Development of simultaneous analysis for marker constituents in Hwangryunhaedok-tang () and its application in commercial herbal formulas.

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.