Determination of spectinomycin and related substances by HPLC coupled with evaporative light scattering detection

Abstract

Summary. An improved ion-pairing reversed-phase high-performance liquid chromatography method coupled with evaporative light scattering detection (HPLC-ELSD) was developed to determine spectinomycin and its related substances in commercial samples. The method was validated in accordance with International Conference on Harmonization (ICH) guidelines. The specificity of the HPLC-ELSD method was similar to that of the European Pharmacopoeia (Ph. Eur.) method, and repeatability and robustness were markedly improved relative to other reported methods due to our empirical evaluation of separation columns. Indeed, it is a more specific assay of spectinomycin than traditional microbiological techniques. The HPLC-ELSD method was used to evaluate the impurity profiles of eight compounds in seven spectinomycin batches from five different companies. Liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was employed to characterize the structures of these compounds. Though the HPLC-ELSD method was not as sensitive as the Ph. Eur. method, its limit of quantitation (LOQ) (0.16%) was lower than the disregard limit (0.3%) described by the Ph. Eur. 7.0. This suggests that the HPLC-ELSD method is appropriate for routine analysis of spectinomycin and its related substances.

Development of sensitive benzofurazan‐based spectrometric methods for analysis of spectinomycin in vials and human biological samples

“…[1][2][3] SPEC is listed in the US Pharmacopeia and characterized using gas chromatography with a flame ionization detector. [4] In addition, various other methods have been used to determine SPEC, such as spectrophotometry, [5] high performance liquid chromatography (HPLC), [1,[6][7][8] gas chromatography [9] and electrophoresis. [10,11] Several drawbacks have been reported for these methods, such as lack of sensitivity, [5] the need for expensive instrumentation and the use of organic solvents.…”

“…[10,11] Several drawbacks have been reported for these methods, such as lack of sensitivity, [5] the need for expensive instrumentation and the use of organic solvents. [1,[6][7][8] 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is a chemical reagent that reacts with amine moietycontaining drugs forming fluorogenic and chromogenic products under alkaline conditions. [12][13][14][15] The methods presented here represent the first use of NBD-Cl to derivatize SPEC, firstly measuring the product spectrophotometrically at 410 nm and secondly measuring the product spectrofluorimetrically at 530 nm (λ ex at 460 nm).…”