R e s i n a c i d s i n c o m m e r c i a l p r o d u c t s a n d t h e w o r k

e n v i r o n m e n t o f S w e d i s h w o o d p e l l e t s p r o d u c t i o n

Sara Axelsson

Resin acids in commercial products and the work environment of Swedish wood pellets production Analytical methodology, occurrence and exposure

Sara Axelsson

©Sara Axelsson, Stockholm 2012

ISBN 978-91-7447-449-7

Printed in Sweden by US-AB, Stockholm 2012

Distributor: Department of Analytical Chemistry, Stockholm University

Front cover: Pine (Pinus Sylvestris), photo Sara Axelsson

The more you think, the more you realize: there is no simple answer Winnie the Pooh

Abstract

The aims of the work this thesis is based upon were to develop convenient analytical procedures for determining resin acids in biological and environ-mental matrices, and apply them to enhance understanding of the occur-rence, exposure to and uptake by exposed individuals of resin acids. Particular focus has been on the workplace environment of the Swedish wood pellets industry.

Sample extraction procedures and high performance liquid chromatogra-phy/electrospray ionisation-mass spectrometry (HPLC/ESI-MS) methodolo-gies were developed that proved suitable for measuring resin acids in dust, skin and urine samples. Chromatographic separation of abietic (AA) and pimaric acid was achieved by using a polar-embedded C12 stationary phase. The developed HPLC/ESI-MS method afforded 10-fold lower detection limits (based on signal/noise ratios) for AA than the standard MDHS 83/2 gas chromatography/flame ionisation detection (GC/FID) methodology. Furthermore, the HPLC/ESI-MS method avoids undesirable oxidation of AA, which was found to occur during the derivatisation step in the GC/FID method, leading to false observations of both AA and the oxidation product 7-oxodehydroabietic acid (7-OXO).

Personal exposures to resin acids in the Swedish wood pellet production industry were found to be lower, on average, than the British Occupational Exposure Limit for rosin (50 µg/m3). However, air concentrations exceeding the limit were measured in certain areas within the production plants, indi-cating that the exposure can sometimes be high. The oxidised resin acid 7-OXO, was detected in both dust and skin samples. This is of particular health concern as its occurrence indicates the presence of allergenic resin acids. A correlation between air and post-shift urinary concentrations of dehydroabi-etic acid (DHAA), and interestingly a trend towards an increase in urinary 7-OXO during work shifts, were also observed. Whether the increase in 7-OXO was due to direct uptake or metabolism of other resin acids cannot be concluded from the results. Hence, for this, more studies are required.

In addition, an efficient HPLC/UV methodology was developed for screening commercial products for rosin (and thus resin acids), which could be used in laboratories lacking sophisticated, expensive instruments, such as mass spectrometers. Diode-array detection (DAD) was applied to gain selec-tivity without the use of MS. Very high concentrations of free resin acids were detected in depilatory wax strips using the method, and AA was de-

tected at high levels in medical tape mounted on paper liner. A particular concern is that when exposed to air, AA is autoxidised to the strong skin allergen 15-hydroperoxy abietic acid. Individuals sensitized to rosin should thus avoid use of such products.

Populärvetenskaplig sammanfattning

Hartssyror är samlingsnamnet på en grupp ämnen som finns i kådan från barrväxter (till exempel tall och gran). Hartssyrorna är klibbiga och löser olja i vatten, egenskaper som gör att de används i många produkter för både hushåll och industri. De används till lim (bland annat till plåster), färg, lack, produkter för lödning och förekommer naturligt i trä och träbaserade produk-ter som papper och träpellets.

Kolofonium är en produkt som till största delen består av hartssyror, eftersom den utvinns ur kåda. Allergi mot kolofonium är väldigt vanligt och forskningsstudier har visat att det är de ämnen som bildas vid kontakt med luft som är allergiframkallande. Undersökningar av personal som jobbar i olika industrier där kolofonium används har visat på samband mellan an-vändning av kolofonium och påverkan på hälsan som irritation i luftvägar och ögon, astma och eksem.

För att studera hur mycket hartssyror som finns i luften på en arbetsplats tas ett prov genom att pumpa luft genom ett filter och sedan mäta mängden uppsamlade hartssyror på filtret. Traditionellt används en teknik kallad gas-kromatografi1 för analysen. Den metoden kräver förbehandling av provet genom omvandling av hartssyrorna med hjälp av reagens. Nackdelar är att reagensen i sig kan vara hälsoskadliga och att provens hållbarhet efter be-handling inte är så lång. I denna avhandling presenteras därför analysmeto-der baserade på vätskekromatografi2 vilket inte kräver användning av re-agens.

Studierna i denna avhandling visar på att hartssyror finns i mätbara halter i träpelletsindustrin, i form av luftburna ämnen, men även på huden och i urinen bland de människor som arbetar där. Hartssyrorna finns också i kos-metiska produkter. I Sverige finns ännu ingen gräns för hur hög halt luft-burna hartssyror som får finnas i arbetsmiljön, men det högsta uppmätta värdet i denna undersökning var 75% av den gräns som används i Storbri-tannien för kolofonium (50 µg/m3). De uppmätta urinvärdena är i samma storleksordning som de man funnit i urin från personal i brittiska lödningsin-dustrin.

1 Gaskromatografi. En analysmetodik som bygger på att ämnen i ett prov har olika kokpunkt. 2 Vätskekromatografi. En analysmetodik som bygger på att ämnen i ett prov har olika löslig-het i vatten och olja.

List of papers

This thesis is based on the following papers, which are referred to hereafter by the corresponding Roman numerals. For convenience, the studies de-scribed in Papers I-V are sometimes referred to as Studies I-V. All published papers are reproduced with kind permission of the publishers.

I. Exposure to wood dust, resin acids, and volatile organic com-

pounds during production of wood pellets. K Hagström, S Axels-son, H Arvidsson, IL Bryngelsson, C Lundholm, K Eriksson. J Oc-cup Environ Hyg, 2008, 5(5), 296-304.

The author was responsible for developing the analytical methodology for

resin acid analysis, planning and conducting the laboratory work and some

of the writing.

II. Tape-stripping as a method for measuring dermal exposure to

resin acids during wood pellet production. K Eriksson, K Hag-ström, S Axelsson, L Nylander-French. J Environ Monit. 2008, 10(3), 345-352.

In this work the author was responsible for developing the analytical meth-

odology for resin acid analysis, planning and conducting the laboratory

work and part of the writing.

III. Determination of resin acids during production of wood pellets –

a comparison of HPLC/ESI-MS with the GC/FID MDHS 83/2

method. S Axelsson, K Eriksson, U Nilsson. J Environ Monit, 2011, 13(10), 2940-2945.

The author was responsible for developing the rationale, planning and con-

ducting the laboratory work and most of the writing.

IV. HPLC/neg-ESI-MS determination of resin acids in urine from

Swedish wood pellet production plants workers and correlation

with air concentrations. S Axelsson, K Hagström, K Eriksson, I-L Bryngelsson, U Nilsson. Anal Bioanal Chem, Submitted.

The author was responsible for developing the rationale, planning and con-

ducting the laboratory work and most of the writing.

V. SPE and HPLC/UV of resin acids in colophonium-containing

products. U Nilsson, N Berglund, F Lindahl, S Axelsson, T Redeby, P Lassen, AT Karlberg. J Sep Sci, 2008, 31(15),2784-2790.

In this work the author contributed to the development of the HPLC method

and participated in the writing.

Papers by the author not included in this thesis:

Dermal uptake study with 4,4′-diphenylmethane diisocyanate led to active sensitization. H Hamada, M Isaksson, M Bruze, M Engfeldt, I Liljelind, S Axelsson, B Jönsson, H Tinnerberg, E Zimerson. Cont Derm, 2012, 66, 101–105. Exposure to environmental tobacco smoke and health effects among hospi-tality workers in Sweden--before and after the implementation of a smoke-free law. M Larsson, G Boëthius, S Axelsson, SM Montgomery. Scand J Work Environ Health, 2008, 34(4), 267-277. Environmental tobacco smoke (ETS) exposure in a sample of European cit-ies project. M Nebot, MJ López, G Gorini, M Neuberger, S Axelsson, M Pilali, C Fonseca, K Abdennbi, A Hackshaw, H Moshammer, AM Laurent, J Salles, M Georgouli, MC Fondelli, E Serrahima, F Centrich and SK Ham-mond. Tob Control, 2005, 14(1), 60-63. Exposure assessment to α- and β-pinene, ∆3-carene and wood dust in indus-trial production of wood pellets; K Edman, H Löfstedt, P Berg, K Eriksson, S Axelsson, I Bryngelsson and C Fedeli; Ann Occup Hyg, 2003, 47(3), 219-226. The major fluvoxamine metabolite in urine is formed by CYP2D6; O Spigs-et, S Axelsson, Å Norström, S Hägg, R Dahlqvist; Eur J Clin Pharmacol, 2001, 57, 653-658.

Contents

Introduction ................................................................................................15

Background .................................................................................................16 Wood pellet production ..........................................................................................16 Exposure assessment .............................................................................................17

Air measurements..............................................................................................18 Biological monitoring .........................................................................................20

Resin acids................................................................................................................23 Occurrence and properties ...............................................................................23 Health effects......................................................................................................26 Metabolism ..........................................................................................................26 Resin acid analysis.............................................................................................27

Papers I-V ...................................................................................................32 Methodology .............................................................................................................32

Company data, Papers I-IV..............................................................................32 Sampling..............................................................................................................32 Sample preparation ...........................................................................................33

Results and discussion............................................................................................34 Chromatographic separation of AA and PA ...................................................34 Resin acid ESI-MS spectra in HPLC/ESI-MS..................................................35 Method performance .........................................................................................36 Exposure assessments ......................................................................................43 Resin acid levels in beauty products and gum rosin ...................................49

Conclusions...............................................................................................................50 Outlook ......................................................................................................................51

Tack!.............................................................................................................52

References ..................................................................................................54

Abbreviations

7-OXO 7-oxodehydroabietic acid 15-HPA 15-hydroperoxyabietic acid AA Abietic acid CAS RN Chemical Abstract Service registry number DAD Diode array detector d2-DHAA Dehydroabietic acid-6,6-d2 DHAA Dehydroabietic acid ESI Electrospray ionization FID Flame ionization detector GC Gas chromatography HPLC High performance liquid chromatography IS Internal standard LC Liquid chromatography LOD Limit of detection LOQ Limit of quantification ME Methylester MS Mass spectrometry MTBE Methyl tert-butylether Mw Molecular weight OEL Occupational exposure limit PA Pimaric acid PFBE Pentafluorobenzyl ester RIC Reconstructed ion chromatogram RSD Relative standard deviation SD Standard deviation SPE Solid phase extraction TMSE Trimethylsilyl ester

15

Introduction

Resin acids are a group of substances that naturally occur in the resin of co-niferous species and are widely used in various household and industrial products. Allergies to resin acids (rosin) are common, indeed they are re-portedly among the 10 substance groups that most frequently cause contact allergy [1]. Health effects such as asthma and contact dermatitis have been reported after occupational exposure to rosin [2, 3].

