High-resolution Extracted Ion Chromatograms Research Articles

An Efficient Integrated Strategy for Comprehensive Metabolite Profiling of Sakurasosaponin from Aegiceras corniculatum in Rats.

Sakurasosaponin, a primary bioactive saponin from Aegiceras corniculatum, shows potential as an anti-cancer agent. However, there is a lack of information on its in vivo metabolism. This study aims to profile the in vivo metabolites of sakurasosaponin in rat feces, urine, and plasma after oral administration. An efficient strategy using ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry was developed, which combined metabolic prediction, multiple mass defects filtering, and highresolution extracted ion chromatograms for rapid and systematic analysis. Firstly, a theoretical list of metabolites for sakurasosaponin was developed. This was done by considering the metabolic pathways of saponins. Next, the multiple mass defects filtering method was employed to identify potential metabolites in feces and urine, using the unique metabolites of sakurasosaponin as multiple mass defects filtering templates. Subsequently, a high-resolution extracted ion chromatogram was used to quickly determine the metabolites in rat plasma post-identification in feces and urine. Lastly, the analysis of accurate mass, typical neutral loss, and diagnostic ion of the candidate metabolites was carried out to confirm their structural elucidation, and metabolic pathways of sakurasosaponin in vivo were also proposed. In total, 30 metabolites were provisionally identified in feces, urine, and plasma. Analysis of metabolic pathways revealed isomerization, deglycosylation, oxidation, hydroxylation, sulfate conjugation, glucuronide conjugation, and other related reactions as the primary biotransformation reactions of sakurasosaponin in vivo. The findings demonstrate that the designed research strategy effectively minimizes matrix interference, prevents the omission of low-concentration metabolites, and serves as a foundation for the discovery of active metabolites of sakurasosaponin.

Integrated Solid-Phase Extraction, Ultra-High-Performance Liquid Chromatography-Quadrupole-Orbitrap High-Resolution Mass Spectrometry, and Multidimensional Data-Mining Techniques to Unravel the Metabolic Network of Dehydrocostus Lactone in Rats.

Dehydrocostus lactone (DL) is among the representative ingredients of traditional Chinese medicine (TCM), with excellent anticancer, antibacterial, and anti-inflammatory activities. In this study, an advanced strategy based on ultra-high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was integrated to comprehensively explore the metabolic fate of DL in rats. First, prior to data collection, all biological samples (plasma, urine, and feces) were concentrated and purified using solid-phase extraction (SPE) pre-treatment technology. Then, during data collection, in the full-scan (FS) data-dependent acquisition mode, FS-ddMS2 was intelligently combined with FS-parent ion list (PIL)-dynamic exclusion (DE) means for targeted monitoring and deeper capture of more low-abundance ions of interest. After data acquisition, data-mining techniques such as high-resolution extracted ion chromatograms (HREICs), multiple mass defect filters (MMDFs), diagnostic product ions (DPIs), and neutral loss fragments (NLFs) were incorporated to extensively screen and profile all the metabolites in multiple dimensions. As a result, a total of 71 metabolites of DL (parent drug included) were positively or tentatively identified. The results suggested that DL in vivo mainly underwent hydration, hydroxylation, dihydrodiolation, sulfonation, methylation, dehydrogenation, dehydration, N-acetylcysteine conjugation, cysteine conjugation, glutathione conjugation, glycine conjugation, taurine conjugation, etc. With these inferences, we successfully mapped the "stepwise radiation" metabolic network of DL in rats, where several drug metabolism clusters (DMCs) were discovered. In conclusion, not only did we provide a refined strategy for inhibiting matrix effects and fully screening major-to-trace metabolites, but also give substantial data reference for mechanism investigation, in vivo distribution visualization, and safety evaluation of DL.

Comprehensive metabolism study of Tangeretin in rat plasma, urine and faeces using ultra-high performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry.

