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lipid and fatty acid profiling
Posted: Thu Mar 23, 2017 6:24 pm
by arnabroy81
I have only a normal C18 reversed phase column. Can I do lipid or fatty acid analysis with it by LCMS?
I had injected oleic acid few days ago (direct injection) and got signal. But when I ran an LCMS method, I didn't get anything. I had run gradient from 2% ACN to 90% ACN. I am suspecting the hydrophobic tail caused the fatty acids to to stick to the hydrophobic surface very tightly.
Please suggest.
Re: lipid and fatty acid profiling
Posted: Thu Mar 23, 2017 7:53 pm
by HPLC chemist
Because the column is so non-polar I am surprised you got anything under those chromatographic conditions. Try looking first at
http://lipidlibrary.aocs.org/index.cfm . Your sample matrix may be included.
Re: lipid and fatty acid profiling
Posted: Mon Apr 24, 2017 11:23 am
by arnabroy81
Because the column is so non-polar I am surprised you got anything under those chromatographic conditions. Try looking first at
http://lipidlibrary.aocs.org/index.cfm . Your sample matrix may be included.
Thank you for the link. I didn't get anything when I loaded through column. However, when I injected directly to the source I got signal.
Re: lipid and fatty acid profiling
Posted: Mon Apr 24, 2017 12:05 pm
by HPLC chemist
Lipids by their nature are non-polar. Thus, I would try 'normal phase' 1st using a silica column (perhaps amino) and hexane (perhaps chloroform) as both the extraction solvent and mobile phase.
I would not use a C18 column and reverse phase HPLC.
Re: lipid and fatty acid profiling
Posted: Thu Apr 27, 2017 10:33 am
by Hartmut Thomas
Oleic acid works perfectly on a RP18 column. Here are the parameters I once used:
Column: Ascentis Express C18 2.7 µm, 100*2.1 mm
Mobile phases A: water with 1 mL/L formic acid, B: methanol with 1 mL/L formic acid (acetic acid would be better here, but I had additional analytes)
Gradient: 60-100% B in 10 min, 100% B for another 2 min
Flow: 0.4 ml/min, RT ca. 11 min
negative ESI and SIM mode (fatty acids give no fragments) at m/z 281.3
We use a similar method routinely for pelargonic acid:
Zorbax Eclipse Plus C18, 1.8 µm, 50*2.1 mm
A: 0.05% acetic acid in water, B: 0.05% acetic acid in Methanol
40 to 95% B in 5 min @ 0.4 ml/min
ESI neg, SIM, m/z=157
You can even detect intact triglycerides, but you need a stronger eluent like 50% ACN/50% IPA. I used these parameters for triglycerides in rapeseed oil:
Phenomenex Synergi MAX 4 µm, 150*2mm
A: water with 0.1% FA and 5mM Ammonium Formate
B: 50:50 IPA:ACN with 0.1% FA and 5mM Ammonium Formate
90 to 100% B in 10 min @ 0.3 ml/min
Triolein elutes after ca. 5.5 min.
Best regards,
Hartmut