Thermo Electron

Rapid structural elucidation of compounds in complex mixtures is a powerful technique in metabolite, degradation and process control applications. The ability to follow the fragmentation pathway through sequential MS

  transitions provides added confirmation and increases the selectivity for monitoring compounds of interest in a complex mixture. Previously, the limiting factors in applying this technique were sensitivity at MS

  and higher transitions, and the cycle time required to acquire multiple MS

  spectra across a narrow LC peak. Recent developments in trapping, detection efficiencies and scan rates have reduced these limitations and enabled rapid characterization of multiple compounds from single chromatographic runs. 

A mixture of the seven drugs listed in Figure 1 was prepared at a concentration of 1nmol/mL.  			

Gradient: 0-100% acetonitrile with 0.1% formic acid in 10 min.

Injection volume: 1 mL, partial loop injection.

 linear ion trap (Thermo Electron) in positive ion electrospray ionization mode.

 was set to perform one full scan MS experiment followed by 17 automatically generated MS

 of 30%, and an isolation width of 3 amu. In addition, the use of 

 activation (WBA) was employed to further fragment ions that underwent a neutral loss of water. All spectra are the result of one microscan.  

Figure 1 Automated Data Dependent MSn Acquisition Scheme, 18 Scan Events (SE).						

The chromatographic results from the LC/MS

  analysis of the mixture of seven drugs are depicted in Figure 2. Chromatographic peak widths are 10-15 s, with several compounds co-eluting. Identification and confirmation for metabolic profiling were achieved with automated real-time data-dependent acquisition to perform MS

  experiments on the five most intense ions in the MS/MS spectra, and subsequent MS

  on the two most intense ions from the MS

  spectra (Figure 1). This results in information-rich spectra for structural elucidation and confirmation.

In this example, the cycle time to complete the 18 MS

  experiments (scan events) was less than 5 s (Figure 2), generating numerous MS

  acquisitions across the chromatographic peak, even on narrow 10 s wide peaks. In addition, maintaining high sensitivity at fast scan speeds is essential to produce high-information content from single spectra, even on low abundance ions. The MS

  spectra acquired in one 18-scan event cycle (inset in Figure 2) are displayed in Figure 3. The data demonstrate the ability to follow the fragmentation pathways with high spectral quality for the structural elucidation of unknowns and confirmation of known compounds of interest.

Figure 2 Reconstructed Ion Chromatogram of Seven Drugs, Generated from Full Scan MS Base Peak Intensities. Inset depicts 18 Data Points Resulting from Data Dependent Acquisition.			

The spectra in Figure 3 are single spectra that were acquired in the automated data-dependent acquisition. The first spectrum of the series, scan event 1 (SE 1) is a full scan MS spectrum. The next two spectra (SE 2 and SE 3) are MS/MS acquisitions on the most intense ion from SE 1, with and without WBA, respectively. The remaining 15 spectra are MS

  acquisitions following the scan event tree in Figure 1. The spectra for SE 4, SE 7, SE 10, SE 13 and SE 16 are MS

  acquisitions of the five most intense ions, respectively, from the SE 3 spectrum. The remaining spectra are MS

  acquisitions on the first and second most intense ion from the corresponding MS

  spectra. For example, the spectrum for SE 5 is the MS

  acquisition of the most intense ion, m/z 255, from the SE 4 spectrum, while SE 6 is the MS

  acquisition of m/z 446, the second most intense ion in the SE 4 spectrum.

Figure 3 Single Spectra Resulting from 18 Data Dependent MSn Acquisitions for Scan Events Described in Figure 2. All 18 Scan Events (SE) were acquired within 4.8 Seconds.			

In addition to automatically determining the mass on which to perform the MS

  acquisitions, the Dynamic Exclusion feature was applied. This automatically sets aside any masses that had been previously fragmented within defined retention time windows, thus preventing repetitive analysis of predominating ions. This is a powerful technique to analyse for co-eluting ions with lower intensities. An example of this is seen in SE 14. The experiment that we would expect for this scan event should be an MS

  acquisition on m/z 255. However, instead MS

  on m/z 281 was automatically performed because the MS

  on m/z 255 was previously analysed in SE 5 (Figure 3). 

With the advent of high throughput LC-MS/MS systems, the analysis of data becomes the bottleneck in the laboratory. Thus, software that facilitates the rapid analysis of data has become increasingly important. 

 software is one such tool. Figure 4 shows an application of Mass Frontier to help identify and confirm the seven drugs in the experimental mixture. 

Figure 4 Fragmentation Pathway for Ketoconazole using Mass Frontier Software. Fragment Masses Corresponding to Ions from MSn  Spectra in Figure 3 are noted.			

The fast scan cycle of the Finnigan LTQ, in combination with automated data-dependent mode acquisitions and ultra-high sensitivity, provide information-rich, high quality spectral data for identification, structural elucidation and confirmation of complex drug mixtures. These occur even with narrow LC peaks and in the presence of co-eluting compounds 			

This study demonstrates high sensitivity achieved with single scans in MS

  acquisitions. This method demonstrates a means of producing high quality information-rich spectra beyond the 28% low-mass cut-off rule by utilizing the superior sensitivity with higher order MS

Techniques shown here can be applied to drug metabolism, discovery, degradation and process control applications

The authors wish to thank Drs Shichang Miao and Ji Ma of Tularik for their valuable contributions.  

Articles

Rapid structural elucidation of compounds in complex mixtures is a powerful technique in metabolite, degradation and process control applications. The ability to follow the fragmentation pathway through sequential MSn transitions provides added confirmation and increases the selectivity for monitoring compounds of interest in a complex mixture. Previously, the limiting factors in applying this technique were sensitivity at MS4 and higher transitions, and the cycle time required to acquire multiple MSn spectra across a narrow LC peak. Recent developments in trapping, detection efficiencies and scan rates have reduced these limitations and enabled rapid characterization of multiple compounds from single chromatographic runs.