Tangeretin, present in citrus fruits, is a polymethoxy flavone with extensive pharmacological effects. It has been widely used in the clinic, but there were no detailed studies on the in vivo metabolism of tangeretin. This study aimed to establish a rapid and effective strategy to identify the metabolites of tangeretin and evaluate the biotransformation pathways of tangeretin in rats. The ultra-high performance liquid chromatography (UHPLC) equipped with a Q-Exactive Orbitrap mass spectrometer was used to identify the metabolites of tangeretin in plasma, urine and faeces of rats after intragastric administration. Based on high-resolution extracted ion chromatograms (HREICs) and parallel reaction monitoring mode (PRM), metabolites of tangeretin were identified by comparing the accurate mass, chromatographic retention times, diagnostic product ions (DPIs) and neutral loss fragments (NLFs) with those of tangeretin reference standard. Isomers were distinguished by ClogP values. An efficient and integrated strategy was established for the comprehensive screening and characterizing of tangeretin metabolites through Rapid Profiling. Based on this strategy, a total of 52 metabolites were detected and identified, among which 25 metabolites were found in rat plasma, while 48 and 16 metabolites were characterized from rat urine and faeces, respectively. These metabolites were produced by demethylation, demethoxylation, hydroxylation, methoxylation, glucuronidation, glycosylation, sulfation, and their composite reactions. Interestingly, tangeretin is easy to lose methyl in vivo and becomes an intermediate product, and then other phase I and phase II reactions occur. Moreover, the characteristic fragmentation pathways of tangeretin were summarized for the subsequent metabolite identification. The analytical method based on UHPLC-Q-Exactive mass spectrometer has the ability to quickly clarify unknown metabolism. And the the comprehensive metabolism study of tangeretin provided an overall metabolic profile, which will be of great scientific basis for further studies on tangeretin in determining its pharmacokinetics, the bioactivity of the metabolites, and clinical applications.

Comprehensive metabolism study of swertiamarin in rats using ultra high-performance liquid chromatography coupled with Quadrupole-Exactive Orbitrap mass spectrometry

Swertiamarin, a natural ingredient with potent pharmacological activities in the iridoid glycoside family, had been reported to have significant therapeutic effects on a variety of human diseases. In this study, a systematic and efficient strategy based on UHPLC-Q-Exactive Orbitrap mass spectrometry was established to reveal the metabolic profile of swertiamarin in rat urine, plasma, and faeces. First of all, post-acquisition data-mining methods, including multiple mass defect filters (MMDFs) and high-resolution extracted ion chromatograms (HREICs), were developed to screen the metabolite candidates of swertiamarin from the complete mass scan data sets. Second, according to the diagnostic product ions (DPIs), neutral loss fragments (NLFs), chromatographic retention time, accurate mass measurement and calculated Clog P values, all metabolite candidates were rapidly identified. As a consequence, 49 metabolites altogether, including archetype compound, were preliminarily characterised. The corresponding in vivo biotransformation processes, such as dehydration, dehydrogenation, hydroxylation, hydrogenation, methylation, sulphonation, N-acetylcysteine (NAC) formation, N-heterocyclisation and their composite reactions, were all discovered in the study. In conclusion, our results not only detailedly elucidated many new metabolites and metabolic pathways of swertiamarin, but also provided a reference for further study of its pharmacological mechanism and evaluation of its safety.

A comprehensive profiling and identification of liquiritin metabolites in rats using ultra-high-performance liquid chromatography coupled with linear ion trap–orbitrap mass spectrometer

Liquiritin (LQ), a main component of liquorice, exerts various biological activities. However, insufficient attentions have been paid to the metabolism study on this natural compound until now. Our present study was conducted to investigate the LQ metabolites in rats urine, faeces and plasma using UHPLC-LTQ-Orbitrap mass spectrometer in both positive and negative ion modes. Meanwhile, post-acquisition data-mining methods including high-resolution extracted ion chromatogram (HREIC), multiple mass defect filters (MMDFs), neutral loss fragments (NLFs) and diagnostic product ions (DPIs) were utilised to screen and identify LQ metabolites from HR-ESI-MS to ESI-MS n stage. As a result, a total of 49 metabolites were detected and characterised unambiguously or tentatively. These metabolites were presumed to generate through glucuronidation, sulfation, deglucosylation, dehydrogenation, methylation, hydrogenation, hydroxylation, ring cleavage and their composite reactions. Our results not only provided novel and useful data to better understand the biological activities of LQ, but also indicated that the proposed strategy was reliable for a rapid discovery and identification drug-related constituents in vivo.

