development and application of high-throughput...

Development and Application of High-throughput Methods for Analysis of

Persistent Organic Pollutants in Human Blood

Örebro Studies in Chemistry 12

SAMIRA SALIHOVIC

Development and Application of High-throughput Methods for Analysis of

Persistent Organic Pollutants in Human Blood

© Samira Salihovic, 2013

Title: Development and Application of High-throughput Methods for Analysis of Persistent Organic Pollutants in Human Blood.

Publisher: Örebro University 2013

www.publications.oru.se [email protected]

Print: Örebro University, Repro 04/2013

ISSN 1651-4270 ISBN 978-91-7668-936-3

http://www.publications.oru.se/

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Abstract Samira Salihovic (2013): Development and Application of High-throughput Methods for Analysis of Persistent Organic Pollutants in Human Blood. Örebro Studies in Chemistry 12, 70 pp. The general public is continuously exposed to a wide range of environmen-tal pollutants. This thesis focuses on a group of anthropogenic chemicals referred to as persistent organic pollutants (POPs) that have been linked to various adverse health effects in humans.

The main objective of this thesis was focused on the development and application of high-throughput methods for analysis of a broad range of chlorinated, brominated, and fluorinated POPs in human blood.

After establishing that the methods were effective, the two methods were applied to human plasma samples to examine the background levels of a broad range of POPs in human plasma samples among elderly men and women from Sweden and to assess the influence of gender. Levels of a wide range of chlorinated, brominated, and fluorinated compounds were deter-mined in plasma samples collected during 2001-2004 from 1, 016 (50.2% women) 70 year-old participants from the population-based Prospective Study of the Vasculature in Uppsala Seniors (PIVUS). The POPs studied were 16 polychlorinated biphenyls (PCBs), 5 organochlorine pesticides (OCPs), 1 dioxin, 1 brominated flame retardant as well as 14 perfluoroal-kyl substances (PFAS) including structural perfluorooctane sulfonic acid (PFOS) isomers. The majority of the studied compounds were detected in the 70-100% of the participants. The structural PFOS isomers were suc-cessfully quantified in a sub-sample of 25 men and women. Furthermore, gender differences in the concentrations of the POPs studied showed that the majority of chlorinated and brominated compounds were significantly different when comparing men and women in the study, while the concen-trations of the fluorinated compounds were found to be less influenced by gender.

This thesis has, by using the developed high-throughput methods requir-ing only small amounts of human blood, provided background exposure information of a broad range of POPs for an epidemiological study.

Keywords: Human blood; POPs; Perfluoroalkylated substances; Structural isomers; Sample preparation; Column-switch; Mass spectrometry Samira Salihovic, School of Science and Technology, Örebro Univeristy, SE-701 82 Örebro, Sweden, [email protected]

Abbreviations BFR brominated flame retardant CI confidence interval CV coefficient of variation DDT trichlorodichlorophenyl-ethane ECF electrochemical fluorination EPA environmental protection agency ESI electrospray ionization GC gas chromatography GMP global monitoring plan HLB hydrophilic-lipophilic-balanced HPLC high-performance liquid chromatography HRGC high-resolution gas chromatography HRMS high-resolution mass spectrometry IPE ion-pair extraction IS Internal standard LC liquid chromatography LLE liquid-liquid extraction LOD limit of detection LOQ limit of quantification LRMS low-resolution mass spectrometry MDL method detection limit MRM multiple reaction monitoring MS mass spectrometry MS/MS tandem mass spectrometry NIST National Institute for Standards and Technology OC organochlorine PP protein precipitation QA quality assurance QC quality control RSD relative standard deviation SIM single ion monitoring SPE solid-phase extraction SRM standard reference material TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin TEF toxic equivalence factor UNEP United Nations Environment Programme UPLC ultra-performance liquid chromatography Abbreviations of chlorinated, brominated, and fluorinated compounds are presented in Table 1and 2.

Table of Contents 1 INTRODUCTION ................................................................................ 13 1.1 Persistent organic pollutants (POPs)................................................... 14

1.1.1 Chlorinated and brominated compounds ................................. 14 1.1.2 Fluorinated compounds ........................................................... 17

1.2 Sources of exposure ........................................................................... 19 1.2.1 Trends over time ...................................................................... 19

1.3 Relevance for human health ............................................................... 20 1.3.1 Limitations with epidemiological studies ................................. 21

1.4 Aims of this thesis .............................................................................. 22

2 PARTICIPANTS AND PROCEDURES ................................................ 23

3 BACKGROUND TO ANALYTICAL METHODS FOR POPs ANALYSIS IN HUMAN BLOOD ........................................................... 25 3.1 Current principles in sample preparation and instrumental analysis .. 26

3.1.1 Chlorinated and brominated compounds ................................. 26 3.1.2 Fluorinated compounds ........................................................... 27

3.2 Major analysis trends ......................................................................... 28

4 DEVELOPMENT OF NEW HIGH-THROUGHPUT METHODS ....... 29 4.1 Analysis of chlorinated and brominated compounds .......................... 29 4.1.1 Method validation .......................................................................... 32

4.1.1.1 Accuracy and precision ..................................................... 32 4.1.1.2 Throughput and robustness .............................................. 34

4.2 Analysis of fluorinated compounds .................................................... 34 4.2.1 Method validation ................................................................... 38

4.2.1.1 Accuracy and precision ..................................................... 38 4.2.1.2 Throughput and robustness .............................................. 39 4.2.1.3 Isomer specific analysis of PFOS ....................................... 41

5 CONCENTRATIONS OF POPs IN THE PIVUS COHORT ................ 42 5.1 Chlorinated and brominated compounds ........................................... 42 5.2 Fluorinated compounds ..................................................................... 45 5.3 POPs in relation to each other and the influence of gender ................ 48 5.4 Applied POPs analysis and the implications for human health ........... 51

6 CONCLUSIONS ................................................................................... 52

7 FUTURE DIRECTIONS ....................................................................... 53

8 ACKNOWLEDGEMENTS ................................................................... 54

9 REFERENCES ...................................................................................... 56

SAMIRA SALIHOVIC Analysis of persistent organic pollutants in human blood I 13

1 Introduction Persistent organic pollutants (POPs) are a group of chemicals with unique properties that have been shown valuable in the industry, agriculture, and a wide variety of commercial applications (Jones et al., 1999, Lindstrom et al., 2011). However, while these chemicals may be useful and even neces-sary they have also been shown to be toxic to humans (Schecter et al., 2006) and wildlife (Letcher et al., 2010) as well as extremely persistent in the environment. There is, in general, very little an individual can do to affect the background exposure to these pollutants and knowledge of the potential adverse health effects from POPs have still not been completely evaluated (White et al., 2009). What is known, however, is that when POPs enter the human body they accumulate and persist for years before finally being metabolized to more soluble but sometimes more persistent substances (Milbrath et al., 2009). At this point, very little is known about the possible negative health effects in response to the endocrine disrupting properties that many of the POPs have been shown to exhibit (Darnerud, 2008, Diamanti-Kandarakis et al., 2009). Another reason for concern is that while epidemiologists still are trying to understand the human health effects of several of the legacy POPs, new chemicals are rapidly entering the market (Muir et al., 2010). Even less is known about the toxic potential and the possible synergistic or additive effects of the mixtures of chemicals that the human population is exposed to (Kortenkamp, 2008). As a step in that direction, the development and improvement of analytical methods has played a crucial role for the identification and determination of POPs in low background concentrations in blood from general populations (Muir et al., 2006).

Over 40 years of analytical improvements have made it possible to mon-itor POPs and while at the same time allow for a significant increase in the analytical capacity as well as an overall reduction in the amount of human blood required for analysis. At present, novel and specialized automated analytical methods that provide cost-effective analysis of POPs are being developed and the challenges in targeted epidemiology and large scale hu-man biomonitoring studies are being overcome. Nonetheless, there is still a need for selective, specific, and sensitive analytical methods that are able to detect the legacy POPs as well as emerging pollutants in small amounts of human blood. Thus, the main theme of this thesis is to develop and vali-date new methods for analysis of a broad range of POPs in less than 1.0 mL of human blood from participants in an epidemiological study.

14 I SAMIRA SALIHOVIC Analysis of persistent organic pollutants in human blood

1.1 Persistent organic pollutants (POPs) Over the past decades, countries have become increasingly aware of the potential public health risks posed by different environmental pollutants. As a result, in May 2001, the Stockholm Convention on POPs was initiat-ed (UNEP, 2013). The Stockholm Convention defines POPs as man-made chemicals that persist in the environment, bioaccumulate in living organ-isms, are toxic to humans and wildlife, and are capable of being distributed globally via long-range transport. These characteristics combined have led to the well-developed idea that POPs are a group of environmental pollu-tants of particular public health concern. The overall objective of the Stockholm Convention is to reduce and eliminate the releases of POPs to the environment and thus protect its inhabitants. In 2001, more than 100 countries had signed the Stockholm Convention, binding them to eliminate the use of 12 POPs of greatest concern and today this list has been expand-ed to include 21 different classes of POPs and 179 countries have signed the Stockholm Convention (UNEP, 2013).

The compounds included in the thesis were chosen based on the recom-mendations in the global monitoring plan (GMP) for analysis of POPs in human blood issued by the Stockholm Convention (UNEP, 2011). Fur-thermore, the different classes of compounds were also selected based on their increasing or decreasing trends and whenever possible at least one compound of each compound class regulated by the Stockholm Conven-tion was included. More specifically, in the group of fluorinated com-pounds, several were added due to their inherent potentials to become POPs. The POPs studied in this thesis have been categorized into two main groups according to their physico-chemical properties and are listed in Table 1 and 2.

