While an in-depth discussion of method development and optimization for the Echo® MS system is beyond the scope of a community post, here are some points to consider as part of the process:
- The maximum recommended ion spray voltage for prolonged electrode life is 5000 V in positive mode and 4500 V in negative mode.
- Evaluation of peak shape and ionization suppression in the matrix is recommended if the method is initially developed using neat standards. Dilution may be required if the matrix causes unacceptable peak shape or suppression. Note that while it may seem counterintuitive, diluting the sample to dilute the matrix can sometimes help improve overall assay quality and sensitivity.
- Conversely, if the matrix does not cause suppression issues or degradation of the peak shape, a droplet ladder test in the matrix can be performed to evaluate if increasing the droplet count improves sensitivity.
- Pure methanol is generally the carrier solvent of choice for initial method development, but if satisfactory peak shape or sensitivity cannot be initially achieved, sensitivity and signal to noise can be investigated using alternative carrier solvents and electrospray-friendly modifiers. For example, peptides often show better performance in 60:40 acetonitrile:water with 0.1% formic acid by volume.
- When changing solvents, it is important to remember that the carrier solvent pump is calibrated for delivery of methanol. Other solvents may be delivered at rates different than those set in the method, but they will be consistent. Also, the optimal carrier solvent flow rate is solvent-dependent. So, if you do change solvent during method development, flow rate optimization should be performed with the final carrier solvent to get the best performance.
Recommended starting conditions
When optimizing a new compound or beginning method development for a new assay, it’s helpful to have a series of conditions that can be used as a starting point. Note that when developing a new assay for a new compound, it’s recommended to optimize the compound using a mostly aqueous standard first to generate the MRM(s) if they are not already known, and to then refine the acoustic ejection method, which includes selecting the carrier solvent and modifier.
Here, we share a few conditions to help you get started (keep in mind that while these conditions may not be optimal for your final method, they should produce peaks that can be further improved upon):
- Prepare the compound to be optimized in a mostly aqueous solution at a high ng/mL concentration. The concentration necessary for optimization is very compound-dependent. MRM values that have been optimized on another SCIEX Triple Quad or QTRAP 6500+ system can be used and the compound-dependent parameters should not require further tuning.
- For the source conditions, specify 90 for GS1, 65 for GS2, 5000 for ISV and 350°C for Temp.
- Start with methanol as a carrier solvent with a flow rate of 350 µL/min (or optimal flow for methanol if already determined on the system).
- Selection of AQ or SP is not critical at this point and should be evaluated in the matrix.