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Ecotoxicology and Human Environmental Health

Simultaneous analysis of seven biomarkers of oxidative damage to lipids, proteins, and DNA in urine Maria Martinez, and Kurunthachalam Kannan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00883 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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Environmental Science & Technology

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Simultaneous analysis of seven biomarkers of oxidative damage to

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lipids, proteins, and DNA in urine

4 Maria P. Martinez a and Kurunthachalam Kannana,b*

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a

Wadsworth Center, New York State Department of Health, and Department of Environmental

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Health Sciences, School of Public Health, State University of New York at Albany, Empire

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State Plaza, P.O. Box 509, Albany, New York 12201-0509, United States

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b

Biochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia

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*Corresponding author: [email protected]

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For submission to: Environmental Science and Technology

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Abstract

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The determination of oxidative stress biomarkers (OSBs) is useful for the assessment of health

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status and progress of diseases in humans. Whereas previous methods for the determination of

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OSBs in urine were focused on a single marker, in this study, we present a method for the

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simultaneous determination of biomarkers of oxidative damage to lipids, proteins, and DNA. 2,4-

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Dinitrophenylhydrazine (DNPH) derivatization followed by solid phase extraction (SPE) and

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high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) allowed

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the

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malondialdehyde (MDA), and four F2-isoprostane isomers: 8-iso-prostaglandinF2α (8-PGF2α),

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11β-prostaglandinF2α

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prostaglandinF2α (8,15-PGF2α) in urine. Derivatization with DNPH and SPE were optimized to

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yield greater sensitivity and selectivity for the analysis of target chemicals. The limits of

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detection of target analytes in urine were below 30 pg ml-1. The assay intra- and inter-day

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variability was below 16% of the relative standard deviation, and the recoveries of target

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chemicals spiked into synthetic urine were near 100%. The method was applied to the analysis of

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21 real urine samples, and the analytes were found at a detection frequency of 85% for 8-PGF2α

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and 15-PGF2α, 71% for 11-PGF2α, 81% for 8,15- PGF2α and 100% for diY, 8-OHdG, and MDA.

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This method offers simultaneous determination of multiple OSBs of different molecular origin in

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urine samples selectively with high accuracy and precision.

determination

of

8-hydroxy-2'-deoxyguanosine

(11-PGF2α),

(8-OHdG),

15(R)-prostaglandinF2α

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2 ACS Paragon Plus Environment

o-o’-dityrosine

(15-PGF2α),

and

(diY),

8-iso,15(R)-

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Keywords: Biomarker, Oxidative Stress, Urine, Malondialdehyde, F2-isoprostane, o-o’-

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dityrosine, 8-Hydroxy-2’-deoxyguanosine

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Introduction

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The imbalance between antioxidant and oxidant species in the body with radical oxygen species’

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(ROS) exceeding the antioxidant capacity of the organism results in oxidative stress, which has

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been claimed as an indicator for the prediction or progression of diseases, health status, or

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exposure to external stressors. Oxidative stress implies that the excess ROS targets biomolecules,

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resulting in their oxidation. Several compounds have been identified as the products of oxidation

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of lipids, proteins, and DNA and were detected in biospecimens (e.g., urine, blood) as oxidative

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stress biomarkers (OSBs) (Figure 1). The link between oxidative stress and breast cancer1 or

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cardiovascular diseases2 has been reviewed previously. Studies that relate OSBs with Down

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syndrome,3 Maple syrup disease,4 and exposure to external environmental factors, such as

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asbestos,5 exercising,6 smoking,7 organic contaminants,8-10 poisons,11 anesthetics and alcohol

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intake,12 have been reported. Under oxidative stress conditions, F2-isoprostanes (prostaglandin-

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like compounds) are generated as oxidation products of arachidonic acid. Malondialdehyde

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(MDA) is a product of the peroxidation of polyunsaturated fatty acids. These two are the most

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common biomarkers of oxidation of lipids.13 DNA oxidation by ROS produces 8-hydroxy-2’-

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deoxyguanosine (8-OHdG), and o,o’-diTyrosine (diY) is a biomarker of protein peroxidation.

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The determination of these compounds in urine can provide an indication of the level of

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oxidative stress.



