Fast and Sensitive Quantitative LC-MS/MS Methods in Bioanalysis: HPLC versus UPLC

This application brief demonstrates the benefits of UPLC compared to traditional HPLC for the analysis of risperidone, 9-hydroxyrisperidone, and the internal standard, clozapine.

HPLC-MS/MS is a widely utilized technique for quantitative bioanalysis due to its sensitivity and selectivity. There are three main challenges that face LC-MS/MS when developing new methods these are:

• Speed
• Sensitivity
• Resolution

Waters solution to the above challenges is the ACQUITY UltraPerformance LC System.

Speed and Resolution

The UPLC method that was developed resulted in a 70% decrease inanalysis time compared to HPLC (Figure 1) with no significant change in chromatographic resolution (Figure 2), allowing a three-fold increase in sample throughput. The gradient and flow rates were optimized for both HPLC and UPLC and the reduction in analysis time results partly from the use of these conditions with the UPLC column, and partly to the very low system volume in the UPLC hardware. This low system volume also has a benefit in reducing the time required for equilibration when gradient elution is used, therefore further increasing sample throughput and allowing the efficient use of MS/MS. 

Sensitivity

The narrow peak widths produced by the UPLC, typically 2–3 seconds wide at base, result in increased peak intensity and improved signal-to-noise ratios. This allows lower limits of quantification (LLOQ) to be reached compared to HPLC. In this example (Figures 3 and 4), a 3-fold increase in the signal-to-noise was achieved for a 0.1 ng/mL LLOQ. 

UPLC allows the development of fast and sensitive LC-MS/MS methods. When compared to conventional HPLC, significant reductions in analysis time and lower limits of quantification can be achieved, without the need to change the sample preparation method or the MS/MS system being used. In this example, a three-fold increase in both throughput and sensitivity was gained using the ACQUITY UPLC System.