1.1.1 Chlorinated and brominated compounds The polychlorinated biphenyls (PCBs) are polyhalogenated aromatic hy-drocarbons theoretically present in 209 different congeners related to their degree of chlorination (Jensen, 1972). Among the 209 PCB congeners, only twelve non-ortho and mono-ortho substituted congeners have toxicity and structural features which make them similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and they are considered to have dioxin-like properties (UNEP, 2013). From the late 1920s until the late 1970s, PCBs were manu-factured for use in various industrial applications. The PCBs studied in this thesis were nine nondioxin-like congeners (#74, #99, #138, #153, #170,


#180, #194, #206, and #209) and seven dioxin-like congeners (#105, #118, #126, #156, #157, #169, and #189) that have been assigned toxic equivalency factors (TEF) by the World Health Organization (Van den Berg et al., 2006). In total, sixteen PCBs were included in this thesis and they are listed in Table 1.

The organochlorine pesticides (OCPs) are a large group of compounds that have been used as agrochemicals. Their toxicity and capability to con-trol insects in agricultural settings have led to their ubiquitous environmen-tal distribution. Included in the group of OCPs are toxaphene, mirex, hep-tachlor, aldrin, endrin, and dieldrin, but also chlordane, hexachloroben-zene (HCB), and trichlorodichlorophenyl-ethane (DDT).

DDT is one of the most well-known amongst the OCPs and large quan-tities of DDT have been distributed worldwide (Eskenazi et al., 2009). It was not until the 1970s that the elimination of DDT was initiated in Swe-den and several other countries worldwide (Swedish Chemicals Inspectorate, 2006). DDT is easily degraded into two major breakdown products and one of them is 4, 4’-dichlorodiphenyldichloro-ethylene (p, p’-DDE). In contrast to the parent compound, p, p’-DDE is extremely persis-tent and has been detected in general populations worldwide. HCB and chlordane are also included in the group of OCPs and they have been used in various industrial applications. The production of both HCB and chlor-dane started in the end of 1940s. HCB and chlordane were primarily used in agricultural applications, but HCB can also be formed as a by-product or impurity in the production of industrial chemicals. In Sweden, the his-torical use of HCB and chlordane was limited and relatively low amounts were introduced to the environment (Swedish Chemicals Inspectorate, 2006).

In this thesis, analysis was focused on the OCPs that are frequently de-tected in the general populations and the other Stockholm Convention OCPs such as toxaphene, mirex, aldrin, endrin, and dieldrin were, there-fore, not were included due to the low expectancy for detection.


Table 1. Chlorinated and brominated compounds included in the thesis Analyte Abbreviations Polychlorinated biphenyls PCBs Tetrachlorobiphenyl 2,4,4’, 5-tetrachlorobiphenyl PCB #74 Pentachlorobiphenyl 2,2’, 4,4’, 5-pentachlorobiphenyl PCB #99 2,3,3’, 4,4’-pentachlorobiphenyl** PCB #105 2,3’, 4,4’, 5-pentachlorobiphenyl** PCB #118 3,3’, 4,4’, 5-pentachlorobiphenyl * PCB #126 Hexachlorobiphenyl 2,2’, 3,4,4’, 5’-hexachlorobiphenyl PCB #138 2,2’, 4,4’, 5,5’-hexachlorobiphenyl PCB #153 2,3,3’, 4,4’, 5-hexachlorobiphenyl** PCB #156 2,3,3’, 4,4’, 5’-hexachlorobiphenyl** PCB #157 3,3’, 4,4’, 5,5'-hexachlorobiphenyl * PCB #169 Heptachlorobiphenyl 2,2’, 3,3’, 4,4’, 5-heptachlorobiphenyl PCB #170 2,2’, 3,4,4’, 5,5’-heptachlorobiphenyl PCB #180 2,3,3’, 4,4’, 5,5’-heptachlorobiphenyl** PCB #189 Octachlorobiphenyl 2,2’,3,3’,4,4’,5,5’-octachlorobiphenyl PCB #194 Nonachlorobiphenyl 2,2’,3,3’,4,4’,5,5’,6-nonachlorobiphenyl PCB #206 Decachlorobiphenyl 2,2’,3,3’,4,4’,5,5’,6,6’-decachlorobiphenyl PCB #209

Polychlorinated dibenzo-p-dioxin PCDD Octachlorodibenzo-p-dioxin OCDD

Organochlorine pesticides OCPs Hexachlorobenzene HCB trans-chlordane cis-chlordane trans-nonachlor

2,2-Bis(4-chlorophenyl)-1,1-dichloroethene p,p’-DDE

Polybrominated diphenylether PBDE

2,2’, 4,4’-tetrabromodiphenyl ether BDE #47 Note: * non-ortho-substituted congener ** mono-ortho-substituted congener


The polychlorinated dibenzo-p-dioxins (PCDDs) represent the group of compounds that are present in 75 different congeners related to their de-gree of chlorination. PCDDs can be naturally produced by forest fires and volcanic activity. However, they are primarily produced as unintentional by-products during incomplete incineration and combustion processes (UNEP, 2013). PCDDs have been extensively monitored and are still found in the blood from general populations. There are a number of reports on PCDDs and their potency to cause adverse health effects in humans and animals (Schecter et al., 2006, Mocarelli et al., 2008). The most frequently detected PCDD congener, octachlorodibenzo-p-dioxin (OCDD), has also shown to be the major contributor to the total PCDD body burden among all detected congeners (Bates et al., 2004, Harden et al., 2007, Patterson et al., 2009). Although OCDD has a low TEF, concentrations of OCDD can be used as a marker of PCDD exposure and was therefore selected in this thesis.

The polybrominated diphenyl ethers (PBDEs) are a class of compounds collectively referred to as brominated flame retardants (BFRs). PBDEs can be found in 209 different congeners depending on their degree of bromina-tion. Since the 1960s, PBDEs have been shown to be useful in a wide varie-ty of consumer products, for example in computers and textiles used for furnishing where their flame retardant properties have been valuable. About 40 of the 209 PBDE congeners can be found in commercial products in the three major commercial PBDEs mixtures typically produced. In this thesis, the congener BDE #47 was included as a marker for PBDE exposure because it has been widely studied in general populations.

1.1.2 Fluorinated compounds The per- and polyfluoroalkyl substances (PFAS) are a class of compounds consisting of a carbon chain that is fully or partly substituted with fluorine atoms with a functional group attached to one end. Depending on the functional group the PFAS can either be ionic or neutral. The ionic PFAS account for the majority of fluorinated compounds included in this thesis and they are referred to as perfluorinated carboxylic acids (PFCAs) and perfluorinated sulfonic acids (PFSAs). Only one compound is neutral and comes from the group of perfluoroalkane sulfonamides, namely perfluo-rooctane sulfonamide (PFOSA). The PFAS have since the end of 1940s been produced in industrial settings and are widely distributed in the envi-ronment due to extensive usage in various commercial applications such as surface protection products and firefighting foams (Prevedouros et al.,


2005, Lindstrom et al., 2011). Table 2 shows the fluorinated compounds included in this thesis and their abbreviations using recently proposed no-menclature by Buck et al. (2011).

Table 2. Fluorinated compounds included in the thesis Analyte Abbreviations

Perfluorinated carboxylic acids PFCAs Perfluoropentanoic acid PFPeA Perfluorohexanoic acid PFHxA Perfluoroheptanoic acid PFHpA Perfluorooctanoic acid PFOA Perfluorononanoic acid PFNA Perfluorodecanoic acid PFDA Perfluoroundecanoic acid PFUnDA Perfluorododecanoic acid PFDoDA Perfluorotridecanoic acid PFTrDA

Perfluorinated sulfonic acids PFSAs Perfluorobutane sulfonic acid PFBuS Perfluorohexane sulfonic acid PFHxS Perfluorooctane sulfonic acid* PFOS

Linear-perfluorooctane sulfonic acid L-PFOS 1-perfluoromonomethyl-PFOS 1-PFOS 6-, 2-perfluoromonomethyl-PFOS 6/2-PFOS 3-, 4-, 5-perfluoromonomethyl-PFOS 3/4/5-PFOS 4.4-, 4.5-, 5.5- perfluorodimethyl-PFOS 4.4/4.5/5.5-PFOS

Perfluorodecane sulfonic acid PFDS

Perfluoroalkane sulfonamides PFASAs Perfluorooctane sulfonamide* PFOSA Note: *compounds listed in the Stockholm Convention

PFAS are produced in the industrial applications predominantly by two different processes, electrochemical fluorination (ECF) and polymerization (3M., 1999, Kissa, 2001). In recent years, interest has been focused on the distribution of perfluorooctane sulfonate (PFOS) and its mono- and disub-stituted branched perfluoromethyl PFOS isomers. Nonetheless, the struc-tural PFOS isomers have been found important in the source elucidation of PFOS related compounds (Benskin et al., 2010a, Benskin et al., 2010b, Martin et al., 2010). Furthermore, recent findings have suggested that the linear and branched isomers of PFOS exhibit different physico-chemical properties and that the branched mono- and disubstituted isomers are more chemically stable than their linear counterparts (Rayne et al., 2010). This might have an effect on the isomer patterns found in humans. These


findings show that not only does the isomer patterns found in general pop-ulations play an important role for source elucidation of PFOS and its pre-cursors but also that the isomer specific differences in their chemical prop-erties might also have different effects in humans. As yet, no studies have investigated these associations. Therefore, the fluorinated compounds in-cluded in this thesis are 14 PFAS including five structural PFOS isomers.

