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. 2021 Sep 15:629:114295.
doi: 10.1016/j.ab.2021.114295. Epub 2021 Jun 26.

Development of a HPLC-MS/MS method for assessment of thiol redox status in human tear fluids

Jiandong Wu  1 , Austin Sigler  1 , Annalise Pfaff  1 , Nan Cen  2 , Nuran Ercal  3 , Honglan Shi  4
Affiliations

Development of a HPLC-MS/MS method for assessment of thiol redox status in human tear fluids

Jiandong Wu et al. Anal Biochem. .

Abstract

Oxidative stress is reported to be part of the pathology of many ocular diseases. For the diagnosis of ocular diseases, tear fluid has unique advantages. Although numerous analytical methods exist for the measurement of different types of biomolecules in tear fluid, few have been reported for comprehensive understanding of oxidative stress-related thiol redox signaling. In this study, a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to determine a panel of twelve metabolites that systematically covered several thiol metabolic pathways. With optimization of MS/MS parameters and HPLC mobile phases, this method was sensitive (LOQ as low as 0.01 ng/ml), accurate (80-125% spike recovery) and precise (<10% RSD). This LC-MS/MS method combined with a simple tear fluid collection with Schirmer test strip followed by ultrafiltration allowed the high-throughput analysis for efficient determination of metabolites associated with thiol redox signaling in human tear fluids. The method was then applied to a small cohort of tear fluids obtained from healthy individuals. The method presented here provides a new technique to facilitate future work aiming to determine the complex thiol redox signaling in tear fluids for accurate assessment and diagnosis of ocular diseases.

Keywords: Biomarker; Glutathione; HPLC-MS/MS; Ocular disease; Tear fluid; Thiol.

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Figures

Figure 1.
Figure 1.
List of the analytes and their metabolic pathways.
Figure 2.
Figure 2.
Representative overlaid extracted ion chromatograms of: A, standards prepared at 20 ng/mL in mobile phase A, except for GSH and GSSG, which were prepared at 500 ng/mL; B, high abundance analytes in tear fluid sample; C, lower abundance analytes in tear fluid sample.
Figure 3.
Figure 3.
Assessment of stabilities of methionine and reduced thiols in standard solutions and human tear fluids. Homocystine (Hcyss) was below detection limit in tear fluid.
Figure 4.
Figure 4.
Relationship between volumes of water spiked and lengths of wet area on Schirmer test strips.
See this image and copyright information in PMC

References

    1. Halliwell B, Oxidative stress and cancer: have we moved forward?, Biochem J, 401 (2007) 1–11. - PubMed
    1. Patel RS, Ghasemzadeh N, Eapen DJ, Sher S, Arshad S, Ko YA, Veledar E, Samady H, Zafari AM, Sperling L, Vaccarino V, Jones DP, Quyyumi AA, Novel Biomarker of Oxidative Stress Is Associated With Risk of Death in Patients With Coronary Artery Disease, Circulation, 133 (2016) 361–369. - PMC - PubMed
    1. Moriarty SE, Shah JH, Lynn M, Jiang S, Openo K, Jones DP, Sternberg P, Oxidation of glutathione and cysteine in human plasma associated with smoking, Free Radic Biol Med, 35 (2003) 1582–1588. - PubMed
    1. Jones DP, Carlson JL, Mody VC, Cai J, Lynn MJ, Sternberg P, Redox state of glutathione in human plasma, Free Radic Biol Med, 28 (2000) 625–635. - PubMed
    1. Carroll D, Howard D, Zhu H, Paumi CM, Vore M, Bondada S, Liang Y, Wang C, St Clair DK, Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS: An insight into the redox state of hematopoietic stem cells, Free Radic Biol Med, 97 (2016) 85–94. - PMC - PubMed

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