Identification of key metabolites of tectorigenin in rat urine by HPLC-MS(n)

This is a report about the identification of key metabolites of tectorigenin in rat urine using high-performance liquid chromatography-electrospray ionization ion trap tandem mass spectrometric method (HPLC-ESI-MS(n)). Six healthy rats were administered a single dose (80 mg/kg) of tectorigenin by oral gavage. Urine was sampled for 0-24 h and centrifuged at 12,000 rpm for 10 min to obtain the supernatants, then the supernatants were purified by solid-phase extraction with a C(18) cartridge. The chromatographic separation was carried out on a reversed-phase C(18) column with a gradient elution program whereas acetonitrile-0.1% formic acid water was used as mobile phase. Mass spectra were acquired in negative ionization mode and a data-dependant scan was used for the identification of the key metabolites of tectorigenin in the urine samples. As a result, four phase II metabolites and the parent drug tectorigenin were found and identified in rat urine for the first time.     

In vivo metabolism study of polygalic acid in rat using HPLC-ESI-MSn

A very simple and direct method has been established for the determination of polygalic acid and its metabolites in rat urine based on HPLC coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC-ESI-MS(n)). The rats were administered a single dose (100 mg/kg) of polygalic acid by oral gavage. The urine samples were collected and purified through a C(18) solid-phase extraction cartridge, and then these pretreated samples were injected into a reversed-phase C(18) column with a gradient elution program, whereas acetonitrile-0.5% aqueous formic acid was used as mobile phase and detected by an on-line MS/MS system. As a result, the parent drug and its four metabolites were identified and characterized in rat urine for the first time by comparing their changes in molecular mass (ΔM), retention times and full-scan MS(n) spectra with those of the parent drug. A possible metabolic pathway of polygalic acid was investigated and proposed. More importantly, the results demonstrated that the newly developed method (HPLC-ESI-MS(n)) was sensitive, simple and suitable for the determination of polygalic acid and its metabolites in biological samples.     

Metabolism studies of casticin in rats using HPLC‐ESI‐MSn

Casticin (3′,5‐dihydroxy‐3, 4′,6,7‐tetramethoxyflavone) has been revealed to possess various kinds of pharmacological activities, including immunomodulatory, anti‐hyperprolactinemia, anti‐tumor and neuroprotetective activities. In order to gain an understanding of the biotransformation of casticin in vivo, a systematic method based on liquid chromatography–electrospray ionization tandem mass spectrometry (LC‐ESI‐MSⁿ) was developed to identify the metabolites of casticin in rats after oral administration of single dose of casticin at 200 mg/kg. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent drug, the parent compound and 25 metabolites were identified in rat plasma, urine and six selected tissues. This is the first systematic metabolism study of casticin in vivo. The results indicated that methylation, demethylation, glucuronidation and sulfation were the main biotransformation pathways of casticin in vivo. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative analysis of a quaternary ammonium drug: ipratropium bromide by LC/ESI-MS(n) in horse plasma and urine

A quantitative method, using LC/ESI-MS(n) with a quadrupole linear ion trap mass analyzer, has been developed for the analysis of ipratropium cation in horse plasma and urine. The method applies solid-phase extraction with WCX cartridges for plasma and MM2 cartridges for urine, prior to analysis by LC/ESI-MS(n). The efficiency of extraction combined with the sensitivity and the selectivity of MS(n) allows for the quantification of ipratropium cation at picogram per milliliter levels. The analytical capabilities of the method have been successfully checked by the quantitative analysis of ipratropium cation in post-administration samples collected from horses treated by nebulization.     

Antidepressant-like effect of triterpenoids extracts from Poria cocos on the CUMS rats by 16S rRNA gene sequencing and LC–MS metabolomics

Abstract

Poria cocos is an edible medicinal mushroom with the effects of inducing diuresis, excreting dampness, invigorating the spleen and tranquilizing the mind. The triterpenoids of Poria cocos as the main active ingredients have shown health benefits of the central nervous system of the human body. Accordingly, this study aimed at further understanding the antidepressant-like effect and a potential mechanism of the triterpenoids extracts from Poria cocos (TPC). As a result, the TPC significantly ameliorated depression-like behaviors on chronic unpredictable mild stress (CUMS) rats, and restored the levels of brain-derived neurotrophic factor and nerve growth factor in the hippocampus of rats. Gut microiota analysis revealed that TPC could increase the biodiversity and markedly regulate the relative abundance of [Prevotella], Allobaculum and Ochrobactrum of CUMS rats. The cecal contents metabolomics pointed to thirteen biomarkers associated with TPC antidepressant effect, which involved in primary bile acid biosynthesis, taurine and hypotaurine metabolism, arginine and proline metabolism. Correlation analysis further showed that there was a strong correlation relationship between the gut microbiota and the cecal contents metabolites, especially compounds involved in energy metabolism, inflammation and immunity. In conclusion, TPC showed a potential antidepressant effect, which was possibly mediated the gut microbiota and cecal contents metabolism.     

