Calibrators and QCs in Schirmer paper strips were prepared wetting each paper strip with 90 µL of calibrator and QC solutions for each levels of calibration. Calibrators, QCs, tear samples collected on Schirmer strip 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. After adding 200 μL of DPS containing IS, each sample was gently mixed (20 °C, 15 min) in a Thermomixer (Eppendorf®) and then centrifuged (4210 rcf, 20 °C, 30 min). The organic layer (135 µL) was transferred into a new 1.5 mL tube and dried in a SpeedVac for 30 min. The residue was then reconstituted with 90 μL of H2O/MeOH 60:40, gently mixed in a Thermomixer (20 °C, 15 min), briefly centrifuged and finally transferred into polypropylene vial (Waters Corporation, Milford, MA, USA). The vials were capped, gently mixed, and placed in the system autosampler for analysis.