Two chromatographic methods for the determination of corticosteroids in human biological fluids: pharmacokinetic applications

Two techniques, high-performance liquid chromatography (HPLC) and quantitative high-performance thin-layer chromatography coupled with densitometry (HPTLC), were developed for the determination of prednisolone (PL), methylprednisolone (MP), and methylprednisolone sodium succinate (MPSS) in human plasma, saliva, and urine. The HPLC and HPTLC methods shared a single and simple step of an organic extraction procedure and separation of steroids using a normal-phase column or HPTLC plate. The methods allow simultaneous measurement of endogenous cortisol in plasma following the administration of PL and MP. The calibration curves of steroids in all biological fluids were linear over a wide range of concentrations of PL and MP in all biological fluids (0.025-4 micrograms/mL). The limit of detection of both assays for PL and MP was 10 ng/mL in plasma and saliva and 25 ng/mL in urine, and of MPSS was 50 ng/mL in plasma. Both methods were reproducible with an inter- and intra-assay coefficient of variation of less than 10% for all steroids over a wide range of concentrations in all biological fluids. No interference from endogenous steroids was found. The presented methods are simple, rapid, specific, sensitive, reproducible, and economical for the pharmacokinetic study of these steroids. The application of these methods for the pharmacokinetic study of both MP and PL in vivo and the in vitro hydrolysis of MPSS is discussed.