An enhanced strategy integrating offline two-dimensional separation with data independent acquisition mode and deconvolution: Characterization of metabolites of Uncaria rhynchophylla in rat plasma as a case.

The importance to clarify the drug metabolites is beyond doubt in view of their potential efficacy and safety. However, due to the complex matrix interference, relatively low content and the co-eluting effect, it is of a great challenge to comprehensively and systematically characterize the metabolites in vivo, especially for the traditional Chinese medicines (TCMs) due to the numerous types of components. In the present study, a comprehensive off-line two-dimensional separation system combining with data independent acquisition (DIA) mode and multi-dimensional data deconvolution method was established for chromatographic separation, data acquisition and data procession of indole alkaloids in rat plasma after intragastrically administrated with the extract of Uncaria rhynchophylla at the dose of 1 g/kg. The orthogonality of the off-line 2D separation system consisting of HILIC for first-dimensional separation and the PRLC for second-dimensional separation was valuated with the "asterisk" equations, and the results showed that off-line 2D separation system had passable orthogonality (A0 = 53.3%). Furthermore, the DIA mode was applied to capture MS/MS spectra in view of its advantage in acquiring MS data, and an effective multi-dimensional deconvolution method integrating the calculation of chemical formula, the extraction of diagnostic ion, the filter of ring double bond (RDB) and the judgement of neutral loss was established to parse the spectra for the complicated DIA data for comprehensive analysis of metabolites in rat plasma. Ultimately, a total of 127 indole alkaloids were tentatively characterized, and the main metabolic pathways were inferred as demethylation, dehydrogenation, hydroxylation and deglycosylation. The off-line two-dimensional separation system was applied for the comprehensive characterization of metabolites in vivo for the first time. This study suggested a new approach to enable the enrichment, separation and analysis of the low content components in vivo.

Simultaneous determination of five alkaloids from Rauvolfia vomitoria in rat plasma by LC-MS/MS: Application to a comparative pharmacokinetic study in normal and type 2 diabetic rats.

Rauvolfia vomitoria is widely distributed in the tropical regions of Africa and Asia, and has been used in traditional folk medicine in China. Indole alkaloids were found to be major bioactive components, while the effects of diabetes mellitus on the pharmacokinetic parameters of the components have not been reflected in vivo. In this study, an efficient and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of five ingredients of R. vomitoria in rats. Detection was implemented in multiple-reaction-monitoring mode with an electrospray positive-ionization source. Validation parameters were all in accordance with the current criterion. The established method was effectively employed to compare the pharmacokinetic behaviors of five alkaloids (reserpine, yohimbine, ajmaline, ajmalicine, and serpentine) between normal and type 2 diabetic rats. The single-dose pharmacokinetic parameters of the five alkaloids were determined in normal and diabetic rats after oral administration of 100 and 200 mg/kg body weight. The results indicated that diabetes mellitus significantly altered the pharmacokinetic characteristics of yohimbine, ajmaline, and ajmalicine after oral administration in rats. This is an attempt to provide some evidence for clinicians that may serve as a guide for the use of antidiabetic medicine in clinical practice.

