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-1 and 33.21 ng·mL-1 respectively,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.
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