Kim, Jung Soo
(Biomedical Research Institute of Chonbuk National University Hospital)
,
Kim, Yunjeong
(Biomedical Research Institute of Chonbuk National University Hospital)
,
Han, Song-Hee
(Biomedical Research Institute of Chonbuk National University Hospital)
,
Jeon, Ji-Young
(Biomedical Research Institute of Chonbuk National University Hospital)
,
Hwang, Minho
(Biomedical Research Institute of Chonbuk National University Hospital)
,
Im, Yong-Jin
(Biomedical Research Institute of Chonbuk National University Hospital)
,
Kim, Jung Hyun
(Biomedical Research Institute of Chonbuk National University Hospital)
,
Lee, Sun Young
(Biomedical Research Institute of Chonbuk National University Hospital)
,
Chae, Soo-Wan
(Biomedical Research Institute of Chonbuk National University Hospital)
,
Kim, Min-Gul
(Biomedical Research Institute of Chonbuk National University Hospital)
A rapid, sensitive and selective analytical method was developed and validated for the determination of compound K, a major intestinal bacterial metabolite of ginsenosides in human plasma. Liquid-liquid extraction was used for sample preparation and analysis, followed by liquid chromatography tandem...
A rapid, sensitive and selective analytical method was developed and validated for the determination of compound K, a major intestinal bacterial metabolite of ginsenosides in human plasma. Liquid-liquid extraction was used for sample preparation and analysis, followed by liquid chromatography tandem spectrometric analysis and an electrospray-ionization interface. Compound K was analyzed on a Phenomenex Luna C18 column ($100{\times}2.00$ mm, 3 ${\mu}m$) with the mobile phase run isocratically with 10 mM ammonium acetate-methanol-acetonitrile (5:47.5:47.5, v/v/v) at a flow rate of 0.5 mL/min. The method was validated for accuracy (relative error <12.63%), precision (coefficient of variation <9.14%), linearity, and recovery. The assay was linear over the entire range of calibration standards i.e., a concentration range of 1 ng/mL to 1,000 ng/mL ($r^2$ >0.9968). The recoveries of compound K after liquid-liquid extraction at 1, 2, 400, and 800 ng/mL were $106.00{\pm}0.08%$, $103.50{\pm}0.19%$, $111.45{\pm}5.21%$, and $89.62{\pm}34.46%$ for intra-day and $85.40{\pm}0.08%$, $94.50{\pm}0.09%$, $112.50{\pm}5.21%$, and $95.87{\pm}34.46%$ for inter-day, respectively. The lower limit of quantification of the analytical method of compound K was 1 ng/mL in human plasma. The developed method was successfully applied to a pharmacokinetic study of compound K after oral administration in ten of healthy human subjects.
A rapid, sensitive and selective analytical method was developed and validated for the determination of compound K, a major intestinal bacterial metabolite of ginsenosides in human plasma. Liquid-liquid extraction was used for sample preparation and analysis, followed by liquid chromatography tandem spectrometric analysis and an electrospray-ionization interface. Compound K was analyzed on a Phenomenex Luna C18 column ($100{\times}2.00$ mm, 3 ${\mu}m$) with the mobile phase run isocratically with 10 mM ammonium acetate-methanol-acetonitrile (5:47.5:47.5, v/v/v) at a flow rate of 0.5 mL/min. The method was validated for accuracy (relative error <12.63%), precision (coefficient of variation <9.14%), linearity, and recovery. The assay was linear over the entire range of calibration standards i.e., a concentration range of 1 ng/mL to 1,000 ng/mL ($r^2$ >0.9968). The recoveries of compound K after liquid-liquid extraction at 1, 2, 400, and 800 ng/mL were $106.00{\pm}0.08%$, $103.50{\pm}0.19%$, $111.45{\pm}5.21%$, and $89.62{\pm}34.46%$ for intra-day and $85.40{\pm}0.08%$, $94.50{\pm}0.09%$, $112.50{\pm}5.21%$, and $95.87{\pm}34.46%$ for inter-day, respectively. The lower limit of quantification of the analytical method of compound K was 1 ng/mL in human plasma. The developed method was successfully applied to a pharmacokinetic study of compound K after oral administration in ten of healthy human subjects.
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제안 방법
Analysis and validation were performed using an Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA, USA), interfaced with a mass spectrometer equipped with an electrosparay-ionization source operated in the positive mode (API4000; AB SCIEX, Foster City, CA, USA). Chromatographic separations were performed using a Luna C18 (2.