Development of a UHPLC-MS/MS-based data-mining method for rapid profiling and characterization of magnolol metabolites in rat urine and plasma

Magnolol, an essential bioactive natural lignans isolated from Magnolia Officinalis, has various pharmacological activities, such as antibacterial, anti-inflammatory, antioxidation and anti-tumor. In this study, a practical strategy based on ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) was established to reveal the metabolic fate of magnolol in rat plasma and urine. First of all, in the data-dependent scanning (DDS) acquisition mode, the ESI-MSn data sets of biological samples and reference standard were obtained by using a high-quality online data analysis method. Subsequently, off-line data-mining techniques including multiple mass defect filters (MMDFs) and high-resolution extracted ion chromatograms (HREICs) were applied to screen major-to-trace metabolites of magnolol at the full-scan ESI-MS1 level. Finally, a total of 177 metabolites (prototype compound included) were preliminarily observed and characterized according to accurate mass measurement, the characteristic fragmentation patterns, chromato-graphic behaviors, and corresponding Clog P values. The results showed that magnolol experienced a variety of biotransformation reactions in rats, including hydroxylation, methoxylation, dehydrogenation, carboxylation, sulfonation, glucuronide conjugation, glucose conjugation, N-acetylcysteine (NAC) conjugation, glutathione conjugation and their composite reactions. In conclusion, this study not only greatly expanded the understanding of the therapeutic material basis and pharmacological mechanism of magnolol, but also provided new ideas for its toxicity evaluation, safety monitoring and drug delivery forms design.

Detection and Identification of Catalpol Metabolites in the Rat Plasma, Urine and Faeces Using Ultra-high Performance Liquid Chromatography-Q Exactive Hybrid Quadrupole-orbitrap High-resolution Accurate Mass Spectrometry.

Catalpol, an iridoid glycoside, is one of the richest bioactive components present in Rehmannia glutinosa. More and more metabolites of drugs have exhibited various pharmacological effects, thus providing guidance for clinical application. However, few researches have paid attention to the metabolism of catalpol. This study aimed to establish a rapid and effective method to identify catalpol metabolites and evaluate the biotransformation pathways of catalpol in rats. In this study, catalpol metabolites in rat urine, plasma and faeces were analyzed by UHPLC-Q-Exactive MS for the characterization of the metabolism of catalpol. Based on high-resolution extracted ion chromatograms (HREICs) and parallel reaction monitoring mode (PRM), metabolites of catalpol were identified by comparing the diagnostic product ions (DPIs), chromatographic retention times, neutral loss fragments (NLFs) and accurate mass measurement with those of catalpol reference standard. A total of 29 catalpol metabolites were detected and identified in both negative and positive ion modes. Nine metabolic reactions, including deglycosylation, hydroxylation, dihydroxylation, hydrogenation, dehydrogenation, oxidation of methylene to ketone, glucuronidation, glycine conjugation and cysteine conjugation, were proposed. A rapid and effective method based on UHPLC-Q-Exactive MS was developed to mine the metabolism information of catalpol. Results of metabolites and biotransformation pathways of catalpol suggested that when orally administrated, catalpol was firstly metabolized into catalpol aglycone, after which phase I and phase II reactions occurred. However, hydrophilic chromatography-mass spectrometry is still needed to further find the polar metabolites of catalpol.

