School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China.
J Ethnopharmacol. 2013 Jun 21;148(1):266-70. doi: 10.1016/j.jep.2013.04.024. Epub 2013 Apr 30.
This study aimed to develop a specific HPLC-MS method for simultaneous quantification of four flavones of Glycyrrhiza in rat plasma after oral administration and to describe the pharmacokinetics of four flavones in rat plasma.
A simple, sensitive and selective method for simultaneous determination of four flavones of Glycyrrhiza in rat plasma, i.e., liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin, by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS) with negative electrospray ionization mode, was developed and validated. The method was applied to investigate the pharmacokinetics of four flavones in rat plasma after oral administration of Glycyrrhiza flavones. Chromatographic separation was accomplished on an Agilent TC-C18 column (4.6mm×250mm, and 5μm), with gradient elution by using a mixture of methanoic acid (A) and acetonitrile (B) as the mobile phase at a flow rate of 0.8mL/min.
The calibration curves for four flavones had good linearity higher than 0.997 in the measured range. Relative standard deviations (RSDs) of the intra- and inter-day precision at different levels were all less than 4.8%. The pharmacokinetic profile of four flavones in rat plasma was fitted with a two-compartment model detected by a simple, rapid and accurate HPLC-MS method. Time (h) to reach peak concentration (μg/mL) of liquiritin (2.69±0.04), isoliquiritin (10.16±0.02), liquiritigenin (2.83±0.02), and isoliquiritigenin (0.28±0.01) was 2.02±0.23, 1.97±0.20, 0.48±0.02, and 1.93±0.36, respectively. The distribution and elimination half-life (h) and area under the concentration-time curve (μg/mL-h) from t=0 to last time of liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin were 1.02±0.48/2.27±0.53/16.97±0.43, 2.04±1.01/2.38±0.80/69.20±5.24, 0.35±0.10/4.26±0.16/14.83±0.11, and 1.18±0.32/3.04±0.22/2.10±0.09, respectively. Isoliquiritin presented the phenomenon of double peaks and the others appeared together in a single and plateau absorption phase. Isoliquiritigenin had the lowest oral bioavailability because of Cmax and AUC0-∞. Liquiritigenin had the fastest absorption and distribution rate and the lowest elimination rate according to Tmax, t1/2α, and t1/2β.
This paper first reported on identification and determination of four flavones of Glycyrrhiza in rat plasma and their respective pharmacokinetic characteristics. The results provided a meaningful basis for better understanding the absorption mechanism of Glycyrrhiza and evaluating the clinical application of this medicine.
本研究旨在建立一种同时定量测定甘草中四种黄酮类化合物在大鼠血浆中含量的 HPLC-MS 方法，并描述四种黄酮类化合物在大鼠血浆中的药代动力学特征。
采用高效液相色谱-串联质谱（HPLC-MS）法，以负离子电喷雾模式，建立并验证了一种同时测定大鼠血浆中四种黄酮类化合物，即甘草素、异甘草素、甘草苷和异甘草苷的灵敏、特异、简单的方法。采用 Agilent TC-C18 柱（4.6mm×250mm，5μm），以甲醇（A）和乙腈（B）的混合物为流动相，梯度洗脱，流速为 0.8mL/min，进行色谱分离。
四种黄酮类化合物的校准曲线在测定范围内均具有良好的线性关系（r>0.997）。不同浓度日内和日间精密度的相对标准偏差（RSD）均小于 4.8%。采用简单、快速、准确的 HPLC-MS 方法检测到四种黄酮类化合物在大鼠血浆中的药代动力学特征符合二室模型。达峰时间（h）和浓度（μg/mL）分别为甘草素（2.69±0.04）、异甘草素（10.16±0.02）、甘草苷（2.83±0.02）和异甘草苷（0.28±0.01）为 2.02±0.23、1.97±0.20、0.48±0.02 和 1.93±0.36。分布半衰期（h）和消除半衰期（h）以及从 t=0 到最后时间的浓度-时间曲线下面积（μg/mL-h）分别为甘草素 1.02±0.48/2.27±0.53/16.97±0.43、异甘草素 2.04±1.01/2.38±0.80/69.20±5.24、甘草苷 0.35±0.10/4.26±0.16/14.83±0.11 和异甘草苷 1.18±0.32/3.04±0.22/2.10±0.09。异甘草素呈双峰现象，而其他三种则在单一、平台吸收相同时出现。由于 Cmax 和 AUC0-∞，异甘草苷的口服生物利用度最低。根据 Tmax、t1/2α 和 t1/2β，甘草苷具有最快的吸收和分布速率以及最低的消除速率。
本文首次报道了大鼠血浆中四种甘草黄酮类化合物的鉴定和测定及其各自的药代动力学特征。研究结果为更好地理解甘草的吸收机制和评价该药物的临床应用提供了有意义的依据。
