Multi‐analysis strategy for metabolism of Andrographis paniculata in rat using liquid chromatography/quadrupole time‐of‐flight mass spectrometry

Compared with chemical drugs, it is a huge challenge to identify active ingredients of multicomponent traditional Chinese medicine (TCM). For most TCMs, metabolism investigation of absorbed constituents is a feasible way to clarify the active material basis. Although Andrographis paniculata (AP) has been extensively researched by domestic and foreign scholars, its metabolism has seldom been fully addressed to date. In this paper, high‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry was applied to analysis and characterization of AP metabolism in rat urine and feces samples after oral administration of ethanol extract. The differences in metabolites and metabolic pathways between the two biological samples were further compared. The chemical structures of 20 components were tentatively identified from drug‐treated biological samples, including six prototype components and 14 metabolites, which underwent such main metabolic pathways as hydrolyzation, hydrogenation, dehydroxylation, deoxygenation, methylation, glucuronidation, sulfonation and sulfation. Two co‐existing components were found in urine and feces samples, suggesting that some ingredients' metabolic processes were not unique. This study provides a comprehensive report on the metabolism of AP in rats, which will be helpful for understanding its mechanism. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification of metabolites of Helicid in vivo using ultra‐high performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry

Helicid is an active natural aromatic phenolic glycoside ingredient originating from a well‐known traditional Chinese herbal medicine and has the significant effects of sedative hypnosis, anti‐inflammatory analgesia and antidepressant. In this study, we analyzed the potential metabolites of Helicid in rats by multiple mass defect filter and dynamic background subtraction in ultra‐high‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry (UHPLC‐Q‐TOF‐MS). Moreover, we used a novel data processing method, ‘key product ions’, to rapidly detect and identify metabolites as an assistant tool. MetabolitePilot™ 2.0 software and PeakView™ 2.2 software were used for analyzing metabolites. Twenty metabolites of Helicid (including 15 phase I metabolites and five phase II metabolites) were detected by comparison with the blank samples. The biotransformation route of Helicid was identified as demethylation, oxidation, dehydroxylation, hydrogenation, decarbonylation, glucuronide conjugation and methylation. This is the first study simultaneously detecting and identifying Helicid metabolism in rats employing UHPLC‐Q‐TOF‐MS technology. This experiment not only proposed a method for rapidly detecting and identifying metabolites, but also provided useful information for further study of the pharmacology and mechanism of Helicid in vivo. Furthermore, it provided an effective method for the analysis of other aromatic phenolic glycosides metabolic components in vivo.     

Metabolite profiles of epimedin C in rat plasma and bile by ultra‐performance liquid chromatography coupled with quadrupole‐TOF‐MS

Epimedin C is one of the major bioactive constituents of Herba Epimedii. In this study, the metabolite profiles of epimedin C in rat plasma and bile were qualitatively investigated, and the possible metabolic pathways of epimedin C were subsequently proposed. After oral administration of epimedin C at a single dose of 80 mg/kg, rat biological samples were collected and pretreated by protein precipitation. Then these pretreated samples were injected into an Acquity UPLC BEH C₁₈ column and detected by ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry. In all, 12 metabolites were identified in the biosamples. Of these, eight, two from plasma and six from bile, are, to our knowledge, reported here for the first time. The results indicated that epimedin C was metabolized via desugarization, dehydrogenation, hydrogenation, dehydroxylation, hydroxylation, demethylation and glucuronidation pathways in vivo. Thus, this study revealed the possible metabolite profiles of epimedin C in rat plasma and bile. Copyright © 2014 John Wiley & Sons, Ltd.     

