Dear Isabelle,
As previous respondents tried to intimate, analysing 2H-uniformly labelled palmitic acid (or methyl palmitate for that matter) by IRMS is less than ideal.
IRMS was (is) designed to measure stable isotopic composition at near natural abundance level, say < 1 APE. Even if your IRMS instruments permits changing cup resistor values to adjust amplification of the major and minor ion currents according to enrichment, in your case this would not help.
Even with 1H/2H contribution from the methyl group, the level of 2H enrichment will result in the majority of H2 formed being (2H)2; conversion of 1 unit of U-2H labelled methy palmitate may theoretically yield 15 units of (2H)2 (m/z 4), 1 unit of 1H2H (m/z 3) and 2 units of (1H)2 (m/z 2). A more likely scenario is its conversion will yield 14 units of (2H)2 and 3 units of 1H2H.
So, the standard set up of measuring m/z 2 and m/z 3 will not work. Adjusting IRMS settings to capture m/z 3 and m/z 4 will not help either since the m/z 4 channel will be not just capture (2H)2+ but He+ too.
To check on the purity of your U-2H labelled palmitate you are better off using a standard GC/MS system. The mass spectrum should show a molecular ion M+ at m/z 301 and a fragment ion at m/z 270 through loss of the methoxy derivative CH3O.
For your in-vivo studies I would suggest you analyse your samples (muscle lipid extract) by GC/MS in the first instance. (A) GC/MS is much more sensitive in terms of limit of detection of compound amount (concentration) than GC/IRMS. (B) Even accounting for dilution of U-2H labelled palmitic acid with biogenic palmitic acid and subsequent breakdown and recycling within the body, the level of 2H enrichment in your target compound may still be too high for IRMS.
Regards,
Wolfram
-----Original Message-----
From: Stable Isotope Geochemistry [mailto:[log in to unmask]] On Behalf Of SUBSCRIBE ISOGEOCHEM Isabelle Chery
Sent: 21 May 2016 15:49
To: [log in to unmask]
Subject: [ISOGEOCHEM] D31 Palmitic Acid
Dear Marylin, Willi, Bill and Brian
Many thanks for your answers.
In fact the D labeling is not 100% and with derivatization there are some H added to the mixture. So the mass 3 is significant and is directly proportional to quantity injected (standard curve over 6 concentrations linear at R2 0.99), even if not representing a 100% of the molecule. Moreover the final analysis will be done on human muscle lipid extracts in which the D31 is really at low concentration in a complex lipid matrix.
The problem Isabelle faces is that the peak she which obtain is not sharp any more and is tailing. It is tailing only for D31 and for non of the other fatty acids of the mixture.
Few weeks ago the peak and shape were perfect. We have changed injector, cut the column, change the pyrolysis oven, the syringe. We are stuck.
Regards
Isabelle
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