柱前衍生化反相高效液相色谱法定量测定重组人促卵泡激素中的单糖

Quantitative determination of monosaccharides in rhFSH by RP-HPLC with pre-column derivatization

目的:建立柱前衍生化反相高效液相色谱法定量测定重组人促卵泡激素(rhFSH)中的单糖。方法:采用20%TFA 水解rhFSH,释放单糖,用邻氨基苯甲酸衍生化,采用反相高效液相色谱法测定。色谱柱为 Jupiter C_(18)柱(250 mm×4.6 mm,5μm);流动相 A 为含0.15%正丁胺、0.25%磷酸、0.5%四氢呋喃的水溶液,流动相 B 为流动相 A 与乙腈比例为1:1的溶液,梯度洗脱,流速0.85 mL·min^(-1);柱温30 ℃;荧光检测器:激发波长为230 nm,发射波长为425 nm。结果:葡萄糖胺、半乳糖、甘露糖、岩藻糖进样量分别在0.39～3.94 nmol(r=0.9994),0.29～5.85 nmol(r=0.9998),0.24～4.90 nmol(r=0.9998),0.05～1.03nmol(r=0.9996)范围内与峰面积呈良好的线性关系。平均加样回收率(n=9)分别为104.1%,104.2%,104.2%,98.0%;RSD 分别为9.3%,1.8%,0.61%,6.0%。结论:该方法灵敏,专属性强,重复性好,可定量测定重组人促卵泡激素中的单糖。

Objective：To establish a method for the quantitative determination of monosaccharides in recombinant human follicle stimulating hormone （rhFSH） by RP - HPLC with pre - column derivatization. Method： Monosaccharides in rhFSH were released by 20% TFA hydrolysis and then derivatised with the 2 - aminobenzoic acid. The derivatives were then separated by RP- HPLC with Jupiter C18（250 mm ×4.6 mm,5 μm） reversed-phase column was used, solvent A consisted of 0. 15 % n - butylamine,0. 5 % phosphoric acid, and 1% tetrahydrofuran in water and solvent B consisted of equal parts solvent A and acetonitrile as mobile phase gradient eluted at a flow rate of 0.85 mL · min^- 1, the column temperature was 30 ℃, with fluorescence detector and the excitation wavelength at 230 nm and emission wavelength at 425 nm. Results：The regression equations for glucosamine, galactose, mannose and fucose showed good linearity in the ranges of 0. 39 -3.94 nmol （r = 0. 9994） ,0.29 -5.85 nmol （r = 0. 9998 ）, 0.24 - 4.90 mnol（ r = 0. 9998 ） ,0.05 - 1.03 nmol （ r = 0. 9996 ）, respectively. The average recoveries （ n = 9 ） were 104. 1%, 104. 2%, 104. 2%, 98.0% with RSDs of 4. 3 %, 1.8 %, 0.61%, 6. 0%, respectively. Conclusion ： The method is sensitive, specific and reproducible. It can be applied for quantitative determination of monosaccharides in rhFSH.