Assay of Human Insulin

Procedure The HPLC is carried out using (a) a Vydac C18 column, for proteins and peptides, maintained at 40 °C, (b) as the mobile phase at a flow rate of 1 ml per minute, a mixture of 48 volumes of mobile phase A and 52 volumes of mobile phase B prepared and maintained at a temperature of not less than 20 °C, and (c) a detection wavelength of 214 nm. 

I 10 pL of soln. (4) The resolution between the peaks corresponding to human and porcine insulin is at least 1.3. (If necessary, adjust the cone, of acetonitrile in the mobile-phase by slight decrease or increase until the required resolution is obtained. 

II Soln. (3) six times The relative standard deviation of the area of the principal peak is at most 2%. 

III 10 pL of each of solns. (1), (2) and (3) The area of the principal peak in the chromatogram with soln. (1) is 10 0.5 times the area of the principal peak in the chromatogram obtained with soln. (2). 

Calculations Calculate the content of human insulin, C257H383N65077S6, from the peak areas and using the declared content of C257H383N65077S6 in human insulin EPCRS. 

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Tanaka T, R. Sato R, Kamiya S, Obata K, Tamiya S, Matsunaga T. (2000). Fully-automated heterogeneous immuno assay of human insulin using protein A-bacterial magnetic particle complexes. Anal. Chem. 72,3518- 3522... 

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