1.2 Sources of exposure Chlorinated, brominated, and fluorinated POPs are widespread chemicals that are found in various biological and environmental media around the world. In the general population, a large part of the POP exposure arises through the dietary exposure (Darnerud et al., 2006, Haug et al., 2011a). In addition to food, drinking water has also been found to be an important source of exposure to the fluorinated compounds (Domingo et al., 2012). The general population might further be exposed through the surrounding air, dust, and other products in the home environment (Johnson et al., 2010, Haug et al., 2011b, Ericson Jogsten et al., 2012). The exposure of small children is often greater than the rest of the population, because in-fants are exposed to large amounts of POPs via breastfeeding (Kärrman et al., 2007a, LaKind et al., 2009, Thomsen et al., 2010). In Sweden, con-sumption of heavily contaminated fish from the Baltic Sea has been report-ed to be a significant source of exposure (Darnerud et al., 2006, Isosaari et al., 2006).

1.2.1 Trends over time Human exposure to chlorinated compounds has been investigated in sever-al contexts (Patterson et al., 2009, Linderholm et al., 2010). The presence of the chlorinated POPs often reflects historical use, since several have been banned already decades ago. Similarly, PBDEs have been detected in gen-eral populations (Sjodin et al., 2008, Fangstrom et al., 2008, Foster et al., 2011). Concentrations of BDE #47 in North America are generally around 10-20 times higher when compared to levels in Europe (Betts, 2002, Petreas et al., 2003, Foster et al., 2011). In contrast to the concentrations of several chlorinated compounds which showed the highest levels in the 1980s, the concentrations of PBDEs in the general population of Sweden increased until 2005 before they levelled off or started to decrease (Noren et al., 2000, Fangstrom et al., 2008). The decrease might be explained by


the inclusion of PBDEs in several regulation agreements including the Stockholm Convention in 2009.

Most temporal trend studies up to date have indicated a declining trend of chlorinated and brominated compounds in the general population (Link et al., 2005, Patterson et al., 2009, Hardell et al., 2010). The declining concentrations of the legacy POPs is the evidence of the effectiveness of the regional restrictions and banns and finally worldwide regulation through the Stockholm Convention.

The fluorinated compounds, on the other hand, have been discovered more recently (Hansen et al., 2001) and it was first in May 2009 that PFOS was included in the Stockholm Convention. Recent temporal studies from Sweden (Sundström et al., 2011, Glynn et al., 2012) and the U.S. (Kato et al., 2011, Wang et al., 2011) indicate that the serum concentra-tions of the fluorinated compounds, such as PFOS and PFOA, are also declining. However, a recent Swedish temporal study (1995-2010) found a significant increase of the fluorinated short-chain compounds perfluorobu-tanoic acid (PFBS) and perfluorohexanoic acid (PFHxA) (Glynn et al., 2012). Moreover, a recent temporal study (1960-2009) from the U.S. showed and increasing trend in the concentrations of longer chain com-pounds such as perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) (Wang et al., 2011). The reasons for these decreasing and increasing trends are suggested to be the shift in the industrial production where, for example, the short-chain fluorinated compounds, among others PFBS, are being produced as replacement chemicals to PFOS and PFOA (3M., 2002, Ehresman et al., 2007, Olsen et al., 2009).

1.3 Relevance for human health The emphasis of epidemiological research is to provide knowledge about the health effects from possibly harmful substances in humans. Recently, epidemiological studies have indicated that chlorinated, brominated, and fluorinated compounds might be related to negative health effects in the general population (Hardell et al., 2003, Turyk et al., 2007, Humblet et al., 2008, Uemura et al., 2009, Lind et al., 2012a). From this perspective, an increased body of research on these issues has over the years showed that POPs cannot be ruled out as possible mediators in the development of disease. Although inconsistent findings have been reported (White et al., 2009, Sergeev, 2010), studies have shown that the chlorinated POPs could also be important for the development of cardiovascular disease, type-2


diabetes, and obesity (Lee et al., 2007a, Lee et al., 2007b, White et al., 2009, Son et al., 2010, Goncharov et al., 2010). Using data from the U.S. National Health and Nutrition Survey Ha et al. (2007) showed that expo-sure to chlorinated POPs might be important in the development of cardio-vascular disease. However, this association was only observed for women in the study, which suggests a gender difference in cardiovascular health outcomes and POP exposure. Furthermore, elevated exposure to fluorinat-ed compounds, such as PFOS and PFOA, were recently found to be associ-ated with reduced immunological function (Grandjean et al., 2012) and another study suggested that in utero exposure to PFOA may affect male semen quality and reproductive hormone levels (Vested et al., 2013). In, general, there is a growing concern regarding the low dose background exposure to endocrine disrupting chemicals and further epidemiological studies are required to understand the human exposure to POPs and possi-ble negative health effects.

1.3.1 Limitations with epidemiological studies Although informative, epidemiological studies have several drawbacks. One drawback is that they require a large number of samples. In order to obtain adequate statistical power, a great number of participants are re-quired including the laborious chemical analysis of the large number of samples. Another drawback is often the limited amount of biological sam-ples (i.e. human blood) that is available for analysis in epidemiological studies where blood samples are used for numerous other clinical tests. Since the concentrations of POPs are present in (ultra)-trace-levels this becomes an analytical challenge and, in response, advanced analytical methods are required. In particular, the methods should include the analy-sis of compounds with toxicological relevance. From this perspective, un-targeted screening may seem to be a way forward, however, it has still not been found to be an alternative to targeted trace-level analysis.

Thus, the major limitations with large epidemiological studies are often related to the high cost and the analytical capacity required in terms of the selectivity, sensitivity, and the overall laboratory throughput.


1.4 Aims of this thesis In order to overcome some of the drawbacks related to POPs analysis in epidemiological research the main objective of this thesis was to develop and apply two high-throughput methods for analysis of a broad range of persistent organic pollutants in human blood. More specifically, the goal was (a) to develop and validate methods suitable for the analysis of POPs regulated by the Stockholm Convention including the perfluorinated com-pounds with special attention to sensitivity and high-throughput and; (b) to apply the developed methods to 1, 016 plasma samples for an epidemiolog-ical study and assess the concentrations of selected chlorinated, brominat-ed, and fluorinated compounds from a gender perspective.

Paper I, II, and III address the method development and validation studies, while Paper III and IV use the developed methods to determine concentrations in human blood and evaluate possible gender differences in exposure.


2 Participants and procedures Background. The background for this thesis originates from a research question that was raised over a decade ago. The Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) was initiated in 2001 as collaboration between the Department of Medicine, University Hospital in Uppsala, Sweden and AstraZeneca, Mölndal, Sweden. The primary focus of the research collaboration was to evaluate cardiovascular function in an elderly population using different methods (Lind et al., 2005). Since 2001, several secondary objectives have also been added to the PIVUS study and a large number of academic groups have been engaged in the evaluation of the cohort studying different health aspects. This thesis provides a step in that direction by developing analytical methods to perform analysis of a large number of samples using less than 1.0 mL of human blood.

The PIVUS study. The participants in the PIVUS study were randomly selected from the general population in the community of Uppsala. Invita-tion letters were sent between April 2001 and June 2004 and within two months of each of the participants 70th birthday. The target sample was 2, 025 participants out of which 1, 016 participated. Thus, the overall partic-ipation rate was estimated at 50%. Two longitudinal follow-ups were per-formed. The first reinvestigation was performed when the participants turned 75 years (2006-2009) with a follow up rate of 80%. The second reinvestigation is being performed at present when the participants are 80 years (2011-2014). This design makes the PIVUS study one of the best characterized epidemiological studies today.

Participants and blood sample collection. In this thesis, the participants were from the first investigation and they were all 70-years old. The 1, 016 participants, of whom 50.2% were female, went through a complete medi-cal investigation where more than seventeen different measurements were performed ranging from cardiovascular function to self-reported history of diseases and medication. In addition, life-style information, such as exer-cise, smoking, alcohol intake, education, social network and so forth was also collected.

Clinical tests such as blood serum and plasma were collected in the morning after an overnight fast over-night fast. After the blood plasma samples were collected (1-2 mL vials), the vials were placed in freezers (-20 °C) until used for chemical analysis. The study was approved by the Ethics Committee of the University of Uppsala and the participants gave written informed consent.


Lipid normalization. Chlorinated and brominated POPs are lipophilic and bioaccumulate in the adipose tissue of humans. The individual expo-sure is usually estimated by measuring the POPs in human blood but also other tissues as well. Because the exposure to POPs is equally distributed in the lipid compartments of an individual plasma or serum concentrations are adjusted by dividing the wet weight concentrations (pgmL-1) with the total lipid content, including blood lipids such as cholesterol and triglycer-ides. The effect of lipid adjustment methods was recently evaluated in a study comparing four statistical models of relevance (Schisterman et al., 2005) and it has been suggested that lipid adjustment could introduce bias in epidemiological research, thus both lipid adjusted and un-adjusted con-centrations are reported in Paper IV. For the chlorinated and brominated compounds, the serum cholesterol and triglycerides were determined using standard enzymatic methods. Finally, an established summation formula was used to calculate the total amount of lipids in each plasma sample (Rylander et al. 2006).

Statistical analysis. To evaluate possible gender differences in POPs con-centrations the final dataset was analyzed using Wilcoxon’s rank-sum (Mann-Whitney). For chlorinated and brominated compounds, differences for which p <0.001 were considered statistically significant. Multiplicity was adjusted using Holm’s method (Holm, 1979). For gender difference analyses of the fluorinated compounds (unpublished data) a lower interval was allowed p <0.05 and <0.01 and is indicated in the text.


