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 (lambda max) at 360 nm. The HPLC method employs a C8, 3-microm particle size analytical column (150 mm x 4.6 mm), 15-microL injection volume, column temperature controlled at 30 degrees 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).

A reversed-phase high-performance liquid chromatographic method (HPLC) with diode-array detection (DAD) has been evaluated for monitoring trace levels of impurities, such as 4-amino-2-ethoxy-cinnamic acid (impurity A), hydrochloride salt of 4-amino-2-ethoxy-ethyl cinnamate (impurity B), and 4-bromo-3-ethoxy-nitrobenzene (impurity C), in drug substance and 3 different formulation prototypes. These compounds have been highlighted as potential genotoxins and 2-ethoxy-4-amino-cinnamic acid (impurity A) as possible degradant isolated during the synthesis of BI drug substance. HPLC-UV-DAD was found to be more promising, and limits of quantification were between 0.09 and 0.6 microg/mL, which enabled detection limits in drug substance at 2-15 ppm for a 15 mg/mL solution. All three genotoxic impurities are completely resolved from each other as well as from diluent peaks, drug substance, and other related impurities within 40 min. The retention times of impurities A, B, and C were 3.4, 13.1, and 21.3 min. The results demonstrating the specificity, assay precision, recovery, linearity, and range achieved during the method validation experiments are presented in this paper.

Soluplus, a graft copolymer of polyethylene glycol, vinyl caprolactam and vinyl acetate, is designed to solubilize poorly soluble active pharmaceutical ingredients. A straightforward aqueous gel permeation chromatography method that exploits both size exclusion and adsorption modes of separation was used to separate and quantify the related residual vinyl caprolactam monomer and caprolactam impurity present in Soluplus. This methodology offers a single step analysis of caprolactam and the residual vinyl caprolacatam monomer, yielding similar results to reversed-phase chromatography measurements, which are time-consuming and may involve multi-step sample preparation. The results of this study demonstrate that gel permeation chromatography provides a viable option to traditional reversed-phase chromatography in the quantitative analysis of residual caprolactam and vinyl caprolactam monomers and can be extended to other monomer-polymeric systems.

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