• Application Note

Direct Injection of Polar Compounds in Highly Organic Protein Precipitated Plasma Using UPC2-MS/MS

Direct Injection of Polar Compounds in Highly Organic Protein Precipitated Plasma Using UPC2-MS/MS

  • Jennifer L. Simeone
  • Paul D. Rainville
  • Waters Corporation

This is an Application Brief and does not contain a detailed Experimental section.

Abstract

This work demonstrates direct-inject separation of highly polar compounds in highly organic protein precipitated plasma for bioanalysis.

Benefits

By changing MS inlet technology from RPLC to UPC2, it is possible to directly inject polar compounds from highly organic samples for separation without additional sample preparation steps such as evaporation and reconstitution.

Introduction

A majority of bioanalytical methods utilize protein precipitation (PPT) extraction methods due to the simplicity, speed, and low cost of the technique. A typical PPT utilizes a 3:1 ratio of organic solvent to biological sample, producing an extract approximately 75% organic. Traditionally, samples are analyzed using reversed-phase liquid chromatography (RPLC).

For compounds that are highly polar, direct injection of the highly organic extract onto the RPLC system is not possible due to strong solvent effects that produce poor chromatographic peak shape, most notably peak fronting and/or peak splitting. Therefore, additional sample manipulation including evaporation and reconstitution or dilution with water is required prior to injection onto the chromatographic system.

Results and Discussion

The use of UltraPerformance Convergence Chromatography (UPC2), which utilizes supercritical carbon dioxide as the major mobile phase, allows for the direct injection of highly organic extracts due to differences in retention mechanisms compared to RPLC.

In this example, four relatively polar compounds were extracted from rat plasma with a 3:1 PPT with acetonitrile, and directly injected onto an ACQUITY UPC2 System as well as a traditional RPLC system for comparison. The UPC2 analysis was performed on an ACQUITY UPC2 BEH Column using methanol modified with ammonium hydroxide as the co-solvent (mobile phase B). The RPLC analysis was performed on an ACQUITY UPLC System with an ACQUITY UPLC BEH C18 Column, employing water and acetonitrile modified with ammonium hydroxide as the mobile phases.

Figure 1 compares the direct injection of a 1-μL injection and a 3-μL injection of a PPT extract containing caffeine on the ACQUITY UPLC System to a 7-μL injection of the same extract on the ACQUITY UPC2 System. The 1-μL injection on RPLC shows adequate peak shape, but at 3-μL injection volume the peak shape is distorted, as can be observed by peak fronting and splitting. Conversely, the injection performed using UPC2 still shows adequate peak shape at the 7-μL injection volume.

Figure 1. Example chromatograms of a caffeine PPT extract obtained from (a) 1-μL injection, (b) 3-μL injection on a UPLC operating in reversedphase mode, and (c) 7-μL injection of the same caffeine PPT extract on the ACQUITY UPC2 System.

Similar results were observed for other polar molecules tested in the same manner (Table 1). Table 1 also shows the maximum injection volume for all analytes tested in the PPT extract using both RPLC and UPC2.

Table 1. Maximum injection volume determined for UPLC and UPC2. NA = peak shape was not adequate for integration at 1 μL.

These data clearly demonstrate the benefit of analyzing polar compounds in highly organic extracts using the ACQUITY UPC2 System without further sample preparation that would be required to inject the same sample onto an RPLC system, thus simplifying the workflow.

Conclusion

By changing the MS inlet technology from standard RPLC to UPC2, it is possible to directly inject polar compounds from highly organic samples onto the ACQUITY UPC2 System without additional sample preparation steps such as evaporation and reconstitution. The addition of UPC2 in a bioanalytical laboratory provides a simplified methodology for the analysis of polar compounds in highly organic sample preparations.

720004754, July 2013

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