Enhanced resolution triple-quadrupole mass spectrometry for fast quantitative bioanalysis using liquid chromatography/tandem mass spectrometry: investigations of parameters that affect ruggedness

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.