Comprehensive metabolism study of polydatin in rat plasma and urine using ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry

Polydatin is one of the main bioactive constituents isolated from Polygonum cuspidatum Sieb. et Zucc., which possesses various pharmacological activities. In this study, we established an efficient strategy based on ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry to uncover polydatin metabolites in rat urine and plasma. Firstly, multiple mass defect filters (MMDFs) combined with high-resolution extracted ion chromatograms (HREICs) was utilized to perform post-acquisition data-mining in ESI-MS1 stage. Secondly, metabolite candidates of polydatin were expounded systematically on the basis of diagnostic product ions (DPIs), chromatographic retention times, accurate mass, and neutral loss fragments (NLFs). Consequently, a total of 41 metabolites (polydatin included) were detected and identified tentatively in 12 min. These metabolites, including 40 in rat urine and 7 in rat plasma, were presumed to generate through glucuronidation, sulfation, deglucosylation, dehydrogenation, methylation, hydrogenation, hydroxylation and their composite reactions. Meanwhile, metabolite clusters, which were set in the form radially, were found in this study. In conclusion, our study expounded polydatin metabolites in rats and provided a reference for further researches on therapeutic material basis and mechanism of polydatin.

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 15metabolites 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.

Rapid Screening and Identification of Daidzein Metabolites in Rats Based on UHPLC-LTQ-Orbitrap Mass Spectrometry Coupled with Data-Mining Technologies.

Daidzein, the main bioactive soy isoflavone in Nature, has been found to possess many biological functions. It has been investigated in particular as a phytoestrogen owing to the similarity of its structure with that of the human hormone estrogen. Due to the lack of comprehensive studies on daidzein metabolism, further research is still required to clarify its in vivo metabolic fate and intermediate processes. In this study, an efficient strategy was established using UHPLC-LTQ-Orbitrap mass spectrometry to profile the metabolism of daidzein in rats. Meanwhile, multiple data-mining methods including high-resolution extracted ion chromatogram (HREIC), multiple mass defect filtering (MMDF), neutral loss fragment (NLF), and diagnostic product ion (DPI) were utilized to investigate daidzein metabolites from the HR-ESI-MS1 to ESI-MSn stage in both positive and negative ion modes. Consequently, 59 metabolites, including prototype compounds, were positively or tentatively elucidated based on reference standards, accurate mass measurements, mass fragmentation behaviors, chromatographic retention times, and corresponding calculated ClogP values. As a result, dehydration, hydrogenation, methylation, dimethylation, glucuronidation, glucosylation, sulfonation, ring-cleavage, and their composite reactions were ascertained to interpret its in vivo biotransformation. Overall, our results not only revealed the potential pharmacodynamics forms of daidzein, but also aid in establishing a practical strategy for rapid screening and identifying metabolites of natural compounds.

Detection and identification of furan fatty acids from fish lipids by high-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry

Using high-performance liquid chromatography coupled to high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC/ESI-Q-TOF-MS), we have developed a new method for detection and identification of furan fatty acids (F-acids), which are widely distributed in living organisms and foods as minor lipid components and are known to have antioxidant and anti-inflammatory effects. For this purpose, total fatty acids prepared from the testis lipids of Japanese chum salmon (Oncorhynchus keta) were examined without any concentration or isolation of F-acids. In negative ESI mode, F-acids gave a prominent [M−H]− ion, by which individual F-acids could be detected and identified. High-resolution extracted ion chromatograms clearly showed the occurrence of five major F-acid homologs as already reported by GC/MS. The method was successfully applied to several fish samples and revealed the occurrence of F-acids for the first time in the two New Zealand fish, hoki (Macruronus novaezelandiae) and school shark (Galeorhinus galeus).

Rapid profiling and identification of puerarin metabolites in rat urine and plasma after oral administration by UHPLC-LTQ-Orbitrap mass spectrometer.

