Eberlein G A
Matrix Pharmaceutical, Inc., Menlo Park, CA 94025, USA.
J Pharm Biomed Anal. 1995 Sep;13(10):1263-71. doi: 10.1016/0731-7085(95)01559-4.
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 x 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.
具有多波长监测等功能的现代高效液相色谱（HPLC）-二极管阵列检测器能够在较长时间内保持高度的响应重现性。这种重现性表明，可使用检测器响应因子而非基于稀释的标准曲线来测量蛋白质和药物的浓度。使用四种不同的HPLC方法分析单一蛋白质，并使用响应因子测试测量的准确性和精密度。将这些结果与使用标准曲线拟合获得的准确性和精密度进行比较。通过定量氨基酸分析确定蛋白质溶液浓度后，再用HPLC进行分析。通过紫外光谱法以及根据已知氨基酸组成计算得出这些蛋白质溶液在277nm处的消光系数。通过将各个氨基酸的消光系数相加计算得到的理论消光系数与实验值相差在2%以内。使用注入已知量蛋白质产生的峰面积计算215nm和277nm处的响应因子。当使用实验消光系数计算预期的HPLC信号响应（峰面积 = 吸光度×持续时间）时，如果在215nm处测量，蛋白质的回收率（准确性）为100%，而在277nm处测量时回收率在90%至94%之间。回收率的耐用性相对标准偏差在2.6%至4%之间，具体取决于HPLC方法。结果发现，使用响应因子从峰面积计算定量结果时，其准确性至少与使用标准曲线时相当。
