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A. Liu, J. Chang, Z. Shen, P. S. Bernstein; Enhanced Methods for Analysis of Very Long Chain Polyunsaturated Fatty Acids From Retina and RPE. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3410.
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Reduced levels of very long chain polyunsaturated fatty acids (VLCPUFA, C24-C36) have been found in the retinas and skin of animal models of dominant Stargardt macular dystrophy (STGD3) secondary to mutations in the ELOVL4 gene. Further study of the potential role of VLCPUFA in ocular health and disease is hampered, however, by their great length, minor abundance, and lack of reference standards. The aim of this study was to optimize a fast, accurate, and sensitive GC-MS method to analyze VLCPUFA in biological samples.
Lipids from retina and retinal pigment epithelium (RPE) were extracted followed by methyl trans-esterification using 16% hydrochloric acid in methanol. Solid phase extraction (SPE) using hexane and hexane:ether (9:1) as the elution solution was applied to purify the samples, which were analyzed on a Rxi-5MS column within 30 min by Thermo Trace GC Ultra coupled DSQ MS. Subsequently, both liquid chemical ionization (LCI) and electron impact (EI) modes were used to identify VLCPUFA, while selected ion monitoring (SIM) in the LCI mode was applied to quantify the samples.
The optimized SPE procedure effectively removed cholesterol within 5 min with recovery >92.0%. In the EI mode, all VLCPUFA (nc=c>2) showed the same base peak of m/z 79 without any distinct full molecular weight peaks, while in the LCI mode each VLCPUFA demonstrated a base peak corresponding to its molecular weight. Limit of detection (LOD) analysis demonstrated that the LCI mode is ten times more sensitive than EI. With this method, VLCPUFA, which have been thought to be uniquely distributed in neural retina, brain and skin, were also detectable in human and bovine RPE.
We have developed a rapid, accurate, and sensitive GC-MS method to purify, identify, and quantify VLCPUFA in retina and RPE. This will facilitate further progress in elucidating the role of VLCPUFA in ocular health and disease.
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