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F2-isoprostanes, which are generated by the oxidation of arachidonic acid, can produce up

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to 64 possible isomers. Bioactive F2-isoprostane isomers, such as 8-iso-prostaglandin F2α (8-

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PGF2α), 15(R)-prostaglandin F2α, (15-PGF2α), 8-iso-15(R)-prostaglandin F2α (8,15-PGF2α), and

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11β-prostaglandin F2α (11-PGF2α), have been used as OSBs.14 The most significant of the F2-

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isoprostane isomers is 8-PGF2α, which has been the subject of over 200 publications that relate

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human health with levels of PGF2α.7,10,15,16 The determination of F2-isoprostanes in biological

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specimens by enzyme-linked immunosorbent assay (ELISA)3,4,10 or gas chromatography-mass

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spectrometry (GC-MS) after a derivatization step17-20 has been described previously. The lack of

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accuracy of ELISA and the extensive sample preparation steps needed for GC-MS analysis

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highlight the need for alternative methods that include liquid chromatography-mass spectrometry

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(LC-MS) for the analysis of F2-isoprostanes.5,14,21,22

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Malondialdehyde (MDA) is the most important OSB due to its abundance in urine. MDA

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has been used as a biomarker in studies that link oxidative stress to cancer,1,7 asbestos- or silica-

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induced lung diseases,5 and exposure to polycyclic aromatic hydrocarbons (PAHs)23 and coke

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oven dust,24 among others. Traditionally, MDA was quantified by a 2-thiobarbituric (TBA)

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assay, and the MDA-TBA product was detected by fluorescence or spectrophotometry.25

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However, the TBA assay lacks specificity, as the reaction can take place with other aldehydes in

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the sample, yielding unknown TBA derivatives in addition to MDA-TBA, which leads to

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overestimation of MDA.13,25,26 Analyses of MDA in urine after O-(2,3,4,5,6-pentafluorobenzyl)

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hydroxylamine hydrochloride (O-PFB) derivatization followed by GC with electron capture

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detection (GC-ECD),6 pentafluorobenzyl bromide (PFB-Br) derivatization followed by GC-MS

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analysis,13

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dinitrophenylhydrazine (DNPH) derivatization followed by liquid chromatography with

TBA

derivatization

followed

by

LC-fluorescence


detection,23,27

2,4-

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ultraviolet detection (LC-UV),28,29 and solid phase extraction (SPE)-LC-MS24 have been

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reported.

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Oxidative DNA damage is believed to be a trigger for diseases such as cancer. 8-oxo-

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hydroxyguanosine (8-OHdG) is an OSB for DNA oxidation, a product of the reaction of ROS

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with guanosine. 8-OHdG has been evaluated in relation to diseases such as cancer,1,7 Down

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syndrome,3 and diabetes30 and in terms of linking oxidative stress with exposure to organic

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contaminants.8,9,22,23 8-OHdG has been analyzed by ELISA3,6,30 or LC-MS8,23 methods.

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When proteins are the targets of oxidation by ROS, o-o’-dityrosine (diY) and other

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unnatural isomers of p-tyrosine are produced. diY is stable and resistant to enzymatic hydrolysis,

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which makes it a good biomarker of protein oxidation.26 diY has been assessed as an OSB for

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Down syndrome3 and diabetes.30 HPLC with fluorescence detection3 and LC-MS have been used

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for the determination of diY in urine.6,26

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Several studies have examined the association of oxidative stress with various diseases

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and stressors.1,4,8-10,15,23 The majority of these studies have measured only a single OSB.1,15,9,10 A

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few studies have developed analytical methods for simultaneous determination of 8-OHdG, 8-

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PGF2α, 8-nitroguanine (biomarker of nitrative damage to DNA), and 4-hydroxy-2-nonenal-

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mercapturic acid (HNE-MA)22 or the lipid peroxidation products, 8-PGF2α, MDA, and HNE.5

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Limited studies have determined OSBs of lipids, proteins, and DNA simultaneously, but such

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studies often used multiple analytical methods for the determination of each of the

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biomarkers.3,6,11 Determination of OSBs produced by various cellular macromolecules would

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provide a better understanding of the mechanisms and allow for a broader interpretation of the

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results of toxicological studies, including the evaluation of correlations among various

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biomarkers. Based on this background, this study was aimed at the development and validation 5 ACS Paragon Plus Environment


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of an analytical method for simultaneous determination of major OSBs of lipids (viz., MDA and

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PGF2α), proteins (viz., diY), and DNA (viz., 8-OHdG) in urine. The method can be applied in

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epidemiological studies to examine oxidative stress patterns in cohorts inflicted with a disease or

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an environmental condition as well as to evaluate antioxidant treatments. The method developed

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and validated in this study, based on DNPH derivatization and SPE followed by HPLC with

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tandem mass spectrometry (HPLC-MS/MS) detection and quantification, is accurate and precise

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and allows for simultaneous determination of MDA, 8-OHdG, diY, and four F2-isoprostane

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isomers, 8-PGF2α, 15-PGF2α, 8,15-PGF2α, and 11-PGF2α, in human urine.

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Material and methods

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Chemicals

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Malondialdehyde tetrabutyl-ammonium salt (>97% purity), 50% glutaraldehyde solution, 8-

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hydroxy-2’-deoxyguanosine (≥98%) (8-OHdG), acetic acid, hydrochloric acid (HCl), ethanol, n-

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hexane, LC-MS grade methanol (MeOH), acetonitrile (ACN), HPLC grade water, ethyl acetate

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(EtAc), synthetic urine, and 2,6-di tert-butyl-4-methylphenol (≥99%) (BHT) were purchased

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from Sigma Aldrich (St. Louis, MO, USA). 2,4-Dinitrophenylhydrazine (DNPH) was purchased

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from Spectrum (New Brunswick, NJ, USA). 1,1,3,3-Tetraethoxypropane-1,3-d2 (>98%) was

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purchased from C/D/N isotopes (Point-Clair, Quebec, Canada). o,o’-Dityrosine (diY) (>99%)

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was supplied by Toronto Research Chemicals (Toronto, Ontario, Canada). 13C12-o,o’-Dityrosine

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(13C12-diY) (>98%) and

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purchased from Cambridge Isotope Laboratories (Andover, MA, USA). 8-Iso-prostaglandin F2α

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(>99%) (8-PGF2α), 15(R)-prostaglandin F2α (15-PGF2α) (>98%), 8-iso-15(R)-prostaglandin F2α

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N5-8-hydroxy-2’-deoxyguanosine (15N5-8-OHdG) (>95%) were


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(8,15-PGF2α) (>98%), 11β-prostaglandin F2α (11-PGF2α) (>98%), and D4-8-iso-prostaglandin F2α

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(D4-8-PGF2α) (>99%) were obtained from Cayman Chemicals (Ann Arbor, MI, USA).

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Urine Samples

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A pool of urine samples collected from healthy adult volunteers was used in method

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development and validation. Concentration of the target analytes in the urine pool were: 1.29 ng

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ml-1 diY, 3.08 ng ml-1 OHdG, 9.91 ng ml-1 MDA, 0.38 ng ml-1 8-PGF2α, 0.23 ng ml-1 11-PGF2α,

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0.89 ng ml-1 15-PGF2α, and 0.19 ng ml-1 8,15-PGF2α. Urine samples collected from 11 adult

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males and 10 adult females in 2017 in Albany, New York, USA, in 50-ml polypropylene tubes

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were analyzed to demonstrate the feasibility of the developed method. The urine samples stored

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at -20° C until analysis. No additional data other than gender were obtained from urine donors.

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Standard Solutions, Reagents, and Derivatization

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Stock solutions (1000 µg ml-1) of analytical standards were prepared for each OSB. An MDA

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solution was prepared in MeOH and stored in glass vials filled with nitrogen gas. F2-isoprostane

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standards were diluted with ethanol, diY solutions were prepared in MeOH, and 8-OHdG was

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diluted in HPLC water. The working standard solution mixture, which contained all OSBs, was

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prepared at 1000 ng ml-1 in MeOH. All stock and working solutions were stored at -20° C.

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D2-malondialdehyde (d2-MDA) stock solution was prepared by the hydrolysis of 1,1,3,3-

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tetraethoxypropane-1,3-D2 with 0.2 N HCl at room temperature for 2 h, as previously reported.24

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An internal standard (IS) mixture, containing d2-MDA (IS for MDA),

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15

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concentration of 500 ng ml-1 for each compound.

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C12-diY (IS for diY),

N5-8-OHdG (IS for 8-OHdG), and D4-8-PGF2α (IS for PGF2α isomers), was prepared at a



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Derivatization reagent (i.e., DNPH) was prepared at a concentration 0.05M in a

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water:ACN:acetic acid mixture (8:1:1 v/v). To reduce background contamination, DNPH

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solution was extracted twice with 5 ml of n-hexane by shaking the mixture for 5 min. After

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extraction, the organic layer was discarded, and DNPH solution was stored in darkness at 4° C

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until use. For the method development, MDA-DNPH derivative was prepared at 10 µg ml-1 by

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the addition of 200 µl of DNPH reagent to 500 µl of standard solution, incubated for 1 h at 60°

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C, and extracted twice with 1 ml of hexane for 5 min. Extracts were collected, evaporated to

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dryness, and reconstituted with MeOH. A BHT solution was prepared at a concentration of 2%

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in ethanol.

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The derivatization step was optimized by a 3 x 3 x 3 full-factorial design to examine the

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effect of temperatures (25° C, 37° C, 60° C), acidic conditions of DNPH reagent (12M HCl, 1%

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acetic acid, 2% formic acid) and concentrations of DNPH (0.005, 0.015, 0.05M), for a total of 27

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experiments. To determine the optimal conditions, in addition to complete derivatization of

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MDA, the stability of other non-aldehyde target analytes (i.e., 8-OHdG, diY, and four F2-

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isoprostane isomers) was checked under the reaction conditions. For derivatization, 10 ng of

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each IS were added to a 500-µl aliquot of sample. Under optimal conditions, 200 µl of

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derivatization reagent were added to the sample and incubated at room temperature for 30 min.

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Extraction of OSBs from Urine

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Target analytes were extracted from urine after the derivatization step. ABS ElutNexus

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cartridges (60mg, 3ml) (Agilent Technologies, Santa Clara, CA, USA) were used for SPE. For

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the optimization of SPE, performances of water and 0.1% acetic acid (in water) as conditioning

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solvents, water and 5% MeOH (in water) as washing solvents, and ACN, EtAc and MeOH 8 ACS Paragon Plus Environment

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(MeOH) as elution solvents, were studied in a 2 x 2 x 3 full-factorial design of experiments

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(DoEs).

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After conditioning the cartridge with 2 ml of MeOH and equilibration with 2 ml of water,

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samples (previously added with 2 ml of water) were loaded onto the cartridge and washed with 2

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ml of 5% MeOH in water. Then, a vacuum was applied to dry the cartridges for 5 min. The target

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analytes were eluted with 1 ml of MeOH followed by 1 ml of EtAc in the same tube. Eluate was

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evaporated to near-dryness under a nitrogen stream, reconstituted with 150 µl of water:MeOH

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(8:2 v/v), and transferred into vials with a glass insert for HPLC-MS/MS analysis.

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HPLC-MS/MS Analysis

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An HPLC-MS/MS system integrated by Agilent 1100 HPLC coupled with an ABSCIEX 4500

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mass spectrometer (Applied Biosystems, Foster City, CA, USA) was used for identification and

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quantification of target analytes. The chromatographic separation of target analytes was

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accomplished with a Zorbax Aq 3.5 µm column (2.1 × 150 mm) (Agilent, Santa Clara, CA,

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USA). The sample injection volume was 20 µl. The HPLC mobile phase comprised MeOH (A)

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and 0.01% acetic acid (B). The initial mobile phase composition was 100% B, held for 2 min,

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and then decreased to 45% B within 0.5 min. Then the composition was decreased to 25% B

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within 19.5 min, decreased to 0% B in 0.5 min, and held for 1.5 min. Flushing of the HPLC

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column and reverting to initial conditions were accomplished in the last 6 min, with a total run

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time of 30 min. Carryover of target analytes was not detected under these conditions.

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The MS/MS method was split into two time periods, consisting of the first period with a

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positive ionization mode for the detection of diY, 8-OHdG, and MDA, and, after 14 min,

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switched to the negative ionization mode (the second period) for the detection of four F29 ACS Paragon Plus Environment


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isoprostane isomers. Compound specific MS/MS parameters, m/z transitions of multiple reaction

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monitoring, and ion source parameters were optimized by the injection of an individual analyte

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standard solution at 1 µg ml-1 by flow injection analysis. Compound specific MS/MS parameters

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as well as retention times of the target analytes are listed in Table 1. Ion source parameters were

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optimized for improved ionization of the compounds eluted in that time window. Curtain gas

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(CUR), collision activated dissociation gas (CAD), source temperature (TEM), nebulizer gas

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(GS1), heater gas (GS2), and turbo ion spray voltage (IS) were set at 50 psi, 50 psi, 450° C, 50

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psi, 50 psi, and 4500 V for Period 1 and 50 psi, 50 psi, 650° C, 45 psi, 40 psi, and -4500 V for

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Period 2.

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A six-port diverter valve integrated into the ABSCIEX 4500 mass spectrometer was used

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to divert LC effluent to waste (position A) or to MS (position B) for the prevention of

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accumulation of salts and impurities in the ion source. The diverter valve was kept in Position A

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(waste) for up to 5.5 min of the analytical run, changed to B (MS) from 5.6 to 14.0 min (for the

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detection of diY, 8-OHdG and MDA), diverted to waste from 14.1 to 19.4 min, and then to MS

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from 19.5 to 29.0 min (for the detection of PGF2α isomers).

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Quantification

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Matrix-matched calibration solutions were prepared by the addition of increasing concentrations

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from 0.05 to 50 ng ml-1 for each target analyte in urine and processed, following the method

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described above. Linear regression of the response (area) of the ratio of the target compound to

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the corresponding IS versus concentration was used for quantification. Target OSBs were not

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detected in procedure blanks with the exception of MDA, which was found at 0.45 ng ml-1.

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Results

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Sample Preparation for LC-MS

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Extraction

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The extraction of OSBs from urine was first examined by liquid-liquid extraction using hexane,

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EtAc, or the mixture ACN:EtAc (1:1 v/v). This approach was unsuccessful due to the poor

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simultaneous extraction of target compounds in the solvents tested result of their different

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polarities (Table S1). Whereas the optimal solvent for the extraction of MDA-DNPH was

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hexane, F2-isoprostanes were preferably extracted in EtAc. diY was not effectively extracted by

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hexane or EtAc, but the mixture ACN:EtAc was found effective for the extraction of diY (Figure

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S1). Therefore, the SPE procedure was evaluated for the extraction and concentration of diY, 8-

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OHdG, F2-isoprostane isomers, and MDA-DNPH in urine samples. To assess the recovery of

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target analytes through the SPE procedure, urine samples were prepared by the addition of target

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analytes, including MDA-DNPH derivative, at 40 ng ml-1. To simulate experimental conditions,

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500 µl of samples were mixed with 200 µl of derivatization reagent, and 2 ml of water were

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added to reduce the composition of the organic solvent in samples.

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In the data analysis for the optimization of SPE, no statistical differences were found

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between the two washing solvents, but 5% MeOH in water was selected to increase the strength

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of the washing solution. Conditioning of cartridges with water yielded significantly higher

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recoveries for 8-PGF2α (p-value = 0.023). Among the three elution solvents tested, acceptable

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recoveries of OSBs (with minimal interferences) were observed with MeOH, which yielded good

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recoveries of diY (p-value = 0.0001) and 8-OHdG (p-value = 0.017), while EtAc increased the

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recovery of MDA (p-value = 0.005). The use of MeOH and ACN as elution solvents yielded

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similar results, and these two solvents yielded better recoveries than did EtAc for 8-PGF2α (p11 ACS Paragon Plus Environment


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value = 0.019). Mean graphs that show significant differences among various SPE parameters

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are presented in Figure S2, and adjustment coefficients and p-values are listed in Table S2.

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The elution step was further optimized for the combination/volume of elution solvents.

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Five different solvent combinations were studied: 4 ml MeOH, 3 ml MeOH followed by 1 ml

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EtAc, 2 ml MeOH followed by 2 ml EtAc, 1 ml MeOH followed by 3 ml EtAc, and 4 ml EtAc.

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SPE cartridges were eluted with the 4 ml of solvent, which was collected in 1 ml fractions

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(following elution). This test was performed in triplicate. In accordance with the DoEs, MeOH

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increased the recovery of all target analytes except MDA, which showed a better recovery with

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EtAc. The elution with 1 ml of MeOH followed by 1 ml of EtAc recovered more than 94% of the

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target analytes from the cartridge (data not shown).

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Under optimal conditions, the samples after derivatization were added with 2 ml of water

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and extracted using an SPE cartridge, conditioned previously with 2 ml of MeOH and 2 ml of

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water. After the sample addition, the cartridge was washed with 2 ml of 5% MeOH in water and

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vacuum dried for 5 min. Elution was performed with 1 ml of MeOH followed by 1 ml of EtAc.

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Both eluents were collected in the same tube and evaporated to near-dryness under a nitrogen

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stream, reconstituted with 150 µl of water:MeOH (8:2 v/v), and transferred into a vial for HPLC-

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MS/MS analysis.

