Original research paper LC-MS/MS determination of 4-hydroxynimesulide, an active metabolite of nimesulide and application to bioequivalence study in Indian subjects Dhiman Halder 1 , Shubhasis Dan 1,2 , Pradipta Sarkar 1 , Dibya Das 2 , Umesh Chandra Halder 3 and Tapan Kumar Pal 1 Abstract 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 bioana- lytical 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 (75mm  4.6 mm, particle size 3.5 mm) 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 mL 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. Keywords Nimesulide and 4-hydroxy-nimesulide, LC-MS/MS, method validation, pharmacokinetics, bioequivalence study Received 20 August 2018; accepted 9 December 2018 Introduction Nimesulide (4-nitro-2-phenoxymethanesulfonanilide) is a nonsteroidal anti-inflammatory drug (NSAID) with a non-acidic property (pKa ¼ 6.5). 1,2 It is a selective inhibitor of cyclo-oxygenase-2 (COX-2) and effectively provides relief from a wide variety of pain (like anal- gesic and antipyretic) and inflammatory conditions (like anti-inflammatory). 3–6 Apart from this potent anti-inflammatory effect, it has been stated that long- term administration of NSD caused hepatotoxicity 7,8 and gastrointestinal toxicity 9 in human being. NSD has various mechanisms of action in its pharmaco- logical activities. It is a concern that the major mech- anisms are the selective inhibition of COX-2 with 5- to 16-fold selectivity for COX-2. COX-1 regulates gastric cytoprotection and vascular haemostasis in many tissue expressions. 10,11 Unwanted gastrointestinal and renal adverse effects are occurred due to the synthesis of cyto- protective compounds which are reduced by the inhib- ition of COX-1 like prostacyclin. Selective inhibition of COX-2 reduces the production of pro-inflammatory prostaglandins, and its beneficial effects in inflamma- tion and pain relief, with modest gastrointestinal (GI) toxicity. 12–14 Ex vivo determination in human whole blood after oral administration of 100 mg nimesulide (NSD) causes complete suppression of COX-2 activity and partial reduction in COX-1 activity. In vitro, in the bronchial tree during prostaglandin synthesis it does not cause any affects and in case of constitutive COX-1 which exerts a bronchoprotective role in the gastric mucosa, where COX-1 preserves mucosal 1 Department of Pharmaceutical Technology, Bioequivalence Study Centre, Jadavpur University, Kolkata, India 2 TAAB Biostudy Services, Kolkata, India 3 Department of Chemistry, Organic Chemistry Section, Jadavpur University, Kolkata, India Corresponding author: Tapan Kumar Pal, Department of Pharmaceutical Technology, Bioequivalence Study Centre, Jadavpur University, Kolkata 700 032, India. Email: tkpal@pharma.jdvu.ac.in European Journal of Mass Spectrometry 0(00) 1–13 ! The Author(s) 2019 Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/1469066718822621 journals.sagepub.com/home/ems