Identification and structural characterization of in vivo metabolites of ketorolac using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS)

In vivo metabolites of ketorolac (KTC) have been identified and characterized by using liquid chromatography positive ion electrospray ionization high resolution tandem mass spectrometry (LC/ESI‐HR‐MS/MS) in combination with online hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, blood urine and feces samples were collected after oral administration of KTC to Sprague–Dawley rats. The samples were prepared using an optimized sample preparation approach involving protein precipitation and freeze liquid separation followed by solid‐phase extraction and then subjected to LC/HR‐MS/MS analysis. A total of 12 metabolites have been identified in urine samples including hydroxy and glucuronide metabolites, which are also observed in plasma samples. In feces, only O‐sulfate metabolite and unchanged KTC are observed. The structures of metabolites were elucidated using LC‐MS/MS and MSⁿ experiments combined with accurate mass measurements. Online HDX experiments have been used to support the structural characterization of drug metabolites. The main phase I metabolites of KTC are hydroxylated and decarbonylated metabolites, which undergo subsequent phase II glucuronidation pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of circulatory and excretory metabolites of meisoindigo in rat plasma,urine and feces by high‐performance liquid chromatography coupled with positive electrospray ionization tandem mass spectrometry

Meisoindigo has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s. However, information relevant to in vivo metabolism of meisoindigo is absent so far. In this study, in vivo circulatory metabolites of meisoindigo in rat plasma, as well as excretory metabolites in rat urine and feces, were identified by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Integration of multiple reaction monitoring with conventional metabolic profiling methodology was adopted to enable a more sensitive detection of in vivo metabolites. By comparing with the MS/MS spectra and retention times of the in vitro reduced metabolites, the major metabolites in rat plasma were proposed to form from 3,3′ double bond reduction, whereas the minor metabolites were formed from reduction followed by N‐demethylation, and reduction followed by phenyl mono‐oxidation. The major metabolites in the rat urine were proposed to form from reduction followed by phenyl mono‐oxidation, and its glucuronide conjugation and sulfate conjugation, whereas the minor metabolites were formed from 3,3′ double bond reduction, N‐demethylation, reduction followed by N‐demethylation, phenyl di‐oxidation, phenyl mono‐oxidation and its glucuronide conjugation and sulfate conjugation. The major metabolites in the rat feces were proposed to form from reduction followed by phenyl mono‐oxidation, whereas the minor metabolites were formed from reduction followed by N‐demethylation, and reduction followed by phenyl di‐oxidation. The phase I metabolic pathways showed a significant in vitro–in vivo correlation in rat. Copyright © 2010 John Wiley & Sons, Ltd.     

Metabolic profile of phillyrin in rats obtained by UPLC‐Q‐TOF‐MS

Forsythia suspensa Vahl (Oleaceae) is an important original plant in traditional Chinese medicine. The air‐dried fruits of Forsythia suspensa have long been used to relieve respiratory symptoms. Phillyrin is one of the main chemical constituent of Forsythia suspensa. A clear understanding of the metabolism of phillyrin is very important in rational clinical use and pharmacological research. In this study, the metabolism of phillyrin in rat was investigated for the first time using an ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS) method. Bile, urine and feces were collected from rats after single‐dose (10 mg/kg) orally administered phillyrin. Liquid–liquid extraction and ultrasonic extraction were used to prepare samples. UPLC‐Q‐TOF‐MS analysis of the phillyrin samples showed that phillyrin was converted to a major metabolite, M26, which underwent deglucosidation, further dehydration and desaturation. A total of 34 metabolites were detected including 30 phase I and four phase II metabolites. The conjugation types and structure skeletons of the metabolites were preliminarily determined. Moreover, 28 new metabolites were reported for the first time. The main biotransformation route of phillyrin was identified as hydrolysis, oxidation and sulfation. These findings enhance our understanding of the metabolism and the real active structures of phillyrin. Copyright © 2015 John Wiley & Sons, Ltd.     

Metabolic profiling of quercetin in rats using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry

Quercetin, a kind of major flavonoid found in many traditional chinese medicines, is an effective substance for treatments such as lowering blood lipids. However, the studies on quercetin have been mainly focused on its pharmacological effect; the treatment of diseases on a material basis, particularly the metabolites derived from quercetin in vivo , has not been evaluated. In this study, we determined the levels, distributions and types of quercetin's metabolites in plasma, urine, feces and bile of rats after a single oral administration of quercetin at a dose of 80 mg/kg, using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS). A total of 36 metabolites of quercetin were identified, including 11 metabolites in plasma, 34 metabolites in urine, 12 metabolites in feces and 21 metabolites in bile. The results showed that phase I metabolites were reduction metabolites and phase II metabolites mainly included glucuronidation, sulfation and methylation metabolites. These results provide important information on the metabolism of quercetin, which will be helpful for its further development and utilization.     