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Derivatization of MDA Using DNPH

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Quantitative analysis of aldehydes is challenging due to their reactivity, volatility, and low

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molecular weight (Table S1). A derivatization step would increase the stability, molecular

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weight, and ionization of aldehydes. Previous methods of analysis of MDA were based on its

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reaction with TBA, which required a high temperature for the formation of TBA derivatives.27 12 ACS Paragon Plus Environment

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Derivatization, using DNPH followed by LC-MS analysis, has been reported for the

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determination of MDA in urine.24 Nevertheless, the earlier method was not applied for the

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simultaneous analysis of other OSBs in urine.

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For the DoE study, 500 µl of urine (pooled sample) were spiked at 40 ng ml-1 of each

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target analyte. An IS mixture and 200 µl of DNPH were added, vortexed, and incubated for 1 h

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at the temperatures listed above. An incubator was used for the incubation of test solutions at 25°

277

C, 37° C, and 60° C. Samples were then extracted by SPE and analyzed by LC-MS/MS. No

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differences in the recoveries of target chemicals were found between various concentrations of

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DNPH. With regard to the use of different acids during derivatization, acetic acid and formic

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acid yielded similar results, while HCl affected the concentration of non-aldehyde OSBs by

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decreasing the levels of F2-isoprostanes (p-value < 0.0001) and 8-OHdG (p-value < 0.0001) and

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increasing diY levels (p-value < 0.0001). Therefore, acetic acid was selected for further

283

experiments. With the increase in temperature, the concentration of MDA-DNPH increased (p-

284

value = 0.018). 8-OHdG levels also increased (p-value = 0.019), however, while 8-PGF2α, 11-

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PGF2α, 15-PGF2α, and 8,15-PGF2α (p-values = 0.004, 70% of the samples.


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The proposed method represents a major improvement over the previously reported methods

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for the individual OSB determination in terms of specificity and efficiency. ELISA methods

408

were used for the analysis of 8-OHdG or 8-PGF2a and TBA assay was used for MDA analysis,

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but they lacked specificity. Other methods for the analysis of F2-isopostanes by GC-MS require

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extended sample preparation. A major strength of this study lies in the measurement of several

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OSBs in a single extraction and detection method. A limited number of studies have reported a

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method for simultaneous determination of more than one OSB in urine. A method for the

413

analysis of MDA, 8-PGF2α, and hydroxynonenal (HNE; biomarker of lipid peroxidation) in

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exhalated breath condensate, plasma, and urine has been reported.5 Simultaneous analysis of 8-

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OHdG, HNE-MA (hydroxynonenal-mercapturic acid), and 8-PGF2α in urine also has been

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reported.22 Our goal was to optimize and validate a method for the determination of

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concentrations of major OSBs produced by the oxidation of lipids, proteins, and DNA in urine.

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Apart from simultaneous determination of seven OSBs in urine, the LODs of this method are

419

below the actual concentrations found in urine from healthy individuals. As the levels of OSBs

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are expected to be higher in individuals with disease conditions or exposed to stressors, this

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method can be applied to a range of population-based studies. Our method allows for

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simultaneous analysis of degradation products of lipids, proteins, and DNA, which implies that

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mechanisms of toxicity or disease progression can be evaluated by measuring effects on major

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biomolecules present in human cells.

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The proposed method, based on DNPH derivatization followed by SPE-LC-MS/MS, has been

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optimized and validated for the analysis of seven OSBs in urine. Optimization of DNPH

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derivatization ensured maximum yield for MDA-DNPH derivative without affecting the

428

quantification of other OSBs in the same sample. The optimization of SPE for the extraction of 19 ACS Paragon Plus Environment


429

target OSBs was accomplished, and the need for a suitable elution solvent for quantitative

430

recovery of all seven OSBs from the SPE cartridge was enabled. The applicability of the method

431

was tested by the analysis of 21 real urine samples. diY, 8-OHdG, and MDA were found in all

432

samples, and F2-isoprostanes were found in >70% of the urine samples. The proposed method

433

can be applied in epidemiological and toxicological studies that link OSBs with health outcomes

434

from exposure to environmental stressors in human populations.

435

Supporting Information

436

Additional information is provided in the supporting information, as listed above (Table S1–S6,

437

Fig. S1–S6). Tables showing physicochemical properties of target chemicals, SPE method

438

development and optimization results, optimization of derivatization steps, slopes of calibration

439

curves prepared in urine, concentrations of OSBs in individual urine samples, and reported

440

LOQs for target analytes; figures showing recoveries from liquid-liquid extraction, mean plots of

441

SPE optimization and derivatization results, derivatization time on OSB recoveries,

442

derivatization reaction, Box and Whisker plots of ODB distribution in males and females. The

443

Supporting Information is available free of charge on the ACS Publications website.