3 Background to analytical methods for POPs analysis in human blood The development of analyses of POPs in human blood has resulted in high-ly specific, sensitive and robust methods. This is mainly due to the use of effective sample preparation procedures, mass selective detection, and ap-plications of quantitative mass spectrometry including isotope dilution. The classes of POPs initially driving this development in the 1980s were the chlorinated compounds i.e. PCDDs, PCBs, and OCPs. Incidents trigger-ing this development were, for example, the Seveso accident in 1976 (Homberger et al., 1979), the Agent Orange exposure from 1962 to 1971 (Kahn et al., 1988) and discoveries of DDT and PCB in the environment (Nachman et al., 1969, Ahling et al., 1970, Jensen, 1972). Understanding of the environmental effects became an urgent requirement for govern-ments’ world over. As a consequence, the demands on the analytical capac-ity increased and there was a call for dioxin analyses in various types of matrices (breast milk, adipose tissue, and blood) in human biomonitoring studies (Nygren, 1988).

The number of environmental pollutants to be monitored has signifi-cantly increased since the early 1970s and in 2001 twelve compound clas-ses of chlorinated compounds were finally included in the first worldwide regulation through the Stockholm Convention. Since 2009 the Stockholm Convention regulates and encourages biomonitoring of 21 compound clas-ses through the GMP in air and water as well as human milk and blood (UNEP, 2011). Although, over the years, progress in the analytical proce-dure has been made (Focant et al., 2004, Muir et al., 2006, Covaci et al., 2007, van Leeuwen et al., 2007, Jahnke et al., 2009, Patterson Jr et al., 2011) there is still room for improvement. These improvements include developments of simple and cost effective sample preparation, extraction, separation, and detection techniques that are able to monitor background concentration of a broad range of environmental pollutants in the blood from general populations.

In Paper I the background of the analytical methods for analysis of POPs in human blood are discussed together with the current trends in analytical procedures for screening studies for the determination of trace-analysis of POPs in human blood.


3.1 Current principles in sample preparation and instrumental analysis The body burden of POPs is generally determined by extracting the ana-lytes from the blood lipids or from the blood proteins. However, blood (whole, serum, or plasma) is a difficult matrice because it not only contains the compounds of interest but also a wide range of interfering substances that can significantly affect the success of the analysis (Focant et al., 2004, van Leeuwen et al., 2007, Covaci et al., 2007). Therefore, the first step in the analysis of persistent organohalogen compounds is particularly im-portant and involves sample preparation, in terms of pre-treatment, extrac-tion, clean-up, and preconcentration, to enhance the selectivity and sensi-tivity of the subsequent instrumental analysis.

Sample preparation procedures can be developed to extract multiple classes of compounds, if the analytes of interest share similar physico-chemical properties. The chlorinated and brominated compounds in this thesis are lipophilic and therefore distribute into adipose the tissues in the human body, including the blood lipids (Covaci et al., 2006). Whereas the ionic fluorinated compounds, exhibit completely different physico-chemical properties and are primarily bound to the protein components in blood (Han et al., 2003).

3.1.1 Chlorinated and brominated compounds In general, the analytical procedures require between 1-40 mL serum, de-pending on the concentrations of the specific compounds of interest and an overview of the method currently used for analysis is given in Table 1, Paper I. As the table shows the sample pre-treatment for blood samples, used for denaturation of proteins, is commonly performed using formic acid. Furthermore, the extraction of the target analytes is commonly per-formed using solid-phase extraction (SPE) or liquid-liquid extraction (LLE). SPE has been shown to offer many advantages over LLE because it reduces the amounts of organic solvents, is more specific, and is considered to be less labour intensive. The overview also shows that current SPE methods used for analysis of PCBs, OCPs, and PBDEs usually are based on sorbents containing reversed-phase alkyl bonded silica of different particle sized. But, other sorbents such as the polymeric divenylbenzene, hydro-philic-lipophilic-balanced (HLB), reversed-phase sorbents are also used.

After sample pre-treatment and extraction using SPE, an additional clean-up is often required to eliminate any interfering compounds and co-


eluting lipids left in the extract. Clean-up procedures are often based on liquid-solid chromatography using activated silica, active alumina, and active carbon materials. One widely used approach to remove lipids and other interferences is based on mixed (neutral, acidified or basic) activated silica columns (Table 1, Paper I).

The instrumental analysis of chlorinated and brominated compounds is performed using gas chromatography often coupled to low-resolution mass spectrometry instruments (GC-LRMS) while the analysis of PCDDs as well as dioxin-like PCBs generally requires larger sample amounts and is per-formed using GC-HRMS.

3.1.2 Fluorinated compounds In general, the serum concentrations of the fluorinated compounds are on a volume basis, in comparison to the concentrations of the chlorinated and brominated compounds, somewhat higher (Kärrman et al., 2006). There-fore, the analysis of fluorinated compounds usually requires smaller sample amounts (Table 2, Paper I). Fluorinated compounds such as the perfluoro-alkyl acids are amphipathic and bind to the proteins in human blood. Sample pre-treatment for the denaturation of proteins is performed by using formic acid, acetonitrile, or trichloroacetic acid. The extraction pro-cedure predominantly involves SPE, LLE, protein-precipitation (PP), or ion-pair extraction (IPE). For SPE, both ion exchange and reversed-phase sorbent chemistries have been shown effective for extraction. Clean-up strategies to remove lipids and interferences have been performed using dispersed graphitized carbon, centrifugation, and filtration using mem-brane filters (van Leeuwen et al., 2007).

The instrumental analysis of fluorinated compounds is preferably per-formed using high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry instruments (MS/MS) operated in negative elec-trospray ionization (ESI) mode. Challenges in analysis of PFAS due to the matrix effects are well known and may cause either ion suppression or enhancement during ESI) (Martin et al., 2004, Chan et al., 2009, Lindström et al., 2009). In this context, using isotope labeled standard for each of the analytes compensates for the matrix effects, variability in re-coveries, and increases the accuracy of the mass spectrometric quantifica-tion. In addition, matrix matched calibrations can be used as an alternative to further reduce quantification errors from matrix effects.


3.2 Major analysis trends In general, there is a trend in the analysis in various monitoring and epi-demiological studies that moves towards more automated systems and comprehensive procedures using smaller and smaller amount of blood and recently even in dried blood spots (Kato et al., 2009, Patterson Jr et al., 2011). Correspondingly, there is also a demand towards the use of analyti-cal procedures in which several classes of Stockholm Convention POPs are analysed simultaneously. This is illustrated closely in Figure 1 from Paper 1 where the amount of sample used for analysis and the number of com-pounds analyzed from the sample extracts are given with the correspond-ing references.

Figure 1. Amount of blood sample used for analysis in mL (left axis) and the num-ber of analytes (right axis) from the end of the 1980s to 2011.


4 Development of new high-throughput methods One of the main objectives of this thesis was to develop and validate meth-ods suitable for high-throughput analysis of POPs in human blood. As yet, the complete single-injection analysis of the group of chlorinated, bromin-ated, and fluorinated compounds has not been performed and the group of chlorinated and brominated POPs require different experimental strategies compared to the fluorinated POPs. The main features of the developed methods and validation studies in Paper II and Paper III are described below along with the advantages and possible drawbacks of each method.

4.1 Analysis of chlorinated and brominated compounds To facilitate high-throughput analysis of chlorinated and brominated com-pounds in human blood optimization of current methods was necessary. The sample amount required for analysis was found to be critical in the method development considering that epidemiological studies are typically hampered with low sample amounts available because the chemical analy-sis is often in competition with other clinical tests. On the other hand, sample amounts are also related to the method detection limits and the concentrations of the target compounds present in the samples. An addi-tional problem when using small samples amounts (500 µL) is the back-ground contamination in the laboratory environment.

Throughout the work presented in Paper II, several efforts to reduce la-boratory contamination were performed. For the majority of chlorinated compounds, these efforts were satisfactory and the procedural blanks were in general satisfactory except for HCB, PCB #138, and PCB #153 that were found in low levels in the laboratory blank samples. The concentra-tions of HCB and the two PCBs were found to fluctuate from sometimes acceptable levels (less than 10% of the levels in human blood) down to even lower levels between the batches of samples. Consequently, the MDLs for these compounds were somewhat higher when comparing to the rest of the analytes as shown in Table 3. Similar to the observations by Sandau et al. (2003), HCB was consistently detected in procedural blanks over long periods of time as shown in Figure 2. No obvious reason for this contami-nation was found, suggesting a source in the laboratory environment in-cluding the laboratory air. However, the obtained detection limit for HCB was still low enough and below 10% of the concentrations in human plasma and serum samples when using 500 µL sample amount.


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Figure 2. Laboratory contamination of HCB in procedural blanks over time

The background contamination of brominated compounds has previous-ly been reported by Thomsen et al. (2001) observing significant contamina-tion of BDE #47 in the laboratory environment, primarily in the laboratory air. Low concentrations of BDE #47 were also observed in our blanks. However, by introducing extensive washing procedures the background concentrations were brought down to a more acceptable concentration range in relation to the levels present in the human plasma samples. When the background contamination in the laboratory environment was identi-fied further attempts to reduce the sample volume were performed. Similar to the method by Sandau et al. (2003) the work in Paper II initially re-quired 1.0 mL serum, however, the sample volume was finally reduced to 0.5 mL. This reduction in sample volume was tested and the results indi-cated that this volume was just about enough to be able to detect concen-trations of the selected POPs including OCDD as well as BDE #47 that are present in considerably lower concentrations in general populations when compared to the other analytes.

