Innovations on the QTRAP systems
Christie Hunter
SCIEX, USA
Multiple Reaction Monitoring (MRM) experiments are the most common LC-MS/MS method used for high sensitivity robust quantitation in complex matrices. While much of the time, the MRM approach provides the required sensitivity, sometimes detection can be impacted by high background or interferences from the matrix. The unique hybrid triple quadrupole linear ion trap configuration of the QTRAP system enables a novel workflow termed MRM3 which can provide an added level of specificity to a quantitative assay when required. The fundamentals of the workflow are described here.
Mass spectrometry has transformed quantitative analysis to become the method of choice for many assays. More recently, LC-MS/MS has revolutionized quantitative bioanalysis. While single MS filtering offers advantages over non-mass selective techniques, the use of tandem mass spectrometry (MS/MS, or MS2) eliminates interferences and results in a dramatic increase in selectivity which yields a very low baseline, excellent limits of quantification, and very good linearity. As a result, the Multiple Reaction Monitoring (MRM) experiment performed on triple quadrupole mass spectrometers has become the technique of choice for highly sensitive and selective quantification in biological matrices.
Mass spectrometry has transformed quantitative analysis to become the method of choice for many assays. More recently, LC-MS/MS has revolutionized quantitative bioanalysis. While single MS filtering offers advantages over non-mass selective techniques, the use of tandem mass spectrometry (MS/MS) eliminates interferences and results in a dramatic increase in selectivity which yields a very low baseline, excellent limits of quantification, and very good linearity. As a result, the Multiple Reaction Monitoring (MRM) experiment performed on triple quadrupole mass spectrometers has become the technique of choice for highly sensitive and selective quantification in biological matrices.
The speed and efficiency of ion-trap fragmentation has also been greatly enhanced on the QTRAP systems. Collision gas is now introduced through a high speed pulsed gas valve that enables a rapid increase in pressure in the LIT (Figure 2). Together with an increase in the RF drive frequency, this pulsed gas results in increased fragmentation efficiency and reduced excitation time of 25 ms or less.
In addition, the scan speed of the linear ion trap has been increased to a maximum of 20,000 through the use of the faster eQ electronics. This enabling fast MRM3 scan cycles at very high sensitivity.
Because of the configuration of the QTRAP systems, the MRM3 quantification workflow is highly flexible and leverages the sensitivity and selectivity of the system. For the MRM3 workflow, the analyte ion of interest is first isolated in the Q1 quadrupole with a user–selected resolution, usually unit resolution (0.7 Th FWHH). It is then fragmented in the Q2 collision cell, providing a broad range of product ions to be selected in the ion trap.
The in-trap fragmentation is achieved through the application of a single wavelength / narrow band excitation. As shown in Figure 3, this allows very selective fragmentation. The C12 isotope of a product ion can be specifically excited and fragmented to completion with minimal fragmentation of the C13 isotope. This provides further selectivity advantages in the removal of interfering background.
Linear Accelerator trap technology has resulted in ground breaking improvement in the handling of ions inside the linear ion trap of the QTRAP systems, resulting in up to 100x more sensitivity. Trapped ions are manipulated within the linear ion trap through the use of auxiliary DC fields provided by the addition of small electrodes (Figure 4, top). Ions are gently moved toward the extraction region of the linear ion trap during the cooling period by a voltage applied to the trap collar. A potential barrier is created by increasing the potential on the auxiliary electrodes just before the mass scan to complete the ion concentration process. The application of this axial field has a significant effect on sensitivity (Figure 4, bottom left).
In addition, a radio frequency is applied to the exit lens of the Linear Accelerator Trap resulting in further sensitivity gains (Figure 4, bottom right). These two innovations enable better than unit resolution to be obtained in the trap scan modes at these very high scan speeds.
Innovations in scanning speed, selectivity and sensitivity on the QTRAP systems enable successful implementation of the MRM3 workflow for a wide range of analytes 3,4. Sometimes, background noise or interferences can limit the detection of an analyte. Shown in Figure 5 is an example of an interference that has the same MRM transition as Clenbuterol and elutes at the same retention time. Use of MRM3 can completely remove this interference and enable a much lower detection of this analyte.