Determination of oleanolic acid in human plasma and study of its pharmacokinetics in Chinese healthy male volunteers by HPLC tandem mass spectrometry

A highly selective and sensitive HPLC–ESI–MS–MS method was developed for the determination of oleanolic acid in human plasma. The oleanolic acid and glycyrrhetinic acid (internal standard) were recovered from plasma with ethyl acetate liquid–liquid extraction. The organic extracts were dried under a stream of warm nitrogen, reconstituted in mobile phase and injected into a Zorbax-Extend ODS analytical column (150 mm × 4.6 mm i.d., 5 μm), with the mobile phase consisting of methanol–ammonium acetate (32.5 mM) (85:15, v/v) pumped at a flow rate of 1.0 ml/min, and 30% of the eluent was split into a MS system with electrospray ionization tandem mass (ESI–MS–MS) detection in negative ion mode. The tandem mass detection was performed on a Finnigan Surveyor LC-TSQ Quantum Ultra AM tandem mass spectrometer operated in selected reaction monitoring mode. The parent to product ion combinations of m/z 455.4 → 455.4 and 469.3 → 425.2 at 38 V 1.5 mTorr Ar CID were used to quantify oleanolic acid and glycyrrhetinic acid, respectively. The assay was validated in the concentration range of 0.02–30.0 ng/ml for oleacolic acid when 0.5 ml of plasma was processed. The precision of the assay (expressed as relative standard deviation, R.S.D.%) was less than 15% at all concentrations levels within the tested range and adequate accuracy, and the limit of quantification was 0.02 ng/ml. The established method was applied for the pharmacokinetics study of oleanolic acid capsules in 18 healthy male Chinese volunteers with the mean values of Cmax, Tmax, AUC0–48, AUC0–∞, t1/2, CL/F, and V/F of oleanolic acid after p.o. a single 40 mg dose obtained were 12.12 ± 6.84 ng/ml, 5.2 ± 2.9 h, 114.34 ± 74.87 ng h/ml, 124.29 ± 106.77 ng h/ml, 8.73 ± 6.11 h, 555.3 ± 347.7 L/h, and 3371.1 ± 1990.1 L, respectively.