Extraction and separation of lactate dehydrogenase inhibitors from Poria cocos (Schw.) Wolf based on a hyphenated technique and in vitro methods

Stroke is one of the most common diseases worldwide. Lactate dehydrogenase inhibitors are widely used in the treatment of ischemic stroke, with natural products considered a promising source of lactate dehydrogenase inhibitors. In this study, ultrafiltration liquid chromatography coupled with mass spectrometry was used for the screening and identification of lactate dehydrogenase inhibitors from Poria cocos . Five lactate dehydrogenase inhibitors were selected: dehydropachymic acid, pachymic acid, dehydrotrametenolic acid, trametenolic acid, and eburicoic acid. The inhibitors were extracted and isolated with purities of 96.75, 98.15, 97.25, 95.46, and 94.88%, respectively, by using a new “hyphenated” strategy of microwave‐assisted extraction coupled with counter‐current chromatography and centrifugal partition chromatography by a two‐phase solvent system of n‐hexane/ethyl acetate/ethanol/water at the volume ratio 0.965:1.000:0.936:0.826 v/v/v/v. The bioactivity of the isolated compounds was assessed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay in PC12 cells. The results also showed that the hyphenated technique of microwave‐assisted extraction coupled with counter‐current chromatography and centrifugal partition chromatography was an efficient method for the continuous extraction and online isolation of chemical constituents from medicinal herbs. Furthermore, the research route based on the activity screening, extraction, separation, and activity verification of the compounds offered advantages of efficiency, orientation, and objectivity.     

Structural elucidation of rat biliary metabolites of corynoxeine and their quantification using LC‐MSn

Corynoxeine (COR) is one of 4 bioactive oxindole alkaloids in Uncaria species. In this work two phase I metabolites, namely 11‐hydroxycorynoxeine (M1) and 10‐hydroxycorynoxeine (M2), and two phase II metabolites, namely 11‐hydroxycorynoxeine 11‐O‐β‐d ‐glucuronide (M3) and 10‐hydroxycorynoxeine 10‐O‐β‐d ‐glucuronide (M4), were detected in rat bile after oral dose of COR (0.105 mmol/kg), by optimized high‐performance liquid chromatography–tandem mass spectrometry (LC‐MSⁿ) with electrospray ionization in positive ion mode. Structures of M1–4 were determined by LC‐MSⁿ, nuclear magnetic resonance, circular dichroism and high‐resolution MS spectra. COR and its metabolites in rat bile were quantified by LC‐MSⁿ. The LC‐MSⁿ quantification methods for COR and its metabolites yielded a linearity with coefficient of determination ≥0.995 from 5.0 × 10^?10 to 5.0 × 10^?7 m . The recoveries of stability tests varied from 96.80 to 103.10%. Accuracy ranged from 91.00 to 105.20%. Relative standard deviation for intra‐day and inter‐day assay was <5.0%. After the oral dose 0.14% of COR was detected in rat bile from 0 to 8 h, in which in total 97.8% COR biotransformed into M1–4. M1 and M2 yielded 48.1 and 49.7%, which successively glucuronidated to M3 and M4 at 47.2 and 43.8%, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

The profiling and identification of the metabolites of 8‐prenylkaempferol and a study on their distribution in rats by high‐performance liquid chromatography with diode array detection combined with electrospray ionization ion trap time‐of‐flight multistage mass spectrometry

