The Determination and Control of Genotoxic Impurities in APIs - Pharmaceutical Technology

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The Determination and Control of Genotoxic Impurities in APIs
The authors provide an overview of methods for the quantitative determination of genotoxic impurities (GTIs) in active pharmaceutical ingredients.


Pharmaceutical Technology
Volume 35, pp. s24-s30

Method development and validation

The analytical technique developed to determine GTIs should take into account origin, control, and suitability at detection limits. The authors developed methods to identify and quantify the genotoxic impurities in APIs, including levetiracetam, salmeterol xinafoate, and montelukast sodium. Validation was performed to assess whether each method fulfilled its intended purpose. The parameters of specificity, detection limit, quantification limit, and accuracy were also evaluated.

Reagents. Methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and methyl p-toluenesulfonate (MTS) were purchased from Sigma–Aldrich. Dimethyl sulfoxide, methanol, and acetonitrile were supplied by TCI American. All water used in the experiment was purified by an in-house Milli-Q system (Millipore). All drug substances used for validation and testing were obtained from current projects at Neuland Laboratories and prepared in house.

Instrumentation. A Shimadzu GC-2010 with headspace (HT3, Teledyne Tekmar) and autosampler (AOC-20i, Shimadzu), Perkin Elmer Clarus 600 gas chromatograph with headspace (Turbo matrix 40, Perkin Elmer) and Perkin Elmer Clarus 500 gas chromatograph with autosampler were used to determine the presence of selected GTIs in selected APIs.

Methods for determining GTIs in levetiracetam. Based on the route of synthesis for levetiracetam, the expected GTIs were 4-chloro butyryl chloride, methyl 2-bromobutyrate, and 2-bromo butyric acid. An analytical technique must determine these GTIs at 0.5 ppm. These GTIs are rare in APIs.


Figure 2: 0.5 ppm level chromatogram of 4-chloro butylryl chloride.
4-chloro butyryl chloride. 4-chloro butyryl chloride is a clear, colorless to yellow volatile liquid with a boiling point of 174 C and a density of 1.26 g/mL. Based on the properties of the analyte, wall-coated capillary columns of various brands, phases, and dimensions were investigated. A nonpolar HP-005 column (30 m length, 0.53 mm i.d.) with a stationary phase of 5% phenyl dimethyl polysiloxane film of 5.0 μm is suitable for the determination of 4-chloro butyryl chloride at the 0.5 ppm level (see Figure 2). The column fulfills all the requirements of the method (i.e., high sensitivity and short run time). A Perkin Elmer Clarus 600 headspace auto sampler was used for method development and validation. Oven temperature was maintained at 60 C for 5 min, and a linear thermal gradient of 10 C/min to 240 C was used with a final hold of 1 min with 150 C injector temperature and 250 C detector temperature. Helium was used as a carrier gas at constant pressure flow rate of 3.0 psi. Operation mode was splitless. Thermostat time was 20 min at 80 C.

A stock solution of 4-chloro butyryl chloride in dimethyl sulfoxide was prepared and injected. Retention time was 8.289 min with good response. The limit of detection (LOD) and limit of quantification (LOQ) were 0.0494 ppm and 0.163 ppm, respectively. The method gave excellent precision and accuracy, even at the LOQ level.


Figure 3: 0.5 ppm level chromatogram of methyl 2-bromo butyrate.
Methyl 2-bromobutyrate. Methyl 2-bromo butyrate is a brownish liquid with a boiling point of 138 C and density of 1.573 g/mL. A polar DB-FFAP column (30 m length, 0.53 mm i.d.) with a stationary phase of acid-modified polyethylene glycol film of 1.0 μm is suitable for the determination of methyl 2-bromo butyrate at the 0.5-ppm level (see Figure 3). A Shimadzu-2010 with autosampler was used for the method development and validation. Oven temperature was maintained at 80 C for 5 min, and a linear thermal gradient of 5 C/min to 150 C was used with a final hold of 1 min at 170 C injector temperature and 250 C detector temperature. Helium was used as a carrier gas at constant pressure flow rate of 3.0 psi. Operation mode was split (2:1), and injection volume was 2.0 μL.

A stock solution of methyl 2-bromo butyrate in methanol was prepared and injected. Retention time was 12.223 min with good response. LOD and LOQ were 0.047 ppm and 0.156 ppm, respectively. The method gave good precision and accuracy even at the LOQ level.


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