Wood pellets, which comprise a type of processed biofuel, are used on large-scale in industries, district heating plants and private households in Sweden. The pellets are produced by compressing sawdust and/or wood shavings, usually from conifers such as pine and spruce. The work environ-ment at the production plants is often dusty [4], posing potential risks of exposure to resin acids by inhalation and dermal routes. Inhalation exposure is usually measured as the concentration in the breathing zone of a worker, while dust is collected on a filter by pumping air through it at a constant flow rate. A common way of assessing dermal exposure is to collect samples by tape-stripping selected parts of the skin, then analysing the substances col-lected by the tape and determining their concentrations relative to the meas-ured amount of skin sampled.

The major work underlying this thesis has been focused on the develop-ment of convenient analytical procedures and sensitive, selective methods for detecting resin acids. Commonly used methods often lack selectivity or involve use of derivatisation techniques, but in Paper III, derivatisation was shown to promote unwanted oxidation of AA. Key aims of the studies were to develop and apply new analytical procedures to investigate the occur-rence, exposure to and uptake by exposed individuals of resin acids, espe-cially in the workplace environment of the Swedish wood pellet industry.

16

Background

Wood pellet production Wood pellets (Figure 1) are a kind of processed biofuel, and have been pro-duced in Sweden since the 1980’s. In 2010 almost 2.3 million tonnes were sold on the Swedish market (34% to private households). Domestic production has settled at around 1.6 million tonnes yearly and the increase on the market is due to increasing imports. Today, there are around 80 pro-ducers in the country, of which 16 produce more than 50 kilotonnes per year. In 2010 the plants had an over-capacity of about 400 kilotonnes [5].

Figure 1. Wood pellets.

Wood pellets are produced by compressing wood shavings and/or saw-dust. Scotch pine (Pinus sylvestris) and European spruce (Picea abies) shav-ings or sawdust are the most commonly used in Sweden. During production the sawdust is dried before grinding, while shavings are ground directly (Figure 2). The ground wood is then pressed through cylindrical holes in a pellet matrix (Figure 3). Due to friction the temperature here reaches 100°C. Most plants apply steam in the matrix, which increases the natural binding of the lignin and thus makes addition of binding agent unnecessary. After pressing, the wood pellets need cooling, which is usually done in a cooling tower, and then they are stored before transport [4]. Some of the pellets are bagged in small (16 kg) or large (600 kg) bags for sale, while others are

17

transported in bulk to industrial plants, district heating plants or private households [5]. The Swedish standard for fuel pellets (SS 18 71 20) sets requirements for wood pellet properties, such as size, sulphur, chloride and moisture contents and energy value.

Figure 2. Flow diagram of the pellet production process [4] (p 16).

Figure 3. Pellet matrix. The ground wood is pressed through the holes of the matrix.

Exposure assessment Exposure to a substance in the environment may follow different routes, for example via inhalation or contact with the skin. The main factors affecting exposure, and thus that should be considered in exposure determinations are: the physical and chemical properties of the substance, its concentration, du-ration and frequency of exposure [6]. The dose an exposed individual re-ceives, the body burden, is dependent on both environmental and physio-logical factors, such as the concentration of the substance in the environ-ment, the barrier functions of the body, distribution of the substance in the body, its metabolism and elimination. A simplified model of the interactions involved is shown in Figure 4.

Sawdust

Shavings

Grinding Pressing Cooling Storage Drying

18

Figure 4. A simplified model of the interactions between matrices that affect doses individuals receive of emitted substances via various exposure routes.

Air measurements Air concentrations of a substance of interest are usually measured by analys-ing samples collected by pumped or diffusive sampling on a filter, adsorbent or chemosorbent. More specifically, dust particles are commonly trapped on filters, while vapours are collected on adsorbents or chemosorbents. The purpose of the measurements may be to compare the air concentration with an occupational exposure limit (OEL) or to investigate exposure-response relationships in epidemiological studies. When an investigation in relation to the health of an individual is performed, such as compliance to an OEL, the air concentration of the substance of interest is measured in the breathing zone of the worker, “personal measurement”. Such measurements are often accompanied by measurements of the air concentration in general within the premises, to examine whether there are areas where the potential exposure is high. These measurements, where there is no connection to an individual, are referred to as area measurements. Direct-reading instruments can provide real-time or near real-time measurements and thus are powerful tools for identifying high exposure work tasks.

Dust measurements In Sweden, sampling of particulate compounds with health relevance in workplace atmospheres should conform to the standard “Workplace atmos-pheres - Size fraction definitions for measurement of airborne particles” (SS-EN 481). This standard defines the required sampling efficiency for three size fractions of particles: inhalable, thoracic and respirable. Inhalable parti-

19

cles are those that can be inhaled via the nose or mouth and have an aerody-namic diameter of less than 100 µm. The thoracic particle fraction can pass the larynx (less than 10 µm in diameter) and the respirable fraction consists of particles small enough to reach the alveoli (smaller than 4 µm). The term “total dust” is defined (in Sweden) as the dust collected by sampling using an open-faced 25- or 37-mm cassette, as shown in Figure 5. For comparison with OELs, total dust is measured as long as the current legislation does not require measurement of any specific size fraction.

In 2005 the specifications of the Swedish OEL for measured dust were changed from total dust to inhalable dust, but the OEL (2 mg/m3) was not changed [7]. The legislation specifies the dust fraction to be measured, but not the kind of sampler to be used. The sampler developed by the Institute of Occupational Medicine (IOM), illustrated in Figure 5, has been criticised for the apparent risk of sampling very large particles (>100 µm), and thus over-estimating exposure. Other samplers with small openings, for example button samplers, have been thought to have a smaller risk of sampling very large particles. However, a recent field evaluation of a range of samplers for the inhalable dust fraction (IOM and button samplers among others) found no significant differences between them in respect to amount dust sampled [8].

Figure 5. Open-faced 25-mm cassette for total dust sampling (a) and Institute of Occupational Medicine (IOM) sampler for the inhalable dust fraction (b).

(a) (b)

20

Biological monitoring Measuring a xenobiotic (a substance that is foreign to the body or ecological system) in target organs is almost always impossible without invasive meth-ods, such as biopsies. Instead, analyses of more accessible kinds of samples (for example blood, hair, urine, faeces, breast milk, saliva and exhaled air) are preferred. Such analyses are referred to as biological monitoring, and the substance determined in those samples is called a biomarker. Biological monitoring takes into account variation in the exposure, individual behaviour and conditions, as stated by Berglund et al [9]:

“Biological monitoring provides information on the absorbed internal dose, and the total exposure of the individual, integrated over all sources and routes of exposure. It also takes into account inter-individual and intra-individual dif-ferences in intake and uptake, as well as differences in metabolism and physi-cal activity.” (p 85)

Biomarkers can be divided into three groups: biomarkers of exposure, ef-

fect and susceptibility. Biomarkers of exposure indicate the exposure to the substance and the body burden. Biomarkers of effect indicate an effect of exposure to the substance on the target organ, and biomarkers of susceptibil-ity indicate the vulnerability of a person to the exposure. This can be exem-plified with exposure to ethanol. The concentration of ethanol in blood is an example of a biomarker of exposure, i.e. it is related to exposure to the sub-stance. The activity of alanine aminotransferases in blood is a biomarker of effect since it indicates damage to the liver [10] and genetic factors are bio-markers of susceptibility since they influence the susceptibility of individu-als to liver damage by alcohol consumption [11].

Some of the problems involved in biological monitoring, apart from the

(often) invasive sampling procedures, are the unknown links between meas-ured concentrations and both health risks and the metabolic kinetics of the measured substances, which complicate attempts to identify when and what to sample [9].

Skin sampling by tape-stripping Tape-stripping is a relatively non-invasive method that is used to remove a sample of the skin for further investigation. It is traditionally used in derma-tology to remove skin for examination or induce skin disruption [12]. It is widely used in pharmacological studies on skin, and in occupational hygiene the technique has been used to measure exposure to a range of substances, inter alia jet-fuel [13], acrylates [14, 15], pyrene, benzo(a)pyrene [16] and resin acids [Paper II]. Tape-stripping is done by applying adhesive tape to the skin, then removing it after a specified time. A few µm of the outermost layer of the skin, stratum corneum, adheres to the tape-strip, together with chemicals adsorbed by or to the sampled skin. Repeated tape-stripping on

21

the same area will remove subsequent layers of the stratum corneum, until the skin barrier is disrupted and emerging intracellular fluids prevent further sampling. Each tape surface collects a specific layer of the skin and can thus provide information on the depth profiles and penetration of the substance studied.

The amount of skin removed is dependent on the type of tape used, pres-sure applied and duration before removal, speed of tape removal, location of the tape on the body and condition of the skin [16-18]. Changes in the skin by tape-stripping can be examined by measuring variables such as transder-mal water loss and skin hydration. The amount removed by the tape has been quantified by weighing [19, 20], microscopic measurements [21], and col-orimetric keratine quantification [22, 23].