The profiling and identification of the metabolites of (+)‐catechin and study on their distribution in rats by HPLC‐DAD‐ESI‐IT‐TOF‐MSn technique

(+)‐Catechin, a potential beneficial compound to human health, is widely distributed in plants and foods. A high‐performance liquid chromatography with diode array detector and combined with electrospray ionization ion trap time‐of‐flight multistage mass spectrometry method was applied to profile and identify the metabolites of (+)‐catechin in rats and to study the distribution of these metabolites in rat organs for the first time. In total, 51 phase II metabolites (44 new) and three phase I metabolites were tentatively identified, comprising 16 (+)‐catechin conjugates, 14 diarylpropan‐2‐ol metabolites, 6 phenyl valerolactone metabolites and 18 aromatic acid metabolites. Further, 19 phase II metabolites were new compounds. The in vivo metabolic reactions of (+)‐catechin in rats were found to be ring‐cleavage, sulfation, glucuronidation, methylation, dehydroxylation and dehydrogenation. The numbers of detected metabolites in urine, plasma, small intestine, kidney, liver, lung, heart, brain and spleen were 53, 23, 27, 9, 7, 5, 3, 2 and 1, respectively. This indicated that small intestine, kidney and liver were the major organs for the distribution of (+)‐catechin metabolites. In addition, eight metabolites were found to possess bioactivities according to literature. These results are very helpful for better comprehension of the in vivo metabolism of (+)‐catechin and its pharmacological actions, and also can give strong indications on the effective forms of (+)‐catechin in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Metabolic profile and pharmacokinetics of polyphyllin I,an anticancer candidate,in rats by UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS

Polyphyllin I (PPI), a natural steroidal saponin originating from rihzome of Paris polyphylla , is a potential anticancer candidate. Previous pharmacokinetics study showed that the oral bioavailability of PPI was very low, which suggested that certain amount of PPI might be metabolized in vivo . However, to date, information regarding the final metabolic fates of PPI is very limited. In this study, metabolites of PPI and their pharmacokinetics in rats were investigated using UPLC‐QTOF‐MS/MS and LC‐TQ‐MS/MS. A total of seven putative metabolites, including six phase I and one phase II metabolites, were detected and identified with three exact structures by comparison with authentic standards for the first time. Oxidation, deglycosylation and glucuronidation were found to be the major metabolic processes of the compound in rats. The pharmacokinetics of prosapogenin A, trillin and diosgenin, three deglycosylation metabolites of PPI with definite anticancer effects, were further studied, which suggested that the metabolites underwent a prolonged absorption and slower elimination after intragastric administration of PPI at the dose of 500 mg/kg. This study provides valuable and new information on the metabolic fate of PPI, which will be helpful in further understanding its mechanism of action.     

Identification of absorbed constituents and in vivo metabolites in rats after oral administration of Physalis alkekengi var. franchetii by ultra‐high‐pressure liquid chromatography quadrupole time‐of‐flight mass spectrometry

The calyces of Physalis alkekengi var. franchetii (Chinese Lantern, JDL) are well‐known as traditional Chinese medicine owing to its various therapeutic effects. However, the bioactive constituents responsible for the pharmacological effects of JDL and their metabolites in vivo are still unclear to date. In this paper, an ultra‐high‐pressure liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry (UHPLC/Q‐TOF‐MS/MS) method was established to identify absorbed constituents and in vivo metabolites in rat biological fluids after oral administration of JDL. Based on the proposed strategy, 33 compounds were observed in dosed rat biosamples. Twelve of 33 compounds were indicated as prototype components of JDL, and 21 compounds were predicted to be metabolites of JDL. Finally, the metabolic pathways were proposed, which were glucuronidation, sulfation, methylation and dehydroxylation for flavonoid constituents and sulfonation and hydroxylation for physalin consitituents. This is the first systematic study on the absorbed constituents and metabolic profiling of JDL and will provide a useful template for screening and characterizing the ingredients and metabolites of traditional Chinese medicine.     

Screening and identification of metabolites of two kinds of main active ingredients and hepatotoxic pyrrolizidine alkaloids in rat after lavage Farfarae Flos extract by UHPLC‐Q‐TOF‐MS mass spectrometry