For denaturation of proteins, formic acid and water with 3% iso-propanol was added to 0.5 mL plasma samples. Solid-phase extraction was performed using 6 cc 150 mg HLB single use cartridges. Further clean-up of sample lipids and other interferences was performed by using small mul-tilayer silica gel columns consisting of standard glass pipette filled with clean-up materials according to the scheme in Table 3. The most significant advantages over the method by Sandau et al. (2003) and the work present-ed in Paper II, are that this method uses l50mg Oasis HLB cartridges and the clean-up using small multilayer acid silica gel columns which provided


cleaner extracts. The final purified extract was injected on a HRGC-HRMS system and measurements were performed in selective ion mode (SIM) and quantification was performed according to the isotope dilution method using 13C-labeled standards. An overview of the final experimental condi-tions and method characteristics presented in Table 3. Overall, the benefits of the method in Paper II is that it requires less sample volume while at the same time facilitates analysis of three classes of chlorinated and one bro-minated compound.

Table 3. Overview of the experimental conditions of the method presented in Paper II

Exp

erim

enta

l con

diti

ons

Method characteristics Extraction Analytes PCBs (#74, #99, #105, #118, #126, #138,

#153, #156, #157, #169, #170, #180, #189, #194, #206, #209), OCPs (HCB, trans-chlordane, cis-chlordane, trans-nonachlor, p,p’-DDE), BDE #47, and OCDD

Sample matrix 500µL serum or plasma Protein precipitation 1 mL formic acid and 1 mL water (3%

iso-propanol) SPE extraction Oasis HLB (6cc, 150mg) SPE wash solvent Water with 40% methanol and 3% iso-

propanol SPE drying 40 min under pure grade N2 (g) Eluent 6 mL dichloromethane and hexane (1:1)

Clean-up Volume 500µL sample extract Multilayer silica chromatography

56% KOH silica gel, 40% H2SO4 silica gel and activated anhydrous Na2SO4 (2 mL; 1.5g)

Eluent 7.5 mL hexane IS recoveries 46-110% for all analytes

HRGC System Agilent Technologies 6890N GC Injection volume 2 µL Injection temperature 275 °C Analytical column SGE BPX5 30m x 0.25 i.d. x 0.25 µm Temperature ramp Initial: 180 °C (2 min) ramped to 260 °C

(3.5 °C/ min) and finally to 300 °C (6.5 °C/min, 2min)

HRMS System Micromass Autospec Ultima HRMS Ionization EI operated at 35 eV Resolution ≥10, 000 Mass spectral analysis SIM MDLs PCBs; 0.80-118 pgmL-1, OCPs; 5.9-89

pgmL-1, BDE #47; 9 pgmL-1 and OCDD; 1.4 pgmL-1


4.1.1 Method validation Once all parameters were established in the method development, these conditions were applied to quality control (QC) reference plasma, which is pooled human plasma obtained from the Örebro University Hospital in 2003, to determine the accuracy and precision as well as throughput and robustness of the method.

4.1.1.1 Accuracy and precision The method accuracy was determined using concentrations of a well

characterized QC plasma sample previously determined and routinely used in our laboratory. The concentration of this QC sample was determined by using two independent methods, open-column extraction and a SPE meth-od using 8 mL human plasma (Karlsson et al., 2005). This sample was characterized while the laboratory took part in several QA/QC studies. The conformity between the concentrations obtained by the method in Paper II and the Karlsson et al. (2005) method, previously used in our laboratory, was good for the majority of compounds, except for those compounds present in low levels and below the detection limit (Table 3).

To determine the method precision the results from the within-run (re-peatability) and between-run (reproducibility) analysis of QC reference plasma was evaluated. The within-run precision where 5 QC samples were analyzed was for the majority of compounds less than 25% RSD, except for compounds which were present close to the MDL. The reproducibility of the method was good over a period of 12 months based on the analysis of 95 replicates of the QC plasma samples. The reproducibility varied from below 20% for all PCBs present at levels above the MDL to 30% and 54% for the mono ortho PCBs #169 and #126, respectively. The RSDs for OCDD were 56% and this high RSD is mainly due to the fact that the OCDD concentrations in the QC sample is borderline below and above the MDL. For BDE #47 which was just above the MDL, the repeatability was 26% RSD and the reproducibility was 30% RSD.

Surprisingly a relatively large variation was seen for p, p’-DDE (43%), despite the fact that p, p’-DDE is one of the most abundant analytes in the QC samples. Figure 3 shows the method reproducibility is presented in control charts for three POPs.


Figure 3. Control chart for PCB #99, trans-nonachlor, and BDE #47 in in-house QC sample. Solid line indicates the moving average and ±1 SD and ±2 SD are rep-resented by the dashed lines (1 SD) and dotted lines (2 SD).

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The charts were initially showing somewhat larger variation during the first usage and the first batches of samples processed. For the two coplanar PCBs #126 and #169 the RSD was somewhat larger since these compounds were present at very low concentrations and due to the lack of correspond-ing 13C-labeled internal standards which were initially not included. Recov-eries for the internal standards were calculated using a one-point calibra-tion against the instrumental standards included in each batch of 12 sam-ples. The recovery standards were 13C-PCB #81, 13C-PCB #114, and 13C-PCB #178. Recoveries ranged between 46-110% for all target analytes as shown in Table 3.

4.1.1.2 Throughput and robustness After the method optimization and validation studies were completed, the method was evaluated for throughput and robustness. In a typical proce-dure, the first day of a run was dedicated to the sample preparation proce-dure and the second day was used to complete final steps in the analytical procedure and prepare for the instrumental analysis. It is possible for one laboratory technician to prepare about 48 samples during one week. The materials, solvents, and time required for analysis per sample showed that the method enables high-throughput when compared to similar methods employing manual sample handling.

After the initial method optimization studies the method robustness was evaluated during a period of one year when many changes were performed. Changes include materials, solvents, chromatographic columns, and reno-vations of the HRGC-HRMS system. Except for some of the higher RSDs in the QC samples and for the fluctuating blank levels for some com-pounds, as discussed above, the overall method was shown robust and suitable for high throughput studies.

4.2 Analysis of fluorinated compounds For analysis of the fluorinated compounds, the overall performance of the method described in Paper III was found to be influenced by a number of parameters. The first was related to the sample preparation procedure which is based on protein precipitation using a 96-well plate. It was found that both the sample volume and solvent employed affected the extraction. More specifically, the 96-well plate was limited to about 180 µL blood serum or plasma and methanol was a poor extraction solvent that fre-quently led to the sample wells clogging. Therefore, the sample volume was


10% AcN

25% AcN

35% AcN

50% AcN

optimized down to 150 µL and test preformed with acetonitrile and ace-tonitrile with 1% formic showed that the latter provided the best recovery for the PFAS.

Another analytical drawback was related to the peak broadening. In general, considering that the method was based on both large volume injec-tion (250 µL) as well as the procedure involving connection of two UPLC columns, some chromatographic improvements were required. In response, two different trap columns were tested, C18 2.1×20mm, 2.5µm and a C18 2.1×20mm, 10µm, and it was established that the 2.5 µm particle size trap column, to some extent, provided better peak shapes. In addition, it was found that the organic solvent ratio of the injected sample solution was shown to be critical in terms of obtaining acceptable peak shapes of the selected analytes. Therefore, in Paper III replicates of standards in which the solvent ratio of the instrumental standards was varied from 50%, 35%, 25%, and 10% acetonitrile (AcN) in 2mM NH4Ac was performed and the results are shown in Figure 4.

Figure 4. Peak shape effects for PFHxA in final extracts prepared with 50%, 35%, 25%, and 10% acetonitrile and 2 mM NH4Ac in water.


As shown from Figure 4, using 10% acetonitrile in 2mM NH4Ac gave the best peak shapes, however, this low organic solvent ratio in the final extract led to decreased recoveries for some more hydrophobic PFAS such as perfluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoDA), and perfluorotridecanoic acid (PFTrDA). These issues have previously been reported to be the result of adsorption losses (Berger et al., 2011) and by having a higher organic solvent ratio in the final extract the adsorption was reduced and higher recoveries were obtained, however, the peak shapes would then be worse and thus not a good compromise.

Instead, to improve the recovery for the target analytes two different ion pairing agents were tested (2 mM NH4Ac and 5 mM 1-methylpiperidne) and no significant differences in recoveries were observed between the two. The remaining option was then a matrix matched calibration curve to avoid the low recoveries for the more hydrophobic PFCAs and PFSAs caused from to matrix effects during ESI. In addition in Paper III, we also tested to include perfluorotetradecanoic acid (PFTDA), perfluorohexadec-anoic acid (PFHxDA), and perfluorooctadecanoic acid (PFOcDA), despite the fact that they are rarely detected in general populations, but the ob-tained recoveries failed to meet QA/QC criteria because of inconsistent internal standard recoveries.

Another issue was the carry-over typically associated with large volume injection. In this case it was predominantly the more hydrophobic PFCAs such as PFDoDA and PFTrDA, which were observed to produce carry-over in the forthcoming samples and blanks (Paper III). The carry-over was first minimized by using more comprehensive wash solutions and then finally completely reduced by adjustments of the timed events that initiate the injection valve. More specifically, it was found that a 10-cycled injection event programmed at the end of the UPLC gradient when in 100% metha-nol (with 2 mM NH4Ac) removed any remaining analytes trapped in the 250 µL loop.

