利用高效液相層析法測定Inslin製劑效價

DETERMINATION OF INSULIN POTENCY IN PREPARATIONS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

最近，利用逆相高效液相層析法（RP-HPLC）分析蛋白質及蛋白棟的報告相當多，關於HPLC應用於Insulin的分析者自1979年以來發展甚速，但是其中使用於Insulin製劑效價測定的相關報告卻只佔少數。在U.S.P. XX及B.P. 1980版中所較仍是傳統式的生物效價測定法：兔子血糖下降測定法（Rabbit blood glucose assay），小白鼠驚厥試驗法（Mouse convulsion assay）以及小白鼠血糖下降測定法（Monse blood glacose assay）。但是，U. S. P. XXI中已將HPLC價測定法引進部份的Insulin製劑內。以下所欲記述的，乃參考美國Lilly研究實驗室Smith等人的報告，方法上稍作修飾，對於市售Insulin製劑分別以HPLC法及生物效價測定法測出效價，證明了HPLC法的可行性與精確性。 A reversed-phase high performance liquid chromatography (RP-HPLC) method for the determination of insulin potency in preparations has been used. The method takes a unique standard solution (40iu/ml in 0.6mg/ml EDTA) which is stable enough to permit use for >1 month. NPH insulin suspensions are clarified by addition of heparin, lente insulins are clarified by addition of tetrasodium EDTA, and EDTA is added to neutral regular insulins. Chromatography was done using a 0.1M PH 2.0 phosphate/25% CH, CN mobile phase and reversed-phase Zorbax TMS column. The HPLC assay results was approved to agree with the bioassay method.