Department of Oriental Pharmacy and Wonkwang-Oriental Medicines Research Institute, Wonkwang University, Sinyong-Dong, Iksan-si, Jeonbuk, 570-749, Republic of Korea.
J Nat Med. 2013 Apr;67(2):390-8. doi: 10.1007/s11418-012-0687-2. Epub 2012 Aug 2.
This study was performed to develop methods for the chromatographic determination of biomarkers in Hwangryunhaedok-tang (HHT) and the quantitative evaluation of commercial HHT. To develop an analytical method, an RP-amide column (2.7 μm, 4.6 × 100 mm, Halo: Supelco, Bellefonte, PA) was used with a gradient solvent system of mixed acetonitrile and 0.1 % phosphoric acid/water and an ultra performance liquid chromatography-diode array detector. The method was validated by specificity, linearity, accuracy (recovery) and precision tests (repeatability, intra and inter-day). The correlation coefficients (R (2)) of biomarkers were calculated as 0.9998-1.000 and their ranges were as follows: geniposide (62.5-1,000.0 μg/ml), berberine (31.3-500.0 mg/ml), palmatine (31.3-500.0 μg/ml), baicalin (125.0-1,500.0 μg/ml), baicalein (15.6-250.0 μg/ml) and wogonin (5.2-125.0 μg/ml), respectively. The limit of detection was 0.34-4.01 μg/ml, and the limit of quantification was 1.02-12.16 μg/ml. The intra-day and inter-day precision of six components were revealed as 0.02-2.48 % as a relative standard deviation (RSD). The repeatability value of biomarkers in three different concentrations of HHT was 0.29-2.98 % (RSD value) and recovery was 95.72-104.90 %. Among several extraction methods tested, biomarker content was higher with the 20 times extraction (20TE) and mixture of extract powder (MEP) methods than with any other method, and some differences among diverse pharmaceutical medicines were revealed. The validation data indicated that the method developed is suited to the determination of six marker compounds in HHT. The content of biomarkers by simultaneous analysis was evaluated in 20TE, MEP, USA formula and Taiwan formula.
本研究旨在开发用于测定黄云鹤汤（HHT）中生物标志物的色谱方法，并对商业 HHT 进行定量评估。为了开发分析方法，使用了 RP-酰胺柱（2.7μm，4.6×100mm，Halo：Supelco，Bellefonte，PA），并采用混合乙腈和 0.1%磷酸/水的梯度溶剂系统和超高效液相色谱-二极管阵列检测器。该方法通过特异性、线性、准确度（回收率）和精密度（重复性、日内和日间）测试进行了验证。生物标志物的相关系数（R （2））计算为 0.9998-1.000，其范围如下：栀子苷（62.5-1000.0μg/ml）、小檗碱（31.3-500.0mg/ml）、黄连碱（31.3-500.0μg/ml）、黄芩苷（125.0-1500.0μg/ml）、黄芩素（15.6-250.0μg/ml）和汉黄芩素（5.2-125.0μg/ml）。检测限为 0.34-4.01μg/ml，定量限为 1.02-12.16μg/ml。六种成分的日内和日间精密度分别以相对标准偏差（RSD）表示为 0.02-2.48%。在三种不同浓度的 HHT 中，生物标志物的重复性值为 0.29-2.98%（RSD 值），回收率为 95.72-104.90%。在测试的几种提取方法中，20 倍提取（20TE）和提取物粉末混合物（MEP）方法的生物标志物含量高于其他任何方法，并且揭示了不同药物之间的一些差异。验证数据表明，所开发的方法适用于测定 HHT 中六种标记化合物。通过同时分析评估了 20TE、MEP、美国配方和台湾配方中生物标志物的含量。
