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 the 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.