Determination of sialic acid in recombinant human follicle stimulating hormone by high performance liquid chromatography with DMB derivatization

doi:10.19803/j.1672-8629.20230498

Abstract

Abstract: Objective To develop a 4,5-methylenedioxy-1,2-phenylenediamine dihydrochloride (DMB) derivatization method based on high-performance liquid chromatography-fluorescence detector (HPLC-FLD)(DMB derivatization method) that can concurrently quantify both N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac) in rhFSH. Methods A comparative study was undertaken between the o-phenylenediamine derivatization method based on high-performance liquid chromatography(o-phenylenediamine derivatization method) and DMB derivatization method. The optimized DMB derivatization method with refined hydrolysis conditions was established and demonstrated as follows: The rhFSH solution was first subjected to acid hydrolysis, followed by derivatization with DMB. The resulting solution was then separated using an Agilent ZORBAX 300SB-C18 chromatographic column (250 mm×4.6 mm, 5 μm) and analyzed by a fluorescence detector. Results When compared to the o-phenylenediamine derivatization, the optimized DMB derivatization method proves to be more effective in separating Neu5Gc and Neu5Ac related impurities. The optimized DMB derivatization method showcases superior specificity, with both Neu5Gc and Neu5Ac demonstrating a strong linear relationship with peak area within the concentration ranges of 0.5~5.0 μg·mL^-1(r=0.999 9) and 25~250 μg·mL^-1(r=0.999 8). The limit of detection and quantification (LOD and LOQ) for Neu5Gc are impressively low as 11.07 ng·mL^-1and 33.21 ng·mL^-1respectively, comparable to the levels of Neu5Ac (LOD: 10.30 ng·mL^-1, LOQ: 20.60 ng·mL^-1). Besides, the recovery rates for Neu5Gc and Neu5Ac are (99.63±1.33)% (n=9) and (100.62±1.58)% (n=9), with low RSDs of 1.3% (n=9) and 1.5% (n=9), respectively. Conclusion The method demonstrates commendable specificity, exceptional sensitivity, and noteworthy precision and accuracy, which would be ideally suited for the precise quantification of Neu5Gc and Neu5Ac content in rhFSH.

Key words: recombinant human follicle stimulating hormone(rhFSH), high-performance liquid chromatography, DMB derivatization, fluorescence detector, N-glycolylneuraminic acid, N-acetylneuraminic acid, content, sialic acid

CLC Number:

R917

WANG Lyuyin, LI Yi, SUN Yue, HU Xinyue, ZHANG Hui, LI Jing, LIANG Chenggang. Determination of sialic acid in recombinant human follicle stimulating hormone by high performance liquid chromatography with DMB derivatization[J]. Chinese Journal of Pharmacovigilance, 2024, 21(1): 89-93.

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