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Shyam Panthi, Jianzhong CHEN, Landon Wilson, Jason J Nichols; Evaluation of extraction and mass spectrometry methods for detection of possible lipid mediators of inflammation in tears.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):391. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To identify an optimal method for extraction of lipid mediators of inflammation in tears and evaluate possible mass spectrometry (MS) techniques for their analysis.
10 µL tears were collected from each eye of 12 subjects using glass microcapillaries. Six samples were extracted with 500 µl of 15% methanol and 0.1% formic acid. Three samples (10 µl each) in glass vials were centrifuged at 1400 revolutions per minute (rpm) and three samples (10 µl each) in eppendorf tubes were centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected; 1% ammonium hydroxide or ammonium acetate was added and then analyzed using the SCIEX TripleTOF 5600 mass spectrometer in direct infusion (DI) mode. The remaining six samples were extracted using 40µL of methanol and 0.1% formic acid and centrifuged at 17,000 rpm for 10 minutes. Supernatant was collected and analyzed using nano liquid chromatography (LC)-MS. Limits of detection were estimated for prostaglandin E2 (PGE2), thromboxane B2 (TXB2) and leukotriene B4 (LTB4) using standards along with examination of the spectra for the presence of these mediators.
Use of glass vials and 15% methanol and 0.1% formic acid and centrifuging at 1400 rpm or 10,000 rpm for 10 minutes did not produce any precipitate on extraction. It also produced a lot of background noise with salts of sodium and potassium during mass spectrometry. Addition of 1% ammonium acetate gave a cleaner spectrum. Extraction with 100% methanol and 0.1% formic acid with centrifuging at 17,000 rpm for 10 minutes using eppendorf tubes produced visible precipitate. Supernatant from this procedure produced the cleanest spectra using the nano LC-MS. Limits of detection for the three standards, PGE2, TXB2 and LTB4 were estimated to be 280pg/ml, 230 pg/ml and 240pg/ml, respectively. The three mediators of inflammation were not observed in any of the tear samples but other lipid species like wax esters, phospholipids and o-acyl-w-hydroxy fatty acids were seen.
Adding 100% methanol and 0.1% formic acid and centrifuging under 17000 rpm for 10 minutes can be a good method to extract lipid metabolites from tears. SCIEX TripleTOF 5600 mass spectrometer was not sensitive enough for identification of lipid mediators of inflammation in tears. Further analysis with more sensitive mass spectrometers may be considered.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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