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If you encounter a question in connection with your kit usage, sample preparation or software use, our dedicated specialists are available to help you.
MetIDQ software issue
A1 No connection to the oracle database
A2 MS data import to MetIDQ™ failed.
MetIDQ™ requires several Windows® runtime components to process MS data (.wiff or .raw). Please refer to the MetIDQ™ user manual section 1.1 “MetIDQ™ Requirements” and check if they are all installed.
A3 How can inter-plate data normalizations in MetIDQ™ be performed?
Data normalization is strongly recommended for multi-plate experiments to minimize batch effects and improve reproducibility. To normalize a dataset, perform the following steps:
- Create a “Report Context” in MetSTAT and make sure the “Condition” is set to “Pending”.
- Link all samples from all kit plates being analyzed in MetSTAT together with the reference sample that is to be used for normalization (Biocrates® QC or own study pool).
- The reference sample should have been measured in replicates of three or more on each kit plate. The “Minimum number of replicates for normalization” is defined in the “Settings > MetSTAT”.
- If your own study pool sample is beeing used for normalization, the MetIDQ™ Sample Barcode of each replicate must be the same.
- Activate the Data Normalization in MetIDQ™ and select the sample to be used for normalization from the dropdown menu. Please refer to the MetIDQ™ user manual sections 7.1 and 7.2.
A4 How can out of range QC accuracies be interpreted?
The kit performance is usually good even if the accuracy of some metabolites from the QC samples are out of range in MetVAL.
- For metabolites with the Analyte Classification “Quantitative”, QC accuracies within the acceptance ranges are expected.
- For metabolites with the Analyte Classification “Relative Quantitative” or “Quantitative with Restrictions” (1-point calibrated metabolites), QC accuracies out of range can occur. Ionization efficiencies among analytes and internal standards are not identical between different instrument types and may result in reported accuracy measurements that are out of range. This, however, can be compensated by data normalization.
- The classification “quantitative” or “relative quantitative” has no impact on the statistical power of your biological interpretation. The results of the relative quantitative compounds have the same analytical quality with respect to precision (reproducibility) as the results of the quantitative compounds.
- If QC values do not meet the expected concentrations given in MetIDQ and the accuracies are therefore out of range, how can the kit performance be evaluated?
- Check if the QC accuracies are consistent over all QC levels and replicates.
- Check if the reproducibility (CVs) of the analytes above LOD in the measured replicates are OK.
- If points 1 and 2 are fulfilled, the performance is good, and the kit run was successful.
Sample preparation issues
B1 The extracts appear cloudy
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 didn’t observe any wastage of hardware parts.
B2 Extracts didn’t go through the filters or the volumes in the capture plate are low/ inconsistent.
Instead of methanol, water or any other solvent has been used for making the extraction solvent. 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).
B3 Many signals in the blank injection/ many analytes in the zero samples are above LOD
The blank well A1 does not contain any analytes or internal standards. The zero samples only contain internal standards, but no analytes. If there are still peaks the following may have occurred:
- A sample or internal standard mix was accidentally loaded.
- Spillover when loading the extraction solvent onto the kit plate, e.g. the dispensing speed of the repeater was too high.
- Spillover when shaking the plate after loading the extraction solvent.
- Cross-contamination by residues at the bottom of the filter plate when removing the filter plate from the capture plate.
- Cross-contamination when transferring the extracts to make the LC and FIA plates.
- To make sure the contamination comes from the plate and not from the system, inject a blank from an autosampler vial to cross-check
LC-MS performance issues
C1 What should FIA peaks look like and how can the peakshape be improved?
- 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.
C2 Cleaning schedule of optimal performance
- 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 for 20 min at 100% A to get rid of salts, followed by another 20 min at 100% B to get rid of lipids.
- 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.
C3 Loss of sensitivity
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:
- Sample preparation:
- 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 used testmix/test sample 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.
C4 Retention times are shifted
- Make sure the mobile phases are purged and there are no bubbles in the system. Also make sure no air is drawn or injected by the autosampler.
- Make sure the entire LC-MS system is well conditioned and the column is sufficiently equilibrated.
Otherwise, check the following parts with your instrument engineer:
- Mixing chamber (needs to be replaced?)
- Intake/outlet valve at pump head
- Seals at pump head
C5 AbsoluteIDQ® p400 HR Kit does not meet SST criteria from the manual
®The SST criteria given in the manual are based on our Q Exactive Focus connected to a Vanquish UHPLC system (used for kit development and validation). The sensitivity of another Q Exactive instrument, as well as of another Q Exactive Focus can be different, depending on the instrument condition. The following ideas can help to improve the instrument performance. Otherwise please check with your Thermo engineer.
- Make sure the Ring Position is B.
- Clean or replace the ion transfer tube.
- Clean the S-lens.
- Was the customized calibration successful?
- Check if the ESI needle is in the correct position and tight (the inner needle can sometimes slip out at higher flow rates and is then no longer in the optimal position). Clean or replace the ESI electrode if necessary.
- The spray voltage that we give in the protocol gave best results during kit development on our Q Exactive Focus. However, you can try other spray voltages. You can go up to 4 kV and compare if the intensities are getting better.
- Check gas supply.
- Perform instrument service.
- See other FAQs: Q-C2, Q-C3.
Can’t find the answer to your question?
Please provide us with the following information in the message section to help resolve the issue promptly.
- LC-/MS Instrument type
- Kit you are working with
- Short description of the issue
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