Urine samples If the compound of interest is excreted in urine, sampling and subsequent analysis of urine offers a simple and non-invasive methodology to determine uptake and body burden. In an early application (in the 1940’s) Weigher and Mottham detected water-soluble derivatives of benzopyrene in urine [24], which were later identified by Harper and Calcutt as glucuronic acid and sulphate conjugates [25]. It is now known that xenobiotics excreted in the urine are often conjugated with those acidic groups by detoxification sys-tems in the body that increase hydrophobic substances’ water solubility and thus facilitate their elimination. Glucuronic acid conjugation is mediated by a group of enzymes called UDP-glucuronosyltransferases, which are primar-ily found in the liver, but are also present in the oesophagus and small intes-tine [26]. Sulphotransferases have been found in tissue from the small intes-tine, liver, kidney and lungs in humans [27], and are phase II enzymes re-sponsible for catalyzing sulphate group conjugation to xenobiotics. The ana-lytical methods used for monitoring xenobiotics in urine are seldom designed to detect the conjugates formed; instead the urine samples are gen-erally subjected to hydrolysis by acids [28, 29], bases [30] or enzymes [31, 32]. Enzymatic hydrolysis is often performed at pH 4-5 and 37°C, experi-mental conditions that at first impression seem to be less harsh than those involving acid or base hydrolysis (pH often less than 1 or higher than 12 and temperatures of 80-90°C). However, rearrangements of the substance by the enzyme may occur [31-33], to an extent which probably depends (inter alia) on the source of the enzyme [32].

Urine excretion rates vary between individuals, and those of specific indi-viduals vary during the day, depending on their fluid intake and sweating rates, among other factors. Therefore, when comparing urinary concentra-tions of a substance obtained from samples collected on different occasions they are often normalised. The two reference variables most often used for normalisation (which provide correlated results [34]) are the urinary creatinine concentration and specific gravity (SG). Creatinine excretion has

22

been found to depend on gender, age, race and health condition [35-37]. However, the excretion rate for an individual is fairly constant, thus the creatinine concentration can be used to assess the urine dilution [38]. The SG of distilled water is 1.000 (in SI units) and can be measured using a density meter [39] or a refractometer [34]. The difference between the SG of a urine sample and distilled water correlates to the total amount of substances dissolved (or suspended) in the urine, and hence provides a measure of its dilution. Normalisation of a urinary concentration by specific gravity is con-ventionally done using Equation 1. The SG standard value is set either in relation to the studied population or to a regulatory value.

Equation 1. Applied to normalise a measured concentration of a substance in a urine sample using specific gravity.

When using conventional normalisation (Equation 1), it is implicitly as-sumed that the relative mass ratio between the substance measured and total dissolved solids does not change with urinary flow. This model has been shown to be non-valid for a range of substances and an alternative approach has been proposed by Vij and Howell [40], Equation 2.

Equation 2. Alternative SG normalization equation proposed by Vij and Howell [40]. Z is a substance-dependent constant.

The constant Z in Equation 2 is substance-specific and has to be experi-mentally determined [40]. In the absence of a z-value the conventional method is used (Equation 1, in which z=1).

Normalisation of measured urine concentrations, using either of the methods, has drawbacks. When using creatinine concentrations, differences in the renal clearance mechanisms for creatinine and the studied compound should be considered, otherwise the result may be erroneous [37, 41]. There is also a large risk of errors in SG normalisation if the wrong Z-constant is used [40, 42].

1

1standard

−

−

=

measured

measurednormalisedSG

SGCC

Z

measured

measurednormalisedSG

SGCC

−

−

=

1

1standard

23

Resin acids

Occurrence and properties Resin acids are the main constituents of rosin from conifer species. They are also known as diterpenoid acids and are divided into two groups, abietanes and pimaranes, according to their structural properties. Abietanes (Figure 6) contain conjugated double bonds and an isopropyl or isoprenyl group at the C-13 position. Pimaranes (Figure 7) lack conjugated double bonds and are methyl and vinyl substituted at C-13.

Rosin is both antibacterial and antifungal [43], but some bacterial strains can use resin acids as substrates [44]. The resin acid composition of the rosin varies depending on the source species, wood type [45], growth site, and age of the tree [46], as illustrated in Table 1.

Figure 6. Structures of selected resin acids of abietane type.

24

Figure 7. Structures of selected resin acids of pimarane type.

Table 1. Resin acid composition in wood from European spruce (Picea abies) and Scots pine (Pinus sylvestris).

Picea abies Pinus sylvestris % Sapwood Heartwood

Abietic 9.3 8.2 15.8 Dehydroabietic 13.2 35.7 14.4

Levopimaric 28.6 10.5 30.0 Palustric 17.5 16.4 15.1 Pimaric 3.3 4.4 8.1

Isopimaric 11.3 11.8 3.5

Reference [45] [45] [45]

Rosin is a technical product rich in resin acids and it is a constituent of

many commercial products used in diverse applications due to its emulsify-ing and adhesive properties. Depending on the production method, different types of rosin are obtained. Tall-oil rosin is a fraction of tall-oil and a by-product of paper and pulp production, while solvent extraction of pine stumps gives wood rosin and processing resin from living trees produces gum rosin. The different production methods give rosins that are slightly different in composition. For instance gum rosin contains more of AA and less of DHAA than tall-oil rosin. Several terms are used in the literature for more or less the same type of technical products. The term “colophony” corresponds to gum rosin in technical literature and in dermatological litera-ture also wood rosin and tall-oil rosin are included in this term. This is due to the fact that the resins contain the same major chemical components and allergens, and in technical products most often no declaration of the source is found. Throughout this thesis the name “rosin” is used as this stands for all types.

The resin acids investigated in Studies I-V, namely abietic (AA), dehy-droabietic (DHAA), 7-oxodehydroabietic (7-OXO) and pimaric acid (PA), are all weak acids (pKa ≈ 4.6), that are only sparingly soluble in water (2-9 mg/L), see Table 2.

25

Resin acids, such as AA, are highly prone to oxidation, and their autoxidation results in the formation of hydroperoxides, oxides and alcohols (Figure 8) [48-51].

Table 2. Selected properties of the studied resin acids.

Abietic

acid (AA)

Dehydroabietic acid

(DHAA)

7-oxo-dehydroabietic

acid (7-OXO)

Pimaric acid (PA)

CAS RNb 514-10-3 1740-19-8 18684-55-4 127-27-5

C20H30O2 C20H28O2 C20H26O3 C20H30O2 Mw 302.45 300.44 314.42 302.45 Water solubility mg/L

[47] 2.75 5.11 9.1a 2.17

Boiling pointa °C

760 Torr 395-483 391-459 430-520 368-458

pKaa 4.64±0.60 4.66±0.40 4.52±0.40 4.68±0.60

a: Chemical Abstracts Service registry substance properties, calculated using Advanced Che-mistry Development (ACD/Labs) Software V11.02. b: Chemical Abstracts Service Registry Number.

Figure 8. Selected oxidation products of abietic and dehydroabietic acid.

26

Health effects Exposure to resin acids (rosin) has been shown to cause irritation of the up-per respiratory tract, alveolitis and rhinitis (with or without asthma). In the electronics, and other, industries where rosin is heated to around 180–400°C, there is a higher prevalence of asthma among employees than in reference populations [2]. The prevalence of contact allergy to rosin among employees at a tall-oil rosin production plant was found to be about the same as among dermatitis patients [52] and allergic contact dermatitis caused by soldering flux has been reported in the literature [53]. Temporary occupational expo-sures have however been reported as of minor importance for dermatitis [3]. Wood dust from pine and spruce via dermal exposure can cause eczema and the resin acids present in the dust are suspected to be the cause [54]. Contact allergy and irritation of eyes and respiratory tract has been associated with exposure to resin acids, and contact dermatitis is caused by oxidized species [48, 54-57]. AA itself is a very weak allergen or maybe even non-allergenic, but it is considered to be a prehapten because when it is exposed to air potent allergens like 15-hydroperoxyabietic acid (Figure 8) rapidly form [51, 58-60].

Metabolism Little is known about the human metabolism of resin acids. DHAA has been identified in urine following exposure to soldering fumes [28, 61] and wood dust generated in pellet production (Paper IV). The Health and Safety Ex-ecutive (HSE) of the UK suggests the substance has a half-life of around 4 h in humans [28], but this is yet to be confirmed. The metabolism of resin ac-ids in other organisms, including fish, rabbits and microorganisms, is to some extent under investigation. Biodegradation of palustric acid and AA by aerobic resin acid-tolerant bacteria yields DHAA, which is metabolized via 7-OXO to 2-isopropyl malic acid. Anaerobic degradation of abietanes has been shown to result in small amounts of retene, and the pimaranes are pro-posed to be transformed to pimanthrene [44]. The metabolic pathways of the substances in rainbow trout, Salmo gairdneri, exposed to resin acid-containing water vary, and seem to depend (inter alia) on the concentration and length of exposure [62]. After ingestion of DHAA by rabbits, the me-tabolites in urine are reportedly primarily oxidised at the isopropyl sustituent [63, 64], but trace amounts of 7-OXO have also been detected [63].

27

Resin acid analysis Due to their harmful properties, resin acids have been determined in many matrices. A wide range of techniques, such as HPLC, HPLC/APCI-MS, HPLC/pos ESI-MS, HPLC/neg ESI-MS, HPLC/neg ESI-MS/MS, GC/FID and GC/MS, have been used in these analyses, as listed in Table 3. For HPLC, C18 reversed phase systems have been the most common.

Methods based on HPLC/MS techniques have the advantages of high sen-sitivity and selectivity, and in contrast to GC/MS they do not require deriva-tisation. Certain derivatives, such as methyl esters, are prone to hydrolysis, whilst methanol solutions of non-derivatised resin acids are relatively stable (Paper IV). Resin acids are ionized in both positive and negative electric fields [Papers I-IV, 65] in ESI-MS, and MS/MS methodology, if available, can give higher detectability. However, it should be noted that the resin acids do not readily yield daughter ions easily in negative ESI-MS/MS mode [65].

28

Table 3. Matrices and resin acids examined, sample preparation and analytical techniques applied in selected studies, and reported limits of detection (LOD).