Puerarin, a bioactive natural C-glycoside isoflavonoid isolated from Pueraria lobata (Willd.) Ohwi, possesses many potential health benefits. However, the in vivo metabolic fates of puerarin have not been comprehensively clarified yet. In this study, an efficient strategy based on UHPLC-LTQ-Orbitrap mass spectrometer in both positive and negative ion modes was developed to profile and characterize puerarin metabolites in rat urine and plasma. Meanwhile, post-acquisition data-mining methods including high-resolution extracted ion chromatogram (HREIC) and multiple mass defect filtering (MMDF) were utilized to screen potential puerarin metabolites from HR-ESI-MS1 stage. The mass fragmentation behaviors of five reference standards, including puerarin, daidzin, daidzein, genistin, and genistein, were comprehensively studied for construction of diagnostic product ions (DPIs), which could be employed to implement rapid identification of puerarin metabolites. Finally, a total of 66 metabolites (prototype compound included) were tentatively or positively identified based on standard substances, chromatographic retention times, accurate mass measurement, and corresponding ClogP values. Our results demonstrated that puerarin underwent multiple in vivo metabolic reactions including methylation, hydroxylation, dehydroxylation, hydrogenation, deglycosylation, glycosylation, sulfonation, glucuronidation, and their complicated reactions. In conclusion, the newly discovered puerarin metabolites significantly expanded the understanding on its pharmacological effects and built the foundation for further toxicity and safety studies.

An integrated strategy for rapid discovery and identification of the sequential piperine metabolites in rats using ultra high-performance liquid chromatography/high resolution mass spectrometery

Piperine, one of the major bioactive constituents isolated from natural flavorings and medicinal-culinary herbs, possesses various biological activities. In the present study, an integrated strategy based on ultra high-performance liquid chromatography/high resolution mass spectrometry was established to reveal piperine metabolism in rats. First of all, post-acquisition data-mining methods, including high resolution extracted ion chromatograms (HREICs) and multiple mass defect filtering (MMDF), were used to screen piperine metabolite candidates in a full-scan HRMS1 level. Then, parent ion list-dynamic exclusion coupled with data-dependent data-acquisition method was utilized to acquire MSn datasets. In addition, the established reverse molecular assembly (RMA) approach based on paired diagnostic product ions (pDPIs) coupled with neutral loss fragments (NLFs) was used to ascertain and identify the major-to-trace piperine metabolites efficiently. And then, the calculated ClogP values were utilized to distinguish the positional isomers. As a result, a total of 148 piperine metabolites were detected and characterized tentatively. The results demonstrated that piperine mainly underwent hydrogenation, dehydrogenation, hydroxylation, glucuronide conjugation, sulfate conjugation, ring-cleavage, and their composite reactions. Our results not only provided novel and useful data to better understand the safety, toxicity and efficacy of this potential therapeutic agent, but also indicated that the proposed strategy was reliable for a rapid discovery and identification drug-related constituents in vivo.

Profiling and identification of chlorogenic acid metabolites in rats by ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer

1. Chlorogenic acids (CGAs), one kind of major bioactive constituents isolated from Flos Lonicera Japonica, possess many biological activities, such as antibacterial, antioxidant and antiviral activities. In this study, we established an efficient strategy using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) to profile the in vivo metabolic fate of CGAs in rat urine and plasma.2. The extract from Flos Lonicera Japonica was orally administrated to Sprague-Dawley (SD) rats at a dose of 1000 mg/kg body weight. Then, a combination of various post-acquisition data mining methods, including high-resolution extracted ion chromatogram (HREIC) and multiple mass defect filters (MMDFs) and diagnostic product ions (DPIs), were adopted to characterize the known and unknown CGA metabolites in SD rats.3. As a result, a total of 68 CGA metabolites were unambiguously or tentatively screened and characterized. These metabolites, including 18 prototype compounds and 50 metabolites, were deduced to be yielded via methylation, hydrogenation, demethylation, dehydration, sulfate conjugation, glucuronide conjugation, glycosylation conjugation and their composite reactions, which mainly occurred to caffeoylquinic acids, dicaffeoylquinic acids, p-coumaroylquinic acids and feruloylquinic acids.4. In conclusion, this study profiled CGA metabolites, which are useful in understanding the in vivo metabolic fate, effective forms, and pharmacological and toxic actions of CGAs.

Profiling and identification of (-)-epicatechin metabolites in rats using ultra-high performance liquid chromatography coupled with linear trap-Orbitrap mass spectrometer.

