The migration process takes a long time even without raw data and we want to establish a simple and straightforward migration process. Since the raw data makes up the bulk of the data, we refrain from transferring it. This saves the customer days of migration and an average database is migrated in a few hours. If the customer still likes to import the raw data, he can simply upload it to WebIDQ afterwards (without changing the integration).
The data processed under WebIDQ aligns with the data processed under MetIDQ, see PCA plot below. Comparability is further ensured through data normalization in WebIDQ which corrects for deviations in accuracy and batch effects.
Changing to the new software can have a major impact on the routine workflow and requires database migration, training and familiarization of the kit users. Moreover, when publishing results, all data should have been processed with the same software to avoid confusion for the reviewer and reader. To minimize complications and maintain a consistent workflow, we do not recommend updating the workflow management software during any ongoing project. However, before starting with a new project, we strongly encourage you to upgrade to WebIDQ and to migrate your MetIDQ database. As of May 1, 2024, MetIDQ will become obsolete and will no longer be supported.
The lipids are annotated according to head group, total number of carbons in the chains, and total number of double bonds in the chains. For example, PC(16:1/20:5) becomes PC(36:6).The lipid analysis in the p180, p400 HR and MxP Quant 500 kits does not provide specific information regarding either the positions or chain lengths of the fatty acid residues linked to each lipid’s backbone. Consequently, the reported signal is a sum of several isobaric/isomeric lipids. For more detailed information please refer to the list of isobaric lipids for each kit: p180, p400 HR, MxP Quant 500 (available on request)
For AbsoluteIDQ® p180, p400 HR and MxP® Quant 500 kits
When adding water to the methanolic sample extracts to make the LC plate, cloudiness can occur due to residues of pyridine and phenyl isothiocyanate from the derivatization solution. If that happened, perform the following steps:
Extend the shaking time at 600 rpm to 20-30 min.
If the extracts are still cloudy, leave the plate standing for 15 min at room temperature and shake for another 15 min at 600 rpm.
Afterwards, run the plate, even if the extracts are still a bit cloudy. We found that the cloudiness has no impact on the kit performance or on the quality of the data. Also, we did not observe any wastage of hardware parts.
Please be aware that performance-related issues that cannot be resolved by instrument cleaning are not within biocrates’ responsibility and must be handled by the user or the LC-MS engineer. Based on our experience, however, we are happy to share some suggestions:
Make sure the testmix/test sample has been reconstituted or diluted correctly.
Make sure there was no air in the pipette tip when transferring sample to another vial.
Make sure the testmix/test sample used was not expired.
Instrumental setup: double-check all method parameters and make sure the analytical column and mobile phases were prepared and installed correctly.
Connections: check for leaks and retighten all connections.
Injection needle: the injection needle might not be correctly adjusted or draws air. Adjust positioning or replace the needle.
Injector: check for leaks or contaminations in the injector valve and rotor seal. Clean or replace the valve.
Divert valve: set to waste instead of MS. Check setting at valve and in method.
ESI electrode: leak, contamination, or defective. Reassemble ESI probe, clean with different solvents at high flow rate, or replace ESI needle.
MS: perform instrument front-end cleaning (Orifice, QJet, quadrupole, ion transfer tube etc.). Tune and calibrate the instrument.
If the QC accuracies are consistent for all measured analytes, but differ significantly from the expected values (i.e. all metabolites for any particular QC sample are at 50% accuracy), the following may have occurred:
Pipetting issue: More or less than 10 µL of the QC sample was transferred to the plate. An air bubble in the pipette tip, pipette tip missed the center of the filter paper, or excess liquid was attached to the outside of the tip. It could also be that the internal standard mix was not transferred correctly (not applicable to the MxP® Quant 500 kit).
Reconstitution issue: The QC vials were not resuspended in the correct volume. For instance, if 200 µL were used instead of 100 µL, the accuracies would be around 50%.
Mixed QC samples: The QC samples were not pipetted in the correct order.
Calibration standard issue: Check both the FIA and LC parts of the kit. If the problem only occurs in the LC part of the kit, it is likely related to a problem with the calibration curves or calibration standards might have been mixed up during pipetting onto the kit plate (e.g. Cal3 to Cal4 well and vice versa).
Frequent cleaning actions and rinsing methods are strongly recommended to minimize the risk of instrument contaminations or performance loss.
For optimal time-efficiency, clean and rinse the instruments routinely between the kit runs while a new kit is prepared (see recommendations below).
Monitor test sample intensities each time you run the SST and perform troubleshooting when you observe significant signal decrease. Clean and rinse system again if required.
Check immediately after each plate run if all injections have worked and if the peaks are visible and have a good shape. In case of missing peaks or bad shapes, troubleshoot and reinject the plate or affected samples.
The instrument requires a full service at least every 6 months when the kit is used in high throughput.
Please be aware that performance-related issues that cannot be resolved by instrument cleaning are not within biocrates’ responsibility and must be handled by the user or the LC-MS engineer!
Cleaning actions after every plate run (LC or FIA):
Rinse the column for 60 min using wash solvent according to user manual (after LC plate only).
Install 10% methanol (in water) as mobile phase A and wash solvent as mobile phase B. Rinse entire LC-MS system:
15 min at 100% A offline (to waste, MS switched off)
15 min at 100% B offline (to waste, MS switched off)
15 min at 100% A online (to source/ESI needle, MS switched on)
15 min at 100% B online (to source/ESI needle, MS switched on)
Keep the wash solvent as mobile phase B, transfer some wash solvent to autosampler vial, and perform 10 blank injections using 100% B.
Before FIA run:
Install FIA mobile phase for the assay, purge and flush the entire LC-MS system with FIA mobile phase at 1 mL/min until the pressure is stable.
Perform another 10 blank injections using FIA mobile phase as blank solvent. Perform more blank injections to decrease background noise if necessary. Continue with SST according to user manual.
Before LC run:
Install mobile phases A and B for the assay, purge and perform another 10 blank injections using 50% methanol as blank solvent.
Perform more blank injections to decrease background noise if necessary. Continue with SST according to user manual.
After every 2 kits:
Rinse the autosampler for 20 min with 10% methanol (in water) to get rid of salts, followed by another 20 min with wash solvent to get rid of lipids (in the same way as the entire LC-MS system above)
Clean Curtain Plate or Cone according to manufacturer’s instructions.
Wipe the Orifice (without removing the Orifice plate) using a foam rod, first with water, followed by isopropanol and methanol.
Wipe the tip of the ESI electrode using a foam rod, first with water, followed by isopropanol and methanol.
After every 5 kits or more:
Replace ESI electrode after every 5 kits.
Perform instrument front-end cleaning (Orifice and QJet or Q0) after every 15-20 kits.
The best peak shapes are obtained by avoiding unnecessary dead volumes (caused by valves or connection parts) and connecting the injector directly to the mass spectrometer. For this, red PEEK tubing is recommended.
The FIA peaks must be located approximately in the middle of the data acquisition window, rather earlier than later. It is important that the peaks are not cut off or running out of the window.
The peaks must not show “satellite” or large shoulder peaks.
The peaks must have a defined beginning and ending without interruptions. They must not show huge valleys in between or be too jagged (stable spray).
Jagged peaks are typically caused by an instable electrospray. In that case, rinse at high flow rate using different solvents or replace the ESI electrode.
Huge and broad valleys can be caused by ion suppression. In that case, dilute the samples further.
Refer to the user manual for more information and SST criteria.
Inconsistent or lack of volume in the filter plate may occur for several reasons. Below are some suggestions:
Water or another solvent was used for making the extraction solvent instead of Methanol. Perform the following steps:
Transfer the extracts back to the filter plate.
Dry the samples under nitrogen.
Dissolve again using pure methanol (volume according to extraction step in user manual).
The pressure manifold was not set up correctly.
Check that the pressure manifold settings are correct according to the instructions.
see document “Instructions using pressure manifolds for 96-well plates with biocrates kits”
Put the plate back in the manifold and try again.
If the problem persists, increase the low flow pressure up to 30 psi
Droplets of liquid remaining on the underside of the filter plate. It is normal if a small amount of liquid is on the underside of the filter. Internal standards will account for small volume discrepancies. Take care that there is no cross contamination into other wells when removing the top filter plate. If large droplets remain on the underside of the filter plate:
Put the plate back into the pressure manifold and run at a higher pressure (30 psi).
While the filter and capture plate are still secured together by tape, tap the plate lightly on the table to shake the droplet loose.
It is also possible that some amount of volume was lost during the shaking step or when transporting the plate. You can check the wells with lower volume in the capture plate to see if the remaining volume is still in the top filter plate. If the top filter plate is empty, then it’s likely that the volume was spilled at some point during the preparation. Take extra care during shaking and carrying the plate that nothing is spilled when the wells are uncovered!