A sensitive and fast HPLC method for determination of Isosorbide 5-mononitrate in human plasma using ion-pair chromatography: application to bioequivalence study

Abstract

Isosorbide 5-mononitrate (5-ISMN) is a charged, hydrophilic, and low ultraviolet-absorbing molecule which is challenging to determine specifically and sensitively in biological matrices. A sensitive and fast ion-exchange chromatography method for determining 5-ISMN in plasma has been developed. The plasma was deproteinized by one-step clean up procedure using sodium chloride and methanol. Then supernatant was injected into a Chromolith C18 column (4 µm, 3.9 × 150 mm). The mobile phase was made of acetate buffer (30 mM, pH: 4.7): methanol, 77:23 (v/v) and 1 mM tetrabutylammonium hydroxide with a flow rate of 1.2 mL/min. Ultraviolet detection was done at 225 nm. The established method was fully validated and successfully applied for pharmacokinetic and bioequivalence study of two different sustained release formulations of 5-ISMN as Elantan LA (reference product) and Mononitral (test product) in twelve healthy male volunteers. A single dose, randomized, two-period, and crossover study was performed under fasting condition. Isosorbide dinitrate was used as internal standard. 5-ISMN and internal standard were eluted at 2.6 and 3.8 min, respectively. Calibration curve was linear within the concentration range of 10–800 ng/mL for 5-ISMN in plasma. The results revealed that the precision of the method ranged from 2 to 5 and relative errors lied within 0.43–4.2% regardless of whether the analysis was performed intraday or interday. By this method, the limits of quantitation and detection of 10 ng/mL and 5 ng /mL were achieved by a sample size of 430 µL plasma. Main pharmacokinetic parameters for test and reference products were 494 ± 210.2 and 500 ± 202.5 for Cmax and 6359 ± 1235 and 6995 ± 1371 for AUC0-36, respectively. The 90% confidence intervals around ratios (test/reference) from logarithmic transformed exposure values were 99–110 for Cmax and 88–105 for AUC 0–36. It can be concluded that both products are bioequivalent. In vitro dissolution test was also carried out together with bioequivalence study and in vitro/in vivo correlation was successfully established.