8‐Prenylkaempferol is a prenylflavonoid that has various bioactivities and benefits for human health. A high‐performance liquid chromatography with a diode array detector combined with electrospray ionization ion trap time‐of‐flight multistage mass spectrometry (HPLC‐DAD‐ESI‐IT‐TOF‐MSⁿ) method was established to profile and identify the metabolites of 8‐prenylkaempferol in rat in vivo and in vitro, and to study the distribution of these metabolites in rats for the first time. A total of 38 metabolites were detected and tentatively identified, 30 of which were identified as new compounds. The new in vivo metabolic reactions in rats of prenylflavonoids of isomerization, polymerization, sulfation, amino acid conjugation, vitamin C conjugation and other known metabolic reactions were found in the metabolism of 8‐prenylkaempferol. The numbers of detected metabolites in feces, urine, plasma, small intestine, stomach, kidneys, liver, heart, lungs, spleen and hepatic S9 fraction were 31, 19, 1, 20, 13, 8, 7, 3, 3, 1 and 11, respectively. This indicated that small intestine and stomach were the major organs in which the 8‐prenylkaempferol metabolites were distributed. Furthermore, 16 metabolites were determined to have bioactivities based on the literature and ‘PharmMapper’ analysis. These findings are useful for better comprehension of the effective forms, target organs and pharmacological actions of 8‐prenylkaempferol. Moreover, they provide a reference for the study of the metabolism and distribution of prenylflavonoid aglycone compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Serum pharmacochemistry combined with multiple data processing approach to screen the bioactive components and their metabolites in Mutan Cortex by ultra‐performance liquid chromatography tandem mass spectrometry

Root cortex of Paeonia suffruticosa Andrews (Paeoniaceae), known as Moutan Cortex (MC), is known to have anti‐allergic and anti‐inflammatory properties. However, the constituents absorbed into blood after oral administration of MC remain unknown. A sensitive and rapid method by ultra‐high‐pressure liquid chromatography–electrospray ionization–quadrupole‐time‐of‐flight mass spectrometry (UPLC‐ESI‐Q‐TOF‐MS) technology and the MetaboLynx^TM software combined with multiple data processing approach (Mdpa) was established to investigate the absorbed constituents in rats after oral administration of MC, providing unique high‐throughput capabilities for drug metabolism study. A hyphenated electrospray ionization and quadrupole‐time‐of‐flight analyzer was used for the determination of accurate mass of the fragment ion in negative mode, with excellent MS mass accuracy and enhanced data acquisition. This rapid automated analysis method was successfully applied for screening and identification of the constituents absorbed and metabolized studies of MC after oral administration to rats. A total of 46 peaks were obtained from MC, 41 of which were tentatively characterized. In the VIP‐plot of orthogonal partial least‐squares discriminant analysis, 23 interesting ions in serum samples were extracted, and 16 parent components and seven metabolites were detected in vivo. The integrative serum pharmacochemistry technique, UPLC‐ESI‐Q‐TOF‐MS, and Mdpa method were successfully applied for rapid discovery of multiple components from MC. Copyright © 2013 John Wiley & Sons, Ltd.     

Enrichment and separation of antitumor triterpene acids from the epidermis of Poria cocos by pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography

Triterpene acids were extracted from the epidermis of Poria cocos (Schw.) Wolf. These acids were found to inhibit the growth of lung cancer cells in vitro and in vivo. An efficient method for the preparative separation of antitumor triterpene acids was established that involves the combination of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography. We used pH‐zone‐refining counter‐current chromatography to concentrate the triterpene acids using a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (3:7:5:5, v/v/v/v), trifluoroacetic acid (10 mM) was added to the upper phase as a retainer, and ammonia (10 mM) was added to the lower phase as an eluter. As a result, 200 mg concentrate of triterpene acids was obtained from 1.0 g of crude extract. The concentrate was further separated by conventional high‐speed counter‐current chromatography using a solvent system composed of petroleum ether/ethyl acetate/methanol/water (0.8:1.2:1.2:0.9, v/v), yielding 50 mg of poricoic acid A and 5 mg of poricoic acid B from 120 mg concentrate, respectively. The inhibitory activity of the major compound on lung A549 cells was examined and poricoic acid A was found to significantly inhibit the growth of A 549 cells.     

Structural elucidation of in vitro metabolites of emodin by liquid chromatography-tandem mass spectrometry

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was employed to investigate the in vitro metabolism of emodin. Emodin was incubated with rat liver microsomes in the presence of a NADPH-generating system, followed by extraction with ethyl acetate. After separation on a reversed-phase C18 analytical column with a linear gradient elution of methanol and 0.1% formic acid in water, negative electrospray ionization tandem mass spectrometry experiments were performed. As a result, the parent drug and its six metabolites were detected from rat liver microsomal incubations. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. Besides three mono-hydroxylated metabolites (omega-hydroxyemodin, 2-hydroxyemodin, 4-hydroxyemodin), three other metabolites were identified, which were emodic acid, 3-carbomethoxy-6-methoxy-1,8-dihydroxyanthraquinone and physcion, respectively.     

