Bioactive Compounds In Rat Plasma Research Articles

Comparative Pharmacokinetic Study of 5 Active Ingredients after Oral Administration of Zuogui Pill in Osteoporotic Rats with Different Syndrome Types.

Zuogui Pill is a kidney-yin-tonifying formula in traditional Chinese medicine that is widely used to manage osteoporosis with kidney-yin-deficiency in China. Herein, an efficient and accurate high-performance liquid chromatography-tandem mass spectrometry method was developed to determine the concentrations of 5 bioactive compounds in rat plasma following oral administration of Zuogui Pill. Because drug absorption and distribution differ under physiological and pathological conditions, the established method was used to quantify blood components and dynamic change in osteoporotic rats with different syndrome types. Moreover, integrated pharmacokinetic study was conducted to describe the overall pharmacokinetic characteristics of traditional Chinese medicine. The results showed that the absorption, distribution, and metabolism of Zuogui Pill varied widely under different states. The bioavailability of most active components showed significant advantages in osteoporotic rats with kidney-yin-deficiency, which corresponds to the opinion that Zuogui Pill has the effect of nourishing kidney-yin. It is hoped that this finding could interpret the pharmacodynamic substances and mechanism of Zuogui Pill in the treatment of osteoporosis with kidney-yin-deficiency.

Quantification of 10 bioactive components of Yazhangsan in rat plasma by LC-MS/MS and its application.

Yazhangsan (YZS) is a common prescription for the treatment of cough and asthma caused by wind-cold. The purpose of this study was to investigate the pharmacokinetic profiles of 10 bioactive components in YZS. A simple, sensitive and reliable high-performance liquid chromatography coupled with a triple-quadruple mass spectrometry method (LC-MS/MS) was developed and fully validated in this study for the measurement of these 10 bioactive compounds in rat plasma. One-step protein precipitation method using methanol was applied to the treatment of rat plasma samples. Chromatographic separation was conducted on a C18 column by gradient elution, and water (containing 0.1% formic acid) and acetonitrile were chosen as the mobile phase. The analytes were quantified by using a mass spectrometer in multiple reaction monitoring scanning mode, and electrospray ionization was performed in positive and negative ion modes. The established method met the requirements for the quantification of these 10 bioactive compounds in biological samples, and it was successfully applied to the pharmacokinetic study of 10 components in rats after the intragastrical administration of YZS. This study will lay a foundation for the investigation of the mechanism of action of YZS and provide useful data for the rational use of YZS in clinical.

Simultaneous characterization of multiple Psoraleae Fructus bioactive compounds in rat plasma by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry for application in sex-related differences in pharmacokinetics.

A method for the simultaneous quantification of 13 bioactive compounds (psoralen, isopsoralen, isobavachin, bakuchalcone, neobabaisoflavone, bavachin, corylin, psoralidin, isobavachalcone, bavachinin, corylifol A, bavachalcone, and bakuchiol) by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry has been developed and validated in rat plasma. Osthol was used as an internal standard and plasma samples were pretreated with one-step liquid-liquid extraction. These analytes were separated using a gradient mobile phase system of water and acetonitrile at a flow rate of 0.2mL/min on a reverse-phase C18 column and analyzed in the selected multiple reactions monitoring mode. All calibration curves were linear (r>0.9952) over the tested ranges. The intra- and interday accuracy and precisions of these analytes at three different concentration levels were within the acceptable limits of<15% at all concentrations. The mean recoveries of these analytes at three concentrations were more than 60.2% and the matrix effects were in the range of 85-115%. Stability studies proved that the analytes were stable under the tested conditions. The developed method was applied to evaluating the pharmacokinetic study of 13 bioactive compounds after oral administration of Psoraleae Fructus in rat of different genders. Some active compounds in Psoraleae Fructus had sex-related pharmacokinetics.