Once the optimizations were performed the full capacity of the 96-well plate was tested including one matrix matched calibration curve (8 points including matrix blank), four replicates of standard reference material (SRM) 1957 obtained from the National Institute for Standards and Tech-nology (NIST), and seven batches of samples, which in turn consisted of one method blank, one QC reference sample, and ten authentic samples. The instrumental analysis was performed using the column-switching UPLC-MS/MS with a run time for each sample injection of 25 minutes, including the reconditioning of the trap column. It was concluded in Paper


III that the overall advantages of the developed method was the high-throughput sample preparation using the 96-well plates together with col-umn-switching UPLC-MS/MS. The final experimental conditions and method characteristics presented in Paper III are shown in Table 4.

Table 4. Overview of the experimental conditions of the method presented in Paper III

Exp

erim

enta

l con

diti

ons

Method characteristics

Sample preparation

Analytes PFCAs (C5-C13), PFSAs (C4-C10) including structural PFOS isomers, and PFOSA

Sample matrix 150 µL serum or plasma

Protein precipitation

450 µL acetonitrile with 1% formic acid

Extraction Ostro Sample preparation 96-well plate

IS recoveries 61-118% for all analytes against a matrix matched calibration

Column switch

External system Six-port column switch valve with the timed events occurring at 16.00 and 21.5 min.

Flow rate 0.350 mL min-1

Trap column Xbridge C18 BEH 2.1×20mm, 2.5µm

Wash solutions 1 50% iso-propanol in water , 2 95% 0.2% NH4OH methanol in water (pH 8-9),3 5% methanol in 0.2% NH4OH water (pH 8-9)

UPLC System Acquity UPLC Injection volume 250 µL full loop injection Flow rate 0.300 mL min-1 Mobile phase A 10 % methanol in 2 mM NH4Ac water Mobile phase B 100 % 2 mM NH4Ac methanol Gradient Initial 100% A, decreasing to 70% between

0.10 and 0.20 min. A linear decrease to 8%A between 0.2 and 14 min thereafter rapidly changed to 0% A and held until 20 min. At 21.5 min the gradient changed to 100% A.

Analytical column

Acquity C18 BEH 2.1×100 mm, 1.7 μm

MS/MS System Quattro Premier XE MS/MS operated in -ESI mode

Mass spectral analysis

MRM

MDLs 0.01-0.17 ngmL-1

Total method linearity (R2)

Range d between 0.9970-0.9997


4.2.1 Method validation Once the optimizations were finalized in the method development, these conditions were applied to the QC reference human plasma samples and a NIST SRM 1957 sample to determine the accuracy and precision as well as robustness and overall throughput of the method in Paper III.

4.2.1.1 Accuracy and precision The accuracy of the developed method was determined using NIST SRM 1957 samples. The concentrations obtained were then compared to deter-mine the conformity to the NIST report values (Reiner et al., 2011). The obtained values were within a good proximity (Table 2, Paper III) to the reference values in the NIST report for 56 replicates analysed during a period of four months including 15 sample batches. As presented in Figure 5, there was good conformity between the concentrations of the PFCAs and PFSAs in the method in Paper III and values reported by NIST.

Figure 5. Accuracy and precision with error bars showing 95% CI of selected ana-lytes in SRM 1957 comparing this method and the reference values.

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To monitor the within-run and between-run precision of the method, con-centrations of the target analytes were evaluated in 103 replicates of QC reference samples and 56 replicates of NIST SRM 1957 samples processed during a period of four months. Figure 6 shows the measured concentra-tions of detectable compounds and the within-run and between-run preci-sion of the method in the QC reference samples. In general, there was good within-run repeatability, with coefficients of variation ranging from 2% to 20%. Similarly, the between-run reproducibility was also good ranging from 5% to 29% variation.

4.2.1.2 Throughput and robustness It was possible to complete sample preparation and begin instrumental analysis of 88 samples in two days. In a typical procedure, the first day of a run was dedicated to the sample preparation procedure and the second day was used to complete final steps in the analytical procedure and prepare for the instrumental analysis. An estimate of the materials and time re-quired for analysis per sample shows that this method was effective and enabled high-throughput.

During four months of validation the method continued to be unaffected by experimental variations and provided accurate and reproducible results. Thus, the overall robustness of the method presented in Paper III makes it suitable for accurate and precise analysis in studies requiring sensitive high-throughput analysis of a broad range of PFAS using 150 µL human plasma or serum.


Figure 6. Control chart for PFOA, PFHxS, and PFDA in QC samples. Solid red lines indicate the within-run (n=7) moving average and ±1 SD and ±2 SD, and are represented by the dashed lines (1 SD) and dotted lines (2 SD).

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4.2.1.3 Isomer specific analysis of PFOS In Paper III the systematic errors often encountered in the quantification of total PFOS in human blood were discussed. These errors might primarily be an effect of treatment linear versus branched PFOS isomers in MRM and further stresses the need to quantify these analytes separately. To over-come systematic errors and accurately determine the linear and branched PFOS isomers the developed method was validated using NIST SRM 1957 serum. The method was found to be able to separate up to seven PFOS isomers in a technical standard. Validation studies of the method presented in Paper III showed that the precision of the method was satisfactory with RSDs more than 8% for all isomers in 56 sample replicates. The accuracy of the determination of structural PFOS isomers was evaluated comparing this method performance against results reported by Riddell et al. (2009) and the conformity between the two methods is shown in Figure 7. In gen-eral, the developed method was very precise and provided narrow confi-dence intervals when compared to the method by Riddell et al. (2009) However, the only exceptions was the concentration of the 6/2-PFOS iso-mers, in which the method in Paper III obtained significantly lower con-centrations, although the method by Riddell et al. (2009) only reported values for the 6-PFOS isomer. Furthermore, the comparing method did not report values for the 4.4-, 4.5-, 5.5- perfluorodimethyl-PFOS.

Figure 7. Method comparison of structural PFOS isomers with 95% CI in SRM 1957 by monitoring three production ions (m/z 499>80, 499>99, and 499>130).


PCB#74 PCB#99 PCB#105 PCB#118

PCB#126

PCB#138

PCB#153

PCB#156

PCB#157

PCB#169

PCB#170 PCB#180

PCB#189 PCB#194

PCB#206

PCB#209

OCDD

HCB cis-

chlordan

trans-chlordane

trans-nonachlordane

p,p'-DDE

BDE # 47

5 Concentrations of POPs in the PIVUS cohort Several factors may influence the exposure to POPs in the general popula-tion. Social factors include diet, smoking, social group, and occupation while biological factors might include age and gender (Papke, 1998, Glynn et al., 2003, Haug et al., 2009). These factors are important to consider when evaluating concentrations of POPs in the general population. The levels of POPs are often not the same when comparing men and women and it has been shown that men, in general, have higher concentrations of POPs (Patterson et al., 2009, Haug et al., 2009). Another important bio-logical factor is breastfeeding because it lowers the body burden of POPs in women (LaKind et al., 2009, Thomsen et al., 2010).

5.1 Chlorinated and brominated compounds In Paper IV the plasma concentrations of chlorinated and brominated compounds were determined in 992 participants. Wide concentrations of the studied POPs were observed in the cohort. The detection rates for all compounds were relatively high and ranged from 70-100% (Table 1, Paper IV). The high detection rate demonstrates the long-term accumulation and persistence of the POPs studied. The OCPs, trans-nonachlor, HCB, and p, p’-DDE were detected the majority of the 992 participants and p,p’-DDE was found in the highest concentrations as shown in Figure 8.

Figure 8. Distribution of chlorinated POPs (ngg-1 lipid )in 992 participants


Cis-chlordane and trans-chlordane were found to be less abundant in the participants and were only detected in 3% and 10% of the participants, respectively. Among the PCBs measured, the highest median concentrations were observed for congener #153 which contributed up to 30% of the total PCB contamination and a sum of PCB marker congeners #118, #138, #153, and #180 together accounted for 73% of the entire PCB burden in the participants. The concentrations observed in Paper IV were compared to the concentrations found in the Flemish Environment Health Study (FLEHS) of 200 women aged 50-65 years sampled between 2001-2003 (Koppen et al., 2002) and extracted data from 1, 128 participants aged 70 years and older from the fourth report of the United States National Health and Nutrition Exposure Survey (NHANES) sampled in 2004-2004 (CDC, 2009) as shown in Figure 9.

Figure 9. Comparison of the median concentrations of selected legacy POPs in human serum or plasma from three countries.

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Overall, the median concentrations and the pattern of PCBs detected in the PIVUS cohort (Table 2, Paper IV) were comparable to those observed to studies from Belgium as well as other studies from Europe and the U.S. where the participants were sampled during the same time period (Covaci et al., 2002, Koppen et al., 2002). The PCB concentrations were found to be somewhat higher in the PIVUS cohort and about three times higher than the elderly population from the U.S. as shown in Figure 9. However, the median levels of PCBs in the PIVUS cohort are somewhat higher compared to the median levels of other studies of the Swedish population (Weiss et al., 2006, Hardell et al., 2010). This might be due to the participants in the present study being all 70 years of age and therefore have accumulated higher concentrations of POPs as a result from long-term exposure. The concentrations of the OCPs were considerably higher in both the study from Belgium as well as the U.S. which might reflect the lower historical use of DDT and other pesticides in Sweden.

Low concentrations of OCDD and BDE #47 were quantified in 81% and 72% of the participants in the study, respectively, with levels just above the LOD in the majority of samples (Table 1, Paper IV). There were substantial differences in the concentrations of the highest and lowest con-centrations of POPs. A comparison of the concentrations of OCDD and BDE #47 among the studies from the same time period from the U.S. and Belgium is shown in Figure 10.

