Simultaneous Quantiﬁcation of Eight Marker Components in Traditional Herbal Formula, Haepyoyijin-Tang Using HPLC–PDA

: Haepyoyijin-tang (HPYJT) is a traditional herbal prescription that is composed of 12 medicinal herbs. Although HPYJT is frequently used in patients with asthma in Korea, no quality assessment protocols have been developed. In the present study, qualitative and quantitative analyses were performed using high-performance liquid chromatography and liquid chromatography–tandem mass spectrometry on the eight main components (mulberroside A, amygalin, liquiritin apioside, liquiritin, narirutin, hesperidin, rosmarinic acid, and glycyrrhizinic acid) to establish a quality control protocol for HPYJT. The simultaneous analysis method developed was satisfactorily validated with respect to linearity, limit of detection, limit of quantiﬁcation, recovery, and precision. This analytical method thus provides an e ﬃ cient approach for the evaluation of HPYJT quality.

To establish an efficient quality control protocol for HPYJT, we developed and validated the first simultaneous quantitative analysis using high-performance liquid chromatography equipped with photodiode array detector (HPLC-PDA) system on the eight main constituents of HPYJT. Marker components for quality control of HPYJT were selected as follows: mulberroside A (MULA) from Mori Radicis Cortex, amygdalin (AMY) from Armeniacae Semen, liquiritin apioside (LIQA), liquiritin (LIQ), and glycyrrhizinic acid (GA) from Glycyrrhizae Radix et Rhizoma, hesperidin (HES) and narirutin (NAR) from Citri Unshius Pericarpium, and rosmarinic acid (RA) from Perillae Folium.

Chemicals and Reagents
The eight constituents ( Figure S1) used for the quality assessment of HPYJT sample were purchased from natural product suppliers: i. Methanol, acetonitrile, water, and formic acid (FA, 99.0%) were from J.T.Baker (Phillipsburg, NJ, USA) and FUJIFILM Wako Pure Chemical Co. (Osaka, Japan) as a solvent or reagent for HPLC analysis.

Preparation of HPYJT Water Decoction
The 12 herbal medicines constituting HPYJT were mixed at the weight ratio (g, w/w) shown in Table S1. These materials were formulated and extracted with distilled water using a COSMOS-660 electric extractor (Kyungseo E&P, Incheon, Korea) under the following conditions: extraction temperature, 100 • C; extraction time, 2 h; extraction pressure, 98 kPa. The water extract was filtered using the sieve (size 53 µm mesh) and freeze-dried to make a powder sample (703.9 g, 14.08% yield).

Preparations of Test Solution and Stock Solution
For quantitative determination of the eight analytes (MULA, AMY, LIQA, LIQ, NAR, HES, RA, and GA) in the HPYJT decoction sample, 100 mg of the freeze-dried HPYJT sample was dissolved in 10 mL of 70% methanol and then extracted using an ultrasonicator (Branson 8510; Denbury, CT, USA) for 60 min at room temperature. The extract was filtered through a 0.2-µm membrane filter (Pall Life Sciences, Ann Arbor, MI, USA) before injection to HPLC system.
Each stock solution of eight constituents was made with methanol at a concentration of 1.0 mg/mL and kept refrigerated (approximately 4 • C).

Validation of Analytical Procedure
Validation of the established analytical method was performed according to the International Conference on Harmonisation guidelines [21]. Namely, the method was assessed based on linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy (recovery), and intra-and interday precisions.
Linearity in the developed assay was confirmed by measuring the coefficient of determination (r 2 ) based on the calibration curves of each marker component, and LOD and LOQ were calculated by using the equation: LOD = 3.3 × σ/S and LOQ = 10 × σ/S (where σ and S are the standard deviation of the y-intercept and the slope of the calibration curve, respectively).
Recovery tests using the standard addition assay were conducted to verify the accuracy and the results were evaluated using the equation: recovery (%) = found concentration/spiked concentration × 100%.
The precision of the developed HPLC analytical method was determined based on the repeatability, intraday precision, and interday precision and the error was evaluated by calculating the relative standard deviation (RSD) values; the latter were calculated as follows: RSD (%) = standard deviation (SD)/mean × 100%.

