LCT Premier – Routine Automated Exact Mass Measurement

For LC-MS applications, the LCT Premier provides the user with the ability to acquire high resolution MS data and therefore the ability to generate an exact mass measurement to within 3 ppm. Using the LockSpray ion source, traditional problems associated with exact mass measurement during LC-MS acquisitions are overcome - no multi point reference masses, no post-column addition of the lock mass, no ionization interference between your analytes and lockmass, and no nominal mass interferences. Having the ability to acquire an exact mass measurement aids determination of elemental composition for a particular analyte of interest, which provides the analyst a high degree of specificity and therefore a high level of confidence in the analytical result.

Benefits

• Determine the elemental compositions of analytes
• Distinguish between nominally isobaric components
• Distinguish between analyte and matrix related material
• Improve database search specificity
• Increase your confidence in analyte identification

Why Exact Mass?

As with every analytical tool, the more accurate your measurement the more confident you can be with your results. For example, when carrying out a metabolism investigation following an in vivo experiment are you sure that a peak detected at m/z 342.1 is an actual metabolite or a matrix related component or even a solvent contaminant? Mass measuring the peak of interest to 4 decimal places will give you that confidence. By reducing the error on a mass measurement, the possible elemental formulae that can fit a particular measurement significantly reduce, therefore providing better specificity. Figure 1 clearly shows this. Such mass measurement accuracy is not routinely available with typical scanning instruments such as quadrupole or ion trap mass spectrometers. For routine and easy-to-use exact mass measurement, orthogonal acceleration time-of-flight (oa-Tof) mass spectrometry provides the solution.

Exact mass measurement on an oa-Tof can help to: 

• Determine the elemental compositions of analytes
• Distinguish between nominally isobaric components
• Distinguish between analyte and matrix related material
• Improve database search specificity
• Increase your confidence in analyte identification

For LC-MS applications, the Waters Micromass LCT Premier benchtop oa-Tof Mass Spectrometer provides the user with the ability to acquire high resolution MS data and thus generate an exact mass measurement. In addition to the next generation of instrument electronics and oa-Tof optics, LCT Premier is equipped with LockSpray as standard. LockSpray is a novel dual electrospray ion source that was designed for dedicated exact mass measurement. Your LC column is connected to the analyte sprayer while the single point reference mass, used to exact mass measure your data, is introduced into the reference sprayer.

The eluent from each of the two sprayers is kept completely separate by a baffle that is indexed via software control for total data integrity. During an experiment, data from the reference sprayer is typically sampled once every 10 secs and the reference data is automatically applied to the analyte data to provide an exact mass measurement. LockSpray overcomes issues associated with traditional exact mass measurement, i.e. post-column addition of a lock mass and potential ionization interferences between analytes and lock mass etc.

Examples of Mass Measurement Accuracy on LCT Premier

1) Single Analyte Exact Mass Measurement

• LockSpray with positive ion electrospray was used.
• Raffinose (500 pg/μL in 50/50 MeCN/H₂O) was infused into the analyte sprayer over 30 mins at 5 μL/min. The [M+Na]⁺ ion was measured (C₁₈H₃₁O₁₆Na, m/z 527.1588)
• Leucine enkephalin (50 pg/μL in 50/50 MeCN/H₂O + 0.1% formic acid) was infused into the reference sprayer at 5 μL/min. The [M+H]⁺ ion was used as the lock mass (C₂₈H₃₇N₅O₇, m/z 556.2771)
• The reference sprayer was sampled every 10 secs and 10 measurements were taken over the 30 min acquisition
• The spectral resolution was 10,000 FWHM

Table 1 below lists the exact mass measurements along with the calculated mDa and ppm errors from the theoretical. The RMS ppm error for all 10 measurements was calculated at 1.32 ppm.

2) Multiple Analyte Exact Mass Measurement during an LC-MS Experiment

• LockSpray with positive ion electrospray was used
• An 8 component analyte mixture was separated using the following conditions:

HPLC: Waters Alliance HT 2795

Mobile phase A: H₂O + 0.1% formic acid

Mobile phase B: MeCN + 0.1% formic acid

Gradient: 95% A to 5% A over 10 mins

Column: Waters Atlantis dC₁₈, 2.1 x 100 mm, 3 μm

Flow rate: 0.3 mL/min.

Injection volume: 10 μL

• Leucine enkephalin (50 pg/μL in 50/50 MeCN/H₂O + 0.1% formic acid) was infused into the reference sprayer at 5 μL/min. The [M+H]⁺ ion was used as the lock mass (C₂₈H₃₇N₅O₇, m/z 556.2771)
• The reference sprayer was sampled every 10 secs.

Figure 4 shows a typical chromatogram obtained with the retention time for all 8 components. Table 2 lists the exact mass measurements obtained along with the calculated mDa and ppm errors from the theoretical. The RMS ppm error for all measurements was calculated at 1.8 ppm.

This example shows that the LCT Premier provides excellent mass measurement accuracy across a wide range of compounds with varying mass, physical properties and retention time during a reversed-phase LC gradient. This provides improved specificity during an LC-MS experiment providing the analyst with increased confidence in the analytical result.

3) Exact Mass Measurement during Positive/Negative Ion Mode Switching

Having the ability to carry out ionization mode switching in real time provides the analyst with an additional piece of information during the same LC-MS experiment. Ionization mode switching is often regarded as being useful for when the expected mode of ionization for a particular compound is unknown e.g. compound screening experiments. With the additional function of being able to generate an exact mass measurement during ion mode switching experiment in real time, further confidence in that the correct ion has been detected is obtained. 

With significant improvements in electronics and detector hardware, the Waters Micromass LCT Premier now has the possibility to switch ionization mode during a real time LC-MS experiment in 300 msecs - providing an exact mass measurement and a sufficient number data points to define a typical chromatographic peak. The previous oa-Tof instrument, the LCT, was only able to switch ionization mode during a real time experiment in 1 second, due to the stabilization time required when the older generation high voltage electronics were switched polarity.

•  LockSpray with positive/negative ionization mode switching was used
• An Inter Scan Delay (ISD) of 0.3 secs was used
• The reference sprayer was sampled every 5 secs
• Raffinose (C₁₈H₃₂O₁₆, 500 pg/μL in 50/50 MeCN/H₂O) was infused into the analyte sprayer at 5 μL/min

• [M+Na]⁺ = 527.1588 m/z

• [M-H]^- = 503.1612 m/z

• Leucine enkephalin (C₂₈H₃₇N₅O₇, 50 pg/μL in 50/50 MeCN/H₂O + 0.1% formic acid) was infused into the reference sprayer at 5 μL/min

• [M+H]+ = 556.2771 m/z

• [M-H]^- = 554.2615 m/z

Figure 5 shows a typical positive and negative ion spectrum for raffinose that was acquired during the experiment. The exact mass measurements shown are representative of the data that is typically obtained during a positive/negative ion mode switching experiment with LCT Premier.

For LC-MS applications, the LCT Premier provides the user with the ability to acquire high resolution MS data and therefore the ability to generate an exact mass measurement to within 3 ppm. Using the LockSpray ion source, traditional problems associated with exact mass measurement during LC-MS acquisitions are overcome - no multi point reference masses, no post-column addition of the lock mass, no ionization interference between your analytes and lockmass, and no nominal mass interferences. Having the ability to acquire an exact mass measurement aids determination of elemental composition for a particular analyte of interest, which provides the analyst a high degree of specificity and therefore a high level of confidence in the analytical result. 

LCT Premier – exact mass measurement made easy for LC-MS applications.