Matrix Resin acids Sample

preparation Technique LOD Refer-ence

Skin (Human)

Tape stripping

Abietic Dehydroabietic 7-oxodehydro-abietic Pimaric

Methanol extraction

HPLC/ pos ESI-

MS

AA 45 ng/sample

DHAA 142

ng/sample 7-OXO 4.5 ng/sample

PA 86 ng/sample

Paper II

Urine (Human)

Dehydroabietic 7-oxodehydro-abietic

Acid hy-drolysis,

SPE mixed mode anion exchanger

HPLC/neg ESI-MS

DHAA 4 nM

7-OXO 3 nM

Paper IV

Urine (Human)

Dehydroabietic Acid hy-drolysis,

ether extrac-tion, deriva-tisation to

ME.

GC/MS DHAA 54 nM [61]

Bile plasma

(Rainbow

trout)

Abietic Dehydroabietic Isopimaric Levopimaric Neoabietic Palustric Pimaric Sandarocopimaric

Hex-ane/acetone extraction w/wo hy-drolysis,

derivatisa-tion to ME.

GC/FID 0.02 µg/mL

[62]

BIO

LO

GIC

AL

Bile (Brown

trout)

Abietic Dehydroabietic Isopimaric Neoabietic Palustric Pimaric Sandaracopimaric

Hex-ane/acetone extraction, derivatisa-

tion to TMSE

GC/MS 0.01-0.05 µg/ml [73]

29

Table 3. Continued.

Matrix Resin acids Sample

preparation Analytical technique LOD

Refer-ence

Rosin (unmodi-

fied)

Abietic Dehydroabietic Levopimaric Neoabietic Palustric

Dissolution in methanol HPLC/UV Not stated [60]

Bindi adhesive

Abietic Dehydroabietic

Ultrasonica-tion in

methanol SPE C18

HPLC/UV-fluorescence

AA 1.25 ng/sample DHAA 0.5 ng/sample

[66]

Propolis Abietic acid Dehydroabietic

Methanol HPLC/UV-fluorescence

AA 33.3 ng/ml

DHAA 66.7 ng/ml

[67]

Traditional

Chinese medica-

tions

Abietic Dehydroabietic

Acetonitrile and/or diethyl

ether extraction SPE C18

HPLC/UV-fluorescence

AA 0.1 µg/mL

DHAA 0.05 µg/ml

[68]

Printing papers

Adhesives Soldering

flux Paint

Abietic Dehydroabietic

Solvent ex-traction

HPLC/UV

AA 0.001% DHAA 0.015%

[69]

Tall oil rosin

Abietic Dehydroabietic Isopimaric Palustric Neoabietic

Diethyl ether extraction,

derivatisation to ME

GC/FID GC/MS

Not stated [70]

Tall oil fractio-nation

products

Abietic Dehydroabietic Dihydroabietic Isopimaric Neoabietic Palustric Pimaric Primaric Secodehydro-abietic

Derivatisation to ME in pyridine

GC/MS Not stated [71]

PRO

DU

CT

S

Cosmetics

Abietic Dehydroabietic 7-oxodehydro-abietic

Acetonitrile extraction, SPE mixed mode anion exchanger

HPLC/UV DAD

AA 7 µg/g DHAA 19 µg/g 7-OXO 13 µg/g

Paper V

30

Table 3. Continued.

Matrix Resin acids Sample

preparation Analytical technique LOD

Refer-ence

Process water

Abietic Dehydroabietic 12,14-dichloro dehydroabietic Isopimaric Levopimaric Neoabietic Sandarocopimaric

Filtration Methanol dilution

HPLC/ APCI-MS

0.9-3 µg/L [72]

Process water

Abietic Dehydroabietic 12,14-dichloro dehydroabietic Isopimaric Levopimaric Neoabietic Sandarocopimaric

MTBE extrac-tion, derivati-

sation to TMSE

GC/MS 0.004-0.1 µg/L [72]

River water

Abietic Dehydroabietic Isopimaric Pimaric n/a

HPLC/ neg ESI-MS

AA 0.40 µg/L

DHAA 0.40 µg/L IPA 0.30

µg/L PA 0.25

µg/L

[65]

Aquar-ium

water

Abietic Dehydroabietic Isopimaric Neoabietic Palustric Pimaric Sandaracopimaric

Hex-ane/acetone extraction,

derivatisation to TMSE

GC/MS 0.05 µg/L [73]

Process water, pulp

Resin acids MTBE extrac-tion, derivati-

sation to TMSE

GC/FID Not stated [74]

WA

TE

R

Process water

8(14)-abietic Abietic Dehydroabietic Isopimaric Levopimaric O-methyl-podocarpic Neoabietic 7-oxodehydro-abietic Palustric Pimaric Sandarocopimaric

SPE C18 or Lichrolut

EN®, derivati-sation to

PFBE

GC/MS Not stated [75]

31

Table 3. Continued.

Matrix Resin acids Sample

preparation Analytical technique LOD

Refer-ence

Solder fumes

Dehydroabietic 7-hydroxy-dehydroabietic 15-hydroxy-dehydroabietic Isopimaric 7-oxodehydro-abietic Pimaric Sandarocopimaric

Methylene chloride

extraction, derivatisa-tion to ME

GC/MS Not stated [76]

Solder fumes

Solvent soluble rosin solids

Dichloro-methane

extraction

Gravimet-ric

10 µg/samplea [77]

Solder fumes

Abietic Dehydroabietic

Methylene chloride

extraction, derivatisa-tion to ME

GC/MS Not stated [78]

Solder fumes

Resin acids Methylene chloride

extraction

Gravimet-ric Not stated [78]

Solder fumes

Abietic Dehydroabietic Isopimaric Neoabietic Palustric Pimaric Sandarocopimaric

Diethyl ether

extraction, derivatisa-

tion to ME.

GC/FID 0.2 µg/sampleb [79]

Wood dust

Abietic Dehydroabietic 7-oxodehydroabietic Methanol

extraction

HPLC/ pos ESI-

MS

AA 9.4 ng/m3

DHAA 5.2 ng/m3

7-OXO 0.42 ng/m3

Paper III

Wood dust

Abietic Dehydroabietic 7-oxodehydroabietic

Ether extraction, derivatisa-

tion to ME.

GC-FID

AA 89 ng/m3 DHAA

115 ng/m3 7-OXO

24 ng/m3

Paper III

AIR

Wood smoke

Abietic Dehydroabietic

Solvent extraction, derivatisa-

tion to TMSE.

GC/MS

AA 0.5 ng/m3

DHAA 0.6 ng/m3

[80]

a: corresponds to 10 µg/m3 assuming 8 hours sampling at a flow rate of 2 L/min. b: corresponds to 0.2 µg/m3 assuming 8 hours sampling at a flow rate of 2 L/min.

32

Papers I-V

Methodology

Company data, Papers I-IV Four wood pellet production plants in Sweden (hereafter denoted plants A-D) participated in the studies. Their annual pellet production ranged between 12 and 120 kilotonnes. In addition to producing pellets, plant A also pro-duced 48 kilotonnes of briquettes annually. All of the companies had a gen-eral ventilation system, and all but one had also installed a local ventilation system at the bagging station.

Out of a total of 65 personnel employed in wood pellet production, 44 participated in at least one of the studies. Examples of the participating per-sonnel’s work tasks were supervising the automated production process from a control room, maintenance, loading of raw material into the grinder, sweeping the factory floor, cleaning equipment with compressed air, truck driving and bagging wood pellets. The general work attire consisted of pro-tective shoes with socks, trousers, and a long- or short-sleeved shirt. Leather gloves were used during some of the tasks. A few of the workers put on their working clothes and entered the production area for an update from their co-workers before pre-shift sampling.

Sampling

Air sampling (Papers I, III and IV) Personal samples for resin acid determination were collected from the breathing zone of the workers as “total dust”. Repeated measurements were acquired for some of the 44 workers, resulting in a total of 68 measurements. Area measurements, 75 samples, were collected from the control room, bag-ging room, wood pellet storage warehouse, beside the pellet press and near the briquette machine and kiln at the plant that also produces briquettes. In Study III two sets of air samples were collected as area measurements at plant B for method comparison.

33

Manufacture of standardised wood pellet dust (Paper III) Wood pellets, from a Swedish wood pellet production plant (made from an unspecified mixture of pine and/or spruce), were ground in a water-cooled sample grinder and then sieved.

Biological samples (Papers II and IV) Skin of the participating workers was sampled in Study II using a tape-stripping technique, in which strips of Leukosilk® were applied to the skin and removed after 2–3 min using tweezers cleaned in methanol. Four skin areas were sampled: the forehead, the front of the neck, the back of the lower arm and the dorsal side of the hand. Prior to a shift the left hand skin areas were sampled, and the corresponding right hand areas were sampled post-shift. Three consecutive tapes were collected from each skin area. To avoid cross-contamination new gloves were used every time a tape was applied or removed from the skin. The position of the first tape was marked with ink on the skin to facilitate replication of the sampling. Repeated sampling was carried out on some individuals, resulting in a total of 63 personal measure-ments, corresponding to 1472 tape strips. The tape-strip sampling was car-ried out in a room separated from the production area, but within the factory premises. The recoveries of resin acids by tape stripping were evaluated both in vivo and in vitro. In Study IV urine samples were collected from 39 work-ers pre- and post-shift, in connection to the air and skin sampling. Repeated samples were collected from 17 workers, giving a total of 112 samples. The methodologies used for the biological sampling were approved by the Ethi-cal Committee of Umeå University (D-no. 03-335).

Products (Paper V) A selection of cosmetic products (lip gloss, skin foundation and depilatory wax strips), gum rosin (unmodified and maleic anhydride-modified) and unmodified rosin were purchased. The products were selected to represent product groups that are applied to the skin and might contain resin acids.