Chromatographic separations were performed using a Luna C18 (2.0×100 mm inner diameter, 3 μm particle size; Phenomenex, Torrance, CA, USA) at a column temperature of 30℃.
0×100 mm inner diameter, 3 μm particle size; Phenomenex, Torrance, CA, USA) at a column temperature of 30℃. In order to optimize the mobile phase, triplicate LC-MS/MS analyses of a mixture containing 500 ng/mL of compound K and 500 ng/mL of IS in methanol:water (50:50) were performed consecutively using each of the investigated mobile phase combinations. All chromatographic separations were performed using each mobile phase combination at a flow rate 0.
Accuracy was defined as the percent relative error (RE, %) and the assay precision was calculated by the percent coefficient of variation (CV, %). The intra- and inter-day recoveries of compound K in blank plasma were determined by comparing the response of the analytes extracted from the replicate QC samples (n=5) at LLQC, LQC, MQC, and HQC with the response of analytes from post-extracted plasma standard samples at the equivalent concentrations.
The method was validated for selectivity, linearity, precision, accuracy and recovery. The selectivity of the method was investigated by comparing chromatograms of extracted blank plasma obtained from six different human plasma samples spiked with compound K and IS to ensure that it was free of interference at the retention time of compound K.
The purpose of this study was to develop and validate and LC-MS/MS analysis method for the determination of compound K in human plasma, and to evaluate the pharmacokinetics of compound K after oral administration to healthy Korean subjects (n=10).
To evaluate these determination, four points of the calibration curve (at LLQC, low, medium, high concentration) were processed by the aforementioned liquidliquid extraction method and then analyzed in triplicate. The recovery was evaluated by processing 1, 2, 400, and 800 ng/mL of compound K concentration. The recovery was expressed by the percent recovery reported in Table 2.
2 for selectivity. The results of these analyses were used to evaluate the linearity range of quantification and LOQ, set to the lowest point of the calibration curve. The intra-day precision and accuracy for compound K at LLOQ (1 ng/mL) were 8.
63%. To evaluate these determination, four points of the calibration curve (at LLQC, low, medium, high concentration) were processed by the aforementioned liquidliquid extraction method and then analyzed in triplicate. The recovery was evaluated by processing 1, 2, 400, and 800 ng/mL of compound K concentration.
3 ng/mL and LOQ was 1 ng/mL. To quantify for the loss of the compounds during the sample treatment process, the recovery of the method was determined as the ratio of the peak area of extracted QC samples after a full assay process to that from direct injection of equivalent concentrations of compounds in methanol. An eight-point standard curve ranging from 1 ng/mL to 1,000 ng/mL of compound K having calibration standards and used in a manner similar to the peak area ratios of the target ions of compound K to those of the IS was compared with weighted (1/x2) least-squares calibration curves in which the peak area ratios of the calibration standards were plotted versus their concentrations.
대상 데이터
Ten young, healthy males (age, 24.9±2.7 years; weight, 68.1±6.3 kg) participated as study subjects and took a 5 g of fermented Korean red ginseng extract (containing 2.1 mg/g of Rg1 and Rb1, and 0.5 mg/g of compound K) dissolved in 100 mL of water as a single oral dose.
The ethyl acetate used as extraction organic solvent was purchased from Duksan (Ansan, Korea). The ammonium acetate (purity 98.0%) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
CUH 2011- 09-022). The clinical trial was performed in the Clinical Trial Center, Chonbuk National University Hospital in Korea. All participants filled out the informed consent form before participating in this study.
이론/모형
[27] reported ginsenoside metabolite by ultra performance liquid chromatography-time of flight/ mass spactrometry after pretreatment using solid phase extraction. In this study, it was simple and fast pretreatment by protein precipitation method. The retention time of compound K was long at 28.
성능/효과
The precision of the determination was evaluated in both MS/MS acquisition methods according to the calculated percentage (CV) of the replicated compound K sample solutions. The precision and accuracy results are reported in Table 1. Intraday precision (CV) ranged from 1.17% to 8.30%, and intra-day accuracy (RE) ranged from -10.38% to 11.45%. Inter-day precision (CV) ranged from 1.
2. Recovery rates were >85.4%, which was consistent over the concentration range 2 to 800 ng/mL at both intra- and inter-day.
To analysis of many samples, it is required rapid sample preparation and short analysis time. In conclusion, this method demonstrated good linearity, precision, and accuracy, and consumed less time, which is helpful in pharmacokinetic study for fermented Korean red ginseng in humans.
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