Gender differences in plasma pharmacokinetics and hepatic metabolism of geissoschizine methyl ether from Uncaria hook in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Geissoschizine methyl ether (GM), an indole alkaloid from Uncaria hook, is an active ingredient in the traditional Japanese Kampo medicine yokukansan, which is used to treat neurosis, insomnia, irritability, and night crying in children. AIM OF THE STUDY: Recent our pharmacokinetic studies suggested that there may be gender differences in the plasma concentrations of GM in rats, but not in humans. However, the details of this difference remain unverified. The purpose of this study was to clarify the reasons for the gender differences in rats. MATERIALS AND METHODS: GM plasma pharmacokinetics was compared in male and female rats orally administered yokukansan (4 g/kg). To confirm the involvement of cytochrome P450 (CYP) in GM liver metabolism, GM was incubated with male and female rat liver S9 fraction in the absence or presence of 1-aminobenzotriazole (a nonspecific CYP inhibitor). CYP isoforms involved in GM metabolism were estimated using recombinant rat CYP isoforms and anti-rat CYP antibodies. RESULTS: The maximum GM plasma concentrations were significantly higher in female than in male rats. When GM was incubated with rat liver S9 fractions, GM reduction was more striking in male S9 (69.3%) than that in female S9 (10.0%) and was completely blocked with nonspecific CYP inhibitor 1-aminobenzotriazole. Screening experiments using recombinant rat cytochrome P450 (CYP) isoforms showed that CYP1A1, CYP2C6, CYP2C11, CYP2D1, and CYP3A2 were involved in GM metabolism. Of these CYP isoforms, the use of anti-rat CYP antibodies indicated that male-dependent CYP2C11 and CYP3A2 were predominantly involved in the liver microsomal GM metabolism with gender differences. CONCLUSIONS: These results suggest that the cause of gender differences in plasma GM pharmacokinetics in rats is most likely because of male-dependent CYP2C11 and CYP3A2, and provide also useful information to further evaluate the pharmacological and toxicological effects in future. This study is the first to demonstrate that the gender differences in plasma GM pharmacokinetics in rats are caused by the gender-dependent metabolism of GM.

A novel two-dimensional liquid chromatography system for the simultaneous determination of three monoterpene indole alkaloids in biological matrices.

The present paper describes a novel two-dimensional liquid chromatography (2D-LC) system, which is comprised of a first-dimensional ion exchange chromatography (IEX1) column, trap column, and second-dimensional reversed-phase chromatography (RP2) column system. The biological sample is separated by the first-dimensional LC using an IEX column to remove interferences. The analytes are transferred to the trap column after heart-cutting. Then, the analytes are transferred to the second-dimensional LC using an RP2 column for further separation and ultraviolet detection. This 2D-LC system can offer a large injection volume to provide sufficient sensitivity and exhibits a strong capacity for removing interferences. Here, the determination of three monoterpene indole alkaloids (MIAs; gelsemine, koumine, and humantenmine) from Gelsemium in biological matrices (plasma, tissue, and urine) was used this 2D-LC system. After a rapid and easy sample preparation method based on protein precipitation, the sample was injected into the 2D-LC. The method was developed and validated in terms of the selectivity, LOD, LOQ, linearity, precision, accuracy, and stability. The sample preparation time for the three MIAs was 15 min. The LOD for these compounds was 10 ng/mL, which was lower than the developed HPLC methods. The results showed that this method had good quantitation performance and allowed the determination of gelsemine, koumine, and humantenmine in biological matrices. The method is rapid, exhibits high selectivity, has good sensitivity, and is low-cost, thus making it well-suited for application in the pharmaceutical and toxicological analysis of Gelsemium. Graphical abstract.

Pharmacokinetics and safety evaluation in healthy Chinese volunteers of alkaloids from leaf of Alstonia scholaris: A multiple doses phase I clinical trial.

BACKGROUND: Alstonia scholaris (Apocynaceae) was reported to be a rich source of indole alkaloids, which exhibited remarkably bioactivities. The leaf of A. scholaris has been used in 'dai' ethno-medicine for treatment of respiratory diseases, and the defined indole alkaloids from leaf of A. scholaris has been registered as investigational new botanical drug (No. 2011L01436) and was approved for phase I/II clinical trials by China Food and Drug Administration (CFDA). PURPOSE: The aim of the trial is to evaluate the safety and explore the relationship of dosing frequency and pharmacokinetics after oral administration of capsule of alkaloids from leaf of A. scholaris (CALAS) at different doses. METHODS: In this randomized, open-labelled, single-center clinical trial, the safety and pharmacokinetics of CALAS were assessed in eligible healthy Chinese volunteers after oral administration of different doses. Each volunteer (n = 10 per group) received single dose of CALAS from 20 mg, 40 mg, 80 mg to 120 mg orally. The pharmacokinetics of CALAS was investigated in healthy Chinese subjects' plasma by a fully-validated LC-MS/MS method. Safety was assessed biochemically and clinically throughout the study, and drug re-excitation research was conducted to verify the correlation between investigational product and minor adverse events. The trial was registered on August 26, 2015 (http://www.chictr.org.cn/showproj.aspx?proj=11736), number ChiCTR-IPR-15006976. RESULTS: 40 subjects completed the study, and as a result, vallesamine had the highest concentration in plasma of healthy volunteers, and the AUC exposure level in each compounds in turn is vallesamine > scholaricine > 19-epischolaricine > picrinine. For the safety evaluation of CALAS, two cases of minor adverse events were observed during the trial, but the drug re-excitation research indicated that these two adverse events were related to the individual's physiological variation. CONCLUSION: Pharmacokinetic characteristics of each ingredient showed different patterns. 19-epischolaricine, vallesamine and picrinine were match to the linear pharmacokinetic characteristics, but scholaricine conformed to the characteristics of nonlinear pharmacokinetics. The CALAS was safe in healthy subjects under the current dose regimen.

