Simultaneous Quantification of Nine Target Compounds in Traditional Korean Medicine, Bopyeo-Tang, Using High-Performance Liquid Chromatography-Photodiode Array Detector and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Bopyeo-tang (BPT) is composed of six medicinal herbs (Morus alba L., Rehmannia glutinosa (Gaertn.) DC., Panax ginseng C.A.Mey., Aster tataricus L.f., Astragalus propinquus Schischkin, and Schisandra chinensis (Turcz.) Baill.) and has been used for the treatment of lung diseases. This study focused on establishing an analytical method that can simultaneously quantify nine target compounds (i.e., hydroxymethylfurfural, mulberroside A, chlorogenic acid, calycosin-7-O-glucoside, 3,5-dicaffeoylquinic acid, quercetin, kaempferol, schizandrin, and gomisin A) from a BPT sample using high-performance liquid chromatography with a photodiode array detector (HPLC-PDA) and ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). The separation of compounds in both analyses was performed on a C₁₈ reversed-phase column using the gradient elution of water-acetonitrile as the mobile phase. In particular, the multiple reaction monitoring mode was applied for quick and accurate detection in UPLC-MS/MS analysis. As a result of analyzing the two methods, HPLC-PDA and UPLC-MS/MS, the coefficient of determination of the regression equation for each compound was ≥0.9952, and recovery was 85.99-106.40% (relative standard deviation (RSD) < 9.58%). Precision testing of the nine compounds was verified (RSD < 10.0%). The application of these analytical assays under optimized conditions for quantitative analysis of the BPT sample gave 0.01-4.70 mg/g. Therefore, these two assays could be used successfully to gather basic data for clinical research and the quality control of BPT.

Keywords: Bopyeo-tang; HPLC–PDA; UPLC–MS/MS; simultaneous quantification; traditional Korean medicine.

Figure 1

Representative HPLC chromatograms of the mixed standard solution (A) and BPT sample (B). Hydroxymethylfurfural (1), mulberroside A (2), chlorogenic acid (3), calycosin-7-O-glucoside (4), 3,5-dicaffeoylquinic acid (5), quercetin (6), kaempferol (7), schizandrin (8), and gomisin A (9). The concentrations of each compound in the mixed standard solution were as follows: 10.00 μg/mL (hydroxymethylfurfural and calycosin-7-O-glucoside), 20.00 μg/mL (chlorogenic acid, 3,5-dicaffeoylquinic acid, quercetin, and kaempferol), 40.00 μg/mL (mulberroside A), and 50.00 μg/mL (schizandrin and gomisin A).

Figure 2

Representative total ion chromatograms of the standard solution (A) and the BPT sample (B) using the UPLC−MS/MS MRM method. Hydroxymethylfurfural (1), mulberroside A (2), chlorogenic acid (3), calycosin-7-O-glucoside (4), 3,5-dicaffeoylquinic acid (5), quercetin (6), kaempferol (7), schizandrin (8), and gomisin A (9). The concentrations of each compound in the mixed standard solution were as follows: 1250.00 μg/L (hydroxymethylfurfural), 250.00 μg/L (mulberroside A), 1000.00 μg/L (chlorogenic acid), 175.00 μg/L (calycosin-7-O-glucoside and 3,5-dicaffeoylquinic acid), 750.00 μg/L (quercetin), 1000.00 μg/L (kaempferol), and 375.00 μg/L (schizandrin and gomisin A).