Sample preparation Each tape strip or filter used in Studies I and II was extracted with methanol and syringe-filtered prior to HPLC/pos ESI-MS analysis. In Study III three sets of samples were divided into sub-groups prior to extraction in order to compare methodologies. A sub-group of the first set (filter samples) was extracted with methanol containing d2-DHAA (IS), syringe-filtered and di-luted with water prior to HPLC/MS analysis, while a corresponding set was ultrasonicated in ether, evaporated and methylated in toluene before GC/FID analysis. The second sample set (standardised wood pellet dust samples) were divided into three sub-groups. Two thirds of the standardised wood

34

pellet dust samples were ultrasonicaded in ether, or extracted with toluene or methanol and filtered before HPLC/MS analysis. One third of the samples were ultrasonicated in ether, evaporated and then methylated before GC/FID analysis. Half of this sub-group were filtered prior to heating. The third sam-ple set in Study III (filter samples) was divided into sub-samples and one sub-sample set was extracted with methanol, syringe filtered and analysed by HPLC/MS. The second sub-sample set was ultrasonicated in ether, and sy-ringe-filtered prior to derivatisation and then analysed by GC/FID.

The urine samples in Study IV were hydrolysed using HCl, in tubes where the atmosphere was saturated with N2 prior to capping to minimise the risk of oxidation. Cleanup was performed using anion mixed-mode solid-phase extraction (SPE) prior to HPLC/MS. Since the introduction of SPE in the late 1970’s, it has become a widely used preparation technique for clean-ing-up complex samples, such as biological fluids and environmental sam-ples [81-83]. The type of SPE used in Studies IV and V (Oasis® MAX, from Waters) is based on a polyvinylbenzene resin containing trialkylamine groups, a combination of functions that is particularly suitable for cleaning-up samples for resin acid analysis as the acids have both anion and lipophilic properties. The utility of enzymatic hydrolysis of the urine samples using glucuronidase/sulphatase was also evaluated in Study IV.

The beauty products examined in Study V were extracted by ultrasonica-tion in acetonitrile and anion mixed mode SPE was used in a similar proce-dure as for the urine samples. The rosin samples were diluted in acetonitrile before analysis.

Results and discussion

Chromatographic separation of AA and PA As AA and PA have the same molecular weight, chromatographic separation is crucial to separately identify and quantify them by HPLC/MS. According to my knowledge no attempts to use HPLC methods with conventional C18 phases to separate the two compounds have been successful, but this was achieved using the method developed and applied in Studies I-III and V. This was possible due to the unique selectivity of the PRISMTM column. The incorporation of a polar functionality in the alkyl chain of the stationary phase, close to the supporting particle surface, have been reported in the literature to give different selectivity than the corresponding standard-alkylated stationary phase [84, 85]. Such a polar-embedded stationary phase is illustrated in Figure 9. Decreased hydrophobicity, increased phenolic se-lectivity and an element of ion-pairing are factors that distinguish polar-embedded phases from the conventional phases [84, 86]. Various polar func-

35

tionalities are used in commercially available materials, some examples are amides, urea, carbamates and ethers [86]. The selectivity of a phase from manufacturer A can differ from a superficially identical phase from manu-facturer B, due to differences in the supporting phase, coating techniques and optional end-capping [84].

Figure 9. A schematic view of a polar-embedded phase. The spacer is usually a propyl ligand. Examples of polar groups used are amide, urea and carbamate, and the alkyl chain is commonly 8 to 18 carbons long.

The PRISMTM column has higher shape-selectivity than a conventional C18 column, and higher anion exchanging capacity than many other polar embedded phases, moderate phenolic selectivity and lower hydrophobicity [84]. The embedded urea group may interact with the resin acids by hydro-gen bonding or ionic interactions and the improved wettability could con-tribute to the high shape selectivity. The high anion capacity indicates that the PRISMTM phase is prepared using a two-stage reaction, which often re-sults in incomplete coating of the support material, in this case aminopropyl-silica [87]. However, the manufacturer does not disclose the type of reaction they use. The combination of small differences in water solubility, pKa and shape between AA and PA, with the anion capacity and shape selectivity of the PRISMTM stationary phase, leads to the observed separation. However, ionic interaction is probably the main contributor to the observed selectivity, as the formic acid concentration needs to be higher than 0.02% in a metha-nolic mobile phase to achieve separation between AA and PA [Paper V]. An interesting observation is that use of methanol and acetonitrile as mobile phases results in reversed retention orders of AA and PA, possible because free amino functions are blocked by methanol by hydrogen bonding, thus lowering the anion exchanging effect.

Resin acid ESI-MS spectra in HPLC/ESI-MS Electrospray, which was used in this thesis for ionization of resin acids, pro-duces spectra with only a few ions. They ionise in both positive and negative modes, and for the tested acids (7-OXO, DHAA and AA) the signal intensi-

36

ties are equal in both modes. However, a higher signal-to-noise ratio was obtained in negative mode analyses of the urine sample extracts, due to the high selectivity in negative mode ESI [Paper IV]. A fragment ion (m/z 173), previously described in APCI spectra of DHAA [88], was observed in the positive ESI mass spectrum of DHAA, see Figure 10. This ion is formed via a complex multistep fragmentation reaction due to the aromatic ring struc-ture of DHAA [88]. A corresponding ion in the mass spectrum of d2-DHAA at m/z 175 was also detected [Paper III]. However, the corresponding frag-ment ion from 7-OXO, observed in positive mode APCI [88], was not de-tected in ESI-MS analysis, possibly due to the lower energies involved in ESI (since collision-induced dissociation is not applied). Ions of resin acids formed by negative mode ESI are very stable and require unusually high collision energies to be fragmented in MS/MS mode [65]. Application of such high energies results in almost complete fragmentation of no informa-tive value. In positive mode MS/MS fragmentation is more easily achieved, but AA and PA yield fragments of the same m/z value, thus it does not im-prove selectivity (data not shown here or in any of the papers).

Figure 10. Positive ESI mass spectrum of DHAA.

Method performance

LOQs The LOQs of individual resin acids provided by each of the methods used in Studies I-V are listed in Table 4. The lower LOQs in Study III compared to those in Study I can be attributed to both the use of a deuterium-labelled internal standard (d2-DHAA), and higher polarity of the injected samples

37

(due to adding water to them), focusing the peaks on-column so that larger volumes could be injected. The LOD of DHAA estimated in Study IV (4 nM) is about ten-fold lower than that of the previously published GC/MS method [28] and the 7-OXO was detectable at an even lower concentration (3 nM). Both LODs have proven to be sufficiently low to measure DHAA and 7-OXO in urine following wood dust exposure [Paper IV]. Segmented injection with water was used in Study IV, a technique that has been shown to increase the efficiency of conventional columns [89]. The effect can be attributed to reduced band broadening of the sample plug before it enters the column, and formation of an in situ gradient on the column by the water plug. Reconstructed ion chromatograms of a urine sample containing 0.087 µM DHAA and 0.024 µM 7-OXO can be seen in Paper IV, Figure 1.

Table 4 Compilation of LOQs estimated in Studies I-V.

Paper Matrix Resin acid LOQ Method

I air (filter sample)

7-OXO DHAA

AA PA

16 260 156 312

ng/m3 a HPLC/pos ESI-MS

II skin (tape-strip)

7-OXO DHAA

AA PA

15 250 150 300

ng/sample HPLC/pos ESI-MS

III air (filter sample) 7-OXO DHAA

AA

1.4 17 31

ng/m3 a HPLC/pos ESI-MS

III air (filter sample) 7-OXO DHAA

AA

80 380 297

ng/m3 a GC/FID

IV urine 7-OXO DHAA

6 8

nM HPLC/neg ESI-MS

V beauty products 7-OXO DHAA

AA

39 58 22

µg/g HPLC/UV DAD

a: assuming 8 h sampling time at a flow rate of 2 L/min.

Linear range In Studies I-IV were flow rates from 0.3 to 0.35 mL/min used and at flow rates higher than a few µL/min ESI is considered a concentration dependent rather than mass flow sensitive technique. This is due to the mechanisms of ionisation, in which the droplets and analytes are not entirely consumed (sampled by the MS), instead a large portion of the spray is lost. The concen-tration of charges in the droplets affects the efficiency of desorption and thus the sensitivity. The ESI source can be regarded as a constant-current electro-chemical cell, where the current is carried by the ions. The total production of ions is fairly constant, although the type of ions produced varies with the composition of the solution [90]. In a solution in which analyte ions

38

predominate, the linear range of response will be limited by the properties of the analyte. Curvature will appear at the point where the analyte concentra-tion exceeds the limit for their production, often around 10-5 M [91]. If the solution predominantly contains ionisable interfering compounds, the sensi-tivity will be reduced, i.e. ion suppression will occur, and the linear range of response will be narrowed [90]. Due to the nature of the ionisation process in ESI-MS, curvature of response is often observed for analytes at high concen-trations. With the HPLC/ESI-MS method described in Paper III the calibra-tion curves were found to be linear within the intervals 50 ng/mL-10 µg/mL for both 7-OXO and DHAA, and 50 ng/mL to 5 µg/mL for AA. Calibration using blank urine samples spiked with known amounts of DHAA and 7-OXO in methanol in Study IV yielded linear responses in the concentra-tion range 0.025 - 0.83 µM for both 7-OXO and DHAA. The HPLC/UV method used for quantifying resin acids in beauty products and rosin samples in Study V was found give linear results in the concentration range of ap-proximately 1–400 µg/g.

Matrix effects in ESI-MS Matrix effects (which be either positive or negative) are further factors to consider when using HPLC/ESI-MS for quantitative analysis, and were thus investigated in this work. A known positive effect is the enhancement of ionization resulting from adding small amounts of modifiers, such as formic acid or ammonia to the mobile phase. The negative effect, ion suppression, was first described in 1993 [92]. Surface-active additives have the strongest ion suppression effect at a given concentration. However, any interfering substance competing for the excess charge will suppress an analyte’s ion signal if present at a sufficiently high concentration [93].

Effects on the ionization may originate from the sample matrix, solvents or the sample preparation method [94-96]. Matrix effects, such as ion sup-pression, are commonly investigated by infusing the analyte post-column, while analysing a blank sample. Another typical approach is to compare the system’s sensitivity to analytes added in known quantities to blank samples and standard solutions with pure solvent [94]. Matrix effects can often be successfully reduced by a thorough cleanup, using a technique such as solid-phase extraction (SPE) prior to MS analysis [95, 97], or by increasing the efficiency of the HPLC separation. Isotope-labelled internal standards (IS) are frequently used to compensate for ion suppression or enhancement.

