A simple and highly sensitive bioanalytical method was developed and validated for simultaneous quantification of nimesulide (NSD) and its active metabolite 4-hydroxy-nimesulide (M1) in human plasma by liquid chromatography-tandem mass spectrometer (LC-MS/MS) and applied in a bioequivalence study performed on Indian subjects. The bioanalytical method was carried out by LC-MS/MS with celecoxib (CXB) as an internal standard (IS) using liquid–liquid extraction technique. The chromatographic separation was performed on a reversed-phase Agilent eclipse plus C18 (75 mm × 4.6 mm, particle size 3.5 µm) column with a mobile phase of acetronitrile and water containing 5 mM ammonium formate (9:1, v/v). Method validation and clinical sample were analysed as per USFDA and EMA guidelines and results met the acceptance criteria. The lower limit of quantitation of NSD and M1 was found 10 ng/mL with a large linearity range from 10 to 6000 ng/mL for both NSD and M1 using only 100 µL of plasma and reported no matrix effect. The multiple reaction monitoring transitions of m/z 307.20 → 229.20, m/z 323.00 → 245.00 and m/z 380.20 → 316.20 were used to measure NSD, M1 and CXB (IS), respectively. The assay method was successfully applied for the simultaneous quantification of both NSD and M1 in plasma samples after oral administration of nimesulide 100 mg tablet in healthy human subjects.

中文翻译：

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LC-MS/MS 测定 4-羟基尼美舒利，尼美舒利的活性代谢物及其在印度受试者的生物等效性研究中的应用

开发并验证了一种简单且高灵敏度的生物分析方法，可通过液相色谱-串联质谱仪 (LC-MS/MS) 同时定量人血浆中的尼美舒利 (NSD) 及其活性代谢物 4-羟基-尼美舒利 (M1) 并应用在对印度受试者进行的生物等效性研究中。生物分析方法采用液-液萃取技术，采用塞来昔布 (CXB) 作为内标 (IS)，通过 LC-MS/MS 进行。色谱分离在反相 Agilent eclipse plus C18（75 mm × 4.6 mm，粒径 3.5 µm）色谱柱上进行，流动相为乙腈和含有 5 mM 甲酸铵 (9:1, v/v) 的水。根据 USFDA 和 EMA 指南分析方法验证和临床样品，结果符合验收标准。发现 NSD 和 M1 的定量下限为 10 ng/mL，NSD 和 M1 的线性范围为 10 至 6000 ng/mL，仅使用 100 µL 血浆，并且报告没有基质效应。m/z 307.20 → 229.20、m/z 323.00 → 245.00 和 m/z 380.20 → 316.20 的多反应监测跃迁分别用于测量 NSD、M1 和 CXB (IS)。该测定方法成功应用于健康人类受试者口服尼美舒利 100 mg 片剂后血浆样品中 NSD 和 M1 的同时定量。M1 和 CXB (IS)，分别。该测定方法成功应用于健康人类受试者口服尼美舒利 100 mg 片剂后血浆样品中 NSD 和 M1 的同时定量。M1 和 CXB (IS)，分别。该测定方法成功应用于健康人类受试者口服尼美舒利 100 mg 片剂后血浆样品中 NSD 和 M1 的同时定量。