Determination of Isosorbide-5-Mononitrate in Human Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Its Application to a Bioequivalence Study

Abstract

Isosorbide-5-mononitrate (5-ISMN), an organic nitrate vasodilator, has been widely used worldwide to prevent angina pectoris for more than two decades. A simple and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the determination of 5-ISMN in human plasma. 13C6-5-ISMN is an internal standard, and 5-ISMN was extracted from human plasma (50 µL) with ethyl acetate (200 µL) by a simple liquid-liquid extraction method. The chromatographic separation was carried out on LC-20A (Shimadzu, Japan) using an analytical column ZORBAX XDB-C18 (4.6 × 50 mm, 5 µm), coupled with API 4000 tandem mass spectrometers in a multiple reaction monitoring (MRM) mode. The mobile phase was composed of acetonitrile (organic phase A) and 2 mM ammonium acetate in water (aqueous phase B) with an isocratic elution of A/B = 90 : 10 (v/v). The total run time was 3.5 min with a small injection volume (5 µL). This method was fully validated in every aspect of selectivity, linearity, accuracy, precision, matrix effect, extraction recovery, and different stabilities. It was proved that the calibration standards within the 5.00–1000 ng/mL concentration range were linear. The lower limit of quantification was 5.00 ng/mL for 5-ISMN. The intrabatch and interbatch accuracy (RE) ranged from −8.8% to 7.1% with precision between 2.4% and 6.6%. The mean values of 5-ISMN extraction recovery and matrix effect were 87.0% and 102.0%, respectively. The fully validated method was successfully applied for a bioequivalence clinical trial of oral 20 mg 5-ISMN tablets in healthy Chinese subjects.

What is the role of current mass spectrometry in pharmaceutical analysis?

“…The highly sensitive detection has been recently demonstrated by UHPLC/HPLC‐QqQ methods used for the bioequivalence study of dabigatran, which is the active metabolite of the prodrug dabigatran etexilate mesylate (Abd Allah et al, 2022), triamterene and hydrochlorothiazide (Margaryan et al, 2019), sumatriptan (Wichitnithad et al, 2020), and beraprost sodium (Prasaja et al, 2019). MS is also needed when the API does not contain chromophores for UV‐VIS detection, for example, bioequivalence study of isosorbide‐5‐mononitrate (Zhou et al, 2020) or the total amount of the drug in plasma and the unbound fractions are required, for example, bioequivalence study of paclitaxel protein‐bound particles for injectable suspension (Li et al, 2019).…”

Eco-friendly estimation of isosorbide dinitrate and hydralazine hydrochloride using Green Analytical Quality by Design-based UPLC Method

“…Several methods are available to analyze these drugs individually and in combined dosage form using UV, 17 HPLC, 18–22 and LCMS. 23 However, so far, no single method was developed using GAC principles or using a robust AQbD 24 -based UPLC method.…”