Quantitation of proteins using HPLC-detector response rather than standard curve comparison

Abstract

Modern high performance liquid chromatography (HPLC)-diode array detectors with features such as multiple wavelength monitoring are capable of maintaining a high degree of response reproducibility over extended periods of time. This reproducibility suggests that detector response factors, rather than dilution based standard curves, might be used to measure concentrations of proteins and pharmaceuticals. Four different HPLC methods were used to analyze a single protein and to test the accuracy and precision of measurements using response factors. These results were compared to the accuracy and precision obtained using fitting to a standard curve. Protein solutions were analyzed by HPLC after the concentration was determined by quantitative amino acid analysis. The extinction coefficient at 277 nm of these protein solutions was determined by UV spectroscopy as well as calculated based on the known amino acid composition. The theoretical extinction coefficient calculated by summing the extinction coefficient of the individual amino acids was within 2% of the experimental value. Response factors at 215 and 277 nm were calculated using the peak area produced by the injection of a known amount of protein. When the experimental extinction coefficient was used to calculate the expected HPLC-signal response (peak area = absorbance × duration), the recovery of the protein (accuracy) was 100% if measured at 215 nm and between 90 and 94% when measured at 277 nm. The ruggedness of the recovery was between 2.6 and 4% relative standard deviation, depending on the HPLC-method. It was found that the quantitation was at least as accurate when calculated from the peak area using the response factor as when a standard curve was used.