Metabolites study on 5‐O‐methylvisammioside in vivo and in vitro by ultra high performance liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry

5‐O‐Methylvisammioside is one of major chromones of Radix Saposhnikoviae possessing definite pharmacological activities, but there are few reports with respect to the metabolism of 5‐O‐methylvisammioside. In this work, metabolites in vivo were explored in male Sprague‐Dawley rats and in vitro investigated on rat intestinal bacteria incubation model and were identified by using ultra high performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry. An online data acquisition method based on a multiple mass defect filter and dynamic background subtraction was developed to trace all probable metabolites. As a result, 26 metabolites in vivo (including 18, 15, 10, and 10 in rat urine, faece, bile, and blood) and 7 metabolites in vitro were characterized, respectively. Additionally, the main metabolic pathways in vivo and in vitro, including deglycosylation, deglycosylation + demethylation, deglycosylation + oxidation, N‐acetylation, and sulfate conjugation, were summarized by calculating the relative content of each metabolite. The obtained results significantly enriched our knowledge about 5‐O‐methylvisammioside metabolism and will lead to a better understanding of its safety and efficacy.     

Metabolite identification of the antimalarial naphthoquine using liquid chromatography–tandem high‐resolution mass spectrometry in combination with multiple data‐mining tools

Naphthoquine (NQ) is one of important partner drugs of artemisinin‐based combination therapy (ACT), which is recommended for the treatment of uncomplicated Plasmodium falciparum. NQ shows a high cure rate after a single oral administration. It is absorbed quickly (time to peak concentration 2–4 h) and has a long elimination half‐life (255 h). However, the metabolism of NQ has not been clarified. In this work, the metabolite profiling of NQ was studied in six liver microsomal incubates (human, cynomolgus monkey, beagle dog, mini pig, rat and CD1 mouse), seven recombinant CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and rat (plasma, urine, bile and feces) using liquid chromatography tandem high‐resolution LTQ‐Orbitrap mass spectrometry (HRMSⁿ) in conjunction with online hydrogen/deuterium exchange. The biological samples were pretreated by protein precipitation and solid‐phase extraction. For data processing, multiple data‐mining tools were applied in tandem, i.e. background subtraction and followed by mass defect filter. NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the hydrogen/deuterium exchange data and cLogP simulation. As a result, five phase I metabolites (M1–M5) of NQ were characterized for the first time. Two metabolic pathways were involved: hydroxylation and N‐oxidation. This study demonstrates that LC‐HRMSⁿ in combination with multiple data‐mining tools in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs.     

Integrated strategy based on high‐resolution mass spectrometry coupled with multiple data mining techniques for the metabolic profiling of Xanthoceras sorbifolia Bunge husks in rat plasma,urine, and feces

An integrated strategy based on high‐resolution mass spectrometry coupled with multiple data mining techniques was developed to screen the metabolites in rat biological fluids after the oral administration of Xanthoceras sorbifolia Bunge husks. Mass defect filtering, product ion filtering, and neutral loss filtering were applied to detect metabolites from the complex matrix. As a result, 55 metabolites were tentatively identified, among which 45 barrigenol‐type triterpenoid metabolites were detected in the feces, and six flavonoids and four coumarins metabolites were in the urine. Moreover, eight prototype constituents in plasma, 36 in urine and 23 in feces were also discovered. Due to the poor bioavailability of barrigenol type triterpenoids, most of them were metabolized by intestinal flora. Phase I metabolic reactions such as deglycosylation, oxidation, demethylation, dehydrogenation, and internal hydrolysis were supposed to be their principal metabolic pathways. Coumarins were found in all the biosamples, whereas flavonoids were mainly in the urine. Unlike the saponins, they were mainly metabolized through phase II metabolic reactions like glucuronidation and sulfonation, which made them eliminated more easily by urine. This work suggested the metabolic profile of X. sorbifolia husks for the first time, which will be very valuable for its further development.     

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 of sweroside in rat urine using ultra‐high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry and NMR spectroscopy

Sweroside, a major active iridoid in Swertia pseudochinensis Hara, is recognized as an effective agent in the treatment of liver injury. Based on previous reports, the relatively short half‐life (64 min) and poor bioavailability (approximately 0.31%) in rats suggested that not only sweroside itself but also its metabolites could be responsible for the observed hepato‐protective effect. However, few studies have been carried out on the metabolism of sweroside. Therefore, the present study aimed at identifying the metabolites of sweroside in rat urine after a single oral dose (100 mg/kg). With ultra‐high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UHPLC/Q‐TOF‐MS), the metabolic profile revealed 11 metabolites in rat urine, including phase I, phase II and aglycone‐related products. The chemical structures of metabolites were proposed based on accurate mass measurements of protonated or deprotonated molecules and their fragmentation patterns. Our findings showed that the aglycone of sweroside (M05) and its glucuronide conjugate (M06) were principal circulating metabolites in rats. While several other metabolic transformations, occurring via reduction, N‐heterocyclization and N‐acetylation after deglycosylation, were also observed. Two metabolites (M05 and M06) were isolated from the rat urine for structural elucidation and identifcation of reaction sites. Both M05 and M06 were characterized by ¹H, ¹³C and two‐dimensional nuclear magnetic resonance (NMR) spectroscopy. UHPLC/Q‐TOF‐MS analysis has provided an important analytical platform to gather metabolic profile of sweroside. Copyright © 2014 John Wiley & Sons, Ltd.     

Metabolite identification of the antimalarial piperaquine in vivo using liquid chromatography–high‐resolution mass spectrometry in combination with multiple data‐mining tools in tandem

Artemisinin‐based combination therapy is widely used for the treatment of uncomplicated Plasmodium falciparum malaria, and piperaquine (PQ) is one of important partner drugs. The pharmacokinetics of PQ is characterized by a low clearance and a large volume of distribution; however, metabolism of PQ has not been thoroughly investigated. In this work, the metabolite profiling of PQ in human and rat was studied using liquid chromatography tandem high‐resolution LTQ‐Orbitrap mass spectrometry (HRMS). The biological samples were pretreated by solid‐phase extraction. Data processes were carried out using multiple data‐mining techniques in tandem, i.e., isotope pattern filter followed by mass defect filter. A total of six metabolites (M1–M6) were identified for PQ in human (plasma and urine) and rat (plasma, urine and bile). Three reported metabolites were also found in this study, which included N‐oxidation (M1, M2) and carboxylic products (M3). The subsequent N‐oxidation of M3 resulted in a new metabolite M4 detected in urine and bile samples. A new metabolic pathway N‐dealkylation was found for PQ in human and rat, leading to two new metabolites (M5 and M6). This study demonstrated that LC‐HRMSⁿ in combination with multiple data‐mining techniques in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs. Copyright © 2016 John Wiley & Sons, Ltd.     

Rapid identification of phase I and II metabolites of artemisinin antimalarials using LTQ‐Orbitrap hybrid mass spectrometer in combination with online hydrogen/deuterium exchange technique

Artemisinin drugs have become the first‐line antimalarials in areas of multi‐drug resistance. However, monotherapy with artemisinin drugs results in comparatively high recrudescence rates. Autoinduction of CYP‐mediated metabolism, resulting in reduced exposure, has been supposed to be the underlying mechanism. To better understand the autoinduction of artemisinin drugs, we evaluated the biotransformation of artemisinin, also known as Qing‐hao‐su (QHS), and its active derivative dihydroartemisinin (DHA) in vitro and in vivo, using LTQ‐Orbitrap hybrid mass spectrometer in conjunction with online hydrogen (H)/deuterium (D) exchange high‐resolution (HR)‐LC/MS (mass spectrometry) for rapid structural characterization. The LC separation was improved allowing the separation of QHS parent drugs and their metabolites from their diastereomers. Thirteen phase I metabolites of QHS have been identified in liver microsomal incubates, rat urine, bile and plasma, including six deoxyhydroxylated metabolites, five hydroxylated metabolites, one dihydroxylated metabolite and deoxyartemisinin. Twelve phase II metabolites of QHS were detected in rat bile, urine and plasma. DHA underwent similar metabolic pathways, and 13 phase I metabolites and 3 phase II metabolites were detected. Accurate mass data were obtained in both full‐scan and MS/MS mode to support assignments of metabolite structures. Online H/D exchange LC‐HR/MS experiments provided additional evidence in differentiating deoxydihydroxylated metabolites from mono‐hydroxylated metabolites. The results showed that the main phase I metabolites of artemisinin drugs are hydroxylated and deoxyl products, and they will undergo subsequent phase II glucuronidation processes. This study also demonstrated the effectiveness of online H/D exchange LC‐HR/MSⁿ technique in rapid identification of drug metabolites. Copyright © 2011 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 phase I and II metabolites of the new designer drug α‐pyrrolidinohexiophenone (α‐PHP) in human urine by liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC‐QTOF‐MS)

