聚乙二醇(PEG)化尿酸氧化酶中游离PEG的含量测定

Determination of the content of free polyethylene glycol in PEGylated uricase

目的：比较并建立PEG化尿酸氧化酶中游离PEG的测定方法。方法：采用反相高效液相色谱-蒸发光散射检测器（ELSD）联用法和SDS—PAGE碘染的方法。RP—I-IPLC法的色谱柱为C。柱（4．6mm×250mm，5μm）；流动相A为0．1％（v／v）的三氟乙酸-水，流动相B为0．1％（v／v）的三氟乙酸-乙腈，梯度洗脱；流速为1．0mL·man～；柱温30℃；SDS—PAGE碘染采用氯化钡固定，碘-碘化钾染色。结果：RP—HPLC法中，PEG的检测灵敏度为0．2μg，PEG在1～6μ范围内呈良好的二项式回归关系（R^2=0．9993）。低、中、高3个浓度的加样回收率分别为96．2％、97．4％、96．8％。SDS—PAGE碘染法灵敏度为0．1μg。结论：建立的高效液相色谱一蒸发光散射检测器联用法与SDS—PAGE碘染法均可对游离PEG进行测定。前者测定结果准确，重复性好，可用于PEG的精确定量分析，后者操作简单，不需要特殊仪器。但只可用于限度检查。

Objective： To compare and establish a method for the determination of free polyethylene glycol （PEG） content in PEGylated uricase. Methods： The methods of high - performance liquid chromatography coupled with e- vaporative light scattering detector and SDS - PAGE staining with iodine reagents were used. The C4 column （4. 6 mm x 250 mm,5 μm）with a column temperature of 30 ℃ was adopted;the mobile phase A was 0. 1% （v/v） trifluoro- acetic acid - water and mobile phase B was 0. 1% （v/v） trifluoroacetic acid - acetonitrile, with the gradient elution at a flow rate of 1.0 mL · min - 1. Iodine staining of SDS - PAGE was fixed using barium chloride fixed and stained with iodine- potassium iodide. Results： The developed high -performance liquid chromatographic method had a good binomial regression relationship in the range of 1 - 6 μg （R2 = 0. 9993 ） and the sensitivity of PEG detection was 0. 2 μg. The average recoveries at low,medium and high concentrations ware 96. 2% ,97.4% and 96. 8% ,re- spectively. The sensitivity of SDS - PAGE iodine staining was 0. 1 μg. Conclusion： Both the high - performance liquid chromatography coupled with evaporative light scattering detector and the SDS - PAGE iodine staining can be used to determine the content of free PEG. The former is accurate and reproducible, and thus can be used for the precise quantitative analysis. The latter is simple, requiring no special equipment, but can only be used in limit in- spection.