Co-extracted substances in the wood dust samples were found to have only slight ion suppression effects on 7-OXO, DHAA and AA. The detected concentrations (at approximately 3-4 µg of each resin acid) differed from expectations by -0.54 to -2% [Paper III]. Although an SPE clean-up proce-dure was applied to the urine samples in Study IV, the ion suppression ef-fects were still in the range –2 to –20%. As only one deuterated IS (d2-DHAA) was used, the ion suppression effect for 7-OXO could not be com-

39

pensated for. Instead this problem was eliminated by adding a standard solu-tion to blank urine samples.

Recovery and repeatability Filter samples and standardised wood pellet dust

Extraction recoveries from filters were close to 100% for all tested acids, with low within- and between-day variances [Paper I]. The high recoveries were expected as the recovery experiment was designed to investigate the efficiency of extracting free resin acids on a filter by methanol. A recovery of nearly 100% of the total amount resin acids present in wood dust is un-likely with the method used, and actually is not desirable. An ideal extrac-tion process would extract only the resin acids that are accessible to interact with the human body. Those lodged deep in the wood matrix are probably not extractable by body fluids, and thus not relevant to the health effects of wood dust. The results from extraction experiments on standardised wood pellet dust [Paper III] showed that methanol, despite its relatively high polar-ity, might be too strong a solvent for selectively extracting the bioavailable amounts, since there was little or no difference in extraction efficiency be-tween methanol, ether and toluene (Table 5).

Table 5. Amounts of resin acids extracted from samples of standardised wood pellet dust using ethanol, ether and toluene (µg/mg dust).

N=6 Methanola Ethera Tolueneb

7-OXO 0.70±0.02 0.70±0.09 0.80±0.08 DHAA 2.66±0.09 2.6±0.3 2.82±0.18 AA 5.8±0.1 4.6±0.6 4.1±0.2

a: room temperature, b: 75°C 30 min. Tape-strip samples

A tape mounted on a liner is preferred for the tape-stripping method, since it facilitates the pre-sampling procedure. Fixomull® and Mefix® are both textile tapes mounted on paper liners, with adhesives of polyacrylate type, thus no resin acids were expected to be present in the tapes. Both tapes were, how-ever, shown to contain AA at a high level, ca. 50 ng/cm2. The variation found within and between batches of tapes indicated that AA probably mi-grated from the paper liner, despite a non-colophony coating. This finding may explain why persons who are sensibilized by rosin sometimes have an allergic reaction to tape with polyacrylate adhesive. Due to this finding, Leukosilk® without a liner was used for the tape-stripping measurements.

The recoveries of the resin acids from spiked tapes were very high (99-100% at 1 µg/tape) [Paper II], indicating that free resin acids do not react with components of the tape adhesive. As shown in Paper II, Table 1, recov-ery of DHAA was also high in the in vitro tests for all combinations of spik-ing levels (15-16 µg and 1-1.6 µg) and exposure times (2 and 30 min). At the high level, the recovery of 7-OXO was approximately 100% for both dura-

40

tions, but at the low level the recovery decreased with a longer exposure. Quantifiable amounts of these acids were found in all three tapes used to sample the same glass area on the same occasion, although the amounts de-creased with each tape-stripping, indicating that the removal efficiency of the tape might be somewhat low. In contrast, the recovery of AA was ap-proximately 100% only at the high level after a short exposure. Further, about 40-50% of the AA applied at the low concentration was recovered on the first tape and the following tapes had no detectable amounts of this resin acid. The variation in the data was similar for all the resin acids studied, and there is no straightforward reason why the tape removal efficiency would be lower for AA than the other resin acids. Most likely AA had oxidised, since the recovery decreased with increasing duration time.

The recoveries from human skin in vivo [Paper II] were highly variable under all experimental conditions, see Paper II, Table 2. At the high expo-sure concentration (13.8-17.6 µg) and both exposure durations (2 and 30 min), the recoveries of 7-OXO and DHAA were significantly higher than for AA (p<0.001). For both exposure durations at the low concentration (1.5-1.8 µg) the recovery of DHAA was significantly higher (p<0.001) than those of both 7-OXO and AA. The mean recovery of the resin acids at the low expo-sure level was significantly higher compared with that at high exposure (p<0.001), after both 2 and 30 min exposures. There was no significant dif-ference between recoveries from men and women. The lower recoveries of the acids at high levels could not be explained, but the low yields in general may be a result of several factors. For instance, the tapes might have been imprecisely applied onto the exposed area, even though they were marked on the skin. The resin acids could have formed adducts with the proteins present in the stratum corneum, and such structures cannot be detected with the method used. The substances might have diffused deeper into the skin than three consecutive tapes are able to sample, or they may have passed into hair follicles or sweat glands. It is unlikely that the resin acids would have evapo-rated from the skin during the exposure as their boiling points are higher than 300°C, see Table 2. A possible explanation for the low recovery of AA is that may have oxidised to 7-OXO during the exposure. According to sev-eral studies, AA is more prone to oxidation than the other studied acids, as also indicated by the results from the urine hydrolysis presented in Paper IV. Further factors that could have contributed to the high variability of the re-coveries are differences in the skin between individuals. The properties of the skin, such as thickness of the stratum cormeum, density of sweat glands and hair follicles, among other factors, will affect skin permeability and vary between individuals. Differences in the amount of skin that adhered to each tape could also have contributed to the large variance. The amount of skin that is sampled with each tape varies between individuals, due to variations in factors such as cohesion of the cells [98] and hydration of the skin [99].

41

One also has to keep in mind that the studied population was small, limiting the ability to identify the main factors influencing the recoveries.

Urine samples

Intramolecular rearrangements of cyclic monoterpenes, a group of com-pounds that are structurally related to resin acids, have been demonstrated after both enzymatic and acid hydrolysis [31, 100]. Thus, the recovery after enzymatic and acid hydrolysis of non-conjugated resin acids was tested in Study IV. The yield of 7-OXO was satisfactory in both cases, see Table 6. Although enzymatic hydrolysis intuitively seems to be a mild method, it resulted in low recoveries of both DHAA and AA. AA was not detected at all at 0.8 µM and at 1.6 µM only small and highly variable amounts were recovered. The losses of DHAA and AA were initially thought to be a solu-bility issue, but samples treated identically to the enzymatically hydrolyzed samples without addition of the enzymes showed no loss of resin acids. As well as leading to low recovery of AA, the enzymatic treatment yielded high artefactual levels of 7-OXO (equivalent to approximately 20% of the AA added). Acid hydrolysis of AA at a high urine concentration (0.8 µM) yielded much lower levels of 7-OXO (about twice the LOD). Partly for this reason, and partly because the predominant resin acid in air samples was DHAA, the acid hydrolysis method was chosen for the subsequent work.

Table 6. Recoveries, following acid or enzymatic hydrolysis, of resin acids added to blank urine samples, N=3.

Recovery (%) Hydrolytic

method Concentration

(µM) 7-OXO DHAA AA 0.025 78±6 99±5 nd Acid

0.8 80±6 95±5 27±15

0.8 76±8 62±10 nd Enzymatic

1.6 94±6 37±15 17±15

Beauty products

Recoveries of all acids tested at both levels (50-90 µg/g and 135-250 µg/g) were high in the experiments involving spiking a skin foundation with known amounts of resin acids, see Paper V, Tables 1 and 2. Clean-up using the Oasis® MAX mixed-mode anion exchanger SPE was very efficient, which is crucial for cosmetic products since they are usually based on greasy matrices which otherwise might interfere with the analysis.

Storage stability of resin acids No oxidation or any other decomposition of 7-OXO, DHAA or AA was observed during six months of storage in methanol solutions (1 mg/mL) at -20°C [Paper III]. Solutions of resin acids (0.8–500 µg/mL) in 9:1 acetoni-

42

trile:water with 0.2% formic acid were found to be stable at 5°C for a mini-mum of two weeks [Paper V].

The results from the storage stability tests of spiked tape-strips in Study II showed that both DHAA and AA are rapidly degraded if stored in open con-tainers (Table 7). AA oxidised to some extent to 7-OXO, which is reflected in the recovery of 7-OXO (112%). Interestingly, the recovery of DHAA was lower than of AA, indicating that the glue of the tape was involved in the degradation of DHAA, since DHAA is more stable than AA in air. In airtight containers the samples were found to be stable for several weeks at both room temperature (20°C) and in a freezer (-20°C). Similar results were ob-tained with spiked filters (data not shown here or in the papers). The amount of resin acids was quantified in relation to the total amount of dust in the samples, although the Swedish OEL (for wood dust) is for the inhalable frac-tion [7]. The sampler available for inhalable dust, the IOM-sampler with a plastic cassette insert, was found to be unsuitable for resin acids. This is because it requires a 48-h stabilization period in a controlled climate prior to weighing, due to the hygroscopic properties of the sampler insert, and the storage stability tests showed that AA and DHAA degrade under such condi-tions. In contrast, the PVC filters used in Studies I and III are non-hygroscopic and can thus be weighed after a short stabilisation period.

Table 7. Storage stability of resin acids on tape strips under indicated storage con-ditions, 1 µg applied, N=3.

