# Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry Multiple Reaction Monitoring-Based Multi-Component Analysis of Bangkeehwangkee-Tang: Method Development, Validation, and Application to Quality Evaluation

## TL;DR

This study developed a reliable method to analyze 22 compounds in Bangkeehwangkee-Tang, a herbal formula, to ensure consistent quality and effectiveness.

## Contribution

A validated UPLC–MS/MS method for simultaneous determination of 22 marker compounds in Bangkeehwangkee-Tang is developed and applied.

## Key findings

- The UPLC–MS/MS method showed excellent selectivity and linearity for all target compounds.
- Marker compound contents varied significantly across BHT samples, especially for sinomenine, magnoflorine, and glycyrrhizin.

## Abstract

Background/Objectives: Bangkeehwangkee-tang (BHT) is a traditional herbal formula composed of six medicinal herbs: Sinomenii Caulis et Rhizoma, Astragali Radix, Atractylodis Rhizoma Alba, Zingiberis Rhizoma Recens, Zizyphi Fructus, and Glycyrrhizae Radix et Rhizoma. BHT has been widely used for its immunomodulatory and anti-inflammatory effects. This study aimed to develop a reliable analytical method for the simultaneous determination of 22 marker compounds to ensure consistent quality control and to ensure consistent efficacy in both clinical and non-clinical studies of BHT. Methods: An ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method based on multiple reaction monitoring was developed and validated for the simultaneous determination of 22 marker compounds in BHT. The method was evaluated for selectivity, linearity (coefficient of determination, r2), sensitivity (limit of detection (LOD) and limit of quantification (LOQ)), accuracy (recovery), and precision (relative standard deviation (RSD)) in accordance with guidelines. Results: The developed method exhibited excellent selectivity and linearity (r2 ≥ 0.9913) for all target compounds. The LOD and LOQ ranged from 0.09 μg/L to 326.58 μg/L and 0.28 μg/L to 979.75 μg/L, respectively. The recovery ranged from 90.36% to 113.74%, and precision (RSD) was ≤15%, confirming the method’s reliability. The application of the method to various BHT samples revealed substantial variations in the marker compound contents, particularly for sinomenine, magnoflorine, and glycyrrhizin. Conclusions: These findings highlight the necessity for standardized quality control of BHT and demonstrate that the developed UPLC–MS/MS method is a practical and reliable tool for performing quality assessment of traditional herbal formulas.