444

Author Contributions

445

The manuscript was written with contributions from all authors. All authors have given approval

446

of the final version of the manuscript.

447

Notes

448

The authors declare no competing financial interest.

449

Acknowledgments 20 ACS Paragon Plus Environment

Page 20 of 31

Page 21 of 31


450

Research reported in this publication was supported in part by the National Institute of

451

Environmental Health Sciences of the National Institutes of Health under Award Number

452

U2CES026542-01. The content is solely the responsibility of the authors and does not

453

necessarily represent the official views of the National Institutes of Health.

454 455

References

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Figure 1. Schematic of oxidative stress biomarkers formation from proteins, deoxyribonucleic acid (DNA), arachidonic acid and polyunsaturated fatty acids (PUFA).



Figure 2. Total ion chromatogram (TIC) and extracted ion chromatograms of A. Real urine sample from a healthy adult (F8: 0.76 ng ml-1 diY, 5.41 ng ml-1 8-OHdG, 14.1 ng ml-1 MDA, 0.46 ng ml-1 8-PGF2α, 0.33 ng ml-1 11-PGF2α, 0.54 ng ml-1 15-PGF2α, 0.65 ng ml-1 8,15-PGF2α); B. Spiked urine at 0.6 ng ml-1 of target chemicals. Peak identification: 1. diY and 13C12-diY; 2. 8OHdG and 15N5-8-OHdG; 3. MDA and D2-MDA; 4. 8,15-PGF2α, 5. 8-PGF2α and D4-8-PGF2α, 6. 11-PGF2α and 7. 15-PGF2α.


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Page 27 of 31


Table 1. Retention time (RT) and compound dependent tandem mass spectrometric parameters ionization mode, multiple reaction monitoring (MRM) period, MRM transition, declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) used in the analysis of oxidative stress biomarkers in urine.

RT

Ionization

MRM

(min)

mode

Period

diY

7.6

+

1

13

7.6

+

8-OHdG

8.0

15

N5-8OHdG

MRM

DP

EP

CE

CXP

(V)

(V)

(V)

(V)

353.0 > 315.0

45

10

23

10

1

373.0 > 327.0

56

10

23

10

+

1

284.0 > 168.0

25

3

18

4

8.0

+

1

289.0 > 173.0

25

3

18

4

MDA-DNPH

13.0

+

1

235.0 > 159.0

30

8

30

3

D2-MDA-DNPH

13.0

+

1

237.0 > 159.0

30

8

30

3

8,15-PGF2α

20.4

-

2

353.0 > 193.0

-40

-6

-40

-2

8-PGF2α

21.2

-

2

353.0 > 193.0

-45

-8

-35

-2

D4-8-PGF2α

21.2

-

2

357.0 > 197.0

-45

-8

-35

-2

11-PGF2α

21.7

-

2

353.0 > 193.0

-30

-8

-35

-2

15-PGF2α

22.7

-

2

353.0 > 193.0

-35

-8

-35

-2

Compound

C12-diY

Transition (m/z)



Page 28 of 31

Table 2. Limits of detection (LOD) and quantification (LOQ), repeatability, intra-day precision and accuracy of the method developed for the analysis of oxidative stress biomarkers in urine. Repeatability, intra-day repeatability and recovery were measured at three concentrations: A. 0.1 ng ml-1, B. 1.0 ng ml-1, C. 10 ng ml-1

Compound

LOD,

LOQ,

Repeatability,

Intra-day

ng ml-1

ng ml-1

RSD %

precision RSD %

Recovery, % ± 95%CI

A

B

C

A

B

C

A

B

C

diY

0.030

0.10

9

9

9

10

9

9

109 ± 20

103 ± 20

98 ± 9

8-OHdG

0.030

0.10

7

7

8

9

5

5

92 ± 17

101 ± 22

97 ± 10

MDA

0.024

0.08

10

4

6

10

9

16

109 ± 24

112 ± 16

91 ± 18

8,15-PGF2α

0.013

0.04

8

8

6

12

7

14

105 ± 15

102 ± 5

95 ± 26

8-PGF2α

0.010

0.03

7

7

4

8

9

8

103 ± 23

96 ± 14

93 ± 18

11-PGF2α

0.016

0.05

8

6

4

2

13

9

107 ± 16

96 ± 8

93 ± 11

15-PGF2α

0.012

0.04

7

10

6

13

10

9

94 ± 26

92 ± 23

103 ± 18


Page 29 of 31


Table 3. Stability of oxidative stress biomarkers in urine samples after storage at -20°C, three freeze-thaw cycles or light exposure during derivatization and analysis (expressed as % recovery ± 95% confidence interval). A. No BHT addition. B. BHT addition at urine collection. C. BHT addition at the beginning of analysis.

