To obtain the best, most reproducible results with Echo MS system, it is important both to select the best solvent for your analyte and matrix and to ensure the flow rate is optimized for your solvent.

Considerations for solvent selection:

1. The Echo MS system is calibrated to methanol. If you change to a different solvent, the readback flow rate may not match the actual flow rate, but it will be stable and consistent. When selecting the carrier solvent, you should be aware that there is no gradient, as there is with HPLC. Therefore, non-soluble components in samples can precipitate out in the transfer line and build up over time, causing flow issues. Selection of proper sample and carrier solvents is a key component of method development and will help ensure reliability of the system. Suggested solvents and solvent mixtures are:
   • Methanol and methanol/water mixtures: Depending on the ratio, these mixtures can have a high viscosity, which will limit flow rates
   • Acetonitrile/water mixtures: Acetonitrile has low viscosity and can result in optimally high flow rates
   • 60:40 Acetonitrile: water + 0.1% formic acid: This mixture has been shown to work well for peptide-based analytes
2. Acid or volatile salts can be added to the mobile phase to improve sensitivity or peak shape. Additives may be useful to improve ionization efficiency or to minimize the impact of interactions with the materials of the flow path. The following additives and carrier solvents are recommended:
   • Up to 0.1% formic acid by volume
   • Up to 2 mM ammonium formate
   • Up to 2 mM ammonium fluoride
3. When changing the carrier solvent, the fluidics module should be purged at 1000 µL/min for 5 minutes

Process for optimizing the flow rate:

The optimal flow rate for the system will be dependent on the viscosity of the carrier solvent and should be optimized whenever carrier solvents are changed. This can be done through either the manual control window or the submission of multiple batches. This is best done through the submission of multiple batches, with AE methods of increasing flow rates, of replicate standards so that both signal to noise and %cv can be evaluated at multiple flow rates.

Manual approach

1. Purge the new solvent to be optimized through the system for 5 min at 1000 µL/min
2. Open the MS method for the analyte(s). Click Start at the bottom of the method window to begin acquisition and display the acquired signal. Set the method duration to an appropriate amount of time to test multiple flow rates (at least 5 minutes).
3. Load a plate into the Echo MS system that contains a standard at a concentration known to give a strong signal on the system
4. Open the manual control screen for the Echo MS system and set the flow rate to a low value for the system. This is often in the range of 300 µL/min, but this value is both system- and solvent-dependent.
5. Once the flow rate has stabilized for 1-2 minutes, instruct the system to perform 10 ejections of the standard using the appropriate liquid class (AQ for aqueous-based solutions; SP for less viscous solvent-based solutions). It can be helpful to include a delay time of 2000 ms between ejections to visualize the return to baseline.
6. Repeat step 5 and iteratively increase the flow rate by 50 µL/ Observe the peak shape and separation.
7. Monitor for changes in peak quality and separation. Eventually, the quality of the peaks will degrade and peaks will no longer be separated at baseline. This indicates that the vortex formed at the OPI is suboptimal and the aspiration force of the source gas is not strong enough to match the flow rate of the carrier solvent.
8. Lower the flow rate by 50 µL/min and allow the system to stabilize for 1-2 minutes. Repeat step 5. If the peak quality is restored, the current setting will be the optimal flow rate for the selected carrier solvent.

Batch submission approach

1. Create an AE method with appropriate parameters (droplet count necessary to meet sensitivity needs and AQ or SP liquid class) and a low value flow rate (~300 µL/min)
2. Create a batch of 10 to 20 repeat ejections from the well containing the standard. Select your newly created AE method and set the data filename. Use a filename convention that indicates the flow rate being tested.
3. Submit and acquire the batch
4. Increase the flow rate of the AE method in increments of 50 µL/min while holding other values constant. Change the data file to represent the new flow rate and submit and acquire the batch again.
5. Repeat step 4 until the quality of the data can be visually observed to degrade in the acquisition window, losing separation of individual ejections
6. Process the data for all acquisitions in the Analytics module in SCIEX OS software. Calculate %CV of the peak areas for each flow rate. The flow rate with the lowest calculated %CV will be the optimal flow rate for the solvent evaluated.

RUO-MKT-18-14112-A