(-)-Epicatechin (EC), an optical antipode of (+)-catechin (C), possesses many potential significant health benefits. However, the in vivo metabolic pathway of EC has not been clarified yet. In this study, an efficient strategy based on ultra-high performance liquid chromatography coupled with a linear ion trap-Orbitrap mass spectrometer was developed to profile and characterize EC metabolites in rat urine, faeces, plasma, and various tissues. Meanwhile, post-acquisition data-mining methods including high-resolution extracted ion chromatogram (HREIC), multiple mass defect filters (MMDFs), and diagnostic product ions (DPIs) were utilized to screen and identify EC metabolites from HR-ESI-MS1 to ESI-MSn stage. Finally, a total of 67 metabolites (including parent drug) were tentatively identified based on standard substances, chromatographic retention times, accurate mass measurement, and relevant drug biotransformation knowledge. The results demonstrated that EC underwent multiple in vivo metabolic reactions including methylation, dehydration, hydrogenation, glucosylation, sulfonation, glucuronidation, ring-cleavage, and their composite reactions. Among them, methylation, dehydration, glucosylation, and their composite reactions were observed only occurring on EC when compared with C. Meanwhile, the distribution of these detected metabolites in various tissues including heart, liver, spleen, lung, kidney, and brain were respectively studied. The results demonstrated that liver and kidney were the most important organs for EC and its metabolites elimination. In conclusion, the newly discovered EC metabolites significantly expanded the understanding on its pharmacological effects and built the foundation for further toxicity and safety studies. Copyright © 2017 John Wiley & Sons, Ltd.

Identification of Metabolites of 6'-Hydroxy-3,4,5,2',4'-pentamethoxychalcone in Rats by a Combination of Ultra-High-Performance Liquid Chromatography with Linear Ion Trap-Orbitrap Mass Spectrometry Based on Multiple Data Processing Techniques.

In this study, an efficient strategy was established using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) to profile the in vivo metabolic fate of 6′-hydroxy-3,4,5,2′,4′-pentamethoxychalcone (PTC) in rat urine and feces. The UHPLC-LTQ-Orbitrap method combines the high trapping capacity and MSn scanning function of the linear ion trap along with accurate mass measurements within 5 ppm and a resolving power of up to 30,000 over a wider dynamic range compared to many other mass spectrometers. In order to reduce the potential interferences of endogenous substances, the post-acquisition processing method including high-resolution extracted ion chromatogram (HREIC) and multiple mass defect filters (MMDF) were developed for metabolite detection. As a result, a total of 60 and 35 metabolites were detected in the urine and feces, respectively. The corresponding in vivo reactions such as methylation, hydroxylation, hydrogenation, decarbonylation, demethylation, dehydration, methylation, demethoxylation, sulfate conjugation, glucuronide conjugation, and their composite reactions were all detected in this study. The result on PTC metabolites significantly expanded the understanding of its pharmacological effects, and could be targets for future studies on the important chemical constituents from herbal medicines.

Plasma protein binding, pharmacokinetics, tissue distribution and CYP450 biotransformation studies of fidarestat by ultra high performance liquid chromatography–high resolution mass spectrometry