Quantification and pharmacokinetics of crizotinib in rats by liquid chromatography–tandem mass spectrometry

Crizotinib is a small molecule inhibitor of anaplastic lymphoma kinase (ALK) and can be used to treat ALK‐positive nonsmall‐cell lung cancer. A rapid and simple high‐performance liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of crizotinib in rat plasma using a chemical synthetic compound buspirone as the internal standard (IS). The plasma samples were pretreated by a simple protein precipitation with methanol–acetonitrile (1:1, v/v). Chromatographic separation was successfully achieved on an Agilent Zorbax XDB C₁₈ column (2.1 × 50 mm, 3.5 µm). The gradient elution system was composed of 0.1% formic acid aqueous solution and 0.1% formic acid in methanol solution. The flow rate was set at 0.50 mL/min. The multiple reaction monitoring was based on the transitions of m/z = 450.3 → 177.1 for crizotinib and 386.2 → 122.2 for buspirone (IS). The assay was successfully validated to demonstrate the selectivity, matrix effect, linearity, lower limit of quantification, accuracy, precision, recovery and stability according to the international guidelines. The lower limit of quantification was 1.00 ng/mL in 50 μL of rat plasma. This LC‐MS/MS assay was successfully applied to the quantification and pharmacokinetic study of crizotinib in rats after intravenous and oral administration of crizotinib. The oral absolute bioavailability of crizotinib in rats was 68.6 ± 9.63%. Copyright © 2015 John Wiley & Sons, Ltd.     

Investigation of the in vivo metabolism of harpagoside and distribution of its metabolites in rats by HPLC‐IT‐TOF‐MSn

Harpagoside, an iridoid glycoside, is the major bioactive constituent of the traditional Chinese medicine Scrophulariae Radix. High‐performance liquid chromatography with a diode array detector combined with electrospray ionization ion trap time‐of‐flight multistage mass spectrometry (HPLC‐ESI‐IT‐TOF‐MSⁿ) was used to profile and identify the metabolites of harpagoside in rats in vivo and to study the distribution of these metabolites in rats for the first time. A total of 45 metabolites were identified, 37 of which were postulated to be new compounds. The number of detected metabolites in the heart, liver, spleen, lung, kidney, stomach and small intestine was 2, 9, 6, 16, 4, 16 and 6, respectively, which indicated that the target organs of harpagoside should be spleen, lung and stomach. The main types of metabolic reactions of harpagoside in rats are hydrolysis, reduction, sulfuric acid addition, hydroxylation, methoxylation, sulfate substitution, methylation, glucose conjugation and amino acid conjugation. Furthermore, 23 metabolites were determined to have bioactivities based on the literature and ‘PharmMapper’ analysis. These findings are useful for better comprehension of the effective forms, target organs and pharmacological effects of harpagoside. Moreover, these findings provide a reference for studying the metabolism and distribution of iridoid compounds.     

Identification and characterization of stressed degradation products of metoprolol using LC/Q-TOF-ESI-MS/MS and MS(n) experiments

A rapid, specific and reliable isocratic high-performance liquid chromatography combined with quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) method has been developed and validated for the identification and characterization of stressed degradation products of metoprolol. Metoprolol, an anti-hypertensive drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per ICH-specified conditions. The drug showed extensive degradation under oxidative and hydrolysis (acid and base) stress conditions. However, it was stable to thermal, neutral and photolysis stress conditions. A total of 14 degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on a C(18) column (4.6 × 250 mm, 5 μm). To characterize degradation products, initially the mass spectral fragmentation pathway of the drug was established with the help of MS/MS, MS(n) and accurate mass measurements. Similarly, fragmentation pattern and accurate masses of the degradation products were established by subjecting them to LC-MS/QTOF analysis. Structure elucidation of degradation products was achieved by comparing their fragmentation pattern with that of the drug. The degradation products DP(2) (m/z 153) and DP(14) (m/z 236) were matched with impurity B, listed in European Pharmacopoeia and British Pharmacopoeia, and impurity I, respectively. The LC-MS method was validated with respect to specificity, linearity, accuracy and precision.     

