Simultaneous determination of fourteen components of Gumiganghwal-tang tablet in human plasma by UPLC-ESI-MS/MS and its application to pharmacokinetic study.

Gumiganghwal-tang is a traditional herbal medicine widely used for its anti-inflammatory, analgesic, and antipyretic effects. However, the safety and efficacy of its active ingredients based on an in vivo pharmacokinetic (PK) study have yet been investigated. We have established a sensitive and accurate UPLC-ESI-MS/MS method and conducted a PK study on 14 constituents of Gumiganghwal-tang through human plasma analysis. Analytical conditions were optimized according to the physicochemical properties of the 14 compounds to facilitate efficient separation and eliminate overlap or interference between peaks. KINETEX-C18 and Inertsil-C8 columns were used as UPLC stationary phases, and acetonitrile and aqueous formic acid were used as mobile phases. All the analytes were quantified with a triple quadrupole mass spectrometer using electrospray ionization in multiple reaction monitoring mode. The chromatograms of 14 bioactive compounds showed excellent elution and sensitivity, and each peak was selectively separated and quantified without interference with each other or impurities. The established analytical method was based on international guidelines and was successfully used to perform PK studies of 14 herbal ingredients in humans after oral administration with Gumiganghwal-tang tablets. The oral absorption of most active components of Gumiganghwal-tang was relatively rapid and remained considerably long in the body to be quantified in plasma up to 48 h after administration.

Palmul-Tang, a Korean Medicine, Promotes Bone Formation via BMP-2 Pathway in Osteoporosis.

Osteoporosis is a common skeletal disease in post-menopausal women. Palmul-tang, an herbal medicine, has been treated for gynecological disease such as anemia, anorexia, anti-fatigue, unspecified menstruation and female infertility in East Asia. In this study, ameliorative effects of Palmul-tang soft extracts (PMT), a Korean Medicine, on osteoporosis were investigated. Ovariectomized (OVX) osteoporotic ICR mice were intragastrically administrated PMT for 4 weeks. The level of bone mineral density (BMD) was analyzed in bone tissues by dual X-ray absorptiometry. The bone medullary cavity and deposition of collagen were investigated by histological analysis. In addition, the BMP-2 signaling-related molecules, osteoblastic differentiation and formation markers, were determined in femoral tissues. The levels of BMD and bone mineral content were significantly increased in tibia, femurs and LV by treatment of PMT. PMT replenished bone marrow cavity and increased collagen deposition in bone marrow cells of femur. In addition, administration of PMT recovered serum ALP, bALP, osteocalcin and calcium levels in osteoporotic mice. Moreover, PMT treatment up-regulated the expressions of BMP-2, RUNX2 and OSX with its downstream factors, ALP, OPN and BSP-1, in the femoral tissues. Taken together, PMT restored the bone minerals and improvement of bone integrity by bone-forming BMP-2 signaling pathway. These results demonstrate that PMT could be an ameliorative agent for osteoporosis.

In vivo and in vitro studies of Banhahoobak-tang tablets using UPLC-ESI-MS/MS with polarity switching.

Banhahoobak-tang is the most prescribed herbal drug in East Asia when individuals experience sudden symptoms such as sore throat or neurological symptoms. The low toxicity and high in-vivo safety of this herbal medicine has made it more attractive to patients, and it has recently been formulated as tablets. In addition, Banhahoobak-tang tablets are registered as health insurance drugs in South Korea, and clinical prescriptions and demand are increasing. However, there are very few clinical trial data as well as very little accurate content analysis and results for Banhahoobak-tang tablets. The purpose of this study was to perform in-vitro and in-vivo studies on Banhahoobak-tang tablets, including content analysis, pharmacokinetics in humans, and plasma protein binding. For this study, a UPLC-ESI-MS/MS method with polarity switching was developed for simultaneous analysis of 18 components of Banhahoobak-tang. To separate the analytes, a C8 reverse-phase column was used as the stationary phase, 0.1 % aqueous formic acid and acetonitrile as the mobile phase, and ionization and multiple reaction monitoring for quantification. The developed method was able to isolate and quantify the 18 components with good sensitivity and selectivity and was fully validated according to international analytical standards. Stability tests were also conducted on the analytes. Finally, the method was applied to in-vitro and in-vivo studies of Banhahoobak-tang tablets, and the tablet components were 52.49 ng/g to 91.00 µg/g on average. The detected components showed rapid oral absorption in humans as well as high plasma protein binding ratio overall. These results and methods can be useful not only for effectiveness and safety evaluation but also for quality control of Banhahoobak-tang tablets.

