General recommendations when beginning method development

Objective: The purpose of this document is to provide a quick reference for transferring MRM-based quantification methods from a SCIEX Triple Quad or QTRAP 6500+ system to a SCIEX 7500 system. While the best sensitivity can be obtained by performing a full compound/source/gas optimization, the recommendations presented here will generally suffice for routine assays. The SCIEX 7500 system features the same quadrupole rodsets as the SCIEX Triple Quad and QTRAP 6500+ systems, which makes it easy to transfer methods, and the m/z and collision energies of the compounds will be the same. Analyst software methods can be directly converted into SCIEX OS software methods using the conversion option in SCIEX OS software (Figure 1).

Figure 1. Analyst software methods can be converted into SCIEX OS software methods. To convert Analyst software methods into SCIEX OS software methods, use the conversion option when creating a new MS method in SCIEX OS software.

The main difference between the platforms is in the source and the front end of the systems.

The SCIEX 7500 system is equipped with the fourth generation of the Turbo V ion source: the new OptiFlow Pro ion source. The OptiFlow Pro ion source is equipped with the E Lens probe, which delivers greater sensitivity in electrospray ionization (ESI), with increased field strength and ion generation, through more energetic ESI droplet desolvation. Since the OptiFlow Pro ion source works at a lower ion spray (IS) voltage compared to the Turbo V and IonDrive Turbo V ion sources, some amount of source parameter tuning is required for method transfer.

The recommended initial source conditions for the OptiFlow Pro ion source are listed in Table 1. Note that:

• The source temperature will need to be higher with a higher flow rate and/or higher aqueous composition in the mobile phase.
• CUR should be set as high as possible without incurring significant signal-to-noise loss.
• There are no physical adjustments with the OptiFlow Pro ion source.

Spray voltage optimization

The OptiFlow Pro ion source works at a lower IS voltage compared to the Turbo V and IonDrive Turbo V ion sources. It is very important to reoptimize the IS voltage to achieve the best sensitivity. Ionization voltage optimization can be done using tee infusion, flow injection analysis (FIA) or on-column optimization in the 1,000 to 5,000 range for positive polarity and the -1,000 to -4,500 range for negative polarity with increments of 250 volts. Spray voltage should be set as low as possible without incurring significant signal loss. Spray voltage generally trends toward lower IS at higher flow rates. More details on source tuning can be found in this community post.

 Q0 dissociation optimization

On the SCIEX Triple Quad and QTRAP 6500+ systems, declustering is achieved as a potential difference between the orifice plate and the dual QJet ion guide. On the SCIEX 7500 system, the larger orifice and short residence times lead to the orifice voltage having little to no effect on ions in that region, thus declustering potential (DP) has been removed from tunable parameters. Declustering on the SCIEX 7500 system is achieved with voltage difference between the QJet ion guide and IQ0 (Q0D simple). This is a parameter that can be ramped and is useful for de-tuning signal, reducing interferences and increasing signal-to-noise ratio in some cases.

Q0 dissociation can be enabled using the advanced settings under MS method in SCIEX OS software. The default value for Q0 dissociation is set at simple -10 and can be optimized in the -10 to 200 range for positive polarity and the 10 to -200 range in negative polarity. It is highly recommended to optimize Q0 dissociation on column in matrix samples to achieve the best signal-to-noise ratio and to reduce any isobaric interferences eluting along with analyte peak.

Each transition in a method can be optimized with a different Q0 dissociation value (Figure 2), even to the same precursor. And, as always, dwell time is adjusted to ensure a minimum of 10 data points across the peak. Data from the matrix sample injected with the MS method with different Q0 dissociation values can be processed in the Analytics module in SCIEX OS software, and the value with the best signal-to-noise ratio should be selected for the method.

Figure 2.  Effect of Q0 dissociation (simple) on signal-to-noise ratio and isobaric interferences. Signal-to-noise ratio is shown at different Q0 dissociation values (left), with a chromatogram (overlay XICs) showing minimized isobaric interferences with different Q0 dissociation values (right).

 Chromatography optimization

It is recommended to reoptimize chromatography in cases where a high background is observed in matrix samples on the SCIEX 7500 system compared to the SCIEX Triple Quad and QTRAP 6500+ systems when Q0 dissociation doesn’t help with signal-to-noise improvement. Due to the high sensitivity of the SCIEX 7500 system, isobaric interferences in matrix samples can sometimes show up with a higher signal compared to the SCIEX Triple Quad and QTRAP 6500+ systems, as they are now in a detectable range on the SCIEX 7500 system. In these cases, it is recommended to separate the peak of interest from the isobaric interferences to achieve the best signal-to-noise ratio for the analytes in the matrix sample.

References

1. For more information on the OptiFlow Pro ion source, refer to Source tuning on the OptiFlow pro ion source.
2. For more info on general source tuning across the SCIEX suite of source options, refer to General source/gas settings to use when starting method development.

 

RUO-MKT-18-13581-A