Utilization of Photochemically Induced Fluorescence Detection for HPLC Determination of Genotoxic Impurities in the Vortioxetine Manufacturing Process

Journal of Chromatographic Science, 2016, Vol. 54, No. 9, 1625–1630 doi: 10.1093/chromsci/bmw116 Advance Access Publication Date: 1 July 2016 Article Article Utilization of Photochemically Induced Fluorescence Detection for HPLC Determination of Genotoxic Impurities in the Vortioxetine Manufacturing Process Michal Douša*, Jan Doubský, and Jan Srbek Zentiva, k.s. Praha, a Sanofi Company, U Kabelovny 130, 102 37 Prague 10, Czech Republic *Author to whom correspondence should be addressed. Email: Received 17 September 2015; Revised 21 March 2016 Abstract An analytical reversed-phase high-performance liquid chromatography (HPLC) method for the detection and quantitative determination of two genotoxic impurities at ppm level present in the vortioxetine manufacturing process is described. Applying the concept of threshold of toxicological concern, a limit of 75 ppm each for both genotoxic impurities was calculated based on the maximum daily dose of active pharmaceutical ingredients. The novel reversed-phase HPLC method with photochemically induced fluorescence detection was developed on XSELECT Charged Surface Hybrid Phenyl-Hexyl column using the mobile phase consisted a mixture of 10 mM ammonium formate pH 3.0 and acetonitrile. The elution was performed using an isocratic composition of 48:52 (v/v) at a flow rate of 1.0 mL/min. The photochemically induced fluorescence detection is based on the use of UV irradiation at 254 nm through measuring the fluorescence intensity at 300 nm and an excitation wavelength of 272 nm to produce fluorescent derivatives of both genotoxic impurities. The online photochemical conversion and detection is easily accomplished for two expected genotoxic impurities and provides a sufficiently low limit detection and quantification for the target analysis. Introduction Vortioxetine (VOR) (1-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]piperazine)) is a novel investigational antidepressant with multimodal activity. It has high affinity for the 5-HT transporter (5-HTT) and moderate affinity for the 5-HT1A receptor in vitro (1–5). Recently, a new synthetic route to VOR starting from commercially available thiol (Figure 1(I)) and 1-chloro-2-nitrobenzene (Figure 1(II)) was developed. The VOR framework is built stepwise employing (i) an aromatic nucleophilic substitution, and (ii) a construction of piperazine moiety using bis(2-chloroethyl)amine hydrochloride (BCEA) as a building block (4–7). Thus, the chlorine atom of the derivative (Figure 1(II)), located in the ortho-position to a strong electron-withdrawing nitro-group, was substituted smoothly with a thiolate-anion generated in situ from the compound (Figure 1(I)) in the presence of sodium hydroxide (NaOH). This reaction afforded the desired nitrosulfide (Figure 1(III)) in almost quantitative yield. Subsequently, the nitro-derivative (Figure 1(III)) was hydrogenated to provide the corresponding aminosulfide (Figure 1(IV)) in an excellent yield under conditions avoiding any undesirable sidereactions (for instance, a desulfurization). In the last step, the aminosulfide (Figure 1(IV)) underwent a cyclization reaction with BCEA in the presence of 0.75 equiv. of sodium iodide (NaI) in refluxing toluene to provide highly pure VOR. The European Medicines Agency (EMA) guideline on the limits of the genotoxic impurities (GTIs) (8) has recommended the use a threshold of toxicological concern (TTC) concept for evaluation of carcinogenicity risk. A TTC was originally developed at the Food and Drug Administration (FDA) for food-contact materials (9). A TTC allows a maximum intake of 1.5 µg/day of any one genotoxic impurity over a patient’s lifetime (9). The concentration limit of GTIs in drug substances and drug products can be derived based on the maximum daily dose and the TTC concept as follows (8): concentration limit ( ppm) = [1.5 µg/day]/[dose (g/day)]. While ICH Q3 guidelines provide © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: 1625 1626 Douša et al. Figure 1. Reaction scheme of VOR synthesis. guidance for qualification and control for the majority of the impurities, ICH M7 guidance is provided for those impurities that are DNA reactive (10). The second possible approach is to calculate a compoundspecific acceptable intake based on rodent carcinogenicity potency data such as TD50 value. This procedure is similar to that employed for derivation of the TTC (10) and provides very similar results. Analytical detection of GTIs at the ppm level is not routine procedure. Traditional pharmaceutical analysis typically deals with impurities at levels above 0.05% (equivalent to 500 ppm), where conventional analytical instrumentation is adequate, such as HPLC with UV detection (11). HPLC or UHPLC coupled with tandem mass spectrometry (MS-MS) or fluorescence detector has become the method of choice, as demonstrated in the recent literature (12–15). The involvement of post-column photochemical reactions in quantitative analyses may be categorized as a form of derivatization. In comparison to chemical derivatization, post-column photochemical reactions offer several advantages, which also function to simplify the adaptation of photochemical reactors into the chromatographic system (16, 17). The fundamental purpose of incorporating post-column photochemical reactors into a method of detection is to convert the starting analyte to a product, which have to increase the sensitivity and/or selectivity of response of fluorescence (18), ultraviolet (19), electrochemical (20) and chemiluminescence detectors (21). Some HPLC methods following on-line post-column photochemical derivatization were applied to the quality control of pharmaceutical drugs and preparations (22–24). The method of hydrophilic interaction liquid chromatography coupled with mass spectrometry detection (HILIC-MS) was developed and validated for determination of BCEA in VOR (25). The other GTIs in VOR and their methods of determination have not yet been described. The aminosulfide and nitrosulfide (Figure 1) impurities as potential GTIs were predicted to be mutagenic by DEREK Nexus version 2.0 and subsequently confirmed by Ames bacterial mutagenicity test (26). For VOR with a maximum dose of 20 mg/day maximum concentration limit of GTIs = [(1.5 µg/day)/(0.02 g/day)] is 75 ppm in the drug substance. Since aminosulfide and nitrosulfide impurities were confirmed as genotoxic, the amount of these analytes has to be controlled rigorously in the final drug. The main objective of the current paper is to demonstrate the possibility of using reversed-phase HPLC (RP-HPLC) method with photochemically induced fluorescence (PIF) detection for the analytical control of GTIs in the manufacturing process of VOR. Experimental Reagents and chemicals Acetonitrile HPLC gradient grade and methanol HPLC gradient grade (J.T. Baker, USA) and water purified by Milli-Q system (Merck/Millipore, Czech Republic) were used for preparation (...truncated)