A simple high performance liquid chromatography method for determination of rebamipide in rat urine

Graphical abstract


Method details
Rebamipide urine analysis was carried out using samples obtained from a previous study [5]. In the study, male Sprague-Dawley rats were dosed with rebamipide (30 mg/kg) twice daily for two days via gastric intubation. On day three, animals received one dose of rebamipide in the morning then were transferred to metabolic cages for a 12 h urine collection period.
Analysis of rebamipide concentrations in the urine involved preparation of rebamipide stock solution, development of low and high linear standard calibration curves to compensate for altered rebamipide concentrations noted in urine samples, use of acetonitrile and hydrochloric acid for urinary rebamipide extraction, and HPLC analysis of rebamipide using a Shimadzu HPLC system.
HPLC analysis was carried out using a C 18 analytical column with a flow rate of 0.5 mL/min and a fluorescent detector set at an excitation wavelength of 320 nm with an emission wavelength of 380 nm.

Materials
Rebamipide powder (Tokyo Chemical Industry

Preparation of stock solutions
Rebamipide stock solution was prepared by dissolving 10 mg rebamipide powder in 200 mL methanol for a final concentration of 50,000 ng/mL. Ofloxacin was used as an internal standard (IS).
Ofloxacin stock solution was prepared by dissolving 100 mg ofloxacin powder in 200mL acetonitrile. Standard solution was then vortex mixed until fully dissolved for a final concentration of 500 ng/mL.

Development of calibration curves
To develop new standard calibration curves based on higher rebamipide stock concentrations, the following steps were followed: 1. Drug free rat urine (100 mL) was placed in a clean glass tube. 2. Samples were spiked with 100 mL IS.
8. Following evaporation, samples were reconstituted in 200 mL HPLC mobile phase. 9. Re-suspended solutions (115 mL) were transferred to clean injection vials and 100 mL was injected into HPLC for analysis. 10. The chromatogram was quantified based on area ratios of rebamipide to ofloxacin.

Sample extraction
Extraction of experimental urine samples was carried out similar to the method described for development of calibration curves. Briefly, 100 mL of urine from rats dosed with rebamipide was placed in clean glass tubes and spiked with 100 mL IS. Afterwards, 20 mL of 36% hydrochloric acid was added followed by 2 mL of acetonitrile. Solutions were vortex mixed for 1 min, then centrifuged for 20 min at 5000 Â g. After centrifugation, supernatant was transferred to clean glass tubes and evaporated to dryness then reconstituted in 200 mL mobile phase and placed in clean injection vials for HPLC analysis.

HPLC analysis
HPLC analysis was performed using the Shimadzu LC Solutions software package. Linearity was achieved for both low and high calibration curves by plotting peak area ratios between detected rebamipide and IS against known concentrations of rebamipide. Fig. 1A and B demonstrates the average of three measurements for each concentration. Coefficient of variation was calculated to be 18.9%. Fig. 2A shows the chromatogram of blank rat urine. Elution of IS only samples showed multiple peaks within 4 min time frame, however based on the sensitivity of fluorescence detection, identification of consistent peak areas was achieved at 4.2 min (Fig. 2B). Analysis of calibration samples containing known concentrations of rebamipide demonstrated sufficient detection of rebamipide with peak elution times of 9.1 min (Fig. 3A and B). Analysis of extracted experimental rat samples showed similar elution time for rebamipide at 9.1 min (Fig. 4). As such, rebamipide elution time was determined and concentrations in experimental urine samples were calculated.

Background information
Various prostaglandins function to regulate and control gut health and gastrointestinal (GI) integrity [6]. Rebamipide acts as a prostaglandin inducer and is often used for prevention and treatment of gastro-duodenal ulcers [1]. As such, studies have been performed looking at rebamipide pharmacokinetics in conjunction with known GI irritants [3,7,8]. These studies often utilize human or animal plasma samples for rebamipide detection and analysis under chromatographic conditions [2,4,9]. Recently, we reported on the pharmacokinetic interactions of rebamipide with select nonsteroidal anti-inflammatory drugs known to promote adverse GI side effects [3]. Our study utilized a previously developed method to determine plasma concentrations of rebamipide in experimental plasma samples [4]. Elucidation of rebamipide elimination processes involved the analysis of urinary concentrations of rebamipide in various treatment samples. Through the modification of methods previously developed for plasma examination of rebamipide we were able to successfully develop a simplified protocol for the analysis of urinary concentrations of rebamipide.