A validated HPLC assay for simultaneous analysis of salmon calcitonin and duck ovomucoid.
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abstract
A highly sensitive and selective analytical HPLC method is reported for the simultaneous measurement of salmon calcitonin (sCT) and its enzyme inhibitor, duck ovomucoid (dOVM). The method used a reversed phase C-18 column (4.6 x 250 mm, 5 microm) at room temperature. The elution was achieved using a gradient technique (20-35% B for 10 min, 35-37% B from 10th to 20th min and 37-20% B from 20th to 25th min). The mobile phase used was 0.05% v/v trifluoroacetic acid (TFA) in water and 0.05% v/v TFA in acetonitrile with a flow rate of 1 ml/min. Detection was carried out by UV spectrophotometry at 210 nm. sCT and dOVM were eluted at 7.8 and 15.4 min respectively, free from any interfering endogenous peaks during a run time of 25 min. Linear relationships were observed between the detector response and the concentrations of the analytes (10-100 microg/ml for CT (r2 = 0.996) and 10-100 microg/ml for the dOVM (r2 = 0.999)). The assay was found to be highly selective and sensitive due to the absence of any interfering peaks. The lower C.V. and % error values of the assay indicates that the assay could accurately and precisely quantitate both sCT and dOVM in the examined concentration range. This method can be usedfor the simultaneous quantitative analysis of sCT and dOVM.
