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Suitability evaluation of new endogenous biomarkers for the identification of nitrite-based urine adulteration in mass spectrometry methods


Steuer, Andrea E; Arnold, Kim; Kamber, Dominique; Kraemer, Thomas (2019). Suitability evaluation of new endogenous biomarkers for the identification of nitrite-based urine adulteration in mass spectrometry methods. Drug Testing and Analysis, 11(2):230-239.

Abstract

Urine adulteration to circumvent positive drug testing is a fundamental challenge for toxicological laboratories all over the world. Untargeted mass spectrometry (MS) methods used in metabolomics had previously revealed uric acid (UA), histidine, methylhistidine, and their oxidation products, for example 5-hydroxyisourate (HIU) as potential biomarkers for urine adulteration using potassium nitrite (KNO2 ). These markers should be further evaluated for their reliability, stability, and routine applicability. Influence of KNO2 concentration, urinary pH, reaction time, and stability at room temperature, 4°C, and - 20°C was determined in urine under varying conditions. Analysis was performed after protein precipitation with acetonitrile by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Receiver operating characteristics (ROC) analysis was applied for cut-off evaluation after biomarker quantification (n = 100 per group). Blinded measurements (n = 50) were performed to check the general applicability to identify adulterated samples under routine conditions. The higher the adulterant concentration, the lower the concentrations of histidine, methylhistidine, and UA. In return, amounts of their oxidation products increased. Highest changes were observed under weak acid conditions (pH 4-5). Storage at -20°C ensured sufficient stability for all oxidative markers over one month. ROC evaluated biomarker performance and application to unknown samples revealed satisfying results, with HIU as the most suitable biomarker (positive predictive value (PPV) 100%), followed by UA (PPV 93%). HIU and UA proved suitable markers to identify urine adulteration using KNO2 and are ready for implementation into routine MS procedures.

Abstract

Urine adulteration to circumvent positive drug testing is a fundamental challenge for toxicological laboratories all over the world. Untargeted mass spectrometry (MS) methods used in metabolomics had previously revealed uric acid (UA), histidine, methylhistidine, and their oxidation products, for example 5-hydroxyisourate (HIU) as potential biomarkers for urine adulteration using potassium nitrite (KNO2 ). These markers should be further evaluated for their reliability, stability, and routine applicability. Influence of KNO2 concentration, urinary pH, reaction time, and stability at room temperature, 4°C, and - 20°C was determined in urine under varying conditions. Analysis was performed after protein precipitation with acetonitrile by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Receiver operating characteristics (ROC) analysis was applied for cut-off evaluation after biomarker quantification (n = 100 per group). Blinded measurements (n = 50) were performed to check the general applicability to identify adulterated samples under routine conditions. The higher the adulterant concentration, the lower the concentrations of histidine, methylhistidine, and UA. In return, amounts of their oxidation products increased. Highest changes were observed under weak acid conditions (pH 4-5). Storage at -20°C ensured sufficient stability for all oxidative markers over one month. ROC evaluated biomarker performance and application to unknown samples revealed satisfying results, with HIU as the most suitable biomarker (positive predictive value (PPV) 100%), followed by UA (PPV 93%). HIU and UA proved suitable markers to identify urine adulteration using KNO2 and are ready for implementation into routine MS procedures.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Legal Medicine
Dewey Decimal Classification:340 Law
610 Medicine & health
Uncontrolled Keywords:Analytical Chemistry, Spectroscopy, Pharmaceutical Science, Environmental Chemistry
Language:English
Date:1 February 2019
Deposited On:02 Oct 2018 15:09
Last Modified:22 Feb 2019 02:02
Publisher:Wiley-Blackwell Publishing, Inc.
ISSN:1942-7603
OA Status:Closed
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1002/dta.2481
PubMed ID:30118186

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