A Validated GC-MS Method for the Determination of Genotoxic Impurities in Divalproex Sodium Drug Substance

Journal of Chromatographic Science, 2019, Vol. 57, No. 2, 101–107 doi: 10.1093/chromsci/bmy089 Advance Access Publication Date: 4 October 2018 Article Article A Validated GC-MS Method for the Determination of Genotoxic Impurities in Divalproex Sodium Drug Substance S. Raghavender Reddy1,*, K. Hussain Reddy2, M. Narendra Kumar1, P. Madhava Reddy1, J. Venkata Ramana Reddy1, and Hemant Kumar Sharma1 1 Aurobindo Pharma Limited Research Centre-II, Survey No: 71&72, Indrakaran Village, Kandi Mandal, Sangareddy 502 329, Telangana, India and 2Department of Chemistry, Sri Krishnadevaraya University, Anantapur 515 003, Andhra Pradesh, India * Author to whom correspondence should be addressed. Email: Received 29 September 2017; Revised 13 August 2018; Editorial Decision 21 August 2018 Abstract A specific GC-MS method has been developed, optimized and validated for the determination of five genotoxic impurities namely Methyl bromide (Me.-Br), Ethyl bromide (Et.-Br), Isopropyl bromide (Ipr.-Br), n-Propyl bromide (n-Pr.-Br) and n-Butyl bromide (n-But.-Br) in Divalproex sodium (DPS) drug substance. Chromatographic separation of five genotoxic impurities was achieved on DB-1 column (30 m × 0.32 mm, 3.0 μm), consists of 100% dimethyl polysiloxane as stationary phase and passing helium carrier gas. The mass fragments (m/z) were selected for the quantification of Me.-Br (m/z 94), Et.-Br (m/z 108), Ipr.-Br (m/z 122), n-Pr.-Br (m/z 122) and n-But.-Br (m/z 136). Bromide ion (m/z 79) was the qualifier ion for the analytes [(Me.-Br), (Et.-Br), (Ipr.-Br), (n-Pr.-Br) and (n-But.-Br)]. The performance of the method was assessed by evaluating the specificity, linearity, sensitivity, precision and accuracy experiments. The established limit of detection and limit of quantification values for the genotoxic impurities were in the range of 0.005–0.019 μg mL−1. The correlation coefficient values of the linearity experiment were in the range of 0.9947–0.9983. The average recoveries for the accuracy were in the range of 97.6–111.3%. The results proved that the method is suitable for the determination of Me.-Br, Et.-Br, Ipr.-Br, n-Pr.-Br and n-But.-Br contents in divalproex sodium. Introduction Divalproex sodium (DPS) is a co-ordination complex of the sodium valproate and valproic acid (1). Chemically, it is known as sodium hydrogen bis (2-Propylpentanoate). The molecular formula of DPS is C16H31NaO4 and the molecular weight is 310.41. DPS is an anticonvulsant used to treat several types of seizure disorders, tonic-clonic simple/complex partial seizures (2). It is also used for prophylactic treatment for migraine in children (3), in adults with intellectual disability and aggressive behavior (4), patients with mood disorder (5), rapid-cycling bipolar disorder (6), antikindling agent, gamma-amino butyric acid enhancer and proposed for treating alcohol withdrawal (7). In the synthesis process of DPS drug substance, n-Propyl bromide (n-Pr.-Br) was used as a key raw material. The lower and higher homologous [Methyl bromide (Me.-Br), Ethyl bromide (Et.-Br), Isopropyl bromide (Ipr.-Br) and n-Butyl bromide (n-But.-Br)] of n-Propyl bromide may give corresponding potential impurities in DPS and this was reported in the European Pharmacopoeia (7.4) for Valproic acid. These brominated impurities (Me.-Br, Et.-Br, Ipr.-Br, n-Pr.-Br and n-But.-Br) are generally toxic, cancer suspect agents and come under genotoxic category. The European Agency for the Evaluation of Medicinal products (EMEA), United States Food and Drug Administration (USFDA) © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: 101 102 analytes [(Me.-Br), (Et.-Br), (Ipr.-Br), (n-Pr.-Br) and (n-But.-Br)]. Timed for MS Detector: The MS must be Dector Off after 15 min. Head space conditions (For GC sampler 80 model): Cycle = HSinjection; Syringe = 2.5 mL-HS; Incubation temperature = 60°C; Incubation time = 1200 s; Agitation speed = 500 rpm; Agitation on time = 10 s; Agitation off time = 10 s; Syringe temperature = 100°C; Fill speed = 1000 μL/s; Fill strokes = 0; Pullup delay = 500 ms; Inject to = GC-injection; Inject speed = 1000 μL/s; Pre inj. delay = 0 ms; Post inj. delay = 0 ms; Syringe flushing = 500 s; Analysis time = 2100 s. The samples of DPS drug substance and reference standards (Me.-Br, Et.-Br, Ipr.-Br, n-Pr.-Br and n-But.-Br) were procured from APL Research Centre-II (A division of Aurobindo Pharma Limited). Analytical reagents (AR grade) N-Methyl-2-pyrolidinone was obtained from Spectrochem limited (India) and HPLC grade water was procured from Merck limited. Method To the head space vial, added 0.80 mL of water and 0.20 mL of N-Methyl-2-pyrrolidinone (NMP) and sealed the vial immediately. This sealed vial was used as blank solution. A standard stock solution was prepared by weighing and diluting of Me.-Br, Et.-Br, Ipr.-Br, n-Pr.-Br and n-But.-Br reference standards with N-Methyl-2pyrrolidinone to get the concentration of 0.062 μg mL−1 for each of five impurities. To the head space vial, added 0.80 mL of water and 0.20 mL of standard stock solution and sealed the vial immediately. This sealed vial was used as standard solution. Further, 0.025 g of divalproex sodium sample was transferred into the head space vial and added 0.80 mL of water, 0.20 mL of N-Methyl-2-pyrrolidinone and sealed the vial immediately. This sealed vial was used for sample analysis. Warning: Brominated reference standards are “toxic”; they are generally considered cancer suspect agents. Hence, while handling Brominated reference standards use safety precautions: wear protective clothing, always wear personal protective equipment such as chemical splash goggles and safety gloves. Work in preferably in an environment with a fume extraction system. Results Instrumentation and reagents The analysis was carried on the Agilent GCMS-5977A gas chromatograph equipped with an AOC-5000 combipal auto sampler and a data handling system with HPCHEM solution software. Agilent J&W DB-1, (30 m × 0.32 mm, 3.0 μm) column consists of 100% dimethyl polysiloxane material as a stationary phase. High purity helium gas was used as the carrier gas with the column flow 1.2 mL/min. The initial column oven temperature of 40°C was maintained for 10 min and then increased to 220°C at the rate of 15°C/ min, followed by holding at 220°C for 3 min. The run time was 25 min. The injection volume was 1.0 mL with a split ratio of 3:1. The capillary injector temperature was 140°C, MS Parameters (For Agilent model No. 5977A): MS source = 230°C; MS quad = 150°C; MSD transfer line = 230°C; Detector voltage = Delta EMV. The analytes were monitored by gas chromatography electron ionization mass spectrometry (GC-EI-MS) with SIM mode. The mass fragments (m/z) were selected for the quantification of Me.-Br (m/z 94), Et.-Br (m/z 108), Ipr.-Br (m/z 122), n-Pr.-Br (m/z 122) and n-But.-Br (m/z 136). Bromide ion (m/z 79) was the qualifier ion for all the Met (...truncated)