱-质谱联用技术,对茄梨和红茄梨成熟期果皮进行非靶向代谢组学研究。通过主成分分析和正交偏最小二乘判别分析,构建了多变量统计分析模型,结合模型和变量重要性投影与最大差异倍数值,基于精确质量数、二级碎片以及同位素分布,使用PMDB(Plant Metabolome Database)数据库进行定性,筛选并鉴定出茄梨和红茄梨果皮中显著性变化(P<0.05, VIP(variable importance in project)≥1)的差异代谢物有83种,主要包括酚酸类、黄酮类和氨基酸类物质,涉及类黄酮代谢、氨基酸代谢、苯丙烷类代谢等代谢途径,其中53种物质含量上调,30种物质表达下调。通过KEGG(Kyoto Encyclopedia of Genes and Genomes)数据库进一步对差异代谢物质进行通路富集分析,差异代谢物主要分布在20条代谢途径中,P<0.05的代谢途径有6条,分别是类黄酮生物合成、黄酮和黄酮醇生物合成、苯丙烷生物合成、丁酸酯代谢、苯丙氨酸代谢、酪氨酸代谢。这些差异代谢物的变化可能是导致茄梨和红茄梨果皮色泽不同的原因。该研究从植物代谢组学角度初步揭示了茄梨和红茄梨成熟期果皮的代谢产物差异性。     

Charged Tags for the Identification of Oxidative Drug Metabolites Based on Electrochemistry and Mass Spectrometry

Most of the active pharmaceutical ingredients like Metoprolol are oxidatively metabolized by liver enzymes, such as Cytochrome P450 monooxygenases into oxygenates and therefore hydrophilic products. It is of utmost importance to identify the metabolites and to gain knowledge on their toxic impacts. By using electrochemistry, it is possible to mimic enzymatic transformations and to identify metabolic hot spots. By introducing charged-tags into the intermediate, it is possible to detect and isolate metabolic products. The identification and synthesis of initially oxidized metabolites are important to understand possible toxic activities. The gained knowledge about the metabolism will simplify interpretation and predictions of metabolitic pathways. The oxidized products were analyzed with high performance liquid chromatography-mass spectrometry using electrospray ionization (HPLC-ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. For proof-of-principle, we present a synthesis of one pyridinated main oxidation product of Metoprolol.     

Liquid microjunction surface sampling probe electrospray mass spectrometry for detection of drugs and metabolites in thin tissue sections

A self-aspirating, liquid microjunction surface sampling probe/electrospray emitter mass spectrometry system was demonstrated for use in the direct analysis of spotted and dosed drugs and their metabolites in thin tissue sections. Proof-of-principle sampling and analysis directly from tissue without the need for sample preparation was demonstrated first by raster scanning a region on a section of rat liver onto which reserpine was spotted. The mass spectral signal from selected reaction monitoring was used to develop a chemical image of the spotted drug on the tissue. The probe was also used to selectively spot sample areas of sagittal whole-body tissue from a mouse that had been dosed orally (90 mg/kg) with R,S-sulforaphane 3 h prior to sacrifice. Sulforaphane and its glutathione and N-acetyl cysteine conjugates were monitored with selected reaction monitoring and detected in the stomach and various other tissues from the dosed mouse. No signal for these species was observed in the tissue from a control mouse. The same dosed-tissue section was used to illustrate the possibility of obtaining a lane scan across the whole-body section. In total, these results illustrate the potential for rapid screening of the distribution of drugs and metabolites in thin tissue sections with the liquid micro-junction surface sampling probe/electrospray mass spectrometry approach. Published in 2007 by John Wiley & Sons, Ltd.     