Rapid and sensitive LC‐MS method for pharmacokinetic study of vinorelbine in rats

A rapid and sensitive LC‐electrospray ionization‐MS method was developed for determining vinorelbine in rat plasma. A 100 µL plasma sample was treated using a protein precipitation procedure and was chromatographed within 4 min using an Inertsil ODS‐3 C₁₈ (2.1 × 50 mm, 5 µm) column. The selected ion monitoring ions [M + H]⁺ were m/z 779 and m/z 811 for vinorelbine and vinblastine (internal standard), respectively. The method validation showed that the calibration curve for vinorelbine was linear over a concentration range of 1–1000 ng/mL with lower limit of quantification at 1 ng/mL. The method has been successfully applied to pharmacokinetics in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of piperaquine and its N‐oxidated metabolite in rat plasma using LC‐MS/MS

A sensitive and efficient liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of piperaquine (PQ) and its N ‐oxidated metabolite (PQ‐M) in plasma. A simple protein precipitation procedure was used for sample preparation. Adequate chromatographic retention was achieved on a C₁₈ column under gradient elution with acetonitrile and 2 mm aqueous ammonium acetate containing 0.15% formic acid and 0.05% trifluoroacetic acid. A triple‐quadrupole mass spectrometer equipped with an electrospray source was set up in the positive ion mode and multiple reaction monitoring mode. The method was linear in the range of 2.0–400.0 ng/mL for PQ and 1.0–50.0 ng/mL for PQ‐M with suitable accuracy, precision and extraction recovery. The lower limits of detection (LLOD) were established at 0.4 and 0.2 ng/mL for PQ and PQ‐M, respectively, using 40 μL of plasma sample. The matrix effect was negligible under the current conditions. No effect was found for co‐administrated artemisinin drugs or hemolysis on the quantification of PQ and PQ‐M. Stability testing showed that two analytes remained stable under all relevant analytical conditions. The validated method was successfully applied to a pharmacokinetic study performed in rats after a single oral administration of PQ (60 mg/kg).     

Optimization of Bioprocess Extraction of Poria cocos Polysaccharide (PCP) with Aspergillus niger β-Glucanase and the Evaluation of PCP Antioxidant Property

Poria cocos mushroom is widely used as a food and an herb in East Asian and other countries due to its high nutritional value. Research has demonstrated that Poria cocos polysaccharides (PCP) are the major bioactives and possess antioxidation, anti-inflammation, immunoregulation, and other health promoting properties. However, the efficient preparation of PCP has been a challenge, particularly in large scale for industry. Herein, we investigated the biotransformation of PCP from Poria cocos, catalyzed by β-glucanase from Aspergillus niger and focused on optimizing the most four influencing parameters: Temperature, time, pH, and enzyme dosage in this study. After numerous optimizations with the assistance of response surface optimization methodology, we have established that the optimal conditions for the biotransformation PCP preparation were as following: Enzymolysis temperature 60 °C, time 120 min, pH 5.0 and enzyme dose 20 mL. Under these conditions, the extraction yield of PCP reached as high as 12.8%. In addition, the antioxidant activities of PCP were evaluated by reducing power assay and 1,1-diphenyl-2-picryl-hydrazyl, superoxide anion, and hydroxyl radicals scavenging assays. Resulting data showed that PCP presented outstanding antioxidant capacity. Thus, these findings indicate that PCP could be produced as a natural antioxidant for further development.     

Identification of major metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside by LC/MS/MS

N(6) -(4-hydroxybenzyl) adenine riboside, a novel neuroprotective compound found in Gastrodia elata at trace level, is regarded as a potential drug for the treatment of neural degenerative disease. To understand the metabolism of this compound, the metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside were analyzed by HPLC-ESI-MS/MS after oral administration of this compound. Beside the parent compound, six phase I metabolites and four phase II metabolites in urine were detected by scanning all possible metabolites in extracted ion chromatograms mode. By comparing their product ion spectra and retention times with those of parent compound, these metabolites were identified and proved to be mainly formed via hydrolysis or hydroxylation in phase I, N-sulfation or N-glucuronidation in phase II or their combinations. Similarly, the parent compound, one phase I metabolite and two phase II metabolites were also identified in rat plasma. Therefore, the in vivo metabolic pathways of N(6) -(4-hydroxybenzyl) adenine riboside in rat were proposed.