Farfarae Flos, the dried flower buds of Tussilago farfara L., is usually used to treat coughs, bronchitic and asthmatic conditions as an important traditional Chinese medicine. Tussilagone and methl butyric acid tussilagin ester are seen as representatives of two kinds of active substances. In addition, the pyrrolizidine alkaloids, mainly senkirkine and senecionine, present in the herb can be hepatoxic. In this study, a rapid and sensitive ultra‐high‐performance liquid chromatography coupled with hybrid triple quadrupole time‐of‐flight mass spectrometry method was successfully applied to identify the metabolites of tussilagone, methl butyric acid tussilagin ester, senkirkine and senecionine. A total of 35, 37, 18 and nine metabolites of tussilagone, methl butyric acid tussilagin ester, senkirkine and senecionine in rats were tentatively identified. Hydrolysis, oxidation, reduction and demethylation were the major metabolic reactions for tussilagone and methl butyric acid tussilagin ester. The main biotransformation routes of senkirkine and senecionine were identified as demethylation, N‐methylation, oxidation and reduction. This study is the first reported analysis and characterization of the metabolites and the proposed metabolic pathways might provide further understanding of the metabolic fate of the chemical constituents after oral administration of Farfarae Flos extract in vivo.     

The metabolite profiling of 3,4-dicaffeoylquinic acid in Sprague–Dawley rats using ultra-high performance liquid chromatography equipped with linear ion trap-Orbitrap MS

3,4-Dicaffeoylquinic acid (3,4-DiCQA) is a dicaffeoylquinic acid that possesses antioxidant, anti-inflammatory, antibacterial, antiviral, anticancer, hypoglycemic, hypotensive, and hepatoprotective activities. This study developed a rapid and reliable method using ultra-high performance liquid chromatography equipped with linear ion trap-Orbitrap MS to identify the metabolites of 3,4-DiCQA in rat plasma, urine, feces, and tissues. The metabolic profile of 3,4-DiCQA was determined after an oral administration of 200 mg/kg to rats. A strategy of full scan-parent ions list acquisition coupled to diagnostic product ion analysis for screening and identification of target metabolites was used. A total of 67 metabolites, combined with accurate mass measurement, diagnostic ions, neutral losses, and reference standards, were observed and characterized for the first time. The results indicated that hydrolysis, methylation, hydrogenation, hydration, dehydroxylation, dehydrogenation, sulfate conjugation, and glucuronide conjugation were the major metabolic reactions of 3,4-DiCQA in vivo.     

Biotransformation and metabolic profile of caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐cymaropyranoside with human intestinal microflora by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In our previous studies, caudatin‐2,6‐dideoxy‐3‐O‐methy‐β‐d‐ cymaropyranoside (CDMC) was for the first time isolated from Cynanchum auriculatum Royle ex Wightand and was reported to possess a wide range of biological activities. However, the routes and metabolites of CDMC produced by intestinal bacteria are not well understood. In this study, ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) technique combined with Metabolynx^TMsoftware was applied to analyze metabolites of CDMC by human intestinal bacteria. The incubated samples collected for 48 h in an anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC‐Q‐TOF‐MS within 12 min. Eight metabolites were identified based on MS and MS/MS data. The results indicated that hydrolysis, hydrogenation, demethylation and hydroxylation were the major metabolic pathways of CDMC in vitro. Seven strains of bacteria including Bacillus sp. 46, Enterococcus sp. 30 and sp. 45, Escherichia sp. 49A, sp. 64, sp. 68 and sp. 75 were further identified using 16S rRNA gene sequencing owing to their relatively strong metabolic capacity toward CDMC. The present study provides important information about metabolic routes of CDMC and the roles of different intestinal bacteria in the metabolism of CDMC. Moreover, those metabolites might influence the biological effect of CDMC in vivo, which affects the clinical effects of this medicinal plant. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of the metabolites of gigantol in rat urine by ultra‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Gigantol is a typical bibenzyl compound isolated from Dendrobii Caulis that has been widely used as a medicinal herb in China for the treatment of diabetic cataract, cancer and arteriosclerosis obliterans and as a tonic for stomach nourishment, saliva secretion promotion and fever reduction. However, few studies have been carried out on its in vivo metabolism. In the present study, a rapid and sensitive method based on ultra‐performance liquid chromatography/electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐Q/TOF‐MS) in positive ion mode was developed and applied to identify the metabolites of gigantol in rat urine after a single oral dose (100 mg/kg). Chromatographic separation was performed on an Acquity UPLC HSS T3 column (100 × 2.1 mm i. d., 1.8 µm) using acetonitrile and 0.1% aqueous formic acid as mobile phases. A total of 11 metabolites were detected and identified as all phase II metabolites. The structures of the metabolites were identified based on the characteristics of their MS, MS² data and chromatographic retention times. The results showed that glucuronidation is the principal metabolic pathway of gigantol in rats. The newly identified metabolites are useful to understand the mechanism of elimination of gigantol and, in turn, its effectiveness and toxicity. As far as we know, this is the first attempt to investigate the metabolic fate of gigantol in vivo. Copyright © 2014 John Wiley & Sons, Ltd.     

Study of the in vitro metabolism of TJ0711 using ultra high performance liquid chromatography with quadrupole time‐of‐flight and ultra fast liquid chromatography with quadrupole linear ion trap mass spectrometry

TJ0711 (1‐[4‐(2‐methoxyethyl)phenoxy]‐3‐[2‐(2‐methoxyphenoxy)ethylamino]‐2‐propanol) is a novel β‐adrenoreceptor blocker with vasodilating activity. The aim of this study was to investigate the in vitro metabolic properties of TJ0711 from both qualitative and quantitative aspects using mouse, rat, dog, and human liver microsomes as well as rat hepatocytes. Two modern liquid chromatography with tandem mass spectrometry systems, ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry and ultra fast liquid chromatography with quadrupole linear ion trap mass spectrometry, were utilized for the analysis. To better characterize the metabolic pathways of TJ0711, two major metabolites were incubated under the same conditions as that for TJ0711. TJ0711 was extensively metabolized in vitro, and a total of 34 metabolites, including 19 phase I and 15 phase II metabolites, were identified. Similar metabolite profiles were observed among species, and demethylation, hydroxylation, carboxylic acid formation, and glucuronidation were proposed as the major metabolic routes. Significant interspecies differences were observed in the metabolic stability studies of TJ0711. Furthermore, gender differences were significant in mice, rats, and dogs, but were negligible in humans. The valuable information provided in this work will be useful in planning and interpreting further pharmacokinetic, in vivo metabolism and toxicological studies of this novel β‐blocker.     

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.     

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.     

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.     

Ligand fishing of anti‐neurodegenerative components from Lonicera japonica using magnetic nanoparticles immobilised with monoamine oxidase B

In this work, monoamine oxidase B was immobilised onto magnetic nanoparticles to prepare a new type of affinity solid‐phase extraction adsorbent, which was used to extract the possible anti‐neurodegenerative components from the Lonicera japonica flower extracts. Coupled with high‐performance liquid chromatography with mass spectrometry, two monoamine oxidase B ligands were fished‐out and identified as isochlorogenic acid A and isochlorogenic acid C, which were found to be inhibitors of the enzyme for the first time, with similar half maximal inhibitory concentration values of 29.05 ± 0.49 and 29.77 ± 1.03 μM, respectively. Furthermore, equilibrium‐dialysis dissociation assay of enzyme‐inhibitor complex showed that both compounds have reversible binding patterns to monoamine oxidase B, and kinetic analysis demonstrated that they were mixed‐type inhibitors for monoamine oxidase B, with K_i and K_is values of 9.55 and 37.24 μM for isochlorogenic acid A, 9.53 and 35.50 μM for isochlorogenic acid C, respectively. The results indicated that isochlorogenic acid A and isochlorogenic acid C were the major active components responsible for the anti‐degenerative activity of the flowers of L. japonica, while magnetic nanoparticles immobilised monoamine oxidase B could serve as an efficient solid‐phase extraction adsorbent to specifically extract monoamine oxidase B inhibitors from complex herbal extracts.     

Identification of metabolites of Ginkgolide B in vivo and in vitro using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry

Ginkgolide B is a dietary diterpene with multiple pharmacological activities. However, current research on ginkgolide B is not comprehensive. The current study analyzed the metabolic profile of ginkgolide B in vivo and in vitro using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. To detect and identify the different metabolites in ginkgolide B, a novel data processing method was used as an assistant tool. A total of 53 different metabolites of ginkgolide B (38 phase I metabolites and 15 phase II metabolites) were detected relative to blank samples. The biotransformation route of ginkgolide B was identified as oxidation, dehydroxylation, hydrogenation, decarbonylation, demethylation, sulfate conjugation, glucose conjugation, methylation, and acetylation. The current study demonstrated a method for rapidly detecting and identifying metabolites and provided useful information to further characterize the pharmacology and mechanism of ginkgolide B. A method for the analysis of other diterpene metabolic components in vivo and in vitro was also established.     

