Jemal Mohammed, Ouyang Zheng
Clinical Discovery Analytical Sciences, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 191, New Brunswick, NJ 08903-0191, USA.
Rapid Commun Mass Spectrom. 2003;17(1):24-38. doi: 10.1002/rcm.872.
In order to increase sample analysis throughput, the use of fast liquid chromatography in quantitative bioanalysis based on liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) has become prevalent. Therefore, it is important to increase the specificity of such bioanalytical methods. This can be done by enhancing both the chromatographic and mass resolving power. Increasing the mass spectrometric resolving power to minimize interference from endogenous compounds in the biological matrix is the subject of this paper. We present the results of our experience with developing and validating SRM-based, enhanced resolution bioanalytical methods using a new triple-quadrupole mass spectrometer with enhanced resolution capability. We have shown that SRM bioanalytical methods using better than unit-mass resolution (Q1 FWHM = 0.2 Th, Q3 FWHM = 0.7 Th) can be developed which are as rugged as unit resolution methods (Q1 FWHM = 0.7 Th, Q3 FWHM = 0.7 Th). The enhanced resolution methods require more attention to detail than unit resolution methods. For instance, the mass setting for precursor ion selection is more critical because the mass peak is narrower. Because of this, enhanced resolution methods may be more easily influenced by temperature changes in the laboratory. We have shown that there is good correlation between the shift in the precursor ion mass and the ambient temperature. Other studies carried out to investigate the effects on mass peak shape and response (both in the SIM and SRM mode) as the result of varying the FWHM revealed some interesting results. For instance, the decrease in response with the decrease in the FWHM was larger using SRM compared to that using SIM. However, the decrease in both SRM and SIM response with decreasing FWHM was significantly smaller compared with the decrease obtained using an older generation instrument. We demonstrate that, at concentrations near the limit of detection, the signal specificity can be improved by using an enhanced resolution method. To compare the performance of an enhanced resolution method against a unit resolution method under optimized mass spectrometric conditions, we analyzed calibration standards and quality control samples using a lower limit of quantitation that could be easily achieved by either method. Under these conditions, the two methods were essentially the same, demonstrating that the enhanced resolution method is as accurate, precise and rugged as the unit resolution method. We propose system suitability procedures, based on precursor ion scan, product ion scan, SRM with fractional mass changes, or SIM with a narrow scan width, for the updating of the SRM set masses before the start of analysis. We also recommend that Q1 SRM masses be determined during and at the end of analysis in order to ascertain whether or not the precursor masses have shifted during the course of the analysis.
为了提高样品分析通量，基于液相色谱-串联质谱（LC/MS/MS）的快速液相色谱在定量生物分析中的应用已变得十分普遍。因此，提高此类生物分析方法的特异性很重要。这可以通过提高色谱和质量分辨能力来实现。提高质谱分辨能力以最小化生物基质中内源性化合物的干扰是本文的主题。我们展示了使用具有增强分辨能力的新型三重四极杆质谱仪开发和验证基于选择反应监测（SRM）的增强分辨生物分析方法的经验结果。我们已经表明，可以开发出使用优于单位质量分辨（Q1半高宽 = 0.2 道尔顿，Q3半高宽 = 0.7 道尔顿）的SRM生物分析方法，其耐用性与单位分辨方法（Q1半高宽 = 0.7 道尔顿，Q3半高宽 = 0.7 道尔顿）相当。增强分辨方法比单位分辨方法需要更注重细节。例如，前体离子选择的质量设置更为关键，因为质量峰更窄。因此，增强分辨方法可能更容易受到实验室温度变化的影响。我们已经表明，前体离子质量的偏移与环境温度之间存在良好的相关性。为研究改变半高宽对质量峰形状和响应（在选择离子监测和SRM模式下）的影响而开展的其他研究揭示了一些有趣的结果。例如，与选择离子监测模式相比，SRM模式下响应随半高宽减小而降低的幅度更大。然而，与使用较旧一代仪器相比，SRM和选择离子监测模式下响应随半高宽减小的幅度都显著更小。我们证明，在接近检测限的浓度下，使用增强分辨方法可以提高信号特异性。为了在优化的质谱条件下比较增强分辨方法与单位分辨方法的性能，我们使用两种方法都能轻松达到的较低定量下限分析校准标准品和质量控制样品。在这些条件下，两种方法基本相同，表明增强分辨方法与单位分辨方法一样准确、精密且耐用。我们提出基于前体离子扫描、产物离子扫描、具有分数质量变化的SRM或窄扫描宽度的选择离子监测的系统适用性程序，用于在分析开始前更新SRM设定质量。我们还建议在分析过程中和结束时确定Q1 SRM质量，以确定在分析过程中前体质量是否发生了偏移。