Pyrrolidinophenones represent one emerging class of newly encountered drugs of abuse, also known as ‘new psychoactive substances’, with stimulating psychoactive effects. In this work, we report on the detection of the new designer drug α‐pyrrolidinohexiophenone (α‐PHP) and its phase I and II metabolites in a human urine sample of a drug abuser. Determination and structural elucidation of these metabolites have been achieved by liquid chromatography electrospray ionisation quadrupole time‐of‐flight mass spectrometry (LC‐ESI‐QTOF‐MS). By tentative identification, the exact and approximate structures of 19 phase I metabolites and nine phase II glucuronides were elucidated. Major metabolic pathways revealed the reduction of the ß‐keto moieties to their corresponding alcohols, didesalkylation of the pyrrolidine ring, hydroxylation and oxidation of the aliphatic side chain leading to n‐hydroxy, aldehyde and carboxylate metabolites, and oxidation of the pyrrolidine ring to its lactam followed by ring cleavage and additional hydroxylation, reduction and oxidation steps and combinations thereof. The most abundant phase II metabolites were glucuronidated ß‐keto‐reduced alcohols. Besides the great number of metabolites detected in this sample, α‐PHP is still one of the most abundant ions together with its ß‐keto‐reduced alcoholic dihydro metabolite. Monitoring of these metabolites in clinical and forensic toxicology may unambiguously prove the abuse of the new designer drug α‐PHP. Copyright © 2015 John Wiley & Sons, Ltd.     

In silico methods for predicting metabolism and mass fragmentation applied to quetiapine in liquid chromatography/time‐of‐flight mass spectrometry urine drug screening

Current in silico tools were evaluated for their ability to predict metabolism and mass spectral fragmentation in the context of analytical toxicology practice. A metabolite prediction program (Lhasa Meteor), a metabolite detection program (Bruker MetaboliteDetect), and a fragmentation prediction program (ACD/MS Fragmenter) were used to assign phase I metabolites of the antipsychotic drug quetiapine in the liquid chromatography/time‐of‐flight mass spectrometry (LC/TOFMS) accurate mass data from ten autopsy urine samples. In the literature, the main metabolic routes of quetiapine have been reported to be sulfoxidation, oxidation to the corresponding carboxylic acid, N‐ and O‐dealkylation and hydroxylation. Of the 14 metabolites predicted by Meteor, eight were detected by LC/TOFMS in the urine samples with use of MetaboliteDetect software and manual inspection. An additional five hydroxy derivatives were detected, but not predicted by Meteor. The fragment structures provided by ACD/MS Fragmenter software confirmed the identification of the metabolites. Mean mass accuracy and isotopic pattern match (SigmaFit) values for the fragments were 2.40 ppm (0.62 mDa) and 0.010, respectively. ACD/MS Fragmenter, in particular, allowed metabolites with identical molecular formulae to be differentiated without a need to access the respective reference standards or reference spectra. This was well exemplified with the hydroxy/sulfoxy metabolites of quetiapine and their N‐ and O‐dealkylated forms. The procedure resulted in assigning 13 quetiapine metabolites in urine. The present approach is instrumental in developing an extensive database containing exact monoisotopic masses and verified retention times of drugs and their urinary metabolites for LC/TOFMS drug screening. Copyright © 2009 John Wiley & Sons, Ltd.     

In vivo metabolite identification of acotiamide in rats using ultra‐performance liquid chromatography–quadrupole/time‐of‐flight mass spectrometry

Acotiamide hydrochloride (ACT) is a drug used for the treatment of functional dyspepsia. Understanding which metabolites are likely to be formed in vivo is essential for interpreting pharmacology, pharmacokinetic and toxicology data. The metabolism of ACT has been investigated using a specific and sensitive liquid chromatography positive ion electrospray ionization high‐resolution tandem mass spectrometry method. In vivo samples including rat plasma, urine and feces were collected separately after dosing healthy Sprague–Dawley rats at a dose of 20 mg kg ⁻¹ ACT at different time points up to 24 h. The metabolites were enriched by optimized sample preparation involving protein precipitation using acetonitrile followed by solid‐phase extraction. The mass defect filter technique was used for better detection of both predicted and unexpected drug metabolites with the majority of interference ions removed. The structural elucidation of the metabolites was performed by comparing their [M + H]⁺ ions and their product ions with those of the parent drug. As a result, a total of seven hitherto unknown metabolites were characterized from the biosamples. The only phase I metabolite detected was N‐ despropyl acotiamide, whereas six phase II glucuronide conjugate metabolites were identified.     

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.     