Identification of metabolites of isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate in rats by high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate (IDHP) is an investigational new drug having the capacity for treating ailments in the cardiovascular and cerebrovascular system. In this work, a rapid and sensitive method using high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐ESI‐Q‐TOF‐MS) was developed to reveal the metabolic profile of IDHP in rats after oral administration. The method involved pretreatment of the samples by formic acid–methanol solution (v/v, 5:95), chromatographic separation by an Agilent Eclipse XDB‐C₁₈ column (150 × 4.6 mm i.dx., 5 μm) and online identification of the metabolites by Q‐TOF‐MS equipped with electrospray ionizer. A total of 16 metabolites from IDHP, including four phase I metabolites and 12 phase II metabolites, were detected and tentatively identified from rat plasma, urine and feces. Among these metabolites, Danshensu (DSS), a hydrolysis product of IDHP, could be further transformed to 11 metabolites. These results indicated that DSS was the main metabolite of IDHP in rats and the major metabolic pathways of IDHP in vivo were hydrolysis, O‐methylation, sulfation, glucuronidation and reduction. The results also demonstrated that renal route was the main pathway of IDHP clearance in rat. The present study provided valuable information for better understanding the efficacy and safety of IDHP. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of metabolites in rats after intravenous administration of salvianolic acid for injection by ultra‐performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry

It is an essential requirement to clarify the metabolites of traditional Chinese medicine (TCM) injections, which contain numerous ingredients, to assess their safe and effective use in clinic. Salvianolic acid for injection (SAFI), made from hydrophilic phenolic acids in Salvia miltiorrhiza Bunge, has been widely used for the treatment of cerebrovascular diseases, but information on its metabolites in vivo is still lacking. In the present study, we aimed to holistically characterize the metabolites of the main active ingredients in rat plasma, bile, urine and feces following intravenous administration of SAFI. An ultra‐performance liquid chromatography coupled with quadrupole‐time‐of‐flight mass spectrometry (UPLC/Q‐TOF‐MS) method was developed. Combining information on retention behaviors, multistage mass spectra and literature data, a total of eight prototypes and 52 metabolites were tentatively characterized. Metabolites originated from rosmarinic acid and salvianolic acid B comprised the majority of identified compounds. Meanwhile, four metabolites derived from salvianolic acid D and five from salvianolic acid B are reported for the first time. This study revealed that methylation, sulfation and glucuronidation were the major metabolic pathways of phenolic acids in SAFI in vivo. Furthermore, the developed UPLC/Q‐TOF‐MS method could also benefit the metabolic investigation of extracts and preparations in TCM with hydrophilic ingredients. Copyright © 2016 John Wiley & Sons, Ltd.     

Simultaneous determination of niacin and its metabolites—nicotinamide,nicotinuric acid and N‐methyl‐2‐pyridone‐5‐carboxamide—in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

An LC‐MS/MS method for the simultaneous quantitation of niacin (NA) and its metabolites, i.e. nicotinamide (NAM), nicotinuric acid (NUA) and N‐methyl‐2‐pyridone‐5‐carboxamide (2‐Pyr), in human plasma (1 mL) was developed and validated using nevirapine as an internal standard (IS). Extraction of the NA and its metabolites along with the IS from human plasma was accomplished using a simple liquid–liquid extraction. The chromatographic separation of NA, NAM, NUA, 2‐Pyr and IS was achieved on a Hypersil‐BDS column (150 ¥ 4.6 mm, 5 mm) column using a mobile phase consisting of 0.1% formic acid : acetonitrile (20:80 v/v) at a flow rate of 1 mL/min. The total run time of analysis was 2 min and elution of NA, NAM, NUA, 2‐Pyr and IS occurred at 1.37, 1.46, 1.40, 1.06 and 1.27 min, respectively. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 100–20000 ng/mL for NA; 10–1600 ng/mL for NUA and NAM and 50–5000 ng/mL for 2‐Pyr with mean correlation coefficient of ≥0.99 for each analyte. The method was sensitive, specific, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. The developed assay method was successfully applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.