Simultaneous determination of fourteen components of Gumiganghwal-tang tablet in human plasma by UPLC-ESI-MS/MS and its application to pharmacokinetic study / 药物分析学报

Gumiganghwal-tang is a traditional herbal medicine widely used for its anti-inflammatory,analgesic,and antipyretic effects.However,the safety and efficacy of its active ingredients based on an in vivo pharmacokinetic (PK) study have yet been investigated.We have established a sensitive and accurate UPLC-ESI-MS/MS method and conducted a PK study on 14 constituents of Gumiganghwal-tang through human plasma analysis.Analytical conditions were optimized according to the physicochemical prop-erties of the 14 compounds to facilitate efficient separation and eliminate overlap or interference be-tween peaks.KINETEX-C18 and lnertsil-C8 columns were used as UPLC stationary phases,and acetonitrile and aqueous formic acid were used as mobile phases.All the analytes were quantified with a triple quadrupole mass spectrometer using electrospray ionization in multiple reaction monitoring mode.The chromatograms of 14 bioactive compounds showed excellent elution and sensitivity,and each peak was selectively separated and quantified without interference with each other or impurities.The established analytical method was based on international guidelines and was successfully used to perform PK studies of 14 herbal ingredients in humans after oral administration with Gumiganghwal-tang tablets.The oral absorption of most active components of Gumiganghwal-tang was relatively rapid and remained considerably long in the body to be quantified in plasma up to 48 h after administration.

Simultaneous determination of asarinin, ß-eudesmol, and wogonin in rats using ultraperformance liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies following administration of standards and Gumiganghwal-tang.

Asarinin, ß-eudesmol, and wogonin have common antiangiogenic activities and have the potential for use in chemotherapy. Besides, they are multivalent substances that are combined in various herbal medicines. The purpose of this study was to develop a method for simultaneous analysis of asarinin, ß-eudesmol, and wogonin, which are representative pharmacological components of Asarum heterotropoides, Atractylodes lancea, and Scutellaria baicalensis, respectively, in rat biosamples using ultraperformance liquid chromatography-tandem mass spectrometry. The three components were separated using 5 mm aqueous ammonium acetate containing 0.1% formic acid and acetonitrile as a mobile phase, equipped with a KINETEX core-shell C18 column. The analysis was quantitated on a triple-quadrupole mass-spectrometer employing electrospray ionization, and operated in the multiple reaction monitoring mode. The chromatograms showed high resolution, sensitivity, and selectivity with no interference with plasma, urine, and feces constituents. The developed analytical method satisfied international guidance criteria and could be successfully applied to the pharmacokinetic (PK) studies evaluating oral bioavailability of asarinin, ß-eudesmol, and wogonin after oral and intravenous administration and their urinary and fecal excretion ratios after oral administration to rats. Furthermore, the analysis was extended to PK studies following oral administration of Gumiganghwal-tang. This study was the first simultaneous analysis of the aforesaid three constituents in rat plasma, urine, and feces that also determined their PK parameters.

Simultaneous determination of fourteen main active components in Gumiganghwal-tang tablet by using a newly developed UPLC-ESI-MS/MS method.

The purpose of this study was to develop a method for simultaneous analysis of fourteen major active components in Gumiganghwal-tang tablet widely prescribed for cold related diseases using UPLC-ESI-MS/MS. Twelve of these 14 components were separated using 0.1% formic acid and acetonitrile as a mobile phase by gradient elution at a flow rate of 0.3 mL/min equipped with a KINETEX C18 column (2.1 × 50 mm, 1.7 µm). The remaining two components were separated using 10 mM aqueous ammonium formate containing 0.01% formic acid and acetonitrile as a mobile phase by gradient elution at a flow rate of 0.2 mL/min equipped with an Inertsil C8-3 column (2.1 × 100 mm, 2.0 µm). Quantitation of this analysis was performed on a triple quadrupole mass spectrometer using electrospray ionization technique operating in multiple reaction monitoring mode. Full validation of the analysis method was carried out, including its linearity, selectivity, sensitivity, precision, accuracy, recovery, and stability. Chromatograms showed high resolution, sensitivity, and selectivity without interference by impurities. Calibration curves of all 14 components ranged from 0.5 to 1000 ng/mL, displaying excellent linearity (correlation coefficients >0.99). The relative standard deviations (RSD) of intra- and inter-day were <11.75%. Recoveries were within the range 95.41-103.24% (RSD value of 1.62-9.09%). These results demonstrate that the developed method is simple, rapid, reliable, specific, accurate, and sensitive for the quantification of bioactive components of Gumiganghwal-tang. The developed method was successfully applied to the analysis of Gumiganghwal-tang tablet. The developed UPLC-ESI-MS/MS method could be useful not only for quality control, but also for effectiveness and safety evaluation of Gumiganghwal-tang tablet.

Simultaneous UPLC-MS/MS determination of four components of Socheongryong-tang tablet in human plasma: Application to pharmacokinetic study.

