Quantitative monitoring of a panel of stress-induced biomarkers in human plasma by liquid chromatography–tandem mass spectrometry: an application in a comparative study between depressive patients and healthy subjects
Using a metabolomic approach, we have found that stress can induce oxidative damage by disturbing the creatine/phosphocreatine shuttle system and purinergic pathway, leading to an excessive membrane breakdown. To further validate our findings and to monitor the biological impact of stress in research of clinical psychiatry, a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously determine a panel of biomarkers comprising choline, creatine, purinergic metabolites, neurosteroids, lysophosphatidylcholines, and phosphatidylethanolamines in human plasma. After optimization of the extraction protocol, all the 15 analytes plus 4 internal standards with distinct polarities were extracted into an organic phase using methyl tert-butyl ether/methanol (1:1, v/v). A reversed-phase C8 column under gradient elution consisting of aqueous phase A of 5 mM ammonium acetate buffer solution containing 0.1% formic acid and organic phase B of acetonitrile/2-propanol (3:7, v/v) was utilized for separation. Four sequential periods under positive or negative ion mode were combined for the determination of analytes with specific multiple reaction monitoring transitions. For all analytes, this method exhibited good linearity with coefficients of determination (R2) higher than 0.99. The lower limit of quantification (LLOQ) values ranged from 0.05 to 80.0 ng/mL. Recovery between 70.5 and 97.3% was obtained by spiking standards to plasma samples stripped by powdered activated carbon. The intra- and inter-assay relative standard deviations (RSDs) of the analyses varied between 2.0 and 13.3%. The mean accuracy ranged from 90.6 to 109.0%. The matrix effect ranged from 91.2 to 107.3% with variations less than 9.0%. Stability under different conditions was tested, with mean recoveries varying between 90.4 and 109.7%. Finally, the established method was successfully applied to analyze the plasma samples from a small cohort of 30 patients with major depressive disorder and 30 matched healthy controls.
Liquid chromatography Tandem mass spectrometry Stress Purines Neurosteroids Phospholipids
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This work was supported by Hunan Provincial Natural Science Foundation of China [2017JJ3444 (HLC)] and Nature Science Foundation of China [NSFC81401113 (HLC)].
Compliance with ethical standards
The study was approved by the Ethics Committee of The Second Xiangya Hospital of Central South University, Hunan, China. The patients were recruited at their first admission to the Second Xiangya Hospital after they signed the informed consent. All healthy volunteers were recruited from the same community of MDD patients. Their fasting morning plasma samples were also collected after the informed consent to participate in the study was obtained.
Conflict of interest
The authors declare that there is no conflict of interest.
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