Identification and characterization of stressed degradation products of metoprolol using LC/Q-TOF-ESI-MS/MS and MS n experiments Roshan M. Borkar a,b , B. Raju a , R. Srinivas a,b *, Prashant Patel b and Satheesh Kumar Shetty c ABSTRACT: A rapid, specific and reliable isocratic high-performance liquid chromatography combined with quadrupole time-of- flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) method has been developed and validated for the identification and characterization of stressed degradation products of metoprolol. Metoprolol, an anti-hypertensive drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per ICH-specified condi- tions. The drug showed extensive degradation under oxidative and hydrolysis (acid and base) stress conditions. However, it was stable to thermal, neutral and photolysis stress conditions. A total of 14 degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on a C 18 column (4.6  250 mm, 5 mm). To characterize degradation products, initially the mass spectral fragmentation pathway of the drug was established with the help of MS/MS, MS n and accurate mass measurements. Similarly, fragmentation pattern and accurate masses of the degradation products were established by subjecting them to LC-MS/QTOF analysis. Structure elucidation of degradation products was achieved by comparing their fragmentation pattern with that of the drug. The degradation products DP 2 (m/z 153) and DP 14 (m/z 236) were matched with impurity B, listed in European Pharmacopoeia and British Pharmacopoeia, and impurity I, respec- tively. The LC-MS method was validated with respect to specificity, linearity, accuracy and precision. Copyright © 2011 John Wiley & Sons, Ltd. Keywords: metoprolol; LC-ESI-MS/MS; degradation products; accurate mass measurements Introduction Metoprolol belongs to the class of selective b 1 blocker receptors used in the treatment of several cardiovascular diseases, especially hypertension. It has little or no effect on b 2 blocker receptors except in high doses. Treatment of heart failure by b-adrenergic blocking agent has been intensely investigated (Swedberg et al., 1979). Chemically, metoprolol (Scheme 1) is 1-(iso-propylamino)-3-[4′ (2-methoxyethyl) phenoxy]-2-propanol. In the literature, many LC and LC-MS methods have been reported for the analysis of drug in biological fluids and in the presence of other drugs (Balmér et al., 1987; Albers et al., 2005; Yilmaz et al., 2010; Baranowska and Wilczek, 2009). Although Jasińska et al. (2009) carried out stability studies on expired tablets, the study was limited to identification, characterization and the degradation pathway of the drug. The drug substance monograph on metoprolol in the European Pharmacopoeia (2005) and British Pharmacopoeia (2009) lists nine impurities (A–H and J). Of these, four are also mentioned as related substances in the drug monograph in the United States Pharmacopeia (2009). Impurity I is mentioned on the TLC pharmachem website (http://www.tlcpharmachem. com/tlc_item.php?upc=M-0812&li=&sub=). The main aim of the present study was to investigate the complete degradation behavior of the drug and to characterize the degradation products. This was done by exposing the drug to ICH-recommended stress conditions of hydrolysis, oxidation, thermal and photolysis. The resultant solutions were subjected to optimized LC-MS, MS/MS, MS n and accurate mass measurements to establish the fragmentation pattern of the drug and its degrada- tion products. Experimental Drug and reagents Pure metoprolol succinate was procured from USP India (P) limited, Hyderabad, India. HPLC-grade methanol and acetonitrile used in the * Correspondence to: R. Srinivas, National Centre for Mass Spectrometry, Indian Institute of Chemical Technology, Hyderabad, 500 607, India. E-mail: srini@iict.res.in a National Centre for Mass Spectrometry, Indian Institute of Chemical Technology, Hyderabad, 500 607, India b National Institute of Pharmaceutical Education and Research, Balanagar, Hyderabad, 500 037, India c United States Pharmacopeia–India Private Limited, Research and Development Laboratory, ICICI Knowledge Park, Turkapally, Shameerpet, Hyderabad, 500 078, India Abbreviations used: CID, collision induced dissociation; TOF, time-of-flight. Biomed. Chromatogr. 2012; 26: 720–736 Copyright © 2011 John Wiley & Sons, Ltd. Research article Received 29 June 2011, Accepted 5 September 2011 Published online in Wiley Online Library: 12 October 2011 (wileyonlinelibrary.com) DOI 10.1002/bmc.1721 720