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Use of On-line Size Exclusion Chromatography (SEC) for Sample Clean-up in LC/MS Analyses

Chromatography and Mass Spectrometry Application Report

Tania A. Sasaki and Paul Shieh, Thermo Electron Corporation, San Jose, CA

Use of LC/MSn has experienced a dramatic increase in applications throughout the drug discovery and development process, from high-throughput screening to ADME/Tox studies. The Finnigan LCQ series ion trap mass spectrometers meet the demands for rapid screening of large numbers of drug candidates and their metabolites because of their sensitivity, high selectivity of MSn, ease of use, and ability to tolerate complex biological matrices. Often, compounds analyzed for drug discovery are present in biological matrices, such as plasma and serum. The task of monitoring target drug compounds and/or metabolites in these matrices can present considerable challenges when trying to achieve optimum separation, identification, and detection. It is often necessary to incorporate a time-consuming sample clean-up step into the analytical method. However, because of the growing emphasis on rapid method development and analysis in drug discovery and development, it is desirable to minimize the number of steps and overall time required in the analytical method without compromising data quality.

This report demonstrates the use of SEC as a quick online clean-up step that improves both the ruggedness of LC/MS methods and the quality of data obtained. On-line SEC also decreases HPLC column degradation, adding to the robustness of the overall analytical method. Furthermore, on-line clean-up reduces sample preparation, which saves time and minimizes potential analyte loss.

All analyses were performed using a Finnigan Surveyor MS Pump and Autosampler and a Finnigan LCQ Deca XP Plus mass spectrometer operated in both full-scan and full-scan MS/MS modes. LC conditions are shown in Table 1. The divert valve was used to transfer the first 2 minutes of each experimental run to waste, which prevented HPLC solvent for large matrix proteins, etc. from being loaded onto the LC column. A schematic diagram of the instrument is shown in Figure 1.

Ruggedness studies were performed using benzodiazepines in protein precipitated bovine plasma and tricyclic antidepressants in whole serum. The compounds and parent masses are listed in Table 2.

Results and Discussion
Developments in technology and mass spectrometer design, including orthogonal source design and Ion Sweep technology, have increased the ruggedness of the LCQ Series mass spectrometers. They are capable of withstanding multiple analyses in complex matrices, as can be seen in Figure 2. No decrease in signal was observed over 1000 loop injections of reserpine in protein precipitated plasma using 65:35 10 mM sodium phosphate buffer:MeOH as the mobile phase. Neither the plasma sample matrix nor the non-volatile phosphate salts had detrimental effects on the signal intensity.

By incorporating a size exclusion pre-column into the LC flow path, sample clean-up can be performed on-line, followed by LC/MS analysis. The second (analytical) column is an LC column appropriate for chromatographic separation of the target analytes (e.g. C8, C18, etc.)

Figure 3 shows chromatograms and mass spectra of the benzodiazepines in plasma analyzed on the SEC column only. All the large matrix compounds (proteins, etc.) elute from the column in about a minute.

There is some hydrophobic interaction between the benzodiazepines and SEC column, as they are retained for about 2-3 minutes. This difference in elution time allows adequate time for valve switching with no loss in integrity of the method. Figure 4 shows total ion and extracted ba se peak chromatograms and MS/MS spectra representative of the data obtained by SEC-LC/MS/MS. Figure 5 is a graph of 200 injections of benzodiazepines in plasma analyzed by SEC-LC/MS/MS. As can be seen from the graph, there is very little, if any, decrease in signal intensity for any of the analytes.

To further investigate the ruggedness and advantages of on-line SEC clean-up, three tricyclic antidepressants were spiked into whole serum and directly injected for SECLC/ MS/MS analysis. Direct injection of whole serum is not commonly performed because the matrix can cause problems, such as column degradation. When SEC clean-up is utilized, however, it was possible to perform sixty injections of antidepressants in whole serum without any signal degradation, as shown by the graph in Figure 6.

Without SEC clean-up, signal intensity started to decrease after less than ten injections, most likely due to column degradation from the serum matrix. Figure 7 shows chromatograms and mass spectra of the first and last injections of the antidepressant compounds in serum analyzed by SEC-LC/MS/MS. As can be seen in the figure, there is no decrease in either signal intensity or quality of data obtained.

The advantages of SEC clean-up in full-scan MS mode were also evaluated. Full-scan MS is often used for screening compounds, even though potential interferences can be more predominant in full-scan MS mode versus MS/MS. To evaluate the effectiveness of SEC as an online clean-up tool, forty full-scan SEC-LC/MS injections of the antidepressants in serum were performed. A graph of peak area of each analyte versus injection number ispresented in Figure 8. There is no significant degradation in signal intensity for any target compound over the course of the analysis. The chromatograms and mass spectra in Figure 9 show the stability between the first and last injections. Signal intensity and chromato graphic resolution are maintained, and the quality of the mass spectra is comparable, i.e. the analyte molecular ion (trimipramine) is the base peak in the spectrum. Mass spectral quality is an important factor, especially if Data- Dependent MS/MS information is to be acquired.

This investigation demonstrates the value of coupling the already robust and sensitive LCQ series of mass spectrometers with an on-line SEC clean-up procedure to dramatically increase the productivity in screening for drug candidates and their metabolites in complex biological matrices. No degradation in either signal intensity or quality of data was observed, even with direct injection of whole serum, demonstrating the improvements to overall ruggedness of the technique, especially column lifetime. This improved ruggedness can allow for reduced sample preparation requirements, resulting in less analyte loss, higher sample throughput and increased productivity.

The authors would like to thank Dr. Paul Ross, Richard C. Ludwig, and Irene D. DeGraft of Thermo Electron, Bellefonte, Pennsylvania for their technical assistance.



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