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

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Abstract

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.

Introduction

Oleanolic acid [a, (3β)-3-hydroxyolean-12-en-28-oic acid (Fig. 1)] is one of the best known bioactive pentacyclic triterpenoids, that exists widely in medicinal herbs and plants, in the form of free acid or aglycones for triterpenoid saponins [1]. The traditional uses of plants containing oleanolic acid in folk medicines are multiple, in terms of anti-inflammatory [2], [3], hepato-protection, analgesia, cardiotonic, glucose-lowering [4], tonic effects and enhancement of the body defense systems, etc. It is now marketed in China as an oral drug for human liver disorders.

Approaches for measuring of oleanolic acid in rabbit plasma has been described by using UV–vis spectrophotometry [5]. But the reported method is not suitable for the pharmacokinetics study of oleanolic acid in human. There is not any reported pharmacokinetics data of oleanolic acid in human up until now. Therefore, a sensitive HPLC–ESI–MS–MS method was established for the determination of oleanolic acid in human plasma and the study of its pharmacokinetics.

Section snippets

Materials

The reference substances of oleanolic acid and glycyrrhetinic acid were purchased from the National Institute for the Control of Pharmaceutical and Biological Products, Tiantanxili No. 2, Beijing, China. All chemicals and reagents used were of HPLC grade or analytical grade from the Nanjing Chemical Reagent Company, Yanyao Rd., Nanjing, China. The water was prepared with double distillation.

The oleanolic acid capsules (lot no: 20030301) were supplied by Zhuhai Rundu Pharmaceutical Co.,

Recovery, linearity, precision and accuracy

The Zorbax-Extend ODS analytical column and the mobile phase used for the determination gave a well-defined separation between the drug, internal standard and endogenous components. Typical chromatograms were shown in Fig. 3 with the retention time for glycyrrhetinic acid and oleanolic acid was about 3.0 and 6.3 min, respectively.

The absolute recoveries of both oleanolic acid and glycyrrhetinic acid from the plasma were more than 88% and less than 100%, indicating that most of oleanolic acid in

Conclusions

In summary, a novel sensitive HPLC–ESI–MS–MS method for the determination of oleanolic acid in human plasma has been developed and validated over the concentration range from 0.02 to 30.0 ng/ml. The method has been successfully used for the pharmacokinetics study of oleanolic acid in Chinese healthy male volunteers.

Acknowledgement

The authors would like to thank Dr. Dave G. Watson, Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow G4 0NR, UK for his many helpful contributions during the course of the study and the writing of this manuscript.

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