Zhou Yinping, Liu Aijing, Jia Ranran, Wu Mengyi, Wu Ni, Liu Chunyan, Han Zhihui, Hu Haitang, Wang Hongyun, He Qing
Clinical Pharmacology Research Center, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing Key Laboratory of Clinical PK and PD Investigation for Innovative Drugs, Beijing 100730, China.
Livzon Group Medical Clinical Research Center, Zhuhai 519020, China.
J Anal Methods Chem. 2020 Jul 17;2020:1753265. doi: 10.1155/2020/1753265. eCollection 2020.
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. C-5-ISMN is an internal standard, and 5-ISMN was extracted from human plasma (50 L) with ethyl acetate (200 L) by a simple liquid-liquid extraction method. The chromatographic separation was carried out on LC-20A (Shimadzu, Japan) using an analytical column ZORBAX XDB-C (4.6 × 50 mm, 5 m), 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 (/). The total run time was 3.5 min with a small injection volume (5 L). 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.
单硝酸异山梨酯（5-ISMN）是一种有机硝酸酯类血管扩张剂，二十多年来在全球范围内被广泛用于预防心绞痛。本文建立并验证了一种简单、灵敏的高效液相色谱-串联质谱法（HPLC-MS/MS），用于测定人血浆中的5-ISMN。C-5-ISMN为内标，采用简单的液-液萃取法，用乙酸乙酯（200μL）从人血浆（50μL）中萃取5-ISMN。色谱分离在LC-20A（日本岛津公司）上进行，使用ZORBAX XDB-C分析柱（4.6×50mm，5μm），与API 4000串联质谱仪联用，采用多反应监测（MRM）模式。流动相由乙腈（有机相A）和2mM乙酸铵水溶液（水相B）组成，A/B = 90∶10（v/v）等度洗脱。总运行时间为3.5分钟，进样量小（5μL）。该方法在选择性、线性、准确度、精密度、基质效应、萃取回收率和不同稳定性等方面均得到充分验证。结果表明，5.00 - 1000 ng/mL浓度范围内的校准标准曲线呈线性。5-ISMN的定量下限为5.00 ng/mL。批内和批间准确度（RE）范围为-8.8%至7.1%，精密度在2.4%至6.6%之间。5-ISMN萃取回收率和基质效应的平均值分别为87.0%和102.0%。该充分验证的方法成功应用于健康中国受试者口服20mg 5-ISMN片的生物等效性临床试验。
