Category: Formulation and Quality
Purpose: Hydroxylamine is a known genotoxic impurity compound that needs to be controlled down to ppm level in pharmaceutical processes. It is difficult to detect using conventional analytical techniques due to its structure, low molecular weight and high polarity. European Pharmacopoeia method for residual hydroxylamine (2.2.25) targeted to 5 ppm level and does not always work in presence of complex matrix components that can react with reagents like sulphanilic acid, iodine, thiosulphate or naphtylamine or affect to the clarity of the final solution. The purpose of this study was to develop an analytical method for the determination of residual hydroxylamine in biological formulations. HPLC-based method has been developed to target to the range of 2-8 ppm hydroxylamine in regard to the sample amount, or 0.14-0.57 µg/mL hydroxylamine in the solution. Method has been shown to be specific, precise, accurate and linear. This method can be applied as a generic method to detect hydroxylamine for pharmaceutical process control, drug substance and drug product release.
Methods: The method utilizes derivatization of hydroxylamine to the oxime in presence of vanillin and sodium acetate. Method requires small amount (50 µL sample) to be taken to the derivatization procedure. Separation of the vanillin oxime was performed on HPLC system equipped with a UV detector. Chromatographic separation was carried out on a Waters Acquity UPLC BEH C18 1.7 µm, 2.1 × 100 mm column with a flow rate 0.25 mL/min of gradient program (Mobile Phase A 0.1% TFA in water; Mobile Phase B is 0.1% TFA in Acetonitrile). Other instrument parameters: injection volume 2 µL, column temperature 30ºC, UV detection 255 nm, run time 12 minutes.
Results: Specificity: No blank interference was observed at the retention time of the vanillin oxime (final product of hydroxylamine derivatization). The retention time of vanillin oxime was ~ 6 min. System suitability: system suitability was established at ~0.29 µg/mL of hydroxylamine (corresponds to the 4 ppm in the sample). The %RSD of the working standard (instrument precision) was ~3% (internal requirements for the limit test method NMT 15%). Linearity: the linearity was evaluated across 0.07 µg/mL to 1.42 µg/mL that corresponds to the 1 to 20 ppm levels with regards to the sample amount. The correlation coefficient was 0.9997 (internal requirements for the limit test method NLT 0.98). Limit of quantitation (LOQ): LOQ was established on lowest linearity standard and was 0.07 µg/mL or 1 ppm in regard to the sample. Accuracy: samples provided by the client (formulation buffer and drug product) were spiked with known amount of hydroxylamine to evaluate accuracy (spike-recovery). Recovery was evaluated at Lowest (corresponds to the LOQ), Low (3 ppm), Medium (5 ppm) and High (8 ppm) levels. The % Recovery was within 87-106% across the levels (internal requirements: 50-150% for LOQ, 70-130% for other levels). Precision: precision of the method was evaluated on samples spiked at 5 ppm hydroxylamine. The % RSD was 3% (internal requirements for the limit method NMT 15%).
Conclusion: The test method for determination of residual hydroxylamine in complex biological formulations was successfully developed. The method has been shown to be specific, linear, precise and accurate. The limit of quantitation (LOQ) was 0.07 µg/mL of hydroxylamine (1 ppm in regard to the sample). The range of the method is 0.14-0.57 µg/mL (2-8 ppm in regard to the sample amount). Method has not been validated.