Chromatography Forum

Hello friends,
Has any of you detected phospholipids (DMPC, DMPG, HSPC) and Cholesterol with a Waters LC-MS UPLC/SQD (Single Quadrupole Detector)? I used a Waters UPLC@BEH C18 (2.1 mmX50 mm, 1.7 um), I see some peaks with the desired m/z in the XIC with 99%MeOH/H2O with 0.1% HCOOH (starting with a gradient H2O/MeOH), but there is a lot of background noise in the blank (50%MeOH/H2O), difficult to interpret.
Any input is very much appreciated.

Since you are looking at known compounds, they have known Mol. Wts. and can be located using the "mass chromatogram plot" feature of the data system.

Plot [M + H]+ or [M - H]- as appropriate, for each compound. Sum spectra across the chromatographic peak ONLY where the peak is 37% above baseline.**

Subtract some adjacent background, et voila......

**This 37% is neither an arbitrary number, nor is it off the top of my head. It comes from a paper in Anal. Chem. about 15-20 yrs ago, that I cannot now locate. This number is selected to minimize dilution with spectra of the background.

Cholesterol has monoisotopic mass of 384.4, so initially set [M+H]+ window at m/z 385.0 - 387.5, so that you include the 13C, 2H and 18O isotopes for better sensitivity.

thank you, JMB! The software used with this UPLC/SQD is Empower_Acquity. Is the massplot available in empower?
I am trying to use the "sample tune" feature in Acquity, infuse the pure analyte directly in the detector with Intellistart, and then Start sample tun. I do not know how to do it, I believeI have to put my mass there, but I do not know how to visualize the peak....

Mass Spec. software from ALL manufacturers has the ability to plot the Intensity vs Time of a selected m/z value. Either a single m/z 385.3 or a range, m/z 384-387.5 etc.

When I was searching for the [M+H]+ of an unknown that did not give a strong response, I would typically scan a chromatogram using m/z windows that were 5 amu wide. E.G. m/z 200-204, 205-209, 210-214.....upto m/z 750-754, for example. Suppose that I found a chromatographic peak at m/z 330-334 window---would then sum spectra over the peak to find the [M+H]+ value.

You need to read the instruction manual slowly and carefully--if still in difficulties, contact their Technical Support for help. I am not familiar with the 'Empower' software.

Hi, Just a quick comment - some membrane lipids have to form adducts when they don't ionise easily on their own - we add ammonium hydroxide and formic acid to maximize adducts of one type- compared to scavenging sodium, potassium, chloride etc etc. you'll either see +H or +NH4 in positive, and -H or +formate in negative - this can have a significant impact on detection limits for those lipids. We also prefer to use a normal phase HPLC method for membrane lipids, a diol column with hexane / IPA eluents - then your membrane lipids are separated according to headgroup and not fatty acids... makes like much easier - not sure what cholesterol looks like on that method though, probably isn't retained.
even if you don't change your HPLC method try looking for other adducts that might be there - PG and PC both readily form Na adducts in +ve. good luck





[quote="badescuvo"]Hello friends,
Has any of you detected phospholipids (DMPC, DMPG, HSPC) and Cholesterol with a Waters LC-MS UPLC/SQD (Single Quadrupole Detector)? I used a Waters UPLC@BEH C18 (2.1 mmX50 mm, 1.7 um), I see some peaks with the desired m/z in the XIC with 99%MeOH/H2O with 0.1% HCOOH (starting with a gradient H2O/MeOH), but there is a lot of background noise in the blank (50%MeOH/H2O), difficult to interpret.
Any input is very much appreciated.[/quote]