LC-MS/MS quantification of 20(S)-protopanaxadiol in complex biological matrices for bioanalytical method validation and pharmacokinetic analysis.

20(S)-Protopanaxadiol (PPD) is a saponin derivative of ginsenoside, with more potent biological and pharmacological activities than Rg3 and Rh2. The lack of ionizable centers leads to low mass spectrometry reactions and internal cleavage of three hydroxyl groups, making it challenging to establish highly sensitive PPD mass spectrometry methods. The aim of this study is to establish and validate a quantitative detection method for PPD in multiple matrices using mass spectrometry. The methods used Rh2 as the internal standard and organic solvent liquid-liquid extraction under alkaline conditions for biological sample pretreatment. Isometric separation was achieved through methanol, acetonitrile, and a 10 mmol/L solution of acetic acid (45:45:10, v/v/v) at a flow rate of 0.4 mL/min. Finally, perform mass spectrometry quantification. Comprehensive method validation was conducted on rat plasma samples, and partial method validations were performed on three types of rat tissues (adipose tissue, smooth muscle, and skeletal muscle), bile, urine, fecal samples, and dog plasma samples. The results were in accordance with the requirements of NMPA for bioanalytical method validation, ensuring the accuracy and reliability of our analytical measurements. This study employed a conventional liquid-liquid extraction sample pretreatment scheme, utilizing multiple biological matrices commonly found in a single treatment protocol and liquid chromatography-tandem mass spectrometry detection parameters. The consistency of processing and detection across diverse samples eliminated the need for methodological changes, providing exceptional convenience. Up to 90% of the organic phase and a 50 mm short chromatographic column achieved rapid and effective separation of PPD. A key aspect of our work is the use of a "programmed injection" technique, which significantly reduces the analysis time from 4.2 min during method exploration to 2.4 min. These methods have achieved a relatively low quantification limit of 2.5 ng/mL. The methods established were successfully applied to the kinetic process of PPD in rats, and the pharmacokinetic characteristics of PPD in dogs were studied for the first time.