2.0 ml microcentrifuge tube

Schirmer’s strips imbibed by tears were cut into 2–3 mm paper pieces and transferred into 2.0 mL microcentrifuge tube (Eppendorf^®, Hamburg, Germany), paying attention to wash the required equipment with MeOH before each sample preparation. Lipidomics and targeted metabolomics analysis were performed from the same Schirmer’s strip for each tear sample by the addiction of the extraction solution (PerkinElmer^®, Turku, Finland) containing isotopically labelled internal standards (ISs) (NeoBase Non-derivatized MSMS Kit; PerkinElmer^®, Turku, Finland). After adding 300 µL of the extraction solution containing ISs, each sample was gently mixed (50 °C, 45 min, 700 rpm) in a Thermomixer (Eppendorf^®) and then centrifuged (13,000 rpm, 10 min). The supernatant was divided into three aliquots as follow: 100 µL for AAs and ACs determinations by DIMS analysis, 100 µL for lipidomics investigations by LC-MS/MS analysis, and the remaining volume was stored at −80 °C for further investigation.
For serum metabolite extraction, 3.2 µL of each sample were transferred into 1.5 mL tubes (Eppendorf) and then mixed with 100 µL of the same extraction solution above described, containing ISs (PerkinElmer^®). The samples were centrifuged (15600 rpm at 4 °C for 15 min), and the supernatants were stored at −80 °C before DIMS analysis.

Approximately 5.0 mg of pure standard compounds and 10.0 mg of MDG samples were weighed in a 2.0-mL microcentrifuge tube (Eppendorf, Vienna, Austria). Thereafter, 600 μL of deuterated chloroform, containing 1.0 mg of the reference standard (tCNB; ¹H NMR signal at 7.76 ppm non-overlapping with glycerides, G, and FA), was added into the microcentrifuge tube. Approx. 50 μL of deuterated water (D₂O) was added to the samples to exchange the hydroxyl protons of G. The samples were then dissolved using vortex agitation for 60 s. The tubes were centrifuged at 5000 rpm for 5 min at room temperature for the complete mixing of the samples. The solution was transferred into NMR tubes (Eurisotop, France), and the ¹H NMR spectra were acquired.