Fidarestat, an aldose reductase inhibitor, has been used for the treatment of the diabetic associated complications such as retinopathy, neuropathy and nephropathy. To better understand the metabolism and pharmacokinetics of fidarestat, we have evaluated plasma protein binding, pharmacokinetics, tissue distribution of the drug and its conjugated metabolites and CYP450 biotransformation by liquid chromatography–high resolution mass spectrometry. Effective chromatographic separation of fidarestat and hydrochlorothiazide (IS) in rat plasma and tissues was achieved on Hypersil gold C-18 column in an isocratic elution mode. For detection, a high-resolution Orbitrap mass spectrometer with heated electrospray ionization inlet in the negative ion mode was used. High-resolution extracted ion chromatograms for each analyte were obtained by processing the full-scan MS mode with 5ppm mass tolerance. The impact of plasma protein binding with the drug and conjugated metabolites of the drug on pharmacokinetics has been determined. The study indicated that 9.5% of free form of fidarestat may be pharmacologically active and the Cmax for free fidarestat was found to be 80.30±6.78ng/mL. The AUC0–t and AUC0–∞ were found to be 185.46±32 and 195.92±15.06ngh/mL, respectively. Among tissues, the maximum observed distribution was found to be in kidney followed by liver and heart. Docking experiments and in vitro CYP450 reaction phenotyping revealed that two CYP1A2 and CYP2D6 are involved in the phase I metabolism of fidarestat. Oxidative deamination and N/O glucuronidation are the major phase I and phase II metabolites, respectively. In vitro CYP450 inhibition assay of fidarestat for drug–drug interaction showed weak inhibition and may not alter pharmacokinetics, distribution or clearance of other co-administered drug.

Qualitative–(semi)quantitative data acquisition of artemisinin and its metabolites in rat plasma using an LTQ/Orbitrap mass spectrometer

Artemisinin (QHS) is one of the first-line antimalarials, and autoinduction of CYP-mediated metabolism can result in its reduced exposure. To better understand the autoinduction of QHS, we evaluated the pharmacokinetics of QHS and its phase I metabolites in rats using an liquid chromatography-high resolution mass spectrometry (LC-HRMS) method. The LC separation was improved, allowing the separation of QHS and its metabolites from their diastereomers, and seven metabolites of QHS with relatively high exposure were identified in rat plasma, including deoxyartemisinin (DQHS), three monoyhydroxylated plus deoxyl metabolites (M1-M3) and three monohydroxylated metabolites (M4-M6). For detection, a high-resolution LTQ/Orbitrap mass spectrometer with an electrospray ionization (ESI) inlet in the positive ion mode was used. High-resolution extracted ion chromatograms for each analyte were obtained by processing the full-scan MS dataset with 10 ppm mass tolerance. The plasma samples were pretreated by protein precipitation with acetonitrile. The standard curve was linear (r(2) > 0.99) over the QHS and DQHS concentration range of 5.0-200.0 ng/ml in 50 µl of plasma, which offered sufficient sensitivity and accuracy for the determination of QHS and its metabolites. A 3-day validation approach was used for absolute quantitation of QHS and DQHS. The other six metabolites of QHS were semiquantified based on the calibration curve of QHS. The present method was applied to the pharmacokinetic study of QHS in rats after a single oral administration. The data shown here also suggest that this type of mass analyzer will be capable of a quantitative-qualitative workflow.

Determination of amphetamine-type stimulants, cocaine and ketamine in human hair by liquid chromatography/linear ion trap–Orbitrap hybrid mass spectrometry

An LTQ Orbitrap XL hybrid mass spectrometry method was developed for the determination of illicit drugs and their metabolites, including amphetamine (AP), methamphetamine (MA), dimethylamphetamine (DMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), ketamine (KET), norketamine (NK), cocaine (COC) and benzoylecgonine (BE), in hair. Micropulverized extraction was employed for sample preparation using a small hair sample (2 cm piece or 0.2 mg). Recoveries of the analytes during sample preparation were estimated using fortified hair samples and ranged from 35.5% for COC to 71.7% for AP. High resolution full-scan mass spectra and unit resolution product-ion spectra were obtained with the Orbitrap analyzer and the linear ion-trap analyzer, respectively. High-resolution extracted ion chromatograms at a tolerance of 3 ppm were utilized for quantification. The analytes were identified using the product-ion spectra in combination with the accurate masses of the corresponding protonated molecules observed in the high-resolution mass spectra. Lower limits of quantification obtained from a 0.2 mg hair sample were 0.050 ng mg(-1) (MDMA, KET and BE), 0.10 ng mg(-1) (AP, MA, DMA, NK and COC) and 0.50 ng mg(-1) (MDA). Two reference materials were analyzed for verification, and segmental analysis of single strands of hair specimens from actual cases was performed.