Measurement of unbound ranitidine in blood and bile of anesthetized rats using microdialysis coupled to liquid chromatography and its pharmacokinetic application

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Abstract

To investigate the pharmacokinetics of unbound ranitidine in rat blood and bile, multiple microdialysis probes coupled to a liquid chromatographic system were developed. This study design was parallel in the following groups: the control-group of six rats received ranitidine alone (10 and 30 mg/kg, i.v.), the treated-group rats were co-administered with ranitidine and cyclosporine (P-glycoprotein (P-gp) inhibitor) or quinidine (both organic cation transport (OCT) and P-gp inhibitors) in six individual rats. Microdialysis probes were inserted into the jugular vein and the bile duct for blood and bile fluids sampling, respectively. Ranitidine in the dialysate was separated by a reversed-phase C18 column (Zorbax, 150 mm × 4.6 mm i.d.; 5 μm) maintained at ambient temperature. Samples were eluted with a mobile phase containing acetonitrile–methanol–tetrahydrofuran–20 mM K2HPO4 (pH 7.0) (24:20:10:946, v/v), and the flow rate of the mobile phase was 1 ml/min. The optimal UV detection for ranitidine was set at wavelength 315 nm. Between 20 and 30 min after drug administration (10 or 30 mg/kg), the ranitidine reached the maximum concentration in the bile. The bile-to-blood distribution ratio (AUCbile/AUCblood) was 9.8 ± 1.9 and 13.9 ± 3.8 at the dosages of 10 and 30 mg/kg, respectively. These studies indicate that ranitidine undergoes hepatobiliary excretion which against concentration gradient from bile-to-blood. In addition, the AUC of ranitidine in bile decreased in the treatment of cyclosporine or quinidine, which suggests that the hepatobiliary excretion of ranitidine was partially regulated by P-glycoprotein or organic cation transporter.

Introduction

Ranitidine {N-(3-[{[5-(dimethylamine)methyl-2-furanyl]methyl}thio]ethyl)-N9-methyl-2-nitro-1,1-ethenediamine hydrochloride} is the H2-receptor antagonist used for peptic ulcer [1]. Analysis of ranitidine from biological samples is commonly performed using high-performance liquid chromatography (HPLC) coupled to UV detection [2], [3] and tandem mass spectrometry [4], [5]. Sample preparation of ranitidine from human plasma has been accomplished by deproteination using perchloric acid [3] and solid-phase extraction [5], [6]. On the whole, these methods are time-consuming, and require tedious procedures for the preparation of biological samples. However, only protein-unbound drugs are available for drug distribution to the target site and for therapeutic application. To date, measurement of protein-unbound ranitidine in the blood and bile has not been described. Microdialysis technique provides an in vivo method to monitor unbound drug in various biological fluids, which excludes large molecule out of the dialysis membrane [7].

Since ranitidine interacts with the P-glycoprotein (P-gp), clarification of the transport mechanism may provide important information for studying the pharmacokinetics of ranitidine. It is also to be noted that not all P-glycoprotein substrates are subject to significant biliary excretion. Thus, to obtain more detailed information about the disposition of ranitidine in vivo, this study investigates the pharmacokinetics of unbound ranitidine in rat blood and bile using a microdialysis sampling technique coupled with HPLC. In addition, further exploration of the mechanism concerning the hepatobiliary excretion of ranitidine is also observed by comparing the pharmacokinetics of ranitidine present both with and without cyclosporine, a P-gp inhibitor, and quinidine, an OCT inhibitor, subsequently.

Section snippets

Chemicals and reagents

Ranitidine was purchased from Aldrich (Milwaukee, WI, USA). Cyclosporine (Sandimmun) and quinidine were obtained from Novartis Pharma (Basle, Switzerland) and Sigma (St. Louis, MO, USA), respectively. Triply deionized water from Millipore (Bedford, MA, USA) was used for all preparations.

Animal experimentation

All experimental protocols involving animals were reviewed and approved by the institutional animal experimentation committee of the National Research Institute of Chinese Medicine. Male specific pathogen-free

Results and discussion

The present validated liquid chromatographic method was coupled to the microdialysis technique and employed to determine ranitidine disposition from rat jugular vein blood and bile following drug administration. The method demonstrated excellent chromatographic selectivity with no endogenous interferences at the peak for ranitidine. Retention time of ranitidine was about 6.5 min. The mobile phase contains 5% acetonitrile and 95% phosphate buffer, ranitidine was not separated well from biological

Acknowledgement

This study was supported in part by research grants (NSC93-2113-M-077-005; NSC93-2320-B-077-006) from the National Science Council, Taiwan.

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