Derivatization of secondary amines with 2-naphthalenesulfonyl chloride for high-performance liquid chromatographic analysis of spectinomycin

Abstract

A normal-phase high-performance liquid chromatographic (HPLC) method has been developed for the assay of spectinomycin hydrochloride and spectinomycin sulfate for detection at 254 nm. The method involves pre-column derivatization of secondary amines of spectinomycin with 2-naphthalenesulfonyl chloride (NSCl) using a catalyst. Lincomycin, 1-methylpyrrole, 2-acetyul-l-methylpyrrole, and 2-acetyl-pyrrole act as catalysts for sulfonylation of spectinomycin. Without a catalyst, the derivatization reaction forms a considerable amount of actinospectinoic acid, a degradation compound of spectinomycin, and peak area:weight ratio of the derivative is approximately 15% lower than those with the catalyst. Following derivatization the sample is extracted and chromatographed on a normal-phase silica column with detection at 254 nm. The method is applicable for the analysis of both the hydrochloride and sulfate salt forms of spectinomycin. All the known degradation compounds of spectinomycin such as actinamine, actinospectinoic acid and the biosynthesis intermediates, dihydrospectinomycin diastereoisomers, are completely separated with this method. Mass spectrometric data confirms that spectinomycin is derivatized with NSCl at the secondary amines located at positions 6 and 8 of the ring structure. The standard curves for the HPLC assay of spectinomycin hydrochloride and sulfate are linear with correlation coefficients of 0.9997 and 0.9999, respectively over the range of 0.05 ng/ml to 0.3 mg/ml. The relative standard deviations (R.S.D.) of the HPLC assay methods for spectinomycin hydrochloride and sulfate are 0.67% and 0.86%, respectively. Spectinomycin hydrochloride and sulfate bulk drugs were assayed by the HPLC method and compared to gas—liquid chromatography and microbiological assay results. The HPLC method was used to assay spectinomycin in a veterinary formulation, Linco-Spectin® soluble powder. The sensitivity of the HPLC assay was determined to be approximately 4 ng sample load on the column, which suggests applicability in serum and residue level studies.