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.
Highlights
Isosorbide-5-mononitrate (5-ISMN) is an organic nitrate vasodilator and one of the long-acting metabolites of isosorbide dinitrate [1]. 5-ISMN is orally administered with an elimination half-life of 5 hours and is not subject to first-pass metabolism. 5-ISMN enters the systemic circulation together in the form of the original drug with high bioavailability [2]. 5-ISMN has been extensively used for cardiovascular diseases such as coronary heart disease and angina pectoris to prevent or at least reduce the occurrence of angina for many years [3], and it has been developed into various formulations such as tablets, immediate-release formulations, and sustained-release formulations [4]
Journal of Analytical Methods in Chemistry mL [12]), requiring a large amount of plasma samples (0.2–0.5 mL) [11, 12] or reagents for sample preparation (34 mL) [9, 11], or requiring relatively expensive solid-phase extraction (SPE) accompanied with an Lower limit of quantification (LLOQ) of 9.02 ng/mL [10], which may result in limited application to clinical studies. e mostly reported LC-MS methods to determine 5-ISMN showed relatively low sensitivity [9,10,11,12,13] except for the report by Sun et al [9]
All the reported LC-MS methods did not consider the precision and accuracy of hyperlipidemia/ hemolysis and the stability of whole blood, which may be important in the study of 5-ISMN in patients or in BE studies containing fed condition. ese reliable indicators were performed in our study. e analytical method was abundantly validated and proved to be specific and reproducible, and it was successfully applied to determine the drug concentration of 5-ISMN in the bioequivalence clinical trial of oral 20 mg 5-ISMN tablets in healthy Chinese subjects
Summary
Isosorbide-5-mononitrate (5-ISMN) is an organic nitrate vasodilator and one of the long-acting metabolites of isosorbide dinitrate [1]. 5-ISMN is orally administered with an elimination half-life of 5 hours and is not subject to first-pass metabolism. 5-ISMN enters the systemic circulation together in the form of the original drug with high bioavailability [2]. 5-ISMN has been extensively used for cardiovascular diseases such as coronary heart disease and angina pectoris to prevent or at least reduce the occurrence of angina for many years [3], and it has been developed into various formulations such as tablets, immediate-release formulations, and sustained-release formulations [4]. 5-ISMN has been extensively used for cardiovascular diseases such as coronary heart disease and angina pectoris to prevent or at least reduce the occurrence of angina for many years [3], and it has been developed into various formulations such as tablets, immediate-release formulations, and sustained-release formulations [4] Published analytical methods such as gas chromatography-mass spectrometry (GC-MS) [5,6,7,8] and liquid chromatographytandem mass spectrometry (LC-MS/MS) [9,10,11,12,13] have been established for the identification or quantification of parent isosorbide dinitrate (ISDN) and its two active metabolites isosorbide-2-dinitrate (2-ISMN) or isosorbide-5-monoitrate (5-ISMN) in previous years. All the reported LC-MS methods did not consider the precision and accuracy of hyperlipidemia/ hemolysis and the stability of whole blood, which may be important in the study of 5-ISMN in patients or in BE studies containing fed condition. ese reliable indicators were performed in our study. e analytical method was abundantly validated and proved to be specific and reproducible, and it was successfully applied to determine the drug concentration of 5-ISMN in the bioequivalence clinical trial of oral 20 mg 5-ISMN tablets in healthy Chinese subjects
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