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It is well known that the diversity of fatty acid chain lengths and degrees of unsaturation (number of double bonds) result in differential fragmentation efficiency which impacts quantitation of lipid molecular species. We worked with Avanti Polar Lipids to develop a broad range of internal standards to work with the Lipidyzer Platform that reflects the diversity of the lipid molecular species to normalize lipid quantitative data and their concentrations in human plasma biology. For the phosphatidylcholine (PC) class we labeled the sn1 position with a labeled palmitate, and then we took the sn2 position and changed the fatty acid from a short chain palmitoleic acid all the way to a long chain docosahexaenoic acid. These were mixed at percentages to reflect those found in biology. Standards for the other 12 lipids classes were created following a similar strategy.
The Lipidyzer internal standards were compared to the use of a single internal standard for their ability to accurately calibrate the concentration of total cholesteryl esters (CE) (on the left), and the fatty acid composition of cholesteryl esters expressed as a mole % fatty acid composition (on the right) in human serum. Twenty-five human serum samples with known total CE and CE fatty acid compositions were profiled using the Lipidyzer Platform. If you focus on the figure on the left, you can see the Lipidyzer Platform quantified total CE with less than 10% bias, compared to a 100% bias in the estimate made using a single internal standard. The results indicated that using a single internal standard greatly overestimated the concentration of CE, likely by overestimating the contribution of the major unsaturated fatty acids. The figure on the right shows the individual fatty acid profiles of CE (expressed as a mole % of total CE) when quantified using the Lipidyzer Platform and the single internal standard. The composition is clearly warped by the bias caused by using one internal standard, whereas using a mixture of internal standards provided an accurate fatty acid composition of CE.
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RUO-MKT-18-5511-A
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