An Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometric Method for the Simultaneous Determination of Eighteen Marker Compounds in the Traditional Herbal Formula Bopyeo-Tang

Bopyeo-tang (BPT), comprising six medicinal plants, has been used for the treatment of respiratory diseases such as pulmonary fibrosis and chronic obstructive pulmonary disease. In this study, we developed and validated a quantitative method for the quality assessment of BPT using ultra-performance liquid chromatography with tandem mass spectrometry (UPLC–MS/MS). Eighteen marker compounds were separated on an Acquity UPLC BEH C18 reversed-phase column (2.1 mm × 100 mm, 1.7 μm) via gradient elution with a 0.1% aqueous formic acid–acetonitrile mobile phase. The multiple-reaction monitoring mode was used to improve analysis speed and accuracy. The coefficients of determination, limits of detection, and limits of quantitation of the 18 marker compounds were 0.9991–0.9996, 0.36–24.45 μg/L, and 1.07–73.35 μg/L, respectively. The recovery was 85.19–110.25%, and the relative standard deviation of precision was ≤9.01%. When applied to a typical BPT sample, the method revealed a range of concentrations from below the quantitative limit (one compound only) to a maximum of 3.20 mg/freeze-dried g. This method will be used for quality control of BPT preparations.

COPD is a disease that causes airflow obstruction and breathing problems, with symptoms such as chronic coughing, phlegm production, wheezing, and dyspnea [2,4].The disease kills approximately three million people worldwide a year.Smoking and exposure to harmful substances are the main causes; both are irreversible risk factors from exposure [2,4,5].The worldwide prevalence of COPD is estimated at between 7.5% and 10% [6].
We conducted a study to establish and verify an analytical method for the quality control of traditional herbal formulas using high-performance liquid chromatography or ultraperformance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) [20][21][22][23][24].However, the only analytical study on BPT was conducted by He et al. [18].This is the only study using a high-performance liquid chromatography-diode array detection electrospray ionization-hybrid ion trap-time of flight mass spectrometry method, detecting 89 peaks from BPT and identifying 47 of these [18].In addition, they identified acetylene glycosides (from Cononopsis Radix), flavonoids and saponins (Astragali Radix), lignan derivatives (Schisandrae Fructus), organic acidic and iridoid compounds (Rehmanniae Radix Preparata), organic acidic and peptides compounds (Asteris Radix et Rhizoma), and styrenes (Mori Radicis Cortex).However, they used BPT containing Cononopsis Radix instead of Ginseng Radix, but more importantly, each analysis takes approximately 150 min [18].Therefore, there is scope for a BPT analysis method capable of quantitatively analyzing BPT's major components in a shorter time.

Selection of Marker Compounds for Quality Assessment of BPT Using the UPLC-MS/MS Multiple-Reaction Monitoring (MRM) Method
Most traditional herbal formulas are complex preparations consisting of two or more different herbal medicines, contain numerous ingredients, and show various targets and various effects.Therefore, a scientific approach is needed to evaluate the quality of these traditional herbal formulas.

Validation of the UPLC-MS/MS MRM Method
In the developed analytical method, validation is a very important factor in the standardization of traditional herbal formulas.This is necessary to ensure the ultimate validity, practicality, and reproducibility of the scientific methods used in the developed assay.
The regression equations and values of the coefficient of determination (r 2 ), limit of detection (LOD), and limit of quantitation (LOQ) tested for different concentrations of each marker are summarized in Table 2.The r 2 values spanned from 0.9991 to 0.9996, showing high linearity, and the LOD and LOQ concentrations were 0.36-24.45µg/L and 1.07-73.35µg/L, respectively.Table 3 shows the recovery, precision, and repeatability results for the markers.The recoveries were 85.19-110.25%(relative standard deviation (RSD) ≤ 6.60%), which were within the acceptable range of 80-120%.The RSD value of precision was ≤9.01%, which was within the tolerance level of ±20%.In the stability test, these 18 marker compounds were considered stable for at least 3 days as the change in peak area content was less than 3.0% within 3 days (Table 3).

Simultaneous Determination of the 18 Marker Compounds in a BPT Sample via the UPLC-MS/MS MRM Method
The new method was applied for the simultaneous determination of the 18 compounds in a BPT sample (Table 4).Among these compounds, gomisin A was detected below the LOQ.The other compounds were detected at 0.001-3.20 mg/freeze-dried g of BPT.Hydroxymethylfurfural-a major component of R. glutinosa-was by far the most abundant compound, at 3.20 mg/g, in this BPT sample.

