Table 3.

Troubleshooting of the UHPLC-EISA-TOF method for the quantification of DL-2-HG in biological samples

Step	Problem	Observation	Possible reason	Solution
Derivatization (Step 16)	Derivatization did not work	Standards or samples do not turn into yellow or brownish color	Not all water was evaporated. Alternatively, it could also be possible that the sample is not very concentrated and thus the color is not that noticeable.	Make sure all water has been evaporated before adding L-DATAN solution.
Liquid chromatography instrument (Steps 24–26)	High column backpressure	Pressure near 400 bar and/or low reproducibility of the retention time	Column may be clogged	Clean the column using 2-propanol. If the problem persists, replace the column
	Pressure fluctuation	The pressure is not constant	Valve or pump seal leaks and might be damaged	Replace the check valve and the pump seals
	Loss of chiral separation	The resolution of the peaks is lower than expected	Mobile phase A and B are not prepared correctly or column is defective	Make sure mobile phase A and B are well prepared and/or replace the column
Mass spectrometry instrument (Steps 21–23)	No peaks appear	Ions of the compound are not observed	Some parameters are not set correctly setup and/or the derivatization step was not successful	Check the MS parameters and/or make sure the derivatization was conducted in the proper way
	Poor mass accuracy	Peaks are not observed when they are extracted with a width of m/z ±0.01	The system is not well calibrated	Calibrate the MS detector according to the manufacturer’s instructions
	Loss of sensitivity	Ion counts are too low	The ion source may be contaminated	Clean the ion source, spray shield and the glass capillary following the manufacturer’s instructions