Development of Liquid Chromatographic Methods for Quantification of Malondialdehyde, 8-Oxo-2'-deoxyguanosine, and Hydroxylated Metabolites of Polycyclic Aromatic Hydrocarbons in Urine Open Access

Kartavenka, Kanstantsin (Fall 2018)

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Household air pollution as a result of use of biomass burning for cooking, heating and/or lightning was shown to be associated with numerous adverse health outcomes, including cancer and respiratory disorders, leading to 2.8 million deaths in 2015 worldwide. Despite global awareness, one third of the world population is still relying on biomass, predominantly from low- and middle-developed countries (LMDC). The incomplete combustion of this fuel results in a complex mixture, including polycyclic aromatic hydrocarbons (PAHs). Internal exposure dose to biomass burning is commonly assessed by measuring PAHs metabolites (OH-PAHs) in urine (biomarkers of exposure); however, no single precise biomarker was found for biomass burning exposure. Due to metabolism of PAHs it was suggested to use additionally oxidative stress biomarkers such as malondialdehyde (MDA) and a biomarker of oxidative DNA damage 8-Oxo-2'-deoxyguanosine (8-Oxo-dG) (biomarkers of effect). Most of existing methods are using either non-specific ultraviolet/visible or fluorescence detectors, or highly expensive tandem quadrupole mass detectors (MS/MS). Additionally, to characterize these three group of compounds, commonly three different methods are used. The complication of such analysis prevents the ability of LMDC to understand better exposure and quantify biomarkers of exposure and effect inside their countries. To achieve improvements in analytical capacities, I used liquid chromatographic (LC) system coupled with single quadrupole mass spectrometry (MS) (Aim 1, 2, 3) and due to limitation LC MS, I used LC MS/MS system to allow simultaneous analysis of all three groups of compounds (Aim 4).


While majority of my work is dedicated to urinary measurements, initial method development started with exhaled breath (EBC) condensate with quantification of MDA using LC MS. To validate method applicability and robustness, 205 individual EBC samples were analyzed. Further analysis of MDA in urine samples using LC MS took into the account limitation that were observed in the Aim 1. To validate method for Aim 2, I used two set of pulled urine samples, pooled serum sample and unknown 138 urine samples given by Chaing Mai university. Kinetics of the reaction, autosampler storage stability were assessed. The method was tested with and without solid phase extraction. For Aim 3, we randomly selected sample previously analyzed as a part of cardiopulmonary outcomes and household air pollution trial. For validation of method for Aim 4, where OH-PAHs, MDA, and 8-Oxo-dG were quantified, urine samples collected from pregnant agricultural works in Northern Thailand in 2012 was used.


In Aim 1, we developed a sensitive analytical method with established reagent previously used in low-resolution, fluorescence methods. We adopted the method to mass spectrometry approach which required advanced chromatography to achieve low limit of detection necessary for the specific matrix. In Aim 2, we take limitations from Aim 1 into the account, and developed a new approach to quantify MDA in urine and serum samples focusing on automatization of the protocol. In Aim 3, we developed a sensitive method that is capable for quantification of OH-PAHs in urine, which potentially allow low- and middle-income countries to understand exposure related to household air pollution widely presented there. For the Aim 4, the first analytical method for simultaneous quantification of MDA, 8-Oxo-dG, and OH-PAHs was developed.


Overall, the present work describes a novel approach of using cost-effective single quadrupole mass spectrometry to quantify biomarkers of exposure and effect in urine samples. We showed that several compartments of the analytical instrument can be used more effectively to minimize cost of analysis. We demonstrated advanced application of sample preparation techniques including re-using of solid-phase extraction cartridges and combining analytes of different properties into one single analytical method via derivatization.

Table of Contents

Introduction. 1

Dissertation aims 6


Abstract 8

Keywords 8

Introduction. 9

Material and methods. 11

Results. 15

Discussion. 16

Conclusion. 19

Funding. 19


Abstract 29

Keywords 29

Introduction. 30

Material and methods. 31

Results. 37

Discussion. 43

Conclusion. 45

Acknowledgement 46

References 47


Abstract 51

Keywords 51

Introduction. 52

Material and methods. 54

Results and Discussion. 64

Conclusion. 81

Funding. 82

References 83


Abstract 87

Keywords 87

Introduction. 88

Material and methods. 91

Results. 101

Discussion. 112

Conclusion. 116

Funding. 117

References 118

Conclusions. 121

Future Work. 123

References 124

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