Chemicals and Reagents
The 18 reference marker compounds (Figure S4) used for simultaneous determination in BPT via the UPLC-MS/MS system were purchased from high-purity natural product manufacturers such as EnsolBioSciences (Daejeon, Republic of Korea), Merck KGaA (Darmstadt, Germany), Shanghai Sunny Biotech (Shanghai, China), Biopurify Phytochemicals (Chengdu, China), and Wuhan ChemFaces Biochemical (Wuhan, China).Detailed information on these marker compounds is summarized in Table S1.LC-MS-grade organic solvents (methanol and acetonitrile) and reagents (formic acid) were purchased from Thermo Fisher Scientific (Cleveland, OH, USA).Ultrapure deionized water was obtained using the Elix Technology Inside system (Milli-Q Integral 15, Merck, Millipore, France).

Analytical Conditions for the Simultaneous Quantification of Markers in a BPT Sample via the UPLC-MS/MS MRM Method
The simultaneous quantification of the 18 marker compounds was performed by modifying analytical protocols previously reported to the UPLC-MS/MS system comprising a Waters Acquity UPLC I-Class Plus system and a Xevo TQ-XS triple quadrupole mass spectrometry system (Waters, Milford, MA, USA) [23,24].The chromatographic separation of all markers was carried out using a 0.1% (v/v) aqueous formic acid-acetonitrile mobile phase system and an Acquity UPLC BEH reverse-phase column (Waters) maintained at 45 • C. Detailed operating conditions for the UPLC and mass spectrometry systems are described in Table 5.In addition, the operating parameters for the MRM of each compound are listed in Table 1.Standard stock solutions of each reference standard compound were prepared at a concentration of 1.0 mg/mL using methanol, stored in a refrigerator (approximately 4 • C), and used for analysis.
The sample solution for UPLC-MS/MS analysis was prepared at a concentration of 0.05 mg/mL with 70% methanol.That is, we took exactly 0.5 mg of BPT sample, placed it in a 10 mL volumetric flask, and added 70% methanol.The mixed sample solution was continuously subjected to ultrasonic extraction for 5 min and vortexing for 1 min.The solution was filtered through a 0.22 µm polytetrafluoroethylene hydrophobic membrane filter (catalog No. SSKPTFE13022B, SsolKorea, Daejeon, Republic of Korea) before use in UPLC-MS/MS analysis.

Validation of the UPLC-MS/MS MRM Method
For validation linearity, sensitivities (LOD and LOQ), accuracy, and precision parameters were evaluated using the International Conference on Harmonisation guidelines, specifically the methods for there Q2B validation of analytical procedures [48].Briefly, linearity was evaluated using the r 2 value of the calibration regression equation for each marker compound.The sensitivities, LOD and LOQ, were calculated from signal-to-noise ratios of 3:1 and 10:1, respectively.
Recovery was determined using a standard addition method.In general, it is difficult to source reference samples to be used as blanks in herbal medicine analysis that do not contain marker components.Therefore, in this study, recoveries were calculated by adding the authentic compounds the BPT extract at three concentrations (low, medium, and high) and subtracting the endogenous quantity in the sample.The calculation equation was as follows: Recovery(%) = found amount − original amount spiked amount × 100 Intraday and interday precisions were determined in the same way recovery was, within one day and over three consecutive days, and then evaluated as RSD (%), which was calculated as follows: RSD(%) = standard deviation mean × 100 Finally, the stability of 18 marker compounds was tested for 3 days at room temperature (23 ± 1 • C) using the sample solution and was evaluated using the RSD values.

Conclusions
We developed and validated a fast, sensitive, and accurate UPLC-MS/MS MRM method to quantify the quality of BPT, which is widely prescribed in oriental medicine for respiratory diseases.This method was validated for linearity, sensitivities (LOD and LOQ), accuracy, and precision.In a practical trial with a BPT sample, hydroxymethylfurfural, a major component of R. glutinosa, was detected in the highest abundance.We believe that the knowledge developed in this study will be useful for efficacy studies.

Table 1 .
Parameters for UPLC-MS/MS MRM of the 18 marker compounds.

Table 2 .
Retention times, linear ranges, regression equations, coefficients of determination (r 2 ), limits of detection (LOD), and limits of quantitation (LOQ) for the simultaneous determination of the 18 marker compounds using the UPLC-MS/MS MRM method.

Table 3 .
Recovery, precision, and stability data of the 18 marker compounds for the UPLC-MS/MS MRM method.

Table 4 .
Amounts of the 18 marker compounds in a BPT sample, determined using the UPLC-MS/MS MRM method.

Table 5 .
Operating conditions for the UPLC-MS/MS multiple-reaction monitoring (MRM) simultaneous analysis of BPT.