Tear Lipid extraction: For tear lipid analysis, one tear
specimen each from 3 patients was pooled (tear collected using
Schirmer’s strip) in both control and VKC groups. The analysis was done
in 7 pooled sets in controls and 22 in VKC (NR-VKC: 6 sets; and R-VKC:
16 sets). Briefly, the Schirmer strips were cut and measured volume of
the extraction solvent, CHCl3: Methanol Reagent (1:1)
was added, vortexed and sonicated for 5 minutes. Samples were then
transferred to a fresh vial and nitrogen purged. Extraction solvent was
further added to the sample followed by vortexing for 1 minute. Sample
was centrifuged for 5 min at 10000 rpm. Supernatant (20 µl) was loaded
for analysis in LC-MS/MS.