Main content area

Trace level liquid chromatography tandem mass spectrometry quantification of the mutagenic impurity 2-hydroxypyridine N-oxide as its dansyl derivative A

Ding, Wei, Huang, Yande, Miller, Scott A., Bolgar, Mark S.
Journal of chromatography 2015 v.1386 pp. 47-52
chlorides, derivatization, ingredients, ions, liquid chromatography, monitoring, mutagens, organochlorine compounds, tandem mass spectrometry
A derivatization LC–MS/MS method was developed and qualified for the trace level quantification of 2-hydroxypyridine N-oxide (HOPO). HOPO is a coupling reagent used in the syntheses of active pharmaceutical ingredients (APIs) to form amide bonds. HOPO was recently confirmed to generate a positive response in a GLP Ames bacterial-reverse-mutation test, classifying it as a mutagenic impurity and as such requiring its control in APIs to the threshold of toxicological concern (TTC). The derivatization reagent 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride) was used in a basic solution to convert HOPO into the corresponding dansyl-derivative. The derivative was separated from different APIs and reagents by liquid chromatography. The detection of the HOPO dansyl-derivative was achieved by mass spectrometry in selected reaction monitoring (SRM) mode. The LC–MS/MS method had a reporting limit of 0.1ng/mL HOPO, which corresponds to 0.1ppm HOPO relative to an API at 1mg/mL, and a linearity range of 0.1–25ng/mL HOPO analyte. Recoveries of HOPO standards spiked into three different API matrices at 0.2, 1.2, and 20ppm levels were all within 90–100%. An SRM-based confirmatory methodology using the ratios of two fragment ions at three CID energies was developed to verify the identity of HOPO when present at ≥0.6ppm. This identity confirmation can be employed to prevent potential false positive detection of mutagenic impurities at trace level. It can be broadly applicable for the confirmation of analytes when the analytes generate at least two major fragments in tandem mass spectrometry experiments.