LC-MS/MS detection of citrus unshiu peel-derived flavonoids in the plasma and brain after oral administration of yokukansankachimpihange in rats.

1. Yokukansankachimpihange (YKSCH), a Kampo formulation combining Citrus unshiu peel (CUP) and Pinellia tuber (PT) with yokukansan (YKS), has been recently used to treat the behavioral and psychological symptoms of dementia. Several flavonoids derived from CUP and PT reportedly exhibit psychopharmacological activity, but it remains unclear whether these flavonoids reach the brain after oral administration of YKSCH. 2. In this study, we first measured eight target flavonoids in the plasma and brain in rats orally administered YKSCH. Among these flavonoids, hesperidin, narirutin, nobiletin, and heptamethoxyflavone (HMF) were detected in the plasma, and nobiletin and HMF were detected in the brain. 3. Next, to clarify whether CUP and PT affect the pharmacokinetics of YKS ingredients in YKSCH, the plasma pharmacokinetics of geissoschizine methyl ether (GM) as a representative active ingredient in YKS was examined in rats orally administered YKSCH or YKS. There was no significant difference between the two groups, inferring that the pharmacokinetics of GM may not be affected by the two additional crude drugs. 4. Taken together, this study suggests that the CUP-derived flavonoids nobiletin and HMF may be responsible for the psychopharmacological effects of YKSCH in addition to YKS ingredients.

Pharmacokinetics and bioavailability of gelsenicine in mice by UPLC-MS/MS.

Gelsenicine is an indole alkaloid isolated from Gelsemium elegans Benth. In recent years, the role of G. elegans Benth preparations in anti-tumor, analgesic, dilatation and dermatological treatment has attracted attention, and it has been applied clinically, but it is easy to cause poisoning with its use. An UPLC-MS/MS method was established to determine the gelsenicine in mouse blood, and the pharmacokinetics of gelsenicine after intravenous (0.1 mg/kg) and intragastric (0.5 and 1 mg/kg) administration was studied. Deltalin was used as internal standard; a UPLC BEH C18 column was used for chromatographic separation. The mobile phase consisted of acetonitrile and 10 mmol/L ammonium acetate (0.1% formic acid) with a gradient elution flow rate of 0.4 mL/min. Multiple reaction monitoring mode was used for quantitative analysis of gelsenicine in electrospray ionization positive interface. Proteins from mouse blood were removed by acetonitrile precipitation. A validation of this method was performed in accordance with the US Food and Drug Administration guidelines. In the concentration range of 0.05-100 ng/mL, the gelsenicine in the mouse blood was linear (r > 0.995), and the lower limit of quantification was 0.05 ng/mL. In the mouse blood, the intra-day precision RSD was <12%, the inter-day precision RSD was <15%, the accuracy ranged from 89.8 to 112.3%, the average recovery was >76.8%, and the matrix effect was between 103.7 and 108.4%, which meet the pharmacokinetic research requirements of gelsenicine. The UPLC-MS/MS method is sensitive, rapid and selective, and has been successfully applied to the pharmacokinetic study of gelsenicine in mice. The absolute bioavailability of gelsenicine is 1.13%.

Ultra-Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS)-Based Pharmacokinetics and Tissue Distribution Study of Koumine and the Detoxification Mechanism of Glycyrrhiza uralensis Fisch on Gelsemium elegans Benth.

