The determination of 14 AAs, 35 ACCs, C0, succinylacetone, 2 nucleosides, and 4 lysophospholipids was performed in tear samples by the addition of isotopically labelled internal standards (ISs) for each analyte of interest prior to the extraction, according to the principle of isotope dilution internal standardization.
Schirmer’s strips imbibed by tears were cut and transferred into microcentrifuge tube (Eppendorf®, Hamburg, Germany). After adding the extraction solution (300 µL) containing ISs, each tear samples were shaken in a thermo mixer (750 rpm, 45 °C, 30 min) [35 (link),36 (link)].
The internal standards, as well as the extraction solution, were obtained from the NeoBase 2 Non-derivatized MSMS Kit (Perkin Elmer Life and Analytical Sciences, Turku, Finland). 100 μL for each extracted sample was transferred to a vial and leave at room temperature for an hour for succinylacetone derivatization [37 (link)].
Schirmer’s strips imbibed by tears were cut and transferred into microcentrifuge tube (Eppendorf®, Hamburg, Germany). After adding the extraction solution (300 µL) containing ISs, each tear samples were shaken in a thermo mixer (750 rpm, 45 °C, 30 min) [35 (link),36 (link)].
The internal standards, as well as the extraction solution, were obtained from the NeoBase 2 Non-derivatized MSMS Kit (Perkin Elmer Life and Analytical Sciences, Turku, Finland). 100 μL for each extracted sample was transferred to a vial and leave at room temperature for an hour for succinylacetone derivatization [37 (link)].
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