Recent Control and Testing Strategies for Genotoxic Impurities

A short review of Recent Control and Testing Strategies for Genotoxic impurities

Analytical Method development and routine QC analysis for the determination of Genotoxic impurities (GI) at trace levels present exceptional challenges to pharmaceutical development and QC laboratories. These impurities are required to be controlled at trace levels in new drug substances and drug products. The International Conference on Harmonization’s (ICH) M7 guideline provides recommendations for toxicology assessment, identification, categorization, and control of actual and potential mutagenic impurities that are likely to arise during the manufacturing and long-term storage of a new drug substance and drug product

Numerous separation and detection techniques are available which can be used to determine the levels present in given samples. Selecting the suitable analytical technique depends on the analyte (GI) physicochemical properties, desired sensitivity, as well as matrix interference, which need to be taken into account.

This short review focuses on the recent analytical development and control strategy using technologies in the analytical space for the determination of GTIs.

Gas chromatography /mass spectrometry

A sensitive method for alkyl sulfonate genotoxic impurities determination in drug substances using gas chromatography coupled to triple quadrupole mass spectrometry is developed by Liu. al

The following are commonly used sulfonate esters in drug substance synthesis. Methyl methanesulfonate, Ethyl methanesulfonate, Butyl methanesulfonate, Methyl ethane sulfonate, Ethyl ethanesulfonate, Isopropyl ethanesulfonate, methyl benzenesulfonate, Methyl p-toluenesulfonate , Ethyl p-toluenesulfonate  and Isopropyl methanesulfonate.

The method uses separation by gas chromatography applying stationary phase Agilent HP-5 MS ultra inert capillary column. Followed by a triple Quadrupole mass spectrometer with Electron Ionization (EI) ion source in MRM mode. The limit of quantitation (LOQ) for the nine commonly encountered sulfonate esters in drug substances was within 0.10–1.05 ng mL−1.  Satisfactory recoveries were within 75%˜120% of all the target analytes in API samples All alkyl sulfonates were stable in three diluents up to 36 h.

Uppala et al developed a method for the quantitate determination of hydroxylamine by derivatization with acetone in the presence of pyridine as a base in penicillamine drug substance by GCMS. Hydroxylamine was determined by using Gs BP-624, 30 mts × 0.32 mm × 1.8 µm column followed by employing MS conditions under selective reaction monitoring of m/z: 73 as iacetone oxime derivative at ppm level. The developed GCHS-MS method was validated and resulted to show the developed method is specific, sensitive, linear, accurate, and precise.

Ion Chromatography

Ion Chromatographic Method for Determination of Chloroacetic Acid in Isoproterenol Hydrochloride Drug Substance is developed recently by Rao et al.  An ion chromatograph (930 compacts IC Flex) with conductometric detector was utilized for the trace level determination while separation was achieved on  ASUPP 5 (6.1006.530), 250 mm X 4.0 mm stationary phase along with mobile phase was a mixture of 4 mM of sodium-bicarbonate and 0.5 mM of sodium carbonate. The sensitivity of the method was confirmed by plotting the linearity curve. The predicted LOQ and LOD levels for chloroacetic acid were 0.033 and 0.099 µg/mL, respectively.

Liquid chromatography Mass Spectrometry.

Wang et, al., reported the LC-MS/MS method for the analysis of two genotoxic impurity materials: N-(5-amino-2-methylphenyl)-4-(3-pyridyl)-2-aminopyrimidine (IMA) and N-(2-methyl-5-nitrophenyl)-4-(pyridine-3-yl) pyrimidine-2-amine (IMN).

The separation was performed on HSS T3 C18 (150 × 2.1 mm, 1.7 μm) maintained at 40 °C. The mobile phase consisted of 0.02 M ammonium formate buffer (pH 3.4) and acetonitrile (containing 0.05% formic acid) in gradient elution mode. Detection was achieved by triple quadrupole mass spectrometry fitted with an ESI probe functioning in the positive ion mode and the following m/z 278/106 and 308/262 were used as qualifier and quantifier transitions. The LOD of IMA in drug substances, tablets, and capsules was 0.0039, 0.0043, and 0.0044 ng mL−1, and the LOD of IMN was 0.0034, 0.0035, and 0.0036 ng mL−1.

Two potentially genotoxic impurities, namely, 2-chloromethyl-3,4-dimethoxy-pyridine hydrochloride (impurity A) and pantoprazole sulfone N-oxide (impurity B),  have been determined in Pantoprazole drug substance.  The sensitivity of the proposed method was in the range of 0.6–10.0 ng/mL. Good recoveries were observed in the range of 94.32%–107.43% with RSD values below 6.5%. This new and sensitive LC-MS/MS method with an adequate limit of quantification (LOQ) values was established and validated for the quantitative determination.

Controlling Genotoxic impurities in pharmaceutical compounds is of the utmost importance and is a requirement throughout the clinical development and commercial stage.

Depending on the structure and physicochemical of the analyte (GI) and the desired limit of detection, one can define the appropriate method.

For volatile impurities, headspace or direct injection GC-FID, or GC-MS can be investigated.  While polar and ionic impurities can be analyzed by ion chromatography. For non-volatile, RPLC-UV or RPLC-MS can be investigated


(1) 5.International Conference on Harmonisation. M7: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk. 2013.

(2) Journal of Pharmaceutical and Biomedical Analysis 168 (2019) 23–29

(3) Materials Today Proceedings (in press) online March 2020

(4) Asian Journal of Chemistry;   Vol. 29, No. 10 (2017), 2203-2206

(5) Chromatographia (online) 12 May 2020

(6) Journal of analytical methods in Chemistry Volume 2020 |Article ID 6597363

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