Identification of prednisolone, methylprednisolone and their metabolites in human urine using HPLC (+) ESI-MS/MS and detection of possible adulteration in Indian herbal drug preparations

Objective: To explore the possibility of identifying the maximum number of metabolites of prednisolone and methylprednisolone by LC-MS/MS and to further test the application of this developed method on six Indian herbal drug preparations. Method and Materials: The sample extraction procedure involves enzymatic hydrolysis and liquid-liquid extraction and further analysis using LC-MS/ MS. The excretion profile was performed with four healthy male volunteers after administration of 40 mg and 8 mg of prednisolone and methylprednisolone, respectively. Six herbal drug preparations obtained from All India Institute of Medical Sciences, India were tested to detect the possible adulteration with prednisolone or methylprednisolone. Results: The Analytical method was validated as per the requirement of WADA International Standard of Laboratories (version 6.0). The parent compound and ten urinary metabolites of prednisolone could be identified. The parent, M-1, M-2 and M-3 could be detected up to 72 hours while rest of the metabolites were detectable up to 24 hours. In the case of methylprednisolone, the parent compound and six urinary metabolites were identified. M-1 and M-2 of methylprednisolone were detectable up to 48 hours while the parent drug methylprednisolone and other metabolites were detectable up to 24 hours. Out of the six herbal drugs tested, one showed the presence of prednisolone. Conclusion: The improved detection method developed for the identification and detection of prednisolone and methylprednisolone metabolites would prove highly beneficial in extending the time of detection of drug abuse in athletes and also detecting spiked ayurvedic, homeopathic and unani preparations.