The purpose of this study was to develop a method for simultaneous analysis of schizandrin, ephedrine, paeoniflorin, and cinnamic acid as constituents of Socheongryong-tang tablet in human plasma using UPLC-MS/MS. These four components were separated using water containing 0.01% formic acid and methanol as a mobile phase by gradient elution at a flow rate of 0.3 mL/min with a HALO-C18 column (2.1 mm × 100 mm, 2.7 µm particle size). Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique operated in multiple reaction monitoring mode. Mass transitions were m/z 432.9 → 384.1 for schizandrin, 165.8 → 148.1 for ephedrine, 525.0 → 449.2 for paeoniflorin, 146.8 → 102.9 for cinnamic acid, and 340.0 → 324.0 for papaverine as internal standard. Liquid-liquid extraction and protein precipitation with ethyl acetate-methanol (1:2, v/v) were used to obtain these four components. Chromatograms showed high resolution, sensitivity, and selectivity without interference by plasma constituents. Calibration curves of schizandrin, ephedrine, paeoniflorin, and cinnamic acid in human plasma ranged from 0.02 to 8 ng/mL, 0.5 to 200 ng/mL, 0.2 to 80 ng/mL, and 1 to 400 ng/mL, respectively. Calibration curves of each analyte displayed excellent linearity, with correlation coefficients > 0.99. For all four components, both intra- and inter-day precisions (CV%) were <5.99%. The accuracy was 99.35-103.30% for schizandrin, 98.48-104.38% for ephedrine, 97.06-103.34% for paeoniflorin, and 99.97-104.36% for cinnamic acid. This analytical method developed in this study satisfied the criteria of international guidance. It could be successfully applied to pharmacokinetic studies of schizandrin, ephedrine, paeoniflorin, and cinnamic acid after oral administration of Socheongryong-tang tablet to humans.

Simultaneous Determination of Decursin, Decursinol Angelate, Nodakenin, and Decursinol of Angelica gigas Nakai in Human Plasma by UHPLC-MS/MS: Application to Pharmacokinetic Study.

Coumarins in Cham-dang-gwi, the dried root of Angelica gigas Nakai (AGN), possess pharmacological effects on anemia, pain, infection, and articular rheumatism. The AGN root containes decursin (D), decursinol angelate (DA), nodakenin, and decursinol (DOH), a major metabolite of D and DA. The aim of this study was to develop a simultaneous determination method for these four coumarins in human plasma using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Chromatographic separation was performed on dual columns (Kinetex® C18 column and Capcell core C18 column) with mobile phase consisting of water and acetonitrile at a flow rate of 0.3 mL/min using gradient elution. Multiple reaction monitoring was operated in positive ion mode with precursors to product ion transition values of m/z 328.9→228.8, 328.9→228.9, 409.4→248.8, and 246.8→212.9 to measure D, DA, nodakenin, and DOH, respectively. Linear calibration curves were fitted over concentration range of 0.05⁻50 ng/mL for these four components, with correlation coefficient greater than 0.995. Inter- and intra-day accuracies were between 90.60% and 108.24%. These precisions were within 11.19% for all components. The established method was then applied to a pharmacokinetic study for the four coumarins after usual dosing in Korean subjects.

A sensitive UHPLC-MS/MS method for the simultaneous quantification of three lignans in human plasma and its application to a pharmacokinetic study.

The aim of this study was to develop an analytical method to simultaneously analyze schizandrin, schizandrol B, and gomisin N lignans in human plasma using ultra high performance liquid chromatography with tandem mass spectrometry. The three lignans were separated using a mobile phase of water and acetonitrile containing 0.02% acetic acid equipped with a Kinetex C18 column (2.1 mm × 50 mm, 1.7 µm). This analysis was achieved by multiple reaction monitoring mode in an electrospray interface. The mass transitions were m/z 433.1→384.0 for schizandrin, 398.8→367.8 for schizandrol B, and 400.6→299.8 for gomisin N. Liquid-liquid extraction with methyl tert-butyl ether was used to obtain the three lignans. The chromatograms showed high resolution, sensitivity, and selectivity with no interference with plasma constituents. The calibration curves for the three lignans in human plasma were 0.05-50 ng/mL and displayed excellent linearity with correlation coefficients greater than 0.99. Precision for all three lignans was within 11.23%. The accuracy was 88.3-99.0% for schizandrin, 90.6-103.4% for schizandrol B, and 90.2-103.5% for gomisin N. The developed simultaneous analytical method satisfied the criteria of international guidance and could be successfully applied to the pharmacokinetic study of three lignans after oral administration of Schisandrae Fructus extract powder to humans.

Simultaneous determination of puerarin and its active metabolite in human plasma by UPLC-MS/MS: application to a pharmacokinetic study.

A rapid, selective and sensitive ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry method about the simultaneous determination of puerarin and its major active metabolite, daidzein, in human plasma was developed and validated in order to investigate the pharmacokinetics (PKs) of Gegen after the usual oral dose administration to human. Chromatography was carried out on a Kinetex C18 column (2.1mm×50mm, 1.7µm) using 0.05% acetic acid in water and 0.05% acetic acid in methanol as mobile phase with a gradient elution. Liquid-liquid extraction with ethyl acetate in acidic condition could remove the interference and minimize the matrix effect of human plasma. The lower limit of quantification in human plasma was 0.2ng/mL for both of compounds, puerarin and daidzein. The calibration curves for puerarin and daidzein in human plasma were linear over all the concentration range of 0.2-100ng/mL with correlation coefficients greater than 0.998. This assay procedure was successfully applied to the PKs of puerarin and daidzein, after the usual oral dose of Gegen extract powder (2.56g, containing 9.984mg puerarin) in human subjects.