Figure 10. Comparison of the median concentrations of OCDD and BDE #47 in human serum or plasma from three countries.

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In Figure 1 in Paper IV show that the concentrations of BDE #47 were, for example, about 50-100 times higher in 4% of the study population suggesting a direct exposure via occupation or another significant source of exposure other than that from food as has been previously proposed by van Bavel et al. (2002). The study from Belgium did not include analysis of BDE #47. Interestingly, low concentrations of OCDD (below 1.0 ngg-1 lipid) in all three studies indicate a generally low PCDD contamination. For BDE #47 the concentrations are about 10 times higher among the el-derly participants in the fourth report of NHANES (CDC, 2009). This has been previously observed in other studies and is considered to be due to the larger usage of BFRs in various commercial applications such as household furnishing in the U.S. (Rahman et al., 2001, Betts, 2002, She et al., 2002).

5.2 Fluorinated compounds In Paper III the concentrations of the analysed fluorinated compounds in a sub-population of 95 participants from the PIVUS cohort were presented. In this thesis, the unpublished data for the entire cohort (n=1, 016) is in-cluded (Table 6). The detection rates for the PFAS were relatively high and the majority of the studied compounds were detected in more than 75% of the participants with the exception for PFDoDA, PFTrDA, PFBuS, and PFDS that were detected in the range between 19-43% of the participants. The distribution of the fluorinated compounds among the participants is shown in Figure 11. The plasma concentrations of PFAS were clearly dom-inated by L-PFOS followed by PFOA, PFHxS, PFNA, and PFDA.

Figure 11. Distribution of fluorinated POPs (pgmL-1) in 1, 016 participants

PFBuS PFHpA

PFHxS

L-PFOS

PFOA

PFNA PFDA

PFOSA PFUnDA PFDoDA PFTrDA


The same pattern has also been observed in previous studies of the gen-eral population and the concentrations are comparable to studies from Norway, the U.S., and Sweden (Haug et al., 2009, Kato et al., 2011, Glynn et al., 2012).

Table 6. Concentrations of selected PFAS including structural PFOS isomers in plasma sam-ples from men and women from the PIVUS cohort PIVUS n=1, 016 Analyte Concentration (ngmL-1) Detection rate (%) 25th Median 75th PFHpA 0.047 0.067 0.106 75 PFOA 2.55 3.33 4.39 99 PFNA 0.530 0.713 0.974 99 PFDA 0.261 0.325 0.411 90 PFUnDA 0.257 0.286 0.400 82 PFDoDA 0.036 0.039 0.056 36 PFTrDA 0.035 0.048 0.066 43 PFBuS 0.045 0.085 1.00 19 PFHxS 1.61 2.08 3.45 99 L-PFOS 10.1 13.4 17.8 99 PFOSA 0.078 0.115 0.172 84 PIVUS sub-sample n=25 PFOS isomers Concentration (ngmL-1) Detection rate (%) 25th Median 75th L-PFOS 7.90 11.8 15.4 100 1-PFOS 0.377 0.402 0.631 100 6/2-PFOS 1.42 1.85 2.28 100 3/4/5-PFOS 2.34 2.75 3.50 100 4.4/4.5/5.5-PFOS 0.077 0.095 0.113 100 ∑PFOS 12.7 17.3 21.6 100

It has been suggested that variations in the serum distribution of structural PFOS isomers among individuals could be the result from different routes of exposure (Benskin et al., 2009, Martin et al., 2010). Therefore, in Paper III, isomer specific analysis of branched PFOS isomers was performed to explore the variability among individuals. In total, five groups of branched PFOS isomers were determined in a sub-sample of 25 participants (Table 6). The results showed that the branched isomer constituted from 31% to 37% of the total PFOS concentration. However, there were subtle interin-dividual variations (Figure 2; Paper III) in the concentrations of the differ-ent structural isomers which might suggest different routes of exposure or


different composition of PFOS and its precursors. The isomer pattern of PFOS also showed similarities to that previously observed in Sweden, Aus-tralia, Norway, and Canada in which the mean concentrations of the branched isomers constituted from 30% to 42% of the total PFOS concen-tration (Kärrman et al., 2007b, Haug et al., 2009, Beesoon et al., 2011). Furthermore, the isomer pattern observed in the present study was com-pared to the isomer patterns found in a non-fortified pooled serum from the U.S. collected in 2004 and distributed by NIST (Riddell et al., 2009) and human serum from China (Zhang et al., 2013). A comparison of the results is shown in Figure 12.

Figure 12. Comparison of the median concentrations of mono- and disubstituted PFOS isomers in human serum or plasma using isomer specific concentrations from three countries. Number of samples and time of collection is shown in brackets.

There are differences in the distribution of the structural PFOS isomers when comparing the studies. The proportion of L-PFOS in Paper III is higher than that found in both the U.S. and China and close to the tech-nical product which constituted about 70% L-PFOS. The proportion of L-PFOS was lower in the U.S. (59%) and even lower in China (48%). The lower proportion of L-PFOS and thus larger proportion of branched mono- and disubstituted PFOS isomers has been suggested to be a bi-omarker of increasing exposure to PFOS-precursors (Benskin et al., 2009,

L-PFOS 1-PFOS 6/2-PFOS 3/4/5-PFOS 4.4/4.5/5.5-PFOS

Paper III (n=25, 2001-2004) 68 3 11 17 1

U.S. (pool, 2004) 59 2 20 19 0

China (n=129, 2010) 48 4 12 35 1

0

10

20

30

40

50

60

70

% of total PFOS


PFBuS

PFHpA

PFHxS

L-PFOS

PFOA

PFNA

PFDA

PFOSA

PFUnDA

PFDoDA

PFTrDA

PCB#74 PCB#99

PCB#118

PCB#105

PCB#153

PCB#138 PCB#156

PCB#157 PCB#180

PCB#170

PCB#189

PCB#194 PCB#206

PCB#209 OCDD

PCB#126

PCB#169

BDE #47 HCB

cis-chlordan trans-chlordane

p,p'-DDE

trans-nonachlor

Martin et al., 2010). Although not as likely, it could also be the result from different elimination rates of the isomers (Rayne et al., 2010), however, more research is required to understand the elimination processes fully. Moreover, the differences may also originate from differences in the ECF process used for production of PFOS related compounds in Europe, China, and the U.S., and the ECF process itself might produce different amounts of isomers and homologues not only between the different batches of pro-duction but also between the different plants worldwide (3M., 1999). There are many factors that influence human exposure of PFOS isomers. Further research is required to better understand these factors as well as the implications for human health.

5.3 POPs in relation to each other and the influence of gender In the general population, the serum concentration, a volume basis, of the chlorinated and brominated compounds have been shown to be considera-bly lower in comparison to the levels of fluorinated compounds (Kärrman et al., 2006). The median distribution of all the compounds included in this thesis is given in Figure 12, which gives a good estimate of the circulating levels of all the POPs. The fluorinated compounds account for more than 70% of the total amount in pgmL-1 plasma in the PIVUS study.

Figure 12. Median distribution (n=992) of the all compounds in pgmL-1 plasma.


0

10

20

30

40

50

60

70

80

90Frequency (n=992)

MEN

WOMEN

However, it should be noted that this plasma distribution comparison is based on the plasma concentration in pgmL-1 and does not necessarily re-flect the tissue distribution pattern in the human body as the legacy POPs are primarily stored in the lipids while the fluorinated compounds are mainly present in the blood and liver. Nonetheless, it is interesting to note the distribution pattern in the human body where the group of PFAS is by far the most dominating class of compounds in human blood followed by p,p’-DDE.

Gender differences (Paper IV) in concentrations of several chlorinated, brominated, and fluorinated POPs were observed. More specifically, the chlorinated and brominated POPs were significantly (p<0.0001) higher for the men as shown for PCB #153 in Figure 13. However, levels of some of the compounds were not always lower for women, as could be expected after breastfeeding earlier in life. The PIVUS women showed significantly higher levels of HCB, OCDD, and PCBs #74, #105, and #118.

Figure 13. Frequency histogram showing the gender distribution of PCB #153 expressed in ngg-1lipid.

In addition, when including parity among women (Paper IV), which is another influencing factor since it is related to breastfeeding, the findings showed that women with at least one child had significantly lower concen-trations of PCBs #74, #105, #118, and p,p’-DDE, when compared with


women with no children. In contrast to the chlorinated and brominated compounds, significant gender differences in concentrations of the fluori-nated compounds were only observed for three compounds (unpublished data). The PIVUS men had significantly (p<0.01) higher levels of PFHpA and L-PFOS compared to the women (Figure 14). The PIVUS women had, on the other hand, significantly (p<0.05) higher levels of only PFHxS. These weak differences can be the result from the higher elimination rate of the fluorinated compounds when compared to the chlorinated and bro-minated POPs. It can also, in accordance with previous findings (Thomsen et al., 2010, Glynn et al., 2012) and could be the result of long time be-tween the sampling and the actual breastfeeding of the 70 years aged wom-en PIVUS.

Figure 14. Frequency histogram showing the gender distribution of L-PFOS expressed in pgmL-1.

The exposure and health effects of POPs have been suggested to be affected by a number of factors including gender (Vahter et al., 2007, Gochfeld, 2007). The results show that that gender differences in concentrations of some POPs do exist. Thus, these findings might be important to further the knowledge on differences in health outcomes when comparing men and women.