Gelsemium elegans Benth. (G. elegans), which is a famous Chinese folk medicine, has been commonly used to treat certain types of skin ulcers and alleviate inflammation, headaches, and cancer pain. However, the extensive clinical use of G. elegans has been greatly hampered by its toxicity. As one of the most widely used herbal medicines, Glycyrrhiza uralensis Fisch, has a unique effect on detoxification of G. elegans. In the present study, a rapid and sensitive method using ultra-liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was established and validated for determination of koumine, the most abundant molecule among the alkaloids of G. elegans, in rat plasma, tissue, and liver microsome. The developed method was successfully applied to the pharmacokinetics, tissue distribution, and in vitro metabolism study in rat with or without pre-treated Glycyrrhiza uralensis Fisch extract. Meanwhile, the expression level of CYP3A1 mRNA was analyzed to explain the detoxification mechanism of Glycyrrhiza uralensis Fisch on G. elegans. As a result, our work demonstrated that Glycyrrhiza uralensis Fisch could significantly affect the pharmacokinetics and tissue distribution of koumine in rats. The detoxification mechanism of Glycyrrhiza uralensis Fisch on G. elegans may be its cytochrome enzyme up-regulation effect.

Simultaneous determination of gelsemine and koumine in rat plasma by UPLC-MS/MS and application to pharmacokinetic study after oral administration of Gelsemium elegans Benth extract.

A simple, rapid and sensitive method using UPLC-MS/MS was established and validated for simultaneous determination of gelsemine and koumine in rat plasma after oral administration of Gelsemium elegans Benth extract. Plasma was performed with methanol precipitation and berberine was chosen as the internal standard. Plasma samples were separated on an Acquity UPLC® BEH C18 column (3.0 × 50 mm, 1.7 µm) with gradient elution using acetonitrile and 0.1% formic acid aqueous solution as the mobile phase at a flow rate of 0.4 mL/min. Multiple reaction monitoring mode in positive ion mode was utilized for detection. The calibration curves were linear over the range of 0.2-100 ng/mL for gelsemine and 0.1-50 ng/mL for koumine, with the lower limits of quantification 0.2 and 0.1 ng/mL, respectively. The intra- and inter-precision and accuracy were well within the acceptable ranges. The developed method was successfully applied to an in vivo pharmacokinetic study in rat after oral administration of 10 mg/kg Gelsemium elegans Benth extract.

Development and in-house validation of a sensitive LC-MS/MS method for simultaneous quantification of gelsemine, koumine and humantenmine in porcine plasma.

Three monomers of G. elegans indole alkaloids (gelsemine, koumine and humantenmine) were simultaneously detected in porcine plasma for the first time with the development and validation of a sensitive and reliable LC-ESI-MS/MS method. Using a gradient mobile phase at a constant flow rate of 0.2 mL/min via electrospray ionization (positive ion mode) in a multiple reaction monitoring (MRM) scan, gelsemine, koumine and humantenmine were eluted, separated and detected at an appropriate retention time. The porcine plasma was prepared using protein precipitation with 1% formic acid-acetonitrile: methanol (2:1, v/v). Using matrix-matched calibration curves and weighted least squares linear regression, a good linearity (r2 > 0.99) was achieved with a concentration range of 0.1-200 µg/L for gelsemine, koumine and humantenmine; estimated LOD and LOQ values were 0.10 µg/L and 0.2 µg/L, respectively. The mean of the recoveries was in the range of 82.68-100.35% of porcine plasma at four different levels, and the intra-day and inter-day precision (CV) were lower than 15% with a range of 2.46-8.76% and 2.73-10.83%, respectively. The proposed method has proved to be suitable for accurate, quantitative determination of gelsemine, koumine and humantenmine in porcine plasma.

[Simultaneous Quantitative Analysis of Koumine, Gelsemine and Gelsenicine in Biological Samples by LC-MS/MS].

OBJECTIVES: To establish a LC-MS/MS method which is accurate and sensitive for determination of koumine, gelsemine, and gelsenicine in biological samples and to verify the method. METHODS: Strychnine was used as internal standard. Analytes in blood, urine and liver with 1% sodium hydroxide solution were extracted by ethyl acetate. Chromatographic separation was achieved on a ZORBAX SB-C18 column （150 mm×2.1 mm, 5 µm）, and gradient elution was performed with the buffer solution of methanol-20 mmol/L ammonium acetate （including 0.1% formic acid and 5% acetonitrile） as mobile phase. Qualitative and quantitative analysis was performed in the multiple reaction monitoring mode coupled with an electrospray ionization source under positive ion mode（ESI⁺）. RESULTS: The linearity of koumine, gelsemine and gelsenicine in blood, urine and liver was good within corresponding linear limitation and the correlation coefficients （r）>0.995 0. The limits of detection were 0.1 ng/mL （0.1 ng/g）, 0.1 ng/mL （0.1 ng/g） and 0.01 ng/mL （0.01 ng/g）, respectively. The extraction recovery and accuracy of the alkaloids ranged from 61.9% to 114.6% and 92.4% to 114.3%, respectively. The relative standard deviations of the intra-day and inter-day precisions were not more than 11.0%. CONCLUSIONS: The method is selective, sensitive and suitable for simultaneous determination of koumine, gelsemine and gelsenicine in body fluids and tissues, which offering technical support for clinical diagnosis and treatment and forensic toxicological analysis of Gelsemium elegans poisoning.

Identification of cryptolepine metabolites in rat and human hepatocytes and metabolism and pharmacokinetics of cryptolepine in Sprague Dawley rats.

BACKGROUND: This study aims at characterizing the in vitro metabolism of cryptolepine using human and rat hepatocytes, identifying metabolites in rat plasma and urine after a single cryptolepine dose, and evaluating the single-dose oral and intravenous pharmacokinetics of cryptolepine in male Sprague Dawley (SD) rats. METHODS: The in vitro metabolic profiles of cryptolepine were determined by LC-MS/MS following incubation with rat and human hepatocytes. The in vivo metabolic profile of cryptolepine was determined in plasma and urine samples from Sprague Dawley rats following single-dose oral administration of cryptolepine. Pharmacokinetic parameters of cryptolepine were determined in plasma and urine from Sprague Dawley rats after single-dose intravenous and oral administration. RESULTS: Nine metabolites were identified in human and rat hepatocytes, resulting from metabolic pathways involving oxidation (M2-M9) and glucuronidation (M1, M2, M4, M8, M9). All human metabolites were found in rat hepatocyte incubations except glucuronide M1. Several metabolites (M2, M6, M9) were also identified in the urine and plasma of rats following oral administration of cryptolepine. Unchanged cryptolepine detected in urine was negligible. The Pharmacokinetic profile of cryptolepine showed a very high plasma clearance and volume of distribution (Vss) resulting in a moderate average plasma half-life of 4.5 h. Oral absorption was fast and plasma exposure and oral bioavailability were low. CONCLUSIONS: Cryptolepine metabolism is similar in rat and human in vitro with the exception of direct glucuronidation in human. Clearance in rat and human is likely to include a significant metabolic contribution, with proposed primary human metabolism pathways hydroxylation, dihydrodiol formation and glucuronidation. Cryptolepine showed extensive distribution with a moderate half-life.

Differences of first-pass effect in the liver and intestine contribute to the stereoselective pharmacokinetics of rhynchophylline and isorhynchophylline epimers in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Uncaria rhynchophylla (Miq.) Miq. ex Havil., is a plant species used in traditional Chinese medicine to treat cardiovascular and central nervous system diseases. Rhynchophylline (RIN) and isorhynchophylline (IRN), a pair of epimers, are major alkaloids isolated from U. rhynchophylla and exhibit diverse pharmacological effects. Our previous study demonstrated that the pharmacokinetics of these epimers existed stereoselectivity after oral administration; however, the specific mechanism remains unknown and merits investigation. AIM OF THE STUDY: In the present study, the aim was to elucidate the mechanism underlying stereoselective pharmacokinetic characteristics of RIN and IRN in rats. MATERIALS AND METHODS: The total (F), hepatic (Fh) and intestinal (Fa·Fg) bioavailabilities of each epimer were measured using portal vein cannulated rats following different dosing routes (intravenous, intraportal and intraduodenal) to assess individual contributions of the liver and intestine in stereoselective pharmacokinetics. Then the differences of first-pass metabolism in the liver and intestine between two epimers were evaluated by in vitro incubation with rat liver microsomes, intestinal S9 and gastrointestinal (GI) content solutions, respectively. Meanwhile, the membrane permeability and efflux by P-glycoprotein (P-gp) were examined by in situ single-pass intestinal perfusion with and without P-gp inhibitor verapamil. The configurational interconversion at different pH values and the excretions via feces and urine were also examined. RESULTS: Pharmacokinetic data showed that the total bioavailability of RIN was 5.9 folds higher than that of IRN (23.4% vs. 4.0%). The hepatic availability of RIN was 4.6 folds higher than that of IRN (46.9% vs. 10.3%), whereas the intestinal availability of RIN (48.1%) was comparable to that of IRN (42.7%). In addition, intestinal perfusion showed that IRN possessed higher intestinal permeability than RIN and co-perfusion with verapamil could affect absorption process of RIN but not IRN. Conversely, the metabolism rate of IRN in rat liver microsomes was significantly faster than that of RIN, resulting in a lower systemic exposure of IRN after oral administration. The degradation in GI lumen and epimerization between two epimers also existed but had small contributions. Additionally, the excretions of both epimers via feces and urine were negligible. CONCLUSIONS: Taken together, different first-pass metabolism in the liver was the major factor responsible for the stereoselective pharmacokinetics of RIN and IRN.

Detection and characterization of the metabolites of rutaecarpine in rats based on ultra-high-performance liquid chromatography with linear ion trap-Orbitrap mass spectrometer.

CONTEXT: Rutaecarpine is an active indoloquinazoline alkaloid ingredient originating from Evodia rutaecarpa (Wu-zhu-yu in Chinese), which possesses a variety of effects. However, its metabolism has not been investigated thoroughly yet. OBJECTIVE: This study develops a highly sensitive and effective method for detection and characterization of the metabolites of rutaecarpine in Sprague-Dawley (SD) rats. MATERIALS AND METHODS: In this study, an efficient method was developed using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer (UHPLC-LTQ-Orbitrap MS) to detect the metabolism profile of rutaecarpine in rat plasma. First, a blood sample (1 mL) was withdrawn 2 h after oral administration of rutaecarpine in SD rats (50 mg/kg). Second, the blood was centrifuged at 4000 rpm for 10 min and pretreated by solid-phase extraction method. Third, 2 µL of the plasma was injected into UHPLC-LTQ-Orbitrap MS for analysis. Finally, the metabolites of rutaecarpine were tentatively identified based on accurate mass measurements, fragmentation patterns and chromatographic retention times. RESULTS: A total of 16 metabolites (four new metabolites, viz., dihydroxylation and sulphate conjugation products of rutaecarpine (M8-M11)) as well as parent drug itself, including three phase I and 12 phase II metabolites were detected and identified in rat plasma. Hydroxylation, sulphate conjugation and glucuronidation were confirmed as the primary metabolic pathways for rutaecarpine in rat plasma. DISCUSSION AND CONCLUSION: These results provide an insight into the metabolism of rutaecarpine and also can give strong indications on the effective forms of rutaecarpine in vivo.

Quantification of mesembrine and mesembrenone in mouse plasma using UHPLC-QToF-MS: Application to a pharmacokinetic study.

Sceletium tortuosum, is an indigenous herb of South Africa which is widely used as an herbal supplement in the treatment of anxiety and stress. Mesembrenone and mesembrine are the two main pharmacologically active alkaloids present in the extract. Despite the wide therapeutic applications of Sceletium extract, there are no reports of in vivo pharmacokinetic properties or analytical methods to quantify these two important alkaloids in plasma. Therefore, the current study aimed to develop and validate a simple and sensitive analytical method for simultaneous quantification of mesembrenone and mesembrine in mouse plasma. Ultra-high-performance liquid chromatography-mass spectrometry (UHPLC/QToF-MS) was employed to achieve our objectives. The compounds were extracted using protein precipitation by methanol (100%) with quinine as an internal standard. The lower limit of quantification for both the compounds was 10 ng/mL. The extraction recovery was between 87 and 93% for both compounds with no matrix effects on the analysis. The accuracy was between 89.5 and 106% and precision was <12.6% for all quality control samples. This validated method was successfully applied to evaluate the i.v. plasma pharmacokinetics of mesembrine and mesembrenone in mouse. However, the oral bioavailability of these alkaloids was poor and the plasma levels were below the detection limits.

Qualitative screening of absorbed indoloquinazoline alkaloids and their metabolites in rat plasma after the oral administration of Wu-Zhu-Yu decoction by high-resolution mass spectrometry with multiple data mining algorithms.

A rapid and sensitive liquid chromatography with high-resolution mass spectrometry method with multiple data processing algorithms was developed and applied for the metabolite profiling of evodiamine and its analogous alkaloids in rat plasma after the administration of Wu-Zhu-Yu decoction. All samples were purified using hydrophilic-lipophilic balanced solid-phase extraction cartridges and analyzed by a Sciex TripleTOF 5600(+) mass spectrometer with a 35 min liquid chromatography gradient elution. High-resolution full-scan mass spectrometry and information-dependent acquisition tandem mass spectrometry data were analyzed using multiple data processing approaches. The results indicated that the detected eight prototype alkaloids could be metabolized to 58 metabolites through both phase I and phase II reactions. Oxidation was demonstrated to be the principle metabolic pathway of the parent compounds. The study contributes to the understanding of the absorption and metabolism of the alkaloids in Wu-Zhu-Yu decoction and provides a detailed analysis of scientific data.

Simultaneous quantitation of the diastereoisomers of scholarisine and 19-epischolarisine, vallesamine, and picrinine in rat plasma by supercritical fluid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study.

Dengtaiye tablet has been used to treat chronic bronchitis cough. Scholarisine, 19-epischolarisine, vallesamine, and picrinine are the representative constituents of Dengtaiye. A rapid and sensitive assay based on supercritical fluid chromatography with tandem mass spectrometry has been developed and validated for the determination of the diastereoisomers of scholarisine and 19-epischolarisine, vallesamine, and picrinine in rat plasma using lamotrigine as internal standard. The analysis in a run time of only 6 min was performed on an ACQUITY UPC(2) Trefoil(TM) BEH 2-EP column (3.0 × 150 mm, 2.5 µm) at 50ºC. The mobile phase consisting of carbon dioxide and methanol (2 mM ammonium formate) was performed as follows: 15% methanol (2 mM ammonium formate) maintained at 0-2 min, 15-19% methanol (2 mM ammonium formate) at 2-4 min, 19-15% methanol (2 mM ammonium formate) at 4-6 min. The flow rate was 1.50 mL/min. The assay was linear over the concentration ranges 50-10000 pg/mL for scholarisine, 19-epischolarisine, vallesamine, and picrinine with corresponding lower limits of quantitation of 50 pg/mL. Intra- and interday precisions were in the range 1.42-12.85% with accuracies in the range -11.71-2.48%. The method was successfully applied to a pharmacokinetic study involving a single oral administration of 108 mg/kg Dengtaiye tablet to rats.

Pharmacokinetic study of isocorynoxeine metabolites mediated by cytochrome P450 enzymes in rat and human liver microsomes.

Isocorynoxeine (ICN) is one of the major bioactive tetracyclic oxindole alkaloids found in Uncaria rhynchophylla (Miq.) Jacks. that is widely used for the treatment of hypertension, vascular dementia, and stroke. The present study was undertaken to assess the plasma pharmacokinetic characteristics of major ICN metabolites, and the role of simulated gastric and intestinal fluid (SGF and SIF), human and rat liver microsomes (HLMs and RLMs), and seven recombinant human CYP enzymes in the major metabolic pathway of ICN. A rapid, sensitive and accurate UHPLC/Q-TOF MS method was validated for the simultaneous determination of ICN and its seven metabolites in rat plasma after oral administration of ICN at 40mg/kg. It was found that 18.19-dehydrocorynoxinic acid (DCA) and 5-oxoisocorynoxeinic acid (5-O-ICA) were both key and predominant metabolites, rather than ICN itself, due to the rapid and extensive metabolism of ICN in vivo. The further study indicated that ICN was mainly metabolized in human or rat liver, and CYPs 2C19, 3A4 and 2D6 were the major enzymes responsible for the biotransformation of ICN to DCA and 5-O-ICA in human. These findings are of significance in understanding of the pharmacokinetic nature of tetracyclic oxindole alkaloids, and provide helpful information for the clinical co-administration of the herbal preparations containing U. rhynchophylla with antihypertensive drugs that are mainly metabolized by CYP3A4 and CYP2C19.

Immunochemical analysis of fumigaclavine mycotoxins in respiratory tissues and in blood serum of birds with confirmed aspergillosis.

The ergoline alkaloid fumigaclavine A (FuA) is one of the major mycotoxins produced by Aspergillus fumigatus, the main causative fungal agent of avian aspergillosis. To study in situ production of FuA, post-mortem respiratory tissues of various avian species, as well as blood samples of falcons (Falco sp.), were analysed by enzyme immunoassay (EIA). At a detection limit of 1.5 ng/ml, FuA EIA positive results were obtained for tissue samples from seven (64%) out of 11 birds with confirmed aspergillosis, with a maximum concentration of 38 ng/g, while all controls (n = 7) were negative. No FuA could be detected in blood serum (detection limit 0.7 ng/ml) of 15 falcons, experimentally inoculated with A. fumigatus conidia. Fungal mycelium material from tissue of clinical aspergillosis cases, cultured on malt extract agar, was highly positive in the FuA EIA in milligrams per gram range. Chromatographic analysis of mycelium extracts revealed the co-presence of FuA and the structurally related fumigaclavine C (FuC). Alkaline hydrolysis of FuA and FuC yielded the corresponding deacetylation products, FuB and FuE. This is the first report showing that fumigaclavine alkaloids are produced by A. fumigatus in situ during the course of clinical aspergillosis in birds; however, the role of these compounds in the pathogenesis of this disease is still unknown.

Pharmacokinetics of Active Components of Yokukansan, a Traditional Japanese Herbal Medicine after a Single Oral Administration to Healthy Japanese Volunteers: A Cross-Over, Randomized Study.

CONTEXT: Yokukansan (YKS) is a traditional Japanese herbal medicine called kampo medicine in Japan. Its extract comprises seven crude drugs: Atractylodis lanceae rhizoma, Poria, Cnidii rhizoma, Uncariae uncis cum ramulus, Angelicae radix, Bupleuri radix, and Glycyrrhizae radix. YKS is used to treat neurosis, insomnia, as well as behavioral and psychological symptoms of dementia. OBJECTIVE: To confirm the exposure and pharmacokinetics of the active components of YKS in healthy volunteers. DESIGN, SETTING, AND PARTICIPANTS: A randomized, open-label, 3-arm, 3-period, crossover trial was conducted on 21 healthy Japanese volunteers at the Kochi Medical University between May 2012 and November 2012. INTERVENTIONS: Single oral administration of YKS (2.5 g, 5.0 g, or 7.5 g/day) during each period. MAIN OUTCOME MEASURE: Plasma concentrations of three active compounds in YKS, namely 18ß-glycyrrhetinic acid (GA), geissoschizine methyl ether (GM), and hirsuteine (HTE). RESULTS: The mean maximum plasma concentrations (Cmax) of GM and HTE increased dose-dependently (ranges: 0.650-1.98 ng/mL and 0.138-0.450 ng/mL, respectively). The times to maximum plasma concentration after drug administration (tmax) were 0.500 h for GM and 0.975-1.00 h for HTE. The apparent elimination half-lives (t1/2) were 1.72-1.95 h for GM and 2.47-3.03 h for HTE. These data indicate the rapid absorption and elimination of GM and HTE. On the other hand, the Cmax, tmax, and t1/2 of GA were 57.7-108 ng/mL, 8.00-8.01 h, and 9.39-12.3 h, respectively. CONCLUSION: We demonstrated that pharmacologically active components of YKS are detected in humans. Further, we determined the pharmacokinetics of GM, HTE, and GA. This information will be useful to elucidate the pharmacological effects of YKS. TRIAL REGISTRATION: Japan Pharmaceutical Information Center JAPIC CTI-121811.