Lipids detection by a Waters Single Quadrupole detector

[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.

[JMB]

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.

[badescuvo]

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....

[JMB]

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.

[hfredricks]

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]