July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
A comparison of two methods to analyse and quantify human tear lipids by ultra-high performance liquid chromatography-mass spectrometry (UHPLC_MS)
Author Affiliations & Notes
  • Elena Vecino
    Cell Biology and Histology, University of the Basque Country, Leioa, Spain
  • Arantxa Acera
    Cell Biology and Histology, University of the Basque Country, Leioa, Spain
  • Xandra Pereiro
    Cell Biology and Histology, University of the Basque Country, Leioa, Spain
  • Beatriz Abad-Garcia
    Servicio Central de Análisis, Facultad de Ciencia y Tecnología, University of the Basque Country, Leioa, Spain
  • Yuri Rueda
    Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country, Spain
  • Noelia Ruzafa
    Cell Biology and Histology, University of the Basque Country, Leioa, Spain
  • Carlos Santiago
    Departamento de Química Orgánica II, Facultad de Ciencia y Tecnología, University of the Basque Country, Leioa, Spain
  • Iratxe Barbolla
    Departamento de Química Orgánica II, Facultad de Ciencia y Tecnología, University of the Basque Country, Leioa, Spain
  • Begoña Ochoa
    Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country, Spain
  • Juan A Duran
    Department of Ophthalmology, University of the Basque Country, Leioa, Spain
  • Footnotes
    Commercial Relationships   Elena Vecino, None; Arantxa Acera, None; Xandra Pereiro, None; Beatriz Abad-Garcia, None; Yuri Rueda, None; Noelia Ruzafa, None; Carlos Santiago, None; Iratxe Barbolla, None; Begoña Ochoa, None; Juan Duran, None
  • Footnotes
    Support  Retos-MINECO Fondos FEDER (RTC-2016-48231), Grupos Consolidados UPV/EHU (GIU17/73, PPGA18/18), PUE2018-04 to EV and the Basque Government (IT-971-16) to BO
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4180. doi:
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      Elena Vecino, Arantxa Acera, Xandra Pereiro, Beatriz Abad-Garcia, Yuri Rueda, Noelia Ruzafa, Carlos Santiago, Iratxe Barbolla, Begoña Ochoa, Juan A Duran; A comparison of two methods to analyse and quantify human tear lipids by ultra-high performance liquid chromatography-mass spectrometry (UHPLC_MS). Invest. Ophthalmol. Vis. Sci. 2019;60(9):4180.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : To compare two protocols to prepare samples for quantitative human tear lipid profiling by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS), one involving liquid-liquid extraction with chloroform/methanol (BD) and the other, protein precipitation with isopropanol (IPA).

Methods : The effectiveness of BD and IPA sample preparation to quantitatively and reproducibly recover 19 non-endogenous internal lipid standards was tested in 10 µl tear samples. The lipid coverage and simplicity of execution was also assessed. Lipid profiles of the tear extracts were acquired by UHPLC-MSE and the lipids were identified using SimLipid software.

Results : Both methods were robust techniques producing good lipid coverage and recovery efficiencies. The two protocols identified a 67 feature-tear lipidome that covered 11 lipid classes from six different lipid categories. The main differences in recovery were due to the intrinsic lipid selectivity of each solvent, although both methods were similarly efficient in recovering OAHFAs and non-polar lipids. By contrast, polar lipids were more efficiently recovered by IPA precipitation, which in turn exhibited higher reproducibility. Indeed, IPA precipitation is automatable and it is a simpler method than the BD approach.

Conclusions : The IPA precipitation method is an excellent procedure to extract lipids from small tear volumes for large-scale studies, which will be very useful to determine the lipid classes in the tear of patients with ocular surface pathologies.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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