June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Detection of sex steroids and their metabolites in human tears using LC-MS/MS.
Author Affiliations & Notes
  • Emma Gibson
    School of Optometry and Vision Science, UNSW, Sydney, New South Wales, Australia
  • Martin Bucknall
    School of Optometry and Vision Science, UNSW, Sydney, New South Wales, Australia
    Bioanalytical Mass Spectrometry Facility, UNSW, Sydney, New South Wales, Australia
  • Blanka Golebiowski
    School of Optometry and Vision Science, UNSW, Sydney, New South Wales, Australia
  • James Wolffsohn
    School of Life and Health Sciences, Aston University, Birmingham, United Kingdom
  • Fiona Stapleton
    School of Optometry and Vision Science, UNSW, Sydney, New South Wales, Australia
  • Footnotes
    Commercial Relationships   Emma Gibson, None; Martin Bucknall, None; Blanka Golebiowski, None; James Wolffsohn, None; Fiona Stapleton, None
  • Footnotes
    Support  none
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 486. doi:
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      Emma Gibson, Martin Bucknall, Blanka Golebiowski, James Wolffsohn, Fiona Stapleton; Detection of sex steroids and their metabolites in human tears using LC-MS/MS.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):486.

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

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Abstract

Purpose : Detection of sex steroids in tears is problematic due to low available volumes of tears and trace level analyte concentrations. This study aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect sex steroids in human tears.

Methods : Limits of detection were determined for a series of sex steroids and metabolites in a mixture of standards and this technique was subsequently applied to human tear extracts. Fifteen analytes were analysed in mixtures at concentrations of 0.1, 0.3, 1, 3, 10, 30, 100 and 1000 ng/mL per component. Chromatographic separation was achieved on a Waters Acuity BEH UPLC C18 column (1.7μm, 1mmx150mm) using an acetonitrile-water gradient. Both water and acetonitrile mobile phases contained 10mM ammonium formate. Analysis was performed using alternating positive and negative atmospheric-pressure chemical ionisation (APCI) with selected-ion monitoring (SIM).
Basal tears were collected and pooled from 4 healthy pre-menopausal females (total 100μl). To precipitate protein, cold ethanol was added to tears at a ratio of 6:1 and centrifuged at -2°C for 25 minutes at 18,000g. 100μl of supernatant was removed for analysis (diluted tears). A further 400μl of supernatant was reduced in a vacuum concentrator to 80μl (concentrated tears). Both tear extracts were then analysed as described above.

Results : Both positive and negative ionisation modes were used because single ion mode analysis was not optimal for all analytes. Limits of detection (Signal/Noise > 3:1) for standards on column (10 μl injected) for negative ionisation were: 3α-Diol-G 30pg, ADT-G 30pg, DHEA-S 50pg, 2-hydroxyestradiol 80pg, 2-hydroxyestrone 80pg. For positive ionisation: estriol 30pg, 16-hydroxyestrone 8pg, estradiol 800pg, testosterone & DHEA 100pg total, estrone 15pg, androstenedione 2pg, androsterone 70pg, DHT 8pg, progesterone 0.8pg. DHEA and testosterone are chemically similar and could not be separated. Progesterone, ADT-G and 3α-Diol-G were successfully detected in concentrated tear extract.

Conclusions : A partly successful LC-MS/MS method to measure sex steroids, metabolites and precursors was developed. One sex steroid and two metabolites were detected in concentrated human tears. Further elaboration of these techniques will enable detection of additional analytes in tears. This data shows that LC-MS/MS can detect certain sex steroids and metabolites in concentrated human tear extract.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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