Densitometry and indirect normal‐phase HPTLC–ESI–MS for separation and quantitation of drugs and their glucuronide metabolites from plasma

The present work describes novel methods using densitometry and indirect or off‐line high performance thin‐layer chromatography–mass spectrometry (HPTLC–MS) for the simultaneous detection and quantification of asenapine, propranolol and telmisartan and their phase II glucuronide metabolites. After chromatographic separation of the drugs and their metabolites the analytes were scraped, extracted in methanol and concentrated prior to mass spectrometric analysis. Different combinations of toluene and methanol–ethanol–n‐butanol–iso‐propanol were tested for analyte separation and the best results were obtained using toluene–methanol–ammonia (6.9:3.0:0.1, v/v/v) as the elution solvent. All of the drug–metabolite pairs were separated with a homologous retardation factor difference of ≥22. The conventional densitometric approach was also studied and the method performances were compared. Both of the approaches were validated following the International Conference on Harmonization guidelines, and applied to spiked human plasma samples. The major advantage of the TLC–MS approach is that it can provide much lower limits of detection (1.98–5.83 pg/band) and limit of quantitation (5.97–17.63 pg/band) with good precision (?3.0% coefficient of variation) compared with TLC–densitometry. The proposed indirect HPTLC–MS method is simple yet effective and has tremendous potential in the separation and quantitation of drugs and their metabolites from biological samples, especially for clinical studies.     

Identification of components and metabolites in plasma of type 2 diabetic rat after oral administration of Jiao‐Tai‐Wan using ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry

Jiao‐Tai‐Wan, which is composed of Coptis Rhizoma and Cinnamon Cortex, has been recently used to treat type 2 diabetes. Owing to lack of data on its prototypes and metabolites, elucidation of the pharmacological and clinically safe levels of this formula has been significantly hindered. To screen more potential bioactive components of Jiao‐Tai‐Wan, we identified its multiple prototypes and metabolites in the plasma of type 2 diabetic rats by ultra high performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry. A total of 47 compounds were identified in the plasma of type 2 diabetic rats, including 22 prototypes and 25 metabolites, with alkaloids constituting the majority of the absorbed prototype components. In addition, this is the first study to detect vanillic acid, gallic acid, chlorogenic acid, protocatechuic acid, 2‐hydroxycinnamic acid, 3‐hydroxycinnamic acid, 4‐hydroxycinnamic acid, and 2‐methoxy cinnamic acid after oral administration of Jiao‐Tai‐Wan. The prototypes from Jiao‐Tai‐Wan were extensively metabolized by demethylation, hydroxylation, and reduction in phase Ⅰ metabolic reactions and by methylation or conjugation of glucuronide or sulfate in phase Ⅱ reactions. This is the first systematic study on the components and metabolic profiles of Jiao‐Tai‐Wan in vivo. This study provides a useful chemical basis for further pharmacological research and clinical application of Jiao‐Tai‐Wan.     

Evaluation of analytical performance and reliability of direct nanoLC‐nanoESI‐high resolution mass spectrometry for profiling the (xeno)metabolome

Mass spectrometry (MS) profiling techniques are used for analysing metabolites and xenobiotics in biofluids; however, detection of low abundance compounds using conventional MS techniques is poor. To counter this, nanoflow ultra‐high‐pressure liquid chromatography‐nanoelectrospray ionization‐time‐of‐flight MS (nUHPLC‐nESI‐TOFMS), which has been used primarily for proteomics, offers an innovative prospect for profiling small molecules. Compared to conventional UHPLC‐ESI‐TOFMS, nUHPLC‐nESI‐TOFMS enhanced detection limits of a variety of (xeno)metabolites by between 2 and 2000‐fold. In addition, this study demonstrates for the first time excellent repeatability and reproducibility for analysis of urine and plasma samples using nUHPLC‐nESI‐TOFMS, supporting implementation of this platform as a novel approach for high‐throughput (xeno)metabolomics. Copyright © 2014 John Wiley & Sons, Ltd.     

MALDI‐MS analysis and imaging of small molecule metabolites with 1,5‐diaminonaphthalene (DAN)

1,5‐diaminonaphthalene (DAN) has previously been reported as an effective matrix for matrix‐assisted laser desorption ionization‐mass spectrometry of phospholipids. In the current work, we investigate the use of DAN as a matrix for small metabolite analysis in negative ion mode. DAN was found to provide superior ionization to the compared matrices for MW < ~400 Da; however, 9‐aminoacridine (9‐AA) was found to be superior for a uridine diphosphate standard (MW 566 Da). DAN was also found to provide a more representative profile of a natural phospholipid mixture than 9‐AA. Finally, DAN and 9‐AA were applied for imaging of metabolites directly from corn leaf sections. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.