Abstract

LIQUID CHROMATOGRAPHY METHOD TO ANALYZE GENOTOXIC IMPURITY HYDROXYLAMINE IN PHARMACEUTICAL PRODUCT

Ajit Anerao*, Vikram Dighe, Anand Gupta and Vishal Solse

ABSTRACT

Hydroxylamine (NH2OH) is a known genotoxic impurity that typically needs to be controlled below threshold of toxicological concern in medicinal products. Hydroxylamine, however, is a challenging molecule to analyze using conventional analytical techniques due to its physical and chemical properties like lacking of chromophore, absence of carbon atom, low molecular weight, high polarity and volatility. The research work describes an accurate and sensitive reversed-phase liquid chromatography with derivatization for determination of trace amount of hydroxylamine in pharmaceutical materials. 1-Fluoro-2,4-dinitrobenzene was chosen as the derivatizing reagent and quantification was carried out with RP-HPLC method. Separation was achieved between the analyte and matrix peaks on Cosmosil MS-II, C-18 HPLC column and maximum response of the peak was obtained at UV 380 nm wavelength. The limit of detection of the derivative of hydroxylamine at the optimized wavelength was 0.56 ppm i.e. 0.56 μg/g and LOQ is 3.57 ppm i.e. 3.57 μg/g. The calibration curve was plotted within the concentration range between 3.57 ppm and 26.88 ppm. The regression coefficient R2 was 0.997 and the recoveries were obtained between 88.7 and 98.4%. The proposed method was validated and applied as a generic method to determine hydroxylamine for pharmaceutical process control and drug material release.

Keywords: Hydroxylamine; Genotoxic impurity; HPLC; Derivatization; Trace analysis.