0

10

20

30

40

50

60

70

80

90

100Frequency (n=1016)

WOMEN

MEN


5.4 Applied POPs analysis and the implications for human health A great deal of research has been carried out on the exposure to POPs and their distribution in the general populations. However, little research has been done on the general population to answer the question of whether POPs may be associated with negative health effects.

The data generated by the developed methods in this thesis have facili-tated epidemiological research that have supported and contributed to some of the unanswered questions by previous research. This was accom-plished by studying associations between a life-long exposure to POPs in an elderly population of men and women in the PIVUS study. The findings revealed that the exposure to several PCBs were associated with higher numbers of arteries with plaques among individuals, even after adjustment for multiple cardiovascular risk factors (Lind et al., 2012b). This suggests that PCBs might be associated to negative cardiovascular function. In fur-ther epidemiological investigations, findings have suggested that the elevat-ed exposure to POPs is related to hypertension, obesity, the metabolic syn-drome, and diabetes (Lind et al., 2011a, Lind et al., 2011b, Lind et al., 2011c, Lee et al., 2011, Rönn et al., 2011, Lee et al., 2012, Roos et al., 2012, Lind et al., 2013a, Lind et al., 2013b). In addition, the co-variation among mixtures of POPs and other environmental contaminants such as the metals and plastics associated chemicals were studied (Lampa et al., 2011) and results suggested that the PCBs co-varied depending on their degree of chlorination.


6 Conclusions The main objective of this thesis was achieved by the development of two independent methods facilitating high-throughput analysis of legacy POPs using SPE in combination with HRGC-HRMS and of fluorinated com-pounds using a 96-well plate format together with column-switching UPLC-MS/MS. Only 650 µL of human blood was required for analysis of legacy POPs and PFAS, respectively. For chlorinated and brominated com-pounds, the high-sample throughput made it possible for one laboratory technician to prepare 48 samples during one week. The analysis of fluori-nated compounds was further advanced by using a 96-well plate format which enabled analysis of 88 samples within 2 working days. Also, with emphasis on the fluorinated compounds, the developed method effectively included the structural isomers that are important in the source elucidation of PFOS related compounds.

After establishing that the methods were effective, both methods were successfully applied to 1, 016 human plasma samples for the determination of chlorinated, brominated, and fluorinated POPs in men and women from the PIVUS cohort. Most of the compounds studied were detected in the majority of participants. Furthermore, gender differences in the concentra-tions of the POPs studied showed that the majority of chlorinated and brominated compounds were significantly different when comparing men and women in the study, while the concentrations of the fluorinated com-pounds were found to be less influenced by gender. The structural PFOS isomers were quantified in a sub-sample of 25 men and women and the isomeric patterns suggested a similar source of exposure, although, subtle variations in the concentrations of the branched isomers were observed which might be related to different sources of exposure.

In conclusion, by using the developed high-throughput methods requir-ing only small amounts of human blood, exposure information of a broad range of POPs in a large epidemiological study was acquired. Conventional analytical methods at the time of analysis were not able to handle the small amounts of sample available in the study. Thus, by utilizing these methods, the knowledge on the exposure to POPs provides support to several epide-miological investigations.


7 Future directions Drawing on the findings presented in this thesis and considering the large number of chemicals entering the world market every year, even more ana-lytical developments will be required to investigate the associations be-tween POPs and human health. Due to the unique prospective study de-sign, the two reinvestigations of the PIVUS cohort, with more than 1, 500 samples, will shed light to the complex interplay between POP exposure and health effects in the general population.

Ultimately, future perspectives should support the development of high-throughput analytical methods that will allow simultaneous analysis of recognised POPs as well as emerging pollutants. Additionally there is a need to develop of analytical methods, in which the differences in the phys-ico-chemical properties of the chlorinated brominated, and fluorinated are overcome and simultaneous one single-injection analysis is enabled. More specifically, this way forward could be combined with the use of ultra-performance convergence chromatography (UPC2) which successfully com-bines both liquid- and gas-chromatography applications. This combination would reduce the use of multiple methods for the different classes of Stockholm Convention POPs and thus further speed up the analysis. An-other way forward might be untargeted screening which would offer new ways to study the exposure to POPs and possible health effects in the gen-eral population. However, untargeted screening has still not been found to be as specific as targeted trace-level analysis.

Either way, future research should support analytical improvements be-cause this will eventually result in improvements in epidemiological studies themselves as a greater number of POPs would be determined faster and at a lower cost.


8 Acknowledgements These last four years have been characterized by a lot of professional and personal development for me. This experience has truly been extraordinary and there are many wonderful people that I have meet on my way. All of you have in your own unique way supported me throughout this process and I am very grateful for that.

My supervisor, Bert van Bavel, thank you for introducing me to this field of research. I admire your passion for analytical chemistry and for always thinking outside the box or in a two dimensional way. I am grateful for all your support, encouragement, and for your humorous approach to many things at work. Bert, thank you for helping me see the finish line even when I thought I was miles away. My second supervisor, Lars Lind, thank you for all of your interest in the field of analytical chemistry and for my research. Lars, you have been very patient with me and the deadlines for the data results.

Gunilla Lindström, thank you for giving me a glimpse into the emer-gence of POPs analysis. Thank you for that and for all of your support and encouragement during these four years. Monica Lind, thank you for all of your help, support, and interesting discussions about research and life, I admire your passion for both.

Some people have been by my side during the work presented in this thesis and their contributions have been invaluable. Anna Kärrman, my colleague and friend, words cannot express the gratitude I feel towards you. Thank you for all of your support with the analysis of the fluorinated compounds and for all your support with my kappa. Anna, hopefully, the next Bolinas Sunrise will be on me. Jessika Hagberg, my GC guru, you have taught me everything there is to know about the high-res system and shared both hope and despair in our occasional “challenges” with that one particular project involving high-level dioxin analyses. Jessika, your laugh-ter frequently lifts up our corridors and has lifted my spirit many times. Helena Nilsson, my travel mate and fan, you are an inspiration beyond words, thank you for all of your support and encouragement during these years. Helena, thank you for not giving up on me on that late Easter-Thursday when I was about to do just that. Lisa Mattioli, thank you for making my first year in the lab endurable and for all of the hours of laugh-ter in “our” corner of the dioxin-lab, but above all for your help with the analyses during that time. Erik Lampa, thank you for all of your help with the statistical analyses, because statistics is truly not for the people who are


terrified of statistics. Helen Björnfoth, I had really hoped to work with you by my side and your initial part in this project is not forgotten, thank you.

Some people have made sure that I had a lot of fun both at work and off work. Ingrid Ericson Jogsten, thank you for your great support all the way from the potato-project and during some of the more trying times I spent in the lab. Ingrid, you have spoiled me with your kladdkaka many times thank you for that and for everything else you taught me. Anna Rotander, thank you for inspiring me to always push my limits whether we are at work or on a road somewhere in Värmland. Rotan, we will always have Mullhyttan. Maria Larsson, thank you for all of your motivation and sup-port when I was writing my kappa and when I was running samples in the lab, you were always there with a spare box of brown vials and pipette-tips when mine were out. Ulrika Ericsson, my office mate, other than helping me improve my green fingers and gardening skills you have supported me up-close, thank you. Ulrika, one more thing, your office drawer filled with Kexchoklad has not always been a savior, but also a curse. Viktor Sjöberg, surely over the years I have realized that computers are not your only forte. Viktor, thank you for always keeping an open door and an open ear to all of my “stupid” computer questions and for all of your support during the unsocial working hours spent at the office.

There are also other people at work that have contributed to making this experience a more pleasant one. Especially, Filip Bjurlid, Dawei Geng, and Jenna Davies, we are so lucky to have gotten you guys in our group. Also, big thanks to my MTM colleagues working with ecotoxicology and biogeosphere dynamics for numerous fikas and afterworks.

To my closest friends Aida, Marie, and Miona – “Sometimes you need a second opinion, with doctors, real estate, and with…” – Thank you for all of your second opinions and for reminding me about the important things in life and Mac, thank you for all of your support.

At last, my la familia, Selma, thank you for being my greatest critic and my biggest fan – Sestro, there, I finally put a bow on it. Pappa och Sadika, hvala vam što me volite.

April 2013, Örebro


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Publications in the series Örebro Studies in Chemistry

1. Bäckström, Mattias, On the Chemical State and Mobility of Lead and Other Trace Elements at the Biogeosphere/Technosphere Interface. 2002.

2. Hagberg, Jessica, Capillary zone electrophoresis for the analysis of low molecular weight organic acids in environmental systems. 2002.

3. Johansson, Inger, Characterisation of organic materials from incineration residues. 2003.

4. Dario, Mårten, Metal Distribution and Mobility under alkaline conditions. 2004.

5. Karlsson, Ulrika, Environmental levels of thallium – Influence of redox properties and anthropogenic sources. 2006.

6. Kärrman, Anna, Analysis and human levels of persistent perfluorinated chemicals. 2006.

7. Karlsson Marie, Levels of brominated flame retardants in humans and their environment: occupational and home exposure. 2006.

8. Löthgren, Carl-Johan, Mercury and Dioxins in a MercOx-scrubber. 2006.

9. Jönsson, Sofie, Microextraction for usage in environmental monitoring and modelling of nitroaromatic compounds and haloanisoles. 2008.

10. Ericson Jogsten, Ingrid, Assessment of human exposure to per- and polyfluorinated compounds (PFCs). Exposure through food, drinking water, house dust and indoor air. 2011.

11. Nilsson, Helena Occupational exposure to fluorinated ski wax. 2012.

12. Salihovic, Samira Development and Application of High-throughput Methods for Analysis of Persistent Organic Pollutants in Human Blood. 2013.

development and application of high-throughput...

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