Optimizing Conditions for HPLC Analysis
The optimal chromatographic separation conditions for the simultaneous quantification of eight analytes (MULA, AMY, LIQA, LIQ, NAR, HES, RA, and GA) in HPYJT using HPLC were: SunFire C18 analytical column, column temperature of 40 • C, and distilled water-acetonitrile mobile system (both solvent containing 0.1% (v/v) FA). All analytes eluted under the optimal separation conditions within 40 min with a resolution >4.0. The retention times of the analytes were 12.20, 14.23, 18.75, 19.16, 20.37, 21.55, 23.25, and 36.79 min, respectively. A representative HPLC chromatogram is shown in Figure 1.

Validation of Analytical Procedure
Validation of the established analytical method was performed according to the International Conference on Harmonisation guidelines [21]. Namely, the method was assessed based on linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy (recovery), and intra-and interday precisions.
Linearity in the developed assay was confirmed by measuring the coefficient of determination (r 2 ) based on the calibration curves of each marker component, and LOD and LOQ were calculated by using the equation: LOD = 3.3 × σ/S and LOQ = 10 × σ/S (where σ and S are the standard deviation of the y-intercept and the slope of the calibration curve, respectively).
Recovery tests using the standard addition assay were conducted to verify the accuracy and the results were evaluated using the equation: recovery (%) = found concentration/spiked concentration × 100%.
The precision of the developed HPLC analytical method was determined based on the repeatability, intraday precision, and interday precision and the error was evaluated by calculating the relative standard deviation (RSD) values; the latter were calculated as follows: RSD (%) = standard deviation (SD)/mean × 100%.

Optimizing Conditions for HPLC Analysis
The optimal chromatographic separation conditions for the simultaneous quantification of eight analytes (MULA, AMY, LIQA, LIQ, NAR, HES, RA, and GA) in HPYJT using HPLC were: SunFire C18 analytical column, column temperature of 40 °C, and distilled water-acetonitrile mobile system (both solvent containing 0.1% (v/v) FA). All analytes eluted under the optimal separation conditions within 40 min with a resolution >4.0. The retention times of the analytes were 12.20, 14.23, 18.75, 19.16, 20.37, 21.55, 23.25, and 36.79 min, respectively. A representative HPLC chromatogram is shown in Figure 1.

Validation of the Analytical Procedure
System suitability tests were conducted to ensure the performance of the chromatography system with respect to capacity factor (k ), relative retention (α), theoretical plate number (N), resolution (Rs), Appl. Sci. 2020, 10, 3888 4 of 7 and symmetry factor (S). The system and all parameters were found to be suitable for performing the analysis (Table S2). The r 2 values of the eight marker components showed good linearity from 0.9996 to 1.0000, and LOD and LOQ values of these compounds were also calculated to be 0.09-0.48 µg/mL and 0.27-1.46 µg/mL, respectively. These results are summarized in Table 1. Recovery tests, which were conducted to ensure the validity of the method, showed that all the analytes were found with recovery rates from 97% to 103.50% and RSD values of ≤2.50% (Table 2). To evaluate repeatability, the results were analyzed six times, and the RSD values of retention time and peak area showed excellent repeatability of ≤0.11% and ≤0.71%, respectively. Intra-and interday precisions also showed good RSD values below 2.94% ( Table 3). The validation parameters reported above confirm that the developed HPLC analytical method is suitable for simultaneous quantification of HPYJT.

Simultaneous Determination of the Eight Marker Analytes in HPYJT
The developed and validated HPLC analytical method was applied for the quantitative analysis of the eight analytes in the lyophilized HPYJT sample. Quantification was performed simultaneously by scanning a wavelength range of 190-400 nm with a PDA detector. The wavelength monitored for the quantitative analysis of each component was: 214 nm for AMY, 254 nm for GA, 275 nm for LIQA and LIQ, 280 nm for NAR and HES, 325 nm for MULA, and 330 nm for RA. In the lyophilized HPYJT sample, the eight marker compounds were found to be present in 0.43 mg/g to 2.83 mg/g and, among these, it was confirmed that MULA was the most abundant in the HPYJT sample at 2.83 mg/g (Table 4).

Conclusions
Simultaneous analysis of a HPYJT sample was performed using HPLC-PDA to establish an efficient quality control protocol. The analytical methods were developed and verified with respect to parameters such as linearity, LOD, LOQ, accuracy (recovery), and precision. It is anticipated that the established analytical method will be used to obtain basic data for the quality assessment of HPYJT and related herbal formulas.