Improved method for gas chromatographic-mass spectrometric analysis of 1-(13)C-labeled long-chain fatty acids in plasma samples.
BACKGROUND
Gas chromatographic-mass spectrometric (GC/MS) tracking of stable-isotope-labeled substrates is useful in metabolic studies. However, GC/MS analysis of long-chain fatty acid methyl esters yields results that mostly depend on their concentration in the system. We describe a protocol aimed to obviate this and other drawbacks in plasma [1-(13)C]palmitic and [1-(13)C]oleic acid measurements.


METHODS
Lipoproteins were separated by sequential ultracentrifugation. Free or esterified heptadecanoic acid was used as internal standard. Fatty acids were derivatized to trimethylsilyl (TMS) esters. GC separation was in isothermal mode at 210 degrees C for 27 min. For both TMS-palmitate and TMS-oleate, M and [M + 1] signals were simultaneously acquired with a dual acquisition program in single-ion monitoring mode. Calibration mixtures containing increasing amounts of labeled fatty acids were prepared gravimetrically to construct calibration curves for isotopic enrichment. Likewise, five calibration curves (for increasing concentrations) were constructed for each fatty acid; this allowed selection of the most appropriate curve for the concentration in a plasma sample.


RESULTS
Oleic acid-TMS ester was clearly separated from that of its stereoisomer, elaidic acid. Within a 10-fold concentration range, the isotopic ratio was independent on the amount of the analyte in the sample, with a maximum uncertainty of 0.34% in terms of molar percent excess. In addition, the within- and between-day imprecision (CV) of the method was <1%.


CONCLUSIONS
Results obtained with this method are independent of concentration and sufficiently precise for tracking 1-(13)C-labeled palmitic and oleic acids in biological samples
Hernández-Pérez
José M
JM
Research Unit, Department of Gastroenterology, Hospital Universitari Germans Trias i Pujol, 08916 Badalona, Catalonia, Spain.
Cabré
Eduard
E
Fluvià
Lourdes
L
Motos
Agata
A
Pastor
Cruz
C
Corominas
August
A
Gassull
Miquel A
MA
D002138:Calibration; D002247:Carbon Isotopes; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007202:Indicators and Reagents; D019301:Oleic Acid / Q000097:blood* / Q000737:chemistry; D019308:Palmitic Acid / Q000097:blood* / Q000737:chemistry; D012680:Sensitivity and Specificity; D014297:Trimethylsilyl Compounds; D014461:Ultracentrifugation
D002247:Carbon Isotopes; D007202:Indicators and Reagents; D014297:Trimethylsilyl Compounds; D019301:Oleic Acid; D019308:Palmitic Acid