Jemal M, Xia Y Q, Whigan D B
Bristol-Myers Squibb Pharmaceutical Research Institute, New Brunswick, NJ 08903-0191, USA.
Rapid Commun Mass Spectrom. 1998;12(19):1389-99. doi: 10.1002/(SICI)1097-0231(19981015)12:19<1389::AID-RCM329>3.0.CO;2-T.
Two bioanalytical methods have been developed and validated utilizing high flow high performance liquid chromatography (HPLC) for on-line purification of plasma and serum samples and electrospray tandem mass spectrometry for detection and quantitation. Each plasma or serum sample, after mixing with an aqueous solution of the internal standard, was injected into a small diameter (1 x 50 mm) column packed with large particles of OASIS (30 microns), with a 100% aqueous mobile phase at a high flow rate (3-4 mL/min). The combination of the high linear speed (6-8 cm/s) of the aqueous mobile phase and the large particle size resulted in the rapid passage of the proteins and other large biomolecules through the column while the small-molecule analytes were retained on the column. During this purification period, the HPLC effluent was directed to waste. After the purification step, the HPLC mobile phase was rapidly changed from 100% aqueous to < or = 100% organic, the flow was reduced to 0.5-0.8 mL/min, and the column effluent was directed towards the mass spectrometer. The small molecule analytes were eluted during this period. In the method developed and validated for the quantitative determination of compound I in rat plasma (method A), the same OASIS column (1 x 50 mm, 30 microns) served as the purification and analytical (elution) column. In the method developed for the simultaneous determination of pravastatin and its positional isomer biotransformation product (SQ-31906) in human serum (method B), the purification column was connected to a conventional C18 analytical column (3.9 x 50 mm, 5 microns) to achieve the required chromatographic separation between the two isomers. For method A, where 50 microL of rat plasma mixed 1:1 with water containing the internal standard was injected, the standard curve range was 1 to 1,000 ng/mL. For method B, where 200 microL of a human serum sample mixed 4:1 with water containing the internal standard was injected, the standard curve range was 0.5 to 100 ng/mL. The total analysis time for each method was < or = 5 min per sample. The accuracy, inter-day precision and intra-day precision were within 10% for both methods.
已开发并验证了两种生物分析方法，它们利用高流量高效液相色谱（HPLC）对血浆和血清样品进行在线纯化，并用电喷雾串联质谱进行检测和定量。每个血浆或血清样品与内标水溶液混合后，注入填充有大颗粒OASIS（30微米）的小直径（1×50毫米）柱中，以100%的水性流动相在高流速（3 - 4毫升/分钟）下进行。水性流动相的高线速度（6 - 8厘米/秒）和大颗粒尺寸的组合导致蛋白质和其他大分子快速通过柱子，而小分子分析物则保留在柱上。在这个纯化阶段，HPLC流出物被导向废液。纯化步骤后，HPLC流动相迅速从100%水性变为≤100%有机相，流速降至0.5 - 0.8毫升/分钟，柱流出物被导向质谱仪。在此期间，小分子分析物被洗脱。在开发并验证的用于定量测定大鼠血浆中化合物I的方法（方法A）中，可以使用相同的OASIS柱（1×50毫米，30微米）作为纯化和分析（洗脱）柱。在开发的用于同时测定人血清中普伐他汀及其位置异构体生物转化产物（SQ - 31906）的方法（方法B）中，纯化柱连接到常规的C18分析柱（3.9×50毫米，5微米）上，以实现两种异构体之间所需的色谱分离。对于方法A，注入50微升与含内标水按1:1混合的大鼠血浆，标准曲线范围为1至1000纳克/毫升。对于方法B，注入200微升与含内标水按4:1混合的人血清样品，标准曲线范围为0.5至100纳克/毫升。每种方法的总分析时间为每个样品≤5分钟。两种方法的准确度、日间精密度和日内精密度均在10%以内。
