LC-MS/MS analysis of sphingolipids in tear
The sphingolipid standards were procured from Avanti Polar Lipids
(Alabama, USA). Based on calibration of the LC column and linearity
check, the following standards were finalized for the assay in pooled
specimens of tear in cases and controls (Supplementary Table
2) . LC-ESI-MS/MS experiments were performed using a triple quadrupole
tandem mass spectrometer (4000 Q-Trap, AB Sciex, Foster City, CA, USA)
coupled with high performance liquid chromatography system (HPLC,
Agilent Technologies, 1260 Infinity, Santa Clara, CA, USA) that
consisted of quaternary pump (G1311C), multi-sampler (G7167A), HIBAR (30
x 2.1mm, 2µm) column compartment (G1316A) with variable wavelength UV
detector (G1314F) and online degasser. All the parameters of tandem mass
spectrometer and HPLC were controlled by Analyst software, version 1.5.2
(AB Sciex, Foster City, CA, USA) and OpenLAB control panel software
(Agilent Technologies, 1260 Infinity, Santa Clara, CA, USA),
respectively.