Development and validation of a novel UPLC-MS/MS method for quantification of spironolactone using deuterated internal standard in spiked human plasma Krishnaveni Nagappan 1 * , Bhadram Kalyan Chekraverthy 1 , Hemkumar B 1 , Karthik Rajendran 1 , Sankham Devendran Rajendran 2 1 JSS College of Pharmacy, JSS Academy of Higher Education & Research, India, 2 Scitus Pharma Services Private Limited, Chennai, Tamilnadu Objective: Quantification of spironolactone in clinical pharmacological practise using deuterated internal standard method requires an efficient, sensitive and optimized UPLC-MS/MS based bioanalytical method. Method: A reverse phase UPLC analysis was performed using Zorbax SB C18, 4.6 x 75 mm, 3.5 µm, column as stationary and isocratic delivery of mobile phase (A & B) i.e., A is Methanol and B is10mM Ammonium formate (70:30, v/v) with a flow rate of 0.3 mL/min. A volume of 10 µL was set as injection volume. The mass spectrometric detection was performed using electrospray ionization in positive ion mode as interface, MRM as mode of acquisition. Liquid Liquid extraction technique was used for the extraction of the analytes from the spiked human plasma. Results: The retention of Spironolactone (SL) and Spironolactone (SL-D7) were found to be 6.19 and 6.10 minutes respectively. The selected mass transition ions for analyte, its respective internal standards are as follows, the product ions(m/z) for SL were 187.01, 107.02, 96.92 for SL-d7 189.08, 107.09, 100.16 and the precursor ions (m/z) for SL, SL-d7 were 341.16 and 348.21 respectively. The linearity range was 1- 100 ng/mL for SL with a 0.01 ml sample volume and a runtime of 6.75 min. The mean recoveries of the analyte were ranged between 69 and 72 %. Conclusion: This method fulfilled the validation in accordance with USFDA’s bioanalytical guidelines and could be employed in human plasma assay for pharmacokinetic analysis References 1. Ram, V. R., Dave, P. N. & Joshi, H. S. Development and Validation of a Stability-Indicating HPLC Assay Method for Simultaneous Determination of Spironolactone and Furosemide in Tablet Formulation. Chromatogr. Sci. 50 , 721–726 (2012). 2. Alexander, K. S., Vangala, S. S. K. S. & Dollimore, D. An Improved High-Performance Liquid Chromatography Assay for Spironolactone Analysis. Drug Dev. Ind. Pharm. 24 , 101–107 (1998). 3. Kaukonen, A. M., Vuorela, P., Vuorela, H. & Mannermaa, J.-P. High-performance liquid chromatography methods for the separation and quantitation of spironolactone and its degradation products in aqueous formulations and of its metabolites in rat serum. Chromatogr. A 797 , 271–281 (1998). 4. Ferreira, A. et al. Liquid chromatographic assay based on microextraction by packed sorbent for therapeutic drug monitoring of carbamazepine, lamotrigine, oxcarbazepine, phenobarbital, phenytoin and the active metabolites carbamazepine-10,11- epoxide and licarbazepine. Chromatogr. B 971 , 20–29 (2014). 5. Varin, F. High-performance liquid chromatographic determination of spironolactone and its metabolites in human biological fluids after solid-phase extraction. J Chromatogr. 574 (1), 57-64 (1992). 6. Jankowski, A., Skorek-Jankowska, A. & Lamparczyk, H. Simultaneous determination of spironolactone and its metabolites in human plasma. Pharm. Biomed. Anal. 14 , 1359–1365 (1996).
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