Trace level determination of residues of dimethyl sulfate and diethyl sulfate in pharmaceutical products by Headspace Gas Chromatography and Mass Spectrometry following derivatization

The synthesis of pharmaceutical products often involves the use of reactive reagents for the formation of intermediates and active pharmaceutical ingredient (API). Low-levels of reagents or byproducts may therefore be present in the final API as impurities. Such chemically reactive impurities may have unwanted toxicities such as genotoxicity and carcinogenicity, which have the potential impact on product quality and therefore their risk profile requires consideration and management. Alkylating agents (AAs) are classified as potentially genotoxic impurities (PGI) that require a control strategy in pharmaceutical process development and may require control in API during manufacture and storage. AAs may be used as reagents during manufacturing or can be products of side reactions, when strong acids (e.g. H2SO4) react with alcohols (used as solvents or present as impurities) to form the corresponding ester. AAs are reactive compounds that have a broad range of physico-chemical properties which requires specific analytical methodologies for the determination of each species in drug substances or intermediate stage products. The work presented in this exxperiment describes the development of a systematic approach for the detection and quantification of dimethyl sulfate (DMS) and diethyl sulfate GCMS-PHARMA Author Dr. Padmaja Prabhu Application Specialist – Chromatography Customer Knowledge Centre for Analytical Sciences PerkinElmer (India) Pvt. Ltd. Th ane – 400 615 India. Email: [email protected] (DES) in APIs to provide an insight to these residues in APIs. A static headspace Gas Chromatography (HS-GC) with Electron Impact ionization (EI) mass spectrometry (MS) method based on pentafluorobenzenethiol (PFBT) derivatization employing SIFI i.e. both scan and selected-ion monitoring (SIM) simultaneously has been developed. This developed method is applied successfully to determine the presence of residual dimethyl sulfate and diethyl sulfate in APIs. Both DMS and DES are not stable therefore this method is designed to detect their volatile derivatives which are more stable and can be determined easily by headspace. The resultant products are more volatile, stable, less polar and easier to extract than the parent compounds. The derivatives are therefore more amenable to GC analysis and also contain groups that significantly enhance detection. Headspace sample preparation is very easy and the derivatization step further adds specificity to the method, which could help in making this method generic for the control of residues of alkylating agents in pharmaceutical products. Stock Solution: Hundred milligrams of each of dimethyl sulfate and diethyl sulfate were weighed into the same 100 mL flask and diluted to volume with methyl tertiary butyl ether to make 1000 μg/mL mixture of dimethyl sulfate and diethyl sulfate. Solution A: Ten milliliters of the stock solution were diluted to 100 mL with methyl tertiary butyl ether to result in a 10 μg/mL mixture. PFBT solution: Accurately weighed 0.62 g of PFBT was dispersed in 100 mL of 1M sodium hydroxide and 2 mL of this was added to each of the HS vials for derivatizing dimethyl sulfate and diethyl sulfate in samples and standard. Calibration curve: One milliliter of dimethyl sulphoxide was taken in 6 headspace vials. Varying volumes of 10 μg/ mL mix of dimethyl sulfate and diethyl sulfate (solution A) was spiked to achieve the concentrations ranging from 50 ppb to 1.5 ppm (0.5 to 15 ppm w.r.t sample). Two milliliters of PFBT solution was added to each of the vials for derivatisation and the vial s were immediately sealed. The vials were equilibrated for 15 minutes and the headspace components were injected in to the gas chromatograph Sample introduction: Samples including: two pharmaceutical APIs were obtained from a local pharmaceutical firm. Headspace sample preparation was very simple. Accurately weighed samples (0.1 g ) were placed in headspace vials. One milliliter of DMSO was added to each of the vials followed by two milliliter of PFBT solution and sealed immediately. The vials were incubated at the headspace conditions and analysed. All the samples were freshly prepared and analyzed immediately. Instrumental conditions ClarusTM 600 Gas Chromatograph instrument details Analytical Column PerkinElmer Elite -624 (60 meter, 0.32 mm i.d., 1.8 μm df) GC column Flow 2.0 mL/min Helium at constant fl ow mode GC inlet temperature 220 oC Split ratio Splitless Oven temperature programme 60 oC hold for 1.0 min, 50 oC/min to 250 oC and hold for 15.0 min, runtime is 20 minutes. ClarusTM 600 C mass spectrometer MS parameters Source temperature 200 oC Interface temperature 200 oC Scan range m/z 55-300 SIM mode: ions monitored 214, 228 Scan time 4.7-7.5 min