Rapid quantification of free cholesterol in tears using direct insertion/electron ionization-Mass spectrometry

Wei, Xiaojia Eric, Korth, John, Brown, Simon H.J., Mitchell, Todd W., Truscott, Roger J.W., Blanksby, Stephen J., Willcox, Mark D.P., & Zhao, Zhenjun (2013) Rapid quantification of free cholesterol in tears using direct insertion/electron ionization-Mass spectrometry. Investigative Ophthalmology and Visual Science, 54(13), pp. 8027-8035.

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Abstract

Purpose

  • To establish a simple and rapid analytical method, based on direct insertion/electron ionization-mass spectrometry (DI/EI-MS), for measuring free cholesterol in tears from humans and rabbits.

Methods

  • A stable-isotope dilution protocol employing DI/EI-MS in selected ion monitoring mode was developed and validated. It was used to quantify the free cholesterol content in human and rabbit tear extracts. Tears were collected from adult humans (n = 15) and rabbits (n = 10) and lipids extracted.

Results

  • Screening, full-scan (m/z 40-600) DI/EI-MS analysis of crude tear extracts showed that diagnostic ions located in the mass range m/z 350 to 400 were those derived from free cholesterol, with no contribution from cholesterol esters. DI/EI-MS data acquired using selected ion monitoring (SIM) were analyzed for the abundance ratios of diagnostic ions with their stable isotope-labeled analogues arising from the D6-cholesterol internal standard. Standard curves of good linearity were produced and an on-probe limit of detection of 3 ng (at 3:1 signal to noise) and limit of quantification of 8 ng (at 10:1 signal to noise). The concentration of free cholesterol in human tears was 15 ± 6 μg/g, which was higher than in rabbit tears (10 ± 5 μg/g).

Conclusions

  • A stable-isotope dilution DI/EI-SIM method for free cholesterol quantification without prior chromatographic separation was established. Using this method demonstrated that humans have higher free cholesterol levels in their tears than rabbits. This is in agreement with previous reports. This paper provides a rapid and reliable method to measure free cholesterol in small-volume clinical samples.

Impact and interest:

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ID Code: 68957
Item Type: Journal Article
Refereed: Yes
Keywords: Cholesterol, Human, Mass spectrometry, Rabbit, Tears
DOI: 10.1167/iovs.13-12786
ISSN: 0146-0404
Subjects: Australian and New Zealand Standard Research Classification > CHEMICAL SCIENCE (030000) > ANALYTICAL CHEMISTRY (030100)
Divisions: Current > QUT Faculties and Divisions > Science & Engineering Faculty
Funding:
Copyright Owner: 2013 The Association for Research in Vision and Ophthalmology, Inc
Deposited On: 19 Mar 2014 22:54
Last Modified: 16 Dec 2016 03:00

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