School of Pharmacy, Jilin Medical University, 5 Jilin Street, Fengman District, Jilin, 132013, China.
School of Pharmacy, Jilin Medical University, 5 Jilin Street, Fengman District, Jilin, 132013, China.
J Pharm Biomed Anal. 2019 Jun 5;170:1-7. doi: 10.1016/j.jpba.2019.03.022. Epub 2019 Mar 14.
Huangqi Guizhi Wuwu Decoction (HGWWD), consisting of Radix Astragali, Cinnamomi Ramulus, Paeoniae Radix Alba, Zingiberis Rhizoma Recens and Jujubae Fructus, is a widely used Traditional Chinese Medicine (TCM) formula for the treatment of human blood impediment in China for nearly 2000 years. In order to make good and rational use of this formula in the future, a rapid, sensitive and robust ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous determination of calycosin-7-O-β-D-glucoside, cinnamic acid, paeoniflorin and albiflorin, the main active constituents of HGWWD, in rat plasma using geniposide as internal standard (IS). The plasma samples were extracted by protein precipitation with acetonitrile and separated on a Shim-pack XR-ODS C column (75 mm × 3.0 mm, 2.2 μm) using gradient elution with a mobile phase consisting of water (containing 0.1% formic acid) and acetonitrile at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed on 3200 QTRAP mass spectrometry equipped with electrospray ionization source in negative ionization mode. Quantification was performed using multiple reaction monitoring (MRM) by monitoring the fragmentation of m/z 491.1→282.9 for calycosin-7-O-β-D-glucoside, m/z147.0→103.1 for cinnamic acid, m/z 525.0→120.9 for paeoniflorin, m/z 525.2→121.0 for albiflorin and m/z 433.1→225.1 for IS, respectively. The method was well validated in terms of linearity, precision, accuracy, recovery, matrix effect and stability. All calibration curves had good linearity (r>0.9977) over the concentration range from 0.1-50 ng/mL for calycosin-7-O-β-D-glycoside, 50-25000 ng/mL for cinnamic acid, 5-2500 ng/mL for paeoniflorin and albiflorin. The intra-day and inter-day precisions (relative standard deviation) were within 11.8%, the accuracy (relative error) ranged from -9.4% to 9.1%, and the lower limit of quantification (LLOQ) were 0.1, 50, 5, 5 ng/mL for calycosin-7-O-β-D-glucoside, cinnamic acid, paeoniflorin and albiflorin, respectively. Extraction recovery, matrix effect and stability were satisfactory in rat plasma. The validated method was successfully applied to a pharmacokinetic study of calycosin-7-O-β-D-glucoside, cinnamic acid, paeoniflorin and albiflorin after oral administration of HGWWD to rats.
黄芪桂枝五物汤（HGWWD）由黄芪、桂枝、白芍、生姜和大枣组成，是中国近 2000 年来治疗人体血液淤滞的一种常用中药方剂。为了在未来能够很好地合理使用该方剂，本研究建立了一种快速、灵敏、稳健的超高效液相色谱-串联质谱（UHPLC-MS/MS）法，同时测定大鼠血浆中黄芪甲苷、桂皮酸、芍药苷和白芍苷（HGWWD 的主要活性成分）的含量，以栀子苷为内标（IS）。采用乙腈沉淀蛋白法提取血浆样品，在 Shim-pack XR-ODS C 柱（75mm×3.0mm，2.2μm）上，以含 0.1%甲酸的水和乙腈为流动相进行梯度洗脱，流速为 0.4mL/min。采用电喷雾离子源在负离子模式下，以多反应监测（MRM）进行质谱检测。通过监测 m/z 491.1→282.9 （黄芪甲苷）、m/z147.0→103.1 （桂皮酸）、m/z 525.0→120.9 （芍药苷）、m/z 525.2→121.0 （白芍苷）和 m/z 433.1→225.1 （IS）的碎片离子，分别对各成分进行定量分析。该方法在浓度范围为 0.1-50ng/mL 时，线性良好（r＞0.9977），用于测定 calycosin-7-O-β-D-glucoside 的线性范围为 50-25000ng/mL，用于测定 cinnamic acid 的线性范围为 5-2500ng/mL，用于测定 paeoniflorin 和 albiflorin 的线性范围为 5-2500ng/mL。日内和日间精密度（相对标准偏差）均在 11.8%以内，准确度（相对误差）在-9.4%至 9.1%范围内，定量下限（LLOQ）分别为 0.1、50、5、5ng/mL。在大鼠血浆中，提取回收率、基质效应和稳定性均令人满意。该方法已成功应用于大鼠灌胃给予 HGWWD 后 calycosin-7-O-β-D-glucoside、cinnamic acid、paeoniflorin 和 albiflorin 的药代动力学研究。
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