Simultaneous Determination of Pantoprazole and Its Two Metabolites in Dog Plasma by HPLC

Journal of Chromatographic Science, Vol. 43, May/June 2005 Simultaneous Determination of Pantoprazole and Its Two Metabolites in Dog Plasma by HPLC Zhiyong Xie, Xiaoyan Chen, Fengdan Jin, and Dafang Zhong* Laboratory of Drug Metabolism and Pharmacokinetics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, P.R. China Abstract A simple and sensitive high-performance liquid chromatography (HPLC) method is developed and validated for simultaneous determination of pantoprazole and its two metabolites (pantoprazole sulfone and pantoprazole thioether) in dog plasma and applied to a pharmacokinetic study in Beagle dogs. Following a protein precipitation procedure, the samples are separated using reversed-phase HPLC (C18) by a gradient of acetonitrile and ammonium acetate (pH 6.0) at a flow rate of 1.0 mL/min and quantitated using UV detection at 290 nm. Omeprazole is selected as the internal standard. The method has a lower limit of quantitation of 0.025 µg/mL for pantoprazole and its two metabolites, using 0.1-mL aliquots of plasma. The linear calibration curves are obtained in the concentration range of 0.025–10.0 µg/mL for three analytes. The intra- and interrun precision (relative standard deviation), calculated from quality control (QC) samples, is less than 13% for three analytes. The accuracy determined from QC samples is between –6.4% and 12%. lites in biological fluids. Several high-perf o rmance liquid chromatographic (HPLC) methods have been available for the determination of PAN and its metabolites in biological fluids (8–12). Although three HPLC methods (8–10) have enabled PAN and PAN-SO2 to be determined in plasma or serum, these methods do not determine PAN-S. Furt h e rmore, two methods among them utilized a column-switching system as a sample cleanup procedure. There are two papers that reported separation and determination of PAN enantiomers in human seru m using cellulose-based chiral stationary phase (11) and tris(3,5-dimeth-oxyphenylcarbamate) of amylose phase (12), respectively. But these methods could not be used to determ i n e its metabolites. This paper describes a simple and sensitive gradient HPLC method for the simultaneous determination of PAN, PAN-SO2, and PAN-S in dog plasma and applies the method to a pharmacokinetic study of PAN in Beagle dogs. Experimental Introduction Pantoprazole (PAN) (Figure 1), a selective and long-acting proton pump inhibitor (1), is used in the treatment of acidrelated gastrointestinal disorders such as duodenal and gastric ulcers, reflux esophagitis, and Zollinger–Ellison syndrome (2). Drugs with sulfoxide moieties, such as omeprazole (3), lansoprazole (4), and PAN, can undergo oxidative metabolism by cytochrome P450 or flavin containing monooxygenase (or both) (5,6) and can undergo reductive metabolism by aldehyde oxidase or thioredoxin-linked enzymes (or both) (7). Both liver and gut flora are potential sites for formation of the sulfide metabolite (5–7). These metabolites include pantoprazole sulfone (PAN-SO2), pantoprazole thioether (PAN-S), demethylated PAN-SO2, and demethylated PAN-S. In order to elucidate the pharmacokinetics of PAN and its metabolites after administration of PAN to animals, it is essential to develop a method to determine the concentration of PAN and its metabo- Chemicals PAN, PAN metabolites (PAN-SO2 and PAN-S), and internal standard (IS) (omeprazole) were provided by Institute of Materia Medica, Shenyang Pharmaceutical University (Shenyang, China). Compound purities exceeded 99%. Methanol and acetonitrile were of HPLC grade, and other Figure 1. Structures of PAN, PAN-SO2, PAN-S, and IS (omeprazole). * Author to whom correspondence should be addressed: email . Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission. 271 Journal of Chromatographic Science, Vol. 43, May/June 2005 of the solution was injected into the HPLC system. reagents were commercially available and of analytical grade. Distilled water, prepared from demineralized water, was used throughout the study. Method validation The specificity of the method was demonstrated by comparing chromatograms of six independent plasma samples f rom dogs, each as a blank and spiked sample. Standard curv e s w e re constructed using weighted (w = 1/c 2) linear least-square s regression analysis of the observed peak area ratios of PAN, PAN-SO2, PAN-S, and IS. The unknown sample concentrations of PAN, PAN-SO2, and PAN-S were calculated from the linear regression equation of the peak area ratio against concentrations of the calibration curves. Accuracy and precision were assessed by determining QC samples at four concentration levels (Table I, six replicate samples of each concentration of QC samples) on three different validation runs. The accuracy was expressed as the relative e rror (RE) [(mean concentration found – concentration spiked)/(concentration spiked) × 100%], and the precision was evaluated by the relative standard deviation (RSD) [(standard Chromatographic system The HPLC system (Hewlett Packard, Palo Alto, CA) consisted of a G1313A quaternary pump, a G1314A UV detector, G1313A autosampler, G1316A column oven, and vacuum degasser unit. The mobile phase consisted of a gradient mixed from acetonitrile (A) and ammonium acetate (pH 6.0, 10mM) (B) at a flow rate of 1.0 mL/min. The column was equilibrated with 35% A at time 0. After injection of the plasma sample (50 µL), the A volume ratio was linearly increased to 50% at 6 min, held constant at 50% until 10 min, then reduced to 35% at 12 min, and the column was equilibrated for 3 min before application of the next sample. The total analysis time was 15 min. Separation was accomplished at 25°C on a diamonsil C18 column (4.0 × 250 mm, 5 µm) (Dikma, Beijing, China). The UV detector was set at 290 nm. Standard solutions and quality control samples The stock standard solution of PAN, PANSO2, and PAN-S was prepared by dissolving accurately weighed portions in methanol to give a final concentration of 400.0 µg/mL. The solution was then successively diluted with methanol to achieve standard working solutions at concentrations of 0.025, 0.050, 0.125, 0.250, 1.0, 2.5, 5.0, and 10.0 µg/mL for PAN, PA N - S O 2, and PAN-S, re s p e c t i v e l y. An 8.0 µg/mL IS (omeprazole) working solution was p re p a red by diluting the 400.0 µg/mL stock standard solution of omeprazole with methanol. The standard working solutions (100 µL) were used to spike blank dog plasma (0.1 mL) to give final concentrations in the range of 0.025–10.0 µg/mL. The plasma samples at concentrations of 0.050, 0.250, 2.5, and 8.0 µg/mL for PAN, PA N - S O2, and PAN-S were used as quality control (QC) samples in developing the analytical method and during the pharmacokinetic study. All of the solutions were stored at 4°C and were brought to room temperature before use. Sample preparation One-hundred microliters of IS (8.0 µg/mL omeprazole pr (...truncated)