diY

8-OHdG

MDA

8,15-PGF2α

8-PGF2α

11-PGF2α 15-PGF2α

A

100 ± 12

100 ± 5

100 ± 14

100 ± 11

100 ± 8

100 ± 6

100 ± 13

B

105 ± 20

105 ± 8

105 ± 13

102 ± 1

102 ± 2

104 ± 20

100 ± 2

C

112 ± 14

96 ± 2

98 ± 2

98 ± 11

95 ± 4

88 ± 6

100 ± 2

A

112 ± 10

88 ± 7

90 ± 14

94 ± 13

88 ± 10

106 ± 8

89 ± 12

B

102 ± 15

91 ± 6

87 ± 8

98 ± 21

89 ± 15

112 ± 25

91 ± 18

C

116 ± 13

98 ± 5

84 ± 2

98 ± 10

96 ± 2

116 ± 3

95 ± 5

A

104 ± 13

98 ± 4

96 ± 8

86 ± 10

95 ± 2

116 ± 12

98 ± 14

B

97 ± 12

99 ± 4

92 ± 11

89 ± 10

96 ± 7

112 ± 16

100 ± 5

C

116 ± 9

98 ± 4

97 ± 9

90 ± 10

98 ± 14

106 ± 3

105 ± 2

A

109 ± 14

93 ± 8

43 ± 1

80 ± 13

95 ± 9

138 ± 24

97 ± 22

B

112 ± 10

88 ± 3

42 ± 4

79 ± 12

96 ± 6

146 ± 5

98 ± 4

C

109 ± 24

93 ± 3

43 ± 5

81 ± 13

95 ± 4

138 ± 4

108 ± 4

A

107 ± 16

111 ± 16 130 ± 25

79 ± 1

97 ± 1

108 ± 12

122 ± 12

B

117 ± 15

112 ± 10

120 ± 7

77 ± 6

97 ± 7

120 ± 21

135 ± 12

C

103 ± 27

105 ± 25

130 ± 22

79 ± 19

93 ± 8

102 ± 17

143 ± 10

A

98 ± 15

101 ± 6

104 ± 5

91 ± 15

101 ± 10

93 ± 1

89 ± 2

B

115 ± 5

98 ± 5

103 ± 3

108 ± 18

99 ± 14

96 ± 13

99 ± 10

C

95 ± 5

102 ± 19

116 ± 5

112 ± 18

108 ± 4

105 ± 12

105 ± 9

Storage time Collection (0 day)

1 day

7 days

30 days

Freeze-thaw cycles

Light exposure



Page 30 of 31

Table 4. Concentrations of seven oxidative stress markers (ng ml-1) in urine sample collected from healthy adults, 11 males (M) and 10 females (F) from Albany, New York, USA.

diY

8-OHdG

MDA

8-PGF2α

11-PGF2α

15-PGF2α

8,15-PGF2α

M Median

1.64

12.7

15.1

0.81

0.34

1.34

1.11

M Min

0.54

6.64

8.14

0.26

0.14

0.19

0.30

M Max

4.34

21.8

33.3

2.15

1.06

2.17

1.41

F Median

0.68

5.91

13.18

0.34

0.13

0.36

0.53

F Min

0.20

1.45

1.98

0.28

0.10

0.051

0.048

F Max

1.10

16.93

31.6

0.69

0.33

0.70

0.77

Median

0.99

9.18

14.07

0.55

0.28

0.56

0.76

Min

0.20

1.45

1.98

0.03

0.10

0.051

0.048

Max

4.34

21.8

31.6

2.15

1.06

2.17

1.41

100

100

100

85

71

85

81

Detection frequency, %


Page 31 of 31


TOC_ es-2018-00883t.R1 Simultaneous analysis of seven biomarkers of oxidative damage to lipids, proteins, and DNA in urine

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