Development and Validation of a High-performance Liquid Chromatography Method for Simultaneous Determination of Five Active Compounds in Kleeb Bua Daeng Formula

Abstract

Kleeb Bua Daeng formula is a popular traditional remedy sold by the Chao Phya Abhaibhubejhr Hospital, Thailand. This formula contains Piper nigrum L., Nelumbo nucifera Gaertn. and Centella asiatica L. components. A reliable and accurate analytical method for the determination of five major active compounds in this formula was developed and validated. High performance liquid chromatography with a diode-array detector was used to quantify piperine, quercitin, kaempferol, asiaticoside and madecassoside in Kleeb Bua Daeng formula. The separation was carried out using a hypersil C18 column with detection at wavelengths 210, 280 and 370 nm. Gradient chromatographic conditions using a mixture of 0.05% phosphoric acid and acetonitrile allowed for complete resolution of the 5 active compounds. The validation outcomes for accuracy, precision, linearity, limit of detection, limit of quantitation and robustness demonstrated that this HPLC method was accurate and reliable for the simultaneous determination of active compounds in this formula. Hence, this developed and validated HPLC fingerprint method is appropriate for quality control of Kleeb Bua Daeng formula and could be applied to modified Kleeb Bua Daeng formulations.