Recovery (RSD) (%) Storage

duration Storage

temperature 7-OXO DHAA AA PA 72 ha 20°C 112 (3) 56 (1) 72 (1) 96 (1)

20°C 101 (3) 101 (6) 93 (3) 100 (1)

28 days -20°C 95 (2) 105 (0.7) 106 (9) 92 (3)

48 days -20°C 102 (2) 102 (6) 103 (1) 95 (2)

a: open containers

Comparison of HPLC/ESI-MS and GC/FID for analysing wood dust samples Unfortunately, an unknown substance interfered with the AA peak in the GC/FID method, in all but two samples from sample set 1, Paper III. This problem could not be solved, thus only two samples of that set could be used for comparison regarding AA. The amounts determined with GC/FID in those samples were 1.59 and 1.12 µg, and with HPLC/MS 1.44 and 1.39 µg. The dust loadings on several filters were uneven and some had loose dust. Despite this, the results obtained using the two methods correlated well for both DHAA and 7-OXO, see Figures 2 and 3 in Paper III. However, as shown in Figure 2, the 7-OXO concentrations obtained with the LC/MS method were consistently lower, on average half of those obtained with

43

GC/FID. Different extraction procedures were tested on a standardised wood pellet dust prior to HPLC/MS analysis to investigate if the differences were due to choice of extraction solvent. The only significant difference was ob-served for AA. At room temperature, the extraction was slightly more effi-cient with methanol compared with ether. Methanol was also more efficient than toluene, even if the temperature was increased to 75°C, as shown in Table1 of Paper III. When comparing those results with results obtained using the sample preparation procedure described in the British MDHS 83/2 method, large differences were noticed. A substantially lower level (22%) of AA, but more than twice the concentration of 7-OXO (265%), was obtained with MDHS 83/2. The GC peak purity was confirmed by using two columns with different selectivities (a medium to high polarity DB-225 column and a much less polar HP-5 column). The high 7-OXO content recorded using GC/FID methodology is most likely due to oxidation of AA during sample preparation. This effect decreased by 43% when dust residues were filtered from the extracts before derivatisation. A second sample set was collected, the filters were divided and the HPLC/MS method was compared with the modified MDHS 83/2 method (including filtration prior to derivatisation). The level of DHAA determined in all samples of filter sample set 2 by these two methods was consistent. Only two of the samples in this second sample set had detectable amounts of all investigated resin acids. In those two cases the amounts of 7-OXO recorded with the modified GC/FID method were still much higher than those recorded with the LC/MS method, and the higher 7-OXO amount was accompanied with an approximately equivalent decrease of AA. Oxidation of AA is suggested to be promoted by the combined presence of N,N-dimethylformamide dimethyl acetal (methylating reagent) and unknown soluble substance(s) co-extracted from the wood dust, as no oxidation was observed in the absence of dust (data not shown either here or in the papers), or in the toluene extraction of dust without methylat-ing reagent. This hypothesis is supported by the finding that the two methods gave similar results for samples 3 and 4 of filter sample set 2, which had significantly less dust on the filters, and hence less extractable substances.

Exposure assessments

Air levels – inhalation exposure Air concentrations and personal exposure to resin acids at the four wood pellet production plants were measured in Study I. The personal exposure to resin acids (sum of 7-OXO and DHAA) during the working operations (shift, bagging and daytime) varied between <0.33 and 25 µg/m3, see Table 8. The highest levels and arithmetic mean values for area measurements were recorded at the pellet press (Table 9).

44

Table 8. Personal exposures of dust and resin acids during three work operations.

Personal exposure Work

operations N Resin acidsa

(µg/m3) Dustb

(mg/m3)

Shift 40 <0.33-25 <0.10-5.7

Daytime 20 <0.33-10 0.13-3.5

Bagging 8 <0.35-8.5 0.20-3.4

Total 68 <0.33-25 <0.10-5.7

a, sum of DHAA and 7-OXO; b, total dust.

Table 9. Area measurements of dust and resin acids at six sites.

Area measurements

Site N Resin acidsa (µg/m3) Dustb

(mg/m3)

Control room 21 <0.28-3.2 <0.1-<0.14

Pellet press 18 <0.29-59 <0.10-28

Bagging 12 <0.28-1.1 <0.10-1.1

Storage 12 <0.28-12 <0.10-6.5

Briquette machine 4 <0.71-1.7 0.10-0.70

Kiln 4 0.40-3.4 <0.10

Total 71 <0.28-59 <0.10-28

a, sum of DHAA and 7-OXO; b, total dust.

Most of the monitored 7-OXO and DHAA levels were above the LOQ (personal monitoring, 88% and 66%, respectively; area measurements, 72% and 54%, respectively). In contrast, most recorded AA and PA levels were below the LOQ, both for personal monitoring (85% and 82%, respectively) and area measurements (87% and 88%, respectively). However, when per-sonal exposure measurements for AA and PA were quantified, levels were found to be high (0.55–5.3 and 1.4–37 µg/m3, respectively) compared with those of 7-OXO and DHAA (data not shown either here or in the papers). Both AA and PA have been previously shown to be emitted during wood processing. AA and PA at levels of 7.2 µg/m3and 0.6 µg/m3 (AM), respec-tively, have been measured in Canadian sawmills [101] and AA levels of 0.3 to 2.4 µg/m3 at a plywood mill in New Zealand [102]. However, exposure to 7-OXO and DHAA has not been previously shown at wood processing and handling plants. The measured personal exposures to resin acids were all less than half of the British OEL for rosin (50 µg/m3, [103]), but the measured air levels exceeded the British OEL in some cases, suggesting that exposures to these substances are sometimes high. Since allergenic reactions have been

45

shown to be caused by oxidised species [48, 54-57] the presence of 7-OXO is of health concern. Another source of 7-OXO in wood dust could be mi-crobial degradation of abietiene resin acids, an oxidation pathway that might not proceed via the strongly allergenic hydroperoxides as intermediates [44]. This microbial oxidation could potentially be promoted in sites where wood is stored outdoors.

The Pearson correlation coefficient between levels of resin acids and total dust was 0.76 (p<0.001) for personal exposure and 0.71 (p<0.001) for area measurements. The correlation (r) between the resin acids and total dust measurements was fairly strong. However, 42% (personal exposure) and 50% (area measurements) of the variation in resin acid levels (1–r2) cannot be explained by the variation in total dust levels. This may be partly due to the differences in resin acid composition between wood of different species and growth sites [45, 46], since the production plants report that approxi-mately half of the raw material they use annually originates from spruce, and the other half from pine trees. However, at the time of the measurements they could not specify the proportions of spruce and pine that were proc-essed. Since the correlations varied substantially between the different types of sites, see Figure 11, it is also possible that some of the dust measured was not from wood. The pellet presses are located in a closed area, where a high proportion of the dust probably originates from wood, whereas the bagging operations and storage areas are in the open where trucks drive in and out. In the latter areas, sources of dust may include clay, metal, gravel, plastic from bags, and diesel exhaust. The proportion of resin acids has been used in pre-vious studies to estimate the relative amount of wood in samples collected in a soft lumber mill in British Columbia [101].

46

(a)

(b)

(c)

Figure 11. Pearson correlation plots of total dust and resin acids (sum of 7-OXO and DHAA), from area measurements at a: pellet press (r=0.90,p<0.01), b: storage (r=0.60,p<0.05) , c: bagging ( r=0.16, p>0.5) areas.

47

Dermal exposure Dermal exposure to resin acids was assessed by tape-stripping in Study II. The oxidised resin acid 7-OXO was quantified in 76 pre-shift and 278 post-shift samples, ranges: <7.5-140 ng/tape (pre-shift) and <7.5-840 ng/tape (post-shift), see Table 10. The two areas with the highest amounts were the right lower arm and hand. As can be seen in Table 10, DHAA was quantified in fewer samples than 7-OXO (57 pre-shift and 131 post-shift), but in those cases the amounts were generally higher. The highest amount was detected on a tape from the right lower arm. AA was quantified in only one pre-shift sample and 46 post-shift samples and PA at in even fewer samples (one pre-shift and eight post-shift). The detected amounts generally decreased with each consecutive tape sampled on the same site, but occasionally the amount on the third tape was higher than the amount on the second tape. These anomalies could have been due to a resin acid concentration gradient in the skin. They might also have been due to misplacement of the third tape, even though the sampling area was carefully marked, thus sampling a new skin area. It is also possible that each tape collected a different amount of stratum

corneum. An attempt was made to quantify the amount of skin on each tape using the method described by Chao and Nylander-French [22]. Unfortu-nately, however, the samples were damaged and no results were obtained. Retrospective estimation of the skin amounts would have been difficult, since the amount of skin sampled depends on individual factors, as discussed above.

Table 10. Results of the assessment of dermal exposure to 7-OXO and DHAA.

7-OXO DHAA AA PA

Side of the bodya N

N >

LOQ Range

(ng/tape)

N >

LOQ Range

(ng/tape)

N >

LOQ Range

(ng/tape)

N >

LOQ Range

(ng/tape)

L 183 26 <7.5-140 10 <125-2800

0 - 1 3550 Fore-head

R 186 76 <7.5-520 39 <125-2800

19 <150-930

4 <300-2500

L 183 7 <7.5-140 14 <125-2600

1 321 0 - Front

of neck R 186 63 <7.5-390 29 <125-

3700 11 <150-

590 0 -

L 186 12 <7.5-62 15 <125-3100

0 - 0 - Lower

arm R 185 59 <7.5-840 29 <125-

9700 7 <150-

810 1 390

L 183 31 <7.5-100 18 <125-2400

0 - 0 - Hand

R 180 80 <7.5-700 34 <125-3500

9 <150-1740 3 1330

a: Left side (L) sampled pre-shift and right side (R) sampled post-shift.

48

Assuming that deposition on the skin was equal on the left and right sides of the workers’ bodies, the results indicate that exposure to 7-OXO and DHAA increased over shift, on all skin areas, on the first sampling occasion (Paper II, Tables 5 and 6). On the second sampling occasion, an increase over shift of 7-OXO was observed on the neck and forehead, but only 20 workers were examined, lowering the statistical power of the test used. Some reductions in amounts of the analytes at some sampling sites were also ob-served over shift for some individuals, including 7-OXO on the hand, and DHAA on the front of the neck, the front of the lower arm and hand. These reductions could have been due to washing of the skin areas, for instance before meals, but errors may also have been introduced by non-uniform deposition on the skin. Another possibility is that resin acids in pre-shift samples included residues from previous work-shifts, or pre-shift contamina-tion from work clothes, which since some of the workers put on and entered the production area prior to pre-shift sampling.

