Michael J. Y. Jarvis (Presenter)
Sciex
Authorship: Michael J. Y. Jarvis
Sciex, Concord, Canada
| Short Abstract The measurement of low pg/mL concentrations of estradiol in serum and plasma, by LC-MS/MS, requires a highly sensitive method. To be sure, the sensitivity challenge is significant. Nevertheless this requirement can be readily addressed with the use of high performance tandem mass spectrometry instrumentation, larger sample volumes, and extensive sample preparation. However, those familiar with this analysis will be acutely aware of the more urgent selectivity challenge posed by the presence of ubiquitous, low-level, non-specific chromatographic interferences that frequently mask the presence of the target estradiol peak. In the work presented here, differential ion mobility spectrometry (DMS) has been employed to enhance the selectivity of the analysis, while enabling the measurement of <1 pg/mL estradiol in serum, using a simple one-step liquid liquid extraction sample preparation. |
Long Abstract
Introduction:
The measurement of low pg/mL concentrations of estradiol in serum and plasma, by LC-MS/MS, requires a highly sensitive method. To be sure, the sensitivity challenge is significant. Nevertheless this requirement can be readily addressed with the use of high performance tandem mass spectrometry instrumentation, larger sample volumes, and extensive sample preparation. However, those familiar with this analysis will be acutely aware of the more urgent selectivity challenge posed by the presence of ubiquitous, low-level, non-specific chromatographic interferences that frequently mask the presence of the target estradiol peak. Many researchers have chosen to address this selectivity challenge using LC strategies, i.e. by attempting to chromatographically separate the interference peaks from the estradiol analyte peak, however this generally results in longer run-times and reduced throughput. Recently, one group has demonstrated the use of MRM3 measurements on a hybrid triple quadrupole/linear ion trap instrument, which provides enhanced selectivity by taking advantage of second-order fragmentation. This approach effectively removes interferences, however it unfortunately yields larger CV (%) values, and results in a reduced linear dynamic range for the analysis. In the work presented here, differential ion mobility spectrometry (DMS) has been employed to enhance the selectivity of the analysis of estradiol. This LC-DMS-MS/MS approach removes the majority of interference peaks, yields no compromise in linear dynamic range, and provides accuracy (%) and CV (%) values that are equivalent to, or better than, the comparable values obtained by traditional LC-MS/MS analysis.
Methods:
100uL of internal standard solution (estradiol-d5 in methanol) was added to 200uL of serum in a microcentrifuge tube. 1000uL of MTBE were added, and the sample was vortex mixed for approximately 10 seconds, then centrifuged at 14,000 rpm for 10 minutes. 900uL of the upper, organic layer was transferred to a clean microcentrifuge tube, and the sample was evaporated to dryness under a stream of nitrogen gas. The dried sample was reconstituted in 120uL of 50:50 water:methanol, and then transferred to an LC autosampler vial containing a 200uL insert. 50uL of the prepared sample was directly injected onto the LC-MS/MS system. Calibration standards were prepared using a stock solution obtained from Cerilliant Corporation (Round Rock, Texas). Estradiol was monitored in negative MRM mode on the Sciex (Framingham, MA) 6500+ LC-MS/MS system, using electrospray ionization. The MS conditions are summarized in Table 1. Liquid chromatography separation was accomplished using a Phenomenex Kinetex C18 column (2.6um, 3.0 x 150mm) at a flow rate of 500uL/minute.
Results:
Serum was spiked at various estradiol concentrations, and analyzed using both LC-MS/MS and LC-DMS-MS/MS methods. Using the conventional LC-MS/MS approach, a large number of interfering chromatographic features were observed (Figure 1b), which have the potential to mask the presence of the estradiol analyte. When the same spiked serum samples were analyzed using the LC-DMS-MS/MS method, all of the interference peaks were removed (Figure 1a), and the high background was completely eliminated. Figure 2 illustrates the benefit of the enhanced selectivity of LC-DMS-MS/MS as a function of estradiol concentration. Clearly the LC-DMS-MS/MS method provides superior data quality, particularly at lower concentrations. The clean, interference-free chromatographic peaks reduce the possibility of incorrect peak area assignments, thereby providing more accurate measurements. Furthermore the application of DMS extended the lower limit of quantitation by a factor of approximately 5x.
Conclusions:
Traditional LC-MS/MS methods for the analysis of estradiol suffer from numerous uncharacterized interferences, which prevent the accurate measurement of low pg/mL concentrations of the target analyte. We have explored differential ion mobility spectrometry (DMS) as a means of tackling the interference problem, since it has the potential to enhance analytical selectivity when combined with MS/MS analysis. A novel LC-DMS-MS/MS method has been demonstrated for the analysis of estradiol in serum, which provides both sensitivity and selectivity. The method enabled the measurement of underivatized estradiol at concentrations <1pg/mL in serum, and effectively removed chromatographic interferences, thereby improving the accuracy and reproducibility of the measurements.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Sciex |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |