7
Figure 3. Example of multiplex analysis for Cytochrome P450 assay.
3. Maximise Throughput with a Multiplex Confi guration
Figure 2. Example of carryover of a standard solution of Glucose 6-phosphate. a) Outside needle rinse by dipping into R0 (50 % methanol aq.) b) Outside needle rinse by dipping into R0 (water), internal needle surface and injection port rinse by R1 (mixture of formic acid, methanol, acetonitrile and isopropyl alcohol) [2].
RPLC gradient analysis requires performing various processes, such as the needle rinse, column washing and re-equilibration as preparation for the next sample injection. While these are essential processes to generate meaningful data, they result in additional ‘non-data-acquisition time’ that impacts throughput. Reducing the time where the MS sits idle, waiting for the LC to wash and re-set, could dramatically increase throughput and lead to higher laboratory productivity. Hence, a front-end confi guration that offers to overlap the data acquisition time and washing/equilibration phase by switching between two streams into one LC-MS is an ideal solution to close to double sample throughput in one system. Figure 3 [3] shows an example where this Dual Stream Technology was used for analysis of four biomarkers for the four major molecular species in the Cytochrome P450 family. Analysis could be completed in only 38 seconds compared to 1 minute 22 seconds using the conventional single stream approach, saving 44 seconds per sample.
Conclusion
LC-MS/MS is a powerful technique for quantitative assays, as it offers high selectivity and sensitive detection. For a laboratory to achieve and maintain high sample throughput and gain as much information as possible from each sample, it is essential to carefully consider the characteristics of the front-end UHPLC system. Certainly, sample injection speed and capacity, the autosampler’s ability to reduce carry-over, while maintaining a high-speed injection cycle, and the possibility to perform multiplex analysis should be considered in an advanced UHPLC front- end confi guration, to achieve high effi ciency and a maximum in sample throughput.
References
1. T. Uchikata, D. Vecchietti, Ultra-Fast Analysis of Drugs in Biological Fluids with the SIL-40 Autosampler - Analytical Intelligence Part 5, Shimadzu Technical Report (C190-E228) 2. K. Watanabe, C. Campbell et. al., Prominent Features of Shimadzu UHPLC for an LC/MS Assay, Shimadzu Technical Report (C190-E282) 3. Nexera MX brochure (C190-E190), Shimadzu Corporation, 2020
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