Hagan R L
Analytical Research Laboratory, Clinical Investigation Facility, David Grant USAF Medical Center, Travis Air Force Base, CA 94535-1800.
Am J Hosp Pharm. 1994 Sep 1;51(17):2162-75.
The fundamentals of high-performance liquid chromatography (HPLC), as applied in small-scale studies of drug stability, are presented. Chromatography is the separation of a complex mixture into its individual compounds through partitioning between a mobile phase and a stationary phase. A high-performance liquid chromatograph consists of mobile-phase reservoirs, pumps, a mixer to mix the solvents, a valve into which the sample is injected, a guard column, a column containing the stationary phase, a detector, and a recorder. Once compounds have been separated in the column, they pass into the detector, where an electronic signal corresponding to the amount of compound present is recorded as a peak in a chromatogram. The most common detection method is ultraviolet and visible light spectroscopy. Key concepts in HPLC theory are retention time, the time from injection of the sample to detection of a peak; capacity factor, a measure of retention corrected for the elution of an unretained compound; resolution, a measure of how well two peaks are separated; the selectivity of the method; efficiency, or resolving power; and the degree of symmetry of the peaks produced. Most HPLC separations are performed in the reverse-phase mode, which involves a nonpolar stationary phase and a largely polar mobile phase. Other modes are normal phase, ion exchange, and size exclusion. Before a drug stability study is carried out, an HPLC method must be developed that suits the needs of the proposed experiment. A thorough literature search is essential. Literature procedures serve as useful starting points but may require a great deal of manipulation. After the HPLC separation has been performed, it is necessary to validate the method used. It must be proved that the method is stability indicating, that the chromatographic standards were properly prepared, that the standard curve is acceptable, and that the method is both precise and accurate. Pharmacists who ensure that reliable, reproducible HPLC methods are used throughout studies of drug stability will obtain sound data that may be of great value in pharmacy practice.
介绍了高效液相色谱法（HPLC）在小规模药物稳定性研究中的基本原理。色谱法是通过在流动相和固定相之间进行分配，将复杂混合物分离成其各个化合物。高效液相色谱仪由流动相储液器、泵、用于混合溶剂的混合器、注入样品的进样阀、保护柱、装有固定相的色谱柱、检测器和记录仪组成。化合物在色谱柱中分离后，进入检测器，在那里与存在的化合物量相对应的电信号被记录为色谱图中的一个峰。最常见的检测方法是紫外可见光谱法。HPLC理论中的关键概念包括保留时间，即从进样到检测到峰的时间；容量因子，一种针对未保留化合物洗脱进行校正的保留量度；分离度，衡量两个峰分离程度的指标；方法的选择性；效率或分离能力；以及产生的峰的对称程度。大多数HPLC分离是在反相模式下进行的，该模式涉及非极性固定相和主要为极性的流动相。其他模式有正相、离子交换和尺寸排阻。在进行药物稳定性研究之前，必须开发一种适合拟议实验需求的HPLC方法。全面的文献检索至关重要。文献方法可作为有用的起点，但可能需要大量的操作。在进行HPLC分离后，有必要验证所使用的方法。必须证明该方法是稳定性指示的，色谱标准品制备正确，标准曲线可接受，并且该方法既精确又准确。确保在整个药物稳定性研究中使用可靠、可重现的HPLC方法的药剂师将获得在药学实践中可能具有巨大价值的可靠数据。
