Determination of Regional Intestinal Permeability of Diclofenac and Metoprolol Using a Newly-Developed and Validated High Performance Liquid Chromatographic Method

Purpose: To develop a simple and rapid reversed-phase high performance liquid chromatographic (HPLC) method with UV detection for the simultaneous determination of diclofenac, metoprolol tartrate, phenol red and propyl paraben in intestinal segments.
Methods: The mobile phase consisted of 55 % methanol, 45 % of 12.5 mM potassium dihydrogen phosphate (KH₂PO₄) aqueous solution, adjusted to pH 7.0 using 0.2 N sodium hydroxide (NaOH) solution, and to which 0.3 % (v/v) triethylamine was added. Analysis was run at a flow rate of 1.0 mL/min with a 12 min total run time at ambient temperature. The developed method was successfully applied to determination of the analytes in samples obtained from in situ single pass intestinal perfusion (SPIP) studies.
Results: The calibration curves were linear for all compounds (r > 0.999) with a limit of detection (LOD) of 0.005, 0.1, 0.075 μg/mL, and limit of quantification of 0.1, 0.3, 0.2 μg/mL for metoprolol tartrate, phenol red and diclofenac respectively. The coefficient of variation for intra-day and inter-day precision was < 5 % and accuracy was between 98 and 102 %. Based on SPIP and HPLC studies, the estimated mean permeability in jejunum, ileum and colon was 0.319 ± 0.184, 0.639 ± 0.241 and 0.84 3± 0.517 x 10^-4 cm/sec, respectively, for metoprolol tartrate while the corresponding permeability values were 1.585 ± 0.729, 1.154 ± 0.433 and 1.775 ± 1.576 x 10^-4 cm/sec for diclofenac.
Conclusion: The findings indicate that diclofenac is a highly permeable compound and its absorption °Ccurs throughout the intestinal tract. Furthermore, the developed method is suitable for the analysis of diclofenac and metoprolol in intestinal regions.

Keywords
Biopharmaceutics classification system; Diclofenac; Metoprolol tartrate; Segmental permeability; Intestinal absorption; Validation