Table 4 Common problems and troubleshooting guide.
Problem | Possible reasons | Solutions |
|---|---|---|
Liquid does not pass through TopTips before loading plant samples | The slit on TopTips is too narrow | Increase the centrifugal force to make liquid pass through, or switch to a new TopTip |
Liquid does not pass through TopTips after loading plant samples | Plant debris blocks the TopTip slit | Always try to avoid transferring plant debris into TopTips Increase the centrifugal force to make liquid pass through Use a dissecting probe to remove visible plant debris |
Drift of LC retention time | Injection volume is too large | Evaporate eluate or extract to a smaller volume with vacuum concentrator Reduce the sample volume injected onto the LC |
Change of LC mobile phase | Check that correct, freshly prepared mobile phase solvents are being used Purge the LC lines after changing solvents | |
LC solvents are blocked or leaking | Replace the precolumn filter Check column connections and reinstall if needed Check LC lines for leaking solvent | |
Low yield of labeled compounds, but normal yield of endogenous compounds and/or internal standards | Slow turnover | Increase the labeling time period |
Insufficient intake of labeled precursors | Increase the concentration of labeled precursors in the labeling solution Make sure plants are bathed in sufficient volume of labeling solution | |
Broad/tailed LC peaks | Injection volume is too large | Evaporate eluate or extract to a lower volume with vacuum concentrator Reduce the sample volume injected onto the LC |
The precolumn filter is dirty | Replace the filter | |
The column is dirty | Disconnect the LC line from the MS source and wash the column with solvent according to manufacturer’s instructions | |
Analyte m/z values do not match calculated values | The MS needs to be calibrated | Perform mass calibration with calibration standard mixture according to instrument manual |
Reduced MS sensitivity | The MS needs to be calibrated | See above |
The tune file needs to be modified | Adjust source parameters for optimal ion intensity using standard compounds |