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

<b>Background/Objectives</b>: 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. <b>Methods</b>: 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, <i>r</i>²), sensitivity (limit of detection (LOD) and limit of quantification (LOQ)), accuracy (recovery), and precision (relative standard deviation (RSD)) in accordance with guidelines. <b>Results</b>: The developed method exhibited excellent selectivity and linearity (<i>r</i>² ≥ 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. <b>Conclusions</b>: 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.