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.
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.
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.
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.
Figure 2: 0.5 ppm level chromatogram of 4-chloro butylryl chloride.
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.
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.
Figure 3: 0.5 ppm level chromatogram of methyl 2-bromo butyrate.
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