Development and Validation of HPLC-UV Method for Determination of Bovine Serum Albumin and Myoglobin Proteins

Abstract

A simple HPLC with UV detection method is developed and validated for determination of Bovine Serum Albumin and Myoglobin proteins in a standard solution of the two protein as well as in a plasma spiked with theses two proteins. Separation was achieved on a reversed-phase C18, 5.0 μm, 150mm × 4.6 mm inner diameter column using a mobile phase consisting of solution A (900 mL of water, 100 mL Acetonitrile, 10 mL Trifluroacetic acid) and solution B (900 mL of acetonitrile, 100 mL water, 10 mL Trifluroacetic acid) with gradient elution of 5 to 50% of solution B in 15 minutes. This new method is validated in accordance with requirements for new methods, which include accuracy, precision, selectivity, linearity and range, robustness, limit of detection and limit of quantitation. The method demonstrates good linearity over the range of 1-1000 ppm for the two proteins with r2 greater than 0.998. The percentage recovery of the method is within 97.9 to 102.0% for the two proteins. Precision of this method reflected by relative standard deviation of the area of six replicate injections of each protein at five concentration levels (1.0, 100.0, 300.0, 500, and 1000.0 ppm) was found to be less than 2%. Limit of quantitation of the two proteins is low which enables the determination of these proteins at low concentration.