Biological monitoring In Study IV, DHAA and 7-OXO were quantified in 38 and 10 of the pre-shift urine samples and in 37 and 19 of the post-shift samples, respectively. The median urinary DHAA post-shift levels were highest at plant A, fol-lowed by B and D, and lowest at plant C, see Paper IV, Table 1. The differ-ences in this respect were significant between Plant A and both plants C (p=1·10-5) and D (p=0.02), and between plants B and C (p=0.02). The DHAA concentrations measured in Study IV are in the same range as con-centrations measured in the British soldering industry (147 values, 90th per-centile 0.087 µM, max value 0.478 µM, personal communication, Kate Jones, Health and Safety Laboratories , UK), suggesting that the resin acid body burden for workers in the Swedish wood pellet industry is also of the same magnitude.

The median measured plant urine DHAA concentration reflected the me-dian plant air DHAA concentration (r=0.93, p=0.068), and a significant cor-relation (r=0.50, p=2.9·10-4) between post-shift urinary DHAA concentra-tions and corresponding DHAA air concentrations was observed, see Figure 3 in Paper IV. The positive linear relationship found between post-shift uri-nary DHAA and air DHAA is consistent with the results of the study on soldering workers by the HSL in the UK [28], but the degree of explanation of the variation for the correlation presented in Paper IV is low. In the HSL study, 43% (r2) of the variation in urine DHAA concentration was explained by the variation in the air concentration, whereas the corresponding degree of explanation in Study IV was just 25% (r2). The differences are suggested to be due to properties of the exposure source, such as particle size and com-position. At the sites investigated in Study IV, a larger proportion of dust was probably trapped in the mucus of the workers’ upper airways and subse-quently swallowed, resulting in secondary exposure via the gastro-intestinal

49

tract. The pellet workers’ exposure is also highly variable during the day, as well as from day to day. Without knowledge of the uptake rates and toxi-cokinetics of DHAA, it is not possible to draw any conclusions about whether the measured post-shift urinary concentrations directly reflect exposure from the preceding shift. Some could be residues from earlier ex-posures.

A trend towards an increase in over shift for the urinary concentration of 7-OXO was observed (p=0.063), see Figure 2 in Paper IV, and in samples where 7-OXO was quantified the concentration correlated to the measured DHAA concentration (r=0.88, p=8·10-6). The increase in 7-OXO concentra-tions could have been due to uptake via either the lungs or the gastro-intestinal tract from swallowed particulates. Another possibility is that some of the increase in 7-OXO levels was due to metabolism of other resin acids to 7-OXO. In accordance with this hypothesis, small amounts of 7-OXO as a metabolite in urine have been observed in rabbits after oral ingestion of DHAA [64], and degradation of abietane resin acids via 7-OXO has been observed in microorganisms as described above [44]. However, further stud-ies are needed to draw any robust conclusions regarding the origin of 7-OXO in workers exposed to wood dust.

Resin acid levels in beauty products and gum rosin The results from Study V indicate that the investigated gum rosin had not oxidised, since no 7-OXO was detected. The determined concentrations of AA and DHAA were 26% (by weight) and 6%, respectively, in unmodified gum rosin from Soccer, and 69 and 4%, respectively, in unmodified gum rosin from Fluka. Rosin modified with maleic anhydride showed levels of free AA and DHAA at 7 and 2%, respectively.

Of the beauty products tested in Study V, only the body depilatory wax strips showed quantifiable amounts of resin acids. The concentrations were very high (2.3, 2.5 and 0.028 mg/g for AA, DHAA, and 7-OXO, respec-tively). The relative concentrations of AA and 7-OXO indicate that at least 1% of the AA had oxidized, thus rather high levels of strong allergens could be present in the wax. The oxidation continues during ageing if the product is not stored in air-free conditions, which is not generally the case for beauty products. The product information on the wax strips declared it contained triethylene glycol and glyceryl-modified rosin. Some of the rosin was clearly left unmodified, since free AA, DHAA, and 7-OXO were detected. Com-mercial modifications of rosin are usually interrupted when the desired tech-nical properties are obtained, leaving some rosin unmodified. Thus, contact allergy may also be caused by products claimed to contain only modified rosin.

According to the EU legislation on dangerous substances (Directive 67/548 /EEC, Annex 1), any content of rosin exceeding 1% in a product

50

must be declared and the product must be labelled with risk phrase R 43 (“May cause sensitization by skin contact”). However, rosin is a mixture of many compounds, mainly a large number of resin acids, and its composition varies due to differences in its origin and extraction, handling, storage, and manufacture. The content of DHAA may be 5–10% in gum rosin and around 30% in tall oil rosin. The concentrations of AA in gum rosin and tall oil rosin are normally 30 - 50% and 35 - 40%, respectively. Use of only AA as a tracer for rosin is not suitable, since low levels of AA may be present due to oxidation, resulting in the formation of highly allergenic compounds, such as 15-hydroperoxyabietic acid. Thus, the stable 7-OXO and DHAA should also be measured as additional markers. Although rosin is a common cause of contact allergy, its occurrence in commercial products has not been well studied. The method developed in Study V uses instrumentation available in many laboratories, and provides an easy-to-use method for selective detec-tion and quantification of resin acids in products. However, if LC/MS equipment is available the method is transferrable to such instrumentation.

Conclusions The presented analytical methodologies have been used to successfully de-tect and quantify levels of resin acids in air (wood dust), skin samples (tape-strips), urine and beauty products. AA and PA can be separated due to the unique selectivity of the stationary phase of the PRISMTM column, which is suggested to be mainly due to ionic interaction, as described above. There are probably also other polar-embedded phases exhibiting similar retention mechanisms on the market, but this was not tested in this work.

The measured air concentrations of resin acids in the Swedish wood pellet industry [Paper I] were generally less than half of the British OEL for rosin, but some very high air concentrations exceeding the OEL were detected. For protective purposes, it is thus important to continue the measurements in pellet production sites.

The differences in amounts of AA and 7-OXO in wood dust samples, de-termined by HPLC/ESI-MS compared with MDHS 83/2 (GC/FID), as ob-served in Study III, are mostly due to oxidation of AA to 7-OXO, most likely promoted by the combined presence of N,N-dimethylformamide di-methyl acetal in the methylating reagent and unknown substance(s) co-extracted from the wood dust. These interfering phenomena and the higher detectability of the LC/MS method suggest that this method is more suitable for workplace measurements of resin acids when wood dust is present.

To my knowledge, this is the first time uptake of resin acids in humans with wood dust as exposure source is shown. More investigations and statis-tics are required to be able to draw any conclusions about the suitability of DHAA as a single urinary biomarker for resin acid exposure, although a

51

positive correlation between urinary and air concentrations was observed in this work. An interesting observation is the tendency for urinary 7-OXO levels to increase during a shift, which could be due to the metabolism of abietane resin acids. However, this possibility also requires further investiga-tion.

Abietic acid was detected in medical tape with adhesives of polyacrylate type. The AA probably originates from the paper liner, since rosin is a com-mon constituent of paper. Individuals sensitive to rosin should therefore avoid use of such medical tapes. The same advice should be given regarding depilatory wax. Sensibilisation is common due to repeated skin contact with rosin-containing products, such as papers, cosmetics, adhesives etc. It is therefore important to reduce the occurrence of such products on the market, but to date few measurements of their contents have been performed. In this thesis, convenient methodologies are presented, which could be used for screening commercial products.

Outlook As mentioned above, results of the studies this thesis is based upon raised the possibility that 7-OXO may be formed in humans by metabolism from DHAA. It would be highly interesting to test this hypothesis, especially since a sensitive and selective analytical method is available. We also have access to metabolites of DHAA from rabbits, presented as kind gifts by Takashi Matsumoto and colleagues. The detection and quantification of those me-tabolites in urine from exposed workers could give insights into the meta-bolic pathways of resin acids in humans, and help efforts to develop a suit-able biomonitoring strategy for resin acid exposure.

52

Tack!

Det är många som har bidragit till de arbeten som presenterats i denna avhandling. Jag skulle vilja tacka dem alla, men särskilt: Min handledare Ulrika Nilsson, ingen fråga har någonsin varit för knasig för att ställa till dig och periodvis har de varit många. Vi har haft ett givande samarbete som jag hoppas får en fortsättning i framtiden. Mina biträdande handledare Håkan Westberg och Kåre Eriksson. Håkan för att du inte gav upp utan till slut lyckades övertala en mig om att forskarut-bildning var en bra idé och Kåre för de många intressanta diskussioner vi haft under arbetets gång. Forskningsrådet för Arbetsliv och Socialvetenskap för det forskningsbidrag som har finansierat delar av denna avhandling. Arbets- och miljömedicinska klinikerna i Örebro och Umeå, och avdelnin-gen för analytisk kemi, Stockholms Universitet för att ni bidragit till finan-sieringen av denna avhandling. Pelletsprojektgruppen; Katja Hagström, Eva Andersson och Kåre Eriksson, idéerna bara sprutar när vi träffas. Ofta har ett projektmöte som skulle sam-manfatta vad vi gjort slutat med en lång lista med nya idéer på vad vi skulle kunna göra med det material som ännu inte bearbetats. Alla de som var ute i fält, analyserat prover och kommit med idéer; Helena Arvidson, Krister Berg, Carin Norberg, Mona Svensson, Sofia Loodh, Sib-ylla Robertsson, Lisbet Viklund, Jessica Westerlund och Britt-Marie Isaks-son. Ing-Liss Bryngelsson för hjälp med statistiken. Ann-Therese Karlberg för värdefull hjälp med kolofoniums hälsoeffekter, förekomst och nomenklatur. Pelletsfabrikerna och alla anställda för att ni deltog i studierna.

53

Alla medarbetare på arbets- och miljömedicinska kliniken i Örebro. För nästan precis tolv år sedan började jag på kliniken, ibland känns det som om det var ifjol – tiden går fort när man har roligt. Alla på avdelningen för analytisk kemi, Stockholms Universitet för att ni hjälpt mig med stort och smått de gånger jag varit hos er. Och sist men inte minst, Mikael – tack för att du alltid finns där för mig. Tillsammans klarar vi allt.

54

References

1 Svensson Å. (2001) Omfattningen av allergi och annan överkänslighet i huden - symtom, omfattning, orsaker. Vetenskaplig kunskapssamman-ställning. Folkhