I am using waters TQS systems to quantify cortisol in my method but I also have mass transitions (184>184 and 104>104) to record phospholipid profile. I see too high background for both mass transitions throughout the whole 5 min long chromatogram even in the blank injections. There is high background even if I run the solvents bypassing the column. Any suggestions or comments?
Regards,
Khem
Dear Khem,
The product ions at m/z 184 and 104 (184 minus H2PO3) arise from the phosphocholine polar head present in glycerophosphatidylcholine lipids, one of the major phospholipid class in mammalian fluids and cells.
Considering you have high signal even in blank injections I would say the system is contaminated (HPLC column or MS source). I´m guessing your sample was too concentrated when you injected it. Have you done phosphate quantification to your extract?
Once you have the system cleaned (HPLC column, MS source and injection syringe) try and dilute your sample 1000fold before injection. This will also dilute the cortisol signal but as your using MRM to quantify cortisol then you should be fine.
Hope this helps,
Ana
Dear Ana,
Thank you very much for your comments and suggestions. Both parent and product ions are same either 184(parent)-184(product) or 104(parent)-104(product) in MRM. The signal is there when I am running without column, so contamination (if any) could arise, as you also said, from MS source or injection syringe. I also tested running only solvent A (water) and only solvent B (methanol) without column for 5min, and both give such background but with solvent B the background signal is very high at the beginning which drops gradually for almost 1.5 min and seems to be stable until the whole run time. I really did not understand why the signal was high and why it dropped gradually for 1.5 min while it was running only with 100%B without column. We have two instruments of the same type routinely used for steroid analysis from serum and both instruments give high background for 184 and 104. For my knowledge I am not sure if the instrument was used for phosphate quantification earlier but not at the moment at least. I will try next time with cleaning the MS source and syringe.
Dear Khem,
Based on your description your whole system is dirty (due injection of too concentrated samples).
You´ll need to clean it properly, do gradient wash on your column and MS source. In case you only clean MS source then the HPLC column will contaminate your system, in case you clean HPLC but not the source then you´ll see the contamination downstream in your MS spectra.
Wash your column (off-line) with washing eluent for 1-2 days and then connect to MS and check for signal. While cleaning HPLC column disassemble MS source and wash.
If problem persists then it might be better to call for technical servicing.
MS instruments are very sensitive so they´re valuable to detect and quantify molecules in very small amounts (nmoles or pmoles). Molecules present in mmoles or that easily ionize (as is the case of phosphatidylcholines in biological samples) will easily contaminate the system and lead to lots of work to clean.
You could try and quantitate inorganic phosphorous to get an idea on the amount of phospholipids in your samples and work from there to inject the minimum amount possible to generate a cortisol signal with good S/N ratio. In case the amount of sample needed is too high then probably you should use another detection technique (UV, fluorescence) in your work otherwise you´ll experience the same problem over and over again.
Hope this helps,
Ana
Dear Ana,
Thanks a lot for the detail information. I will check couple of things according to your suggestions. Thank you once again.
Regards,
khem