HPLC-UV method development and validation for the determination of low level formaldehyde in a drug substance

Abstract

A reversed-phase high-performance liquid chromatographic method (HPLC) with diode-array detection is developed and validated for the quantitative determination of formaldehyde in a drug substance. Formaldehyde (HCHO) is reacted with 2,4-dinitrophenylhydrazine (DNPH) to form a Schiff base (HCHO-DNPH derivatization product), which has an absorbing maximum (λmax) at 360 nm. The HPLC method employs a C8, 3-μm particle size analytical column (150 mm × 4.6 mm), 15-μL injection volume, column temperature controlled at 30°C, detection at 360 nm, and a water - acetonitrile (55:45, v/v) mobile phase at a flow rate of 1 mL/min. These conditions resolve the HCHO - DNPH product from unreacted DNPH, the drug substance and related impurities, as well as diluent peaks within 20 min. The retention time of the HCHO - DNPH product is approximately 6.4 min. The method is linear, accurate in the specified range (0.33-333 ppm), and robust based on analyte (HCHO - DNPH derivatization product) stability in standard and sample. Detection limit is 0.03 ng (0.1 ppm).