Characterization of forsythoside A metabolites in rats by a combination of UHPLC‐LTQ‐Orbitrap mass spectrometer with multiple data processing techniques

Forsythoside A (FTA), the main active constituent isolated from Fructus Forsythiae, has various biological functions including anti‐oxidant, anti‐viral and anti‐microbial activities. However, while research on FTA has been mainly focused on the treatment of diseases on a material basis, FTA metabolites in vivo have not been comprehensively evaluated. Here, a rapid and sensitive method using a UHPLC‐LTQ‐Orbitrap mass spectrometer with multiple data processing techniques including high‐resolution extracted ion chromatograms, multiple mass defect filters and diagnostic product ions was developed for the screening and identification of FTA metabolites in rats. As the result, a total of 43 metabolites were identified in biological samples including 42 metabolites in urine, 22 metabolites in plasma and 15 metabolites in feces. These results demonstrated that FTA underwent a series of in vivo metabolic reactions including methylation, dimethylation, sulfation, glucuronidation, diglucuronidation, cysteine conjugation and their composite reactions. The research enhanced our understanding of FTA metabolism and built a foundation for further toxicity and safety studies.     

Identification and quantitative analysis of physalin D and its metabolites in rat urine and feces by liquid chromatography with triple quadrupole time‐of‐flight mass spectrometry

Physalin D is known to show extensive bioactivities. However, no excretion study has elucidated the excretion of physalin D and its metabolites. This study investigates the excretion of physalin D and its metabolites in rats. Metabolites in rat urine and feces were separated and identified by liquid chromatography with triple quadrupole time‐of‐flight mass spectrometry. Furthermore, a validated high‐performance liquid chromatography with tandem mass spectrometry method was developed to quantify physalin D, physalin D glucuronide, and physalin D sulfate in rat feces and urine after the intragastric administration of physalin D. The analyte showed good linearity over a wide concentration range (r  > 0.995), and the lower limit of quantification was 0.0532 μg/mL and 0.226 μg/g for urine and feces, respectively. Nine metabolites, including five phase I and four phase II metabolites, were identified and clarified after dosing in vivo. Only 4.0% of the gavaged dose, including physalin D and its phase II metabolites, was excreted in urine, whereas 10.8% was found in feces in the unchanged form. The results indicate that the extensive and rapid metabolism may be the main factors leading to the short half‐life of physalin D. These results can provide a basis for further studies on the structural modification and pharmacology of physalin D.     

In vivo metabolism study of vaccarin in rat using HPLC‐LTQ‐MSn

In order to illustrate the main biotransformation pathways of vaccarin in vivo, metabolites of vaccarin in rats were identified using a specific and sensitive high‐performance liquid chromatography–electrospray ionization linear ion trap mass spectrometry (LTQ XL?) method. The rats were administered a single dose (200 mg/kg) of vaccarin by oral gavage. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent drug, the parent compound and six metabolites were found in rat urine after oral administration of vaccarin. The parent compound and five metabolites were detected in rat plasma. In heart, liver and kidney samples, respectively, one, four and three metabolites were identified, in addition to the parent compound. Three metabolites, but no trace of parent drug, were found in the rat feces. This is the first systematic metabolism study of vaccarin in vivo. The biotransformation pathways of vaccarin involved methylation, hydroxylation, glycosylation and deglycosylation. Copyright © 2012 John Wiley & Sons, Ltd.     

Liquid chromatography/quadrupole time‐of‐flight mass spectrometry in combination with online hydrogen/deuterium exchange technique for structural elucidation of phase I metabolites of iso‐phenylcyclopentylamine in rat bile

MS/MS experiment and accurate mass measurement are powerful tools in metabolite identification. However, sometimes these data do not provide enough information to assign an unambiguous structure to a metabolite. In combination with MS techniques, hydrogen/deuterium (H/D) exchange can provide additional information for structural elucidation by determination of the number of exchangeable hydrogen atoms in a structure. In this study, the principal phase I metabolites of iso‐phenylcyclopentylamine in rat bile were identified by high‐performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF‐MS). Since N‐oxidation may occur because of the existence of the primary amino group in the structure, it was difficult to differentiate the hydroxylated metabolites from N‐oxides by ESI‐Q‐TOF‐MS alone. Therefore, online H/D exchange technique was applied to solve this problem. Finally, 25 phase I metabolites were detected and structurally described, in which 11 were confirmed to be N‐oxides. This study demonstrated the effectiveness of high‐resolution mass spectrometry in combination with an online H/D exchange technique in rapid identification of drug metabolites, especially in discriminating hydroxylated metabolites from N‐oxides. Copyright © 2014 John Wiley & Sons, Ltd.