Determination of Regional Intestinal Permeability of Diclofenac and Metoprolol Using a Newly-Developed and Validated High Performance Liquid Chromatographic Method|
Kaynak, Mustafa Sinan; Buyuktuncel, Ebru; Caglar, Hatice & Sahin, Selma
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
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.
Biopharmaceutics classification system; Diclofenac; Metoprolol tartrate; Segmental permeability; Intestinal absorption; Validation