I'm new to LCMS technique and need some assistance. Could someone provide a reference or detailed protocol for preparing an isoleucine standard for LCMS analysis? I aim to quantify the isoleucine abundance in bacterial samples and would appreciate guidance on the preparation and validation of the standard. Thank you in advance for your help.
Nishitha Landgey I would suggest making a solution of your isoleucine in 1 mg/ml of water to start. Inject a small portion, like 10 ul. You can then see how the detector reacts to that amount and go from there. You may need to inject more or less on the column. It's important to know the column and injector capacity of material to be injected. You may want to inject only 10 ug of material.
LC-MS/MS Isoleucine Standard Protocol: Prepare certified L-isoleucine stock (0.1–1000 µmol/L) in bacterial matrix, validate linearity (r² >0.99) with internal standards. Use chiral columns (CROWNPAK CR-I(+)) to resolve isomers, optimize mobile phase (0.1% formic acid/acetonitrile), and assess matrix effects via solvent vs. lysate calibration curves.
Thanks Hanieyeh Etezadi , That was very informative and helpful. we have tried to calibrate the curve with BCCA, unable you distinguish between isoleucine and leucine in the peaks, we have used hilic column with ead fragmentation.
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