Simultaneous characterisation of multiple Mahonia fortunei bioactive compounds in rat plasma by UPLC–MS/MS for application in pharmacokinetic studies and anti-inflammatory activity in vitro

The stems of Mahonia fortunei (MF) are commonly used in Chinese Traditional Medicine and contain multiple bioactive compounds, including 3,4,5-trimethoxyphenol-1-O-β-d-glucopyranoside (1), 5-hydroxypicolinic acid methyl ester (2), acortatarin A (3), syringic acid (4), 9-epi-acortatarin A (5), vomifoliol (6), corydaldine (7), noroxyhydrastinine (8), columbamine (9), jatrorrhizine (10), palmatine (11), berberine (12) and schisandrin (13). The pharmacokinetics of these 13 compounds in the rat plasma were assessed using a novel sensitive, rapid, and specific UPLC-ESI-MS/MS method after oral administration of an aqueous extract of MF stems. Carbamazepine was employed as the internal standard (IS) and all samples were precipitated with acetonitrile. Chromatographic separation was performed on a C18 column using a gradient elution at 0.3 mL/min, with the mobile phase consisting of acetonitrile and 0.06 % formic acid and 5 mM ammonium acetate aqueous solution. The calibration curves showed satisfactory linearity in the examination area (r2 ≥ 0.99). The accuracy, precision, extraction recovery, matrix effect, and stability were within acceptable ranges. The method successfully assessed the pharmacokinetics of these 13 compounds. In vitro, compound 12 exhibited potent inhibitory activity against production of nitric oxide (NO) in the RAW264.7 cell line when stimulated by lipopolysaccharide (LPS), while compounds 7, 12, and 13 were the most potent inhibitors of NO production in the BV2 cell line when stimulated by LPS. The IC50 values of compounds 7, 12 and 13 were 42.81, 20.55 and 22.74 μM. We conclude that these compounds have promise for clinical application, although their synergistic action may be more effective than that by any single compound alone.

A UPLC-MS/MS Method for Simultaneous Determination of Six Bioactive Compounds in Rat Plasma, and its Application to Pharmacokinetic Studies of Naoshuantong Granule in Rats

Background: It is urgently needed to clarify the pharmacokinetic mechanism for the multibioactive constituents in Traditional Chinese Medicines for its clinical applications. A rapid, sensitive and reliable ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of Danshensu, Ferulic acid, Astragaloside IV, Naringin, Neohesperidin and Puerarin after oral administration of Naoshuantong Granule using Carbamazepine as internal standard (IS). Methods: The plasma samples were pretreated by liquid-liquid extraction method using ethyl acetate after acidification, and separated on a Waters ACQUITY UPLC® BEH C18 column (50×2.1 mm, i.d., 1.7 µm) by gradient elution with a mobile phase composing of water (containing 0.1% formic acid) and acetonitrile at a flow rate of 0.2 mL/min. Multiple reaction monitoring (MRM) mode with both positive and negative ion mode was operated using an electrospray ionization (ESI) to detect the six compounds. Result: All calibration curves showed good linearity (r&gt;0.99) over a wide concentration range. The intra- and inter-day precision (RSD%) was below 8.4% and the accuracy (RE%) ranged from 91.1% to 107.5%. The extraction recoveries of the six analytes and IS in the plasma were more than 77.9% and no severe matrix effect was observed. Conclusion: The fully validated method was successfully applied to the pharmacokinetics of Naoshuantong Granule.

LC-ESI-MS/MS method for simultaneous determination of eleven bioactive compounds in rat plasma after oral administration of Ling-Gui-Zhu-Gan Decoction and its application to a pharmacokinetics study

A sensitive and robust LC-MS/MS method has been developed and validated to simultaneous determine the concentrations of tumulosic acid, dehydrotumulosic acid, polyporenic acid C, cinnamic acid, Atractylenolide I, Atractylenolide II, Atractylenolide III, glycyrrhizic acid, glycyrrhetinic acid, liquiritigenin and isoliquiritin in plasma from rats who received Ling-Gui-Zhu-Gan Decoction extract oral administration. The samples were prepared by a liquid-liquid extraction procedure using ethyl ether as the extraction solvent and schisandrin as the internal standard. Chromatographic separation was achieved using a Thermo Hypersil GOLD C18 column (2.1 mm × 100 mm, 1.9 µm) and a gradient mobile phase consisting of acetonitrile-water with 0.1% formic acid. All of the analytes were quantified using negative and positive multiple reaction monitoring mode. The method was validated for selectivity, linearity, accuracy, precision, recovery, matrix effect and sample stability under various storage conditions, whose values are all fell in the acceptable limits. We report the lowest limit of quantification for tumulosic acid, dehydrotumulosic acid and polyporenic acid C as 2 ng/mL. This is the first study for simultaneous determination of so many analytes in rat plasma after oral administration of Ling-Gui-Zhu-Gan Decoction. This validated method was successfully used to study the pharmacokinetics of multiple compounds in rat plasma after oral administration of Ling-Gui-Zhu-Gan Decoction.

Development of a HPLC-MS/MS Method to Determine 11 Bioactive Compounds in Tongmai Yangxin Pill and Application to a Pharmacokinetic Study in Rats.

A sensitive and reliable HPLC-MS/MS method has been developed and validated for simultaneous determination of eleven bioactive compounds (rhein, emodin, stilbene glycoside, liquiritin, ononin, verbascoside, gallic acid, schisandrin, liquiritigenin, glycyrrhizic acid, and isoliquiritigenin) in rat plasma after oral administration of Tongmai Yangxin Pill. The collected plasma samples were prepared by liquid-liquid extraction with ethyl acetate after acidification. Eleven compounds were separated on a CORTECS™ C18 column with mobile phases consisting of 0.1% formic acid in deionized water and acetonitrile. The flow rate was 0.3 mL/min. The detection was performed on a tandem mass system with an electrospray ionization (ESI) source in both positive and negative ionization using multiple-reaction monitoring (MRM) mode. The calibration curves were linear over the range of 8-2000 ng/mL for glycyrrhizic acid; 4-1000 ng/mL for liquiritin; 0.8-200 ng/mL for emodin, gallic acid, ononin, schisandrin, and stilbene glycoside; 0.4-100 ng/mL for isoliquiritigenin, liquiritigenin, rhein, and verbascoside, respectively. The intra- and interday precision of the analytes were less than 9.3% and 8.5%. The intra- and interday accuracy were in the range of -14.0% to 10.3% and -6.5% to 9.6%. Meanwhile, the extraction recovery of the analytes in plasma samples ranged from 85.2% to 109.1% and matrix effect from 89.2% to 113.4%. The developed method was successfully applied to the pharmacokinetics of eleven bioactive compounds in rat plasma after oral administration of Tongmai Yangxin Pill prescription.

Simultaneous determination and pharmacokinetics of fourteen bioactive compounds in rat plasma by LC-ESI-MS/MS following intravenous injection of Gegen-Sanqi compatibility solution.

A high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and fully validated for simultaneous determination of puerarin, 3'-hydroxy puerarin, 6″-O-xylosyl puerarin, 3'-methoxy puerarin, mirificin, puerarin-7-O-glucoside, daidzin, daidzein, daidzein-7-O-glucoside, ginsenoside-Rd, notoginsenoside-R1, ginsenoside-Re, ginsenoside-Rg1, ginsenoside-Rb1 in rat plasma after intravenous injection of 5mL/kg Gegen-Sanqi compatibility solution (containing Pueraria flavonid 10.8mg/mL and Panax notoginsenosidum 5.4mg/mL). After addition of internal standard (IS) baicalin, the analytes and IS were recovered from rat plasma by protein precipitation using acetonitrile containing 0.1% formic acid. Chromatographic separation was performed on a Capcell pak MG C18 column (3.0mm×75mm, 3.0μm) at 35°C with a flow rate of 0.75mL/min. Mass spectrometry was conducted using multiple reaction monitoring in positive mode. The method was linear for all of the analytes over 1000 times concentration range with correlation coefficients greater than 0.99. The precision and accuracy of the LC-MS/MS assay based on the three analytical quality control (QC) levels were well within the acceptance criteria from FDA guidance for bioanalytical method validation. Method recoveries were higher than 75% and the matrix effects were minimal. All analytes were stable under tested conditions. It was the first time to study the pharmacokinetics of daidzein-7-O-glucoside in rat plasma. To the best of our knowledge, this is the first study for simultaneous determination of so many analytes in rat plasma after intravenous injection of Gegen-Sanqi compatibility solution. It was expected that the present work would provide some helpful references for the apprehension of the mechanism of action and further clinical efficacy studies of Gegen-Sanqi herb-pair.

Simultaneous determination and pharmacokinetic study of twelve bioactive compounds in rat plasma after intravenous administration of Xuebijing injection by UHPLC-Q-Orbitrap HRMS

Xuebijing injection (XBJ) is a traditional Chinese herbal prescription widely used in the treatment of sepsis. Extensive studies revealed that the major bioactive constituents of XBJ injection, including phenolic acids, flavonoids, monoterpene glycosides, lactones and organic acids, play an important role in the treatment. In this study, a rapid, sensitive and accurate ultra high performance liquid chromatography – Q Exactive hybrid quadrupole – orbitrap high-resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS) method was developed for simultaneous determination of twelve bioactive compounds in rat plasma after intravenous administration of XBJ injection. A gradient elution for separation was achieved on a waters ACQUITY UHPLC® BEH C18 column (2.1mm×50mm, 1.7μm) column with acetonitrile-water (containing 0.1% formic acid) as mobile phase at a flow rate of 0.2mL/min. All compounds and IS were monitored by parallel reaction monitoring assay both in positive and negative ion mode. The developed method was validated for intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Extraction recoveries at three levels of QC concentrations were all more than 80% for all compounds and IS, and matrix effects were found in the range of 80.0–120.0%. Stability results showed that all analytes were stable at the investigated conditions. The validated method was successfully applied to a pharmacokinetic study of Xuebijing injection following intravenous administration of 2.5, 5.0, 10.0mL/kg to rats respectively. And the result indicated that the pharmacokinetic behavior of XBJ injection is positively related to dosage at the range of 2.5–10mg/kg. This study will provide a meaningful basis for evaluating the rationality of XBJ injection for clinical application.

UHPLC-ESI-MS/MS determination and pharmacokinetics of pinoresinol glucoside and chlorogenic acid in rat plasma after oral administration of Eucommia ulmoides Oliv extract.

This study aimed to develop a specific UHPLC-ESI-MS/MS method for simultaneous determination and pharmacokinetics of pinoresinol glucoside and chlorogenic acid in rat plasma after oral administration of Eucommia ulmoides. The chromatographic separation was achieved on a Hypersil GOLD column with gradient elution by using a mixture of 0.1% formic acid aqueous solution and acetonitrile as the mobile phase at a flow rate of 200 μL/min. A tandem mass spectrometric detection was conducted using multiple-reaction monitoring via an electrospray ionization source in negative ionization mode. Samples were pre-treated by a single-step protein precipitation with acetonitrile, and bergenin was used as internal standard. After oral administration of 3 mL/kg E. ulmoides extract in rats, the maximum plasma concentrations of pinoresinol glucoside and chlorogenic acid were 57.44 and 61.04 ng/mL, respectively. The times to reach the maximum plasma concentration were 40.00 and 23.33 min for pinoresinol glucoside and chlorogenic acid, respectively. The intra- and inter-day precision (RSD) values for the two analytes were <2.46 and 5.15%, respectively, and the accuracy (RE) values ranged from -12.76 to 0.00. This is the first study on pharmacokinetics of bioactive compounds in rat plasma after oral administration of E. ulmoides extract.

Development and validation of a sensitive and fast UPLC–MS/MS method for simultaneous determination of seven bioactive compounds in rat plasma after oral administration of Guizhi-gancao decoction

Rapid, sensitive, selective and accurate UPLC–MS/MS method was developed and fully validated for simultaneous determination of cinnamaldehyde, cinnamic acid, 2-methoxy cinnamic acid, glycyrrhizic acid, glycyrrhetinic acid, liquiritigenin and isoliquiritin in rat plasma after oral administration of Guizhi-gancao decoction. Plasma samples were processed with a simple protein precipitation technique using acetonitrile, followed by chromatographic separation using a Thermo Hypersil GOLD C18 column. A 11.0min linear gradient elution was used at a flow rate of 0.2mL/min with a mobile phase of 0.1% acetic acid containing 0.2mM ammonium acetate in water and acetonitrile. The analytes and internal standard, schisandrin, were detected using both positive and negative ion electrospray ionization in multiple reaction monitoring mode. The developed method was validated for intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Matrix effect was found to be minimal. Recovery efficiency of all the analytes was found to be >60%. Stability results showed that the analytes were stable at all the conditions. This validated method was successfully used to study the pharmacokinetics of multiple compounds in rat plasma after oral administration of Guizhi-gancao decoction.

Simultaneous determination by UPLC-MS/MS of seven bioactive compounds in rat plasma after oral administration of Ginkgo biloba tablets: application to a pharmacokinetic study.

A rapid, reliable, and sensitive method was developed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with an electrospray ionization (ESI) source for determination of seven bioactive compounds in rat plasma after oral administration of Ginkgo biloba tablets (GBTs). The method simultaneously detects bilobalide (BB), ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC), quercetin (QCT), kaempferol (KMF), and isorhamnetin (ISR) for pharmacokinetic study. The analytes and internal standard (IS) were extracted from rat plasma by acetidin. An MS/MS detection was conducted using multiple reaction monitoring (MRM) and operating in the negative ionization mode. The calibration curve ranges were 5-500, 5-500, 2.5-250, 1-100, 1-100, 1-100, and 1-100 ng/ml for BB, GA, GB, GC, QCT, KMF, and ISR, respectively. The mean recovery of the analytes ranged from 68.11% to 84.42%. The intra- and inter-day precisions were in the range of 2.33%-9.86% and the accuracies were between 87.67% and 108.37%. The method was used successfully in a pharmacokinetic study of GBTs. The pharmacokinetic parameters of seven compounds were analyzed using a non-compartment model. Plasma concentrations of the seven compounds were determined up to 48 h after administration, and their pharmacokinetic parameters were in agreement with previous studies.

Determination of paeoniflorin, calycosin‐7‐O‐β‐d‐glucoside, ononin, calycosin and formononetin in rat plasma after oral administration of Buyang Huanwu decoction for their pharmacokinetic study by liquid chromatography–mass spectrometry

The purpose of this study was to simultaneously investigate the pharmacokinetics of five bioactive compounds in rat plasma after oral administration of Buyang Huanwu decoction (BYHWD) using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). The separations were performed on a Thermo Hypersil Gold C(18) analytical column (50 × 2.1 mm, 3 µm) with the column temperature kept at 30°C. The quantitative analysis was performed using a quadrupole mass spectrometer detector operated under selected ion monitoring mode. A linear gradient elution of A (0.1% formic acid solution) and B (100% acetonitrile) was used at a flow rate of 0.2 mL/min. The method was validated within the concentration ranges 1.8-450, 6.0-1500, 2.0-500, 1.2-300 and 1.2-150 ng/mL for paeoniflorin, calycosin-7-O-β-d-glucoside, ononin, calycosin and formononetin, respectively. The calibration curves were linear with correlation coefficients > 0.99. The lower limits of quantitations were < 6.0 ng/mL. The method was further applied to assess the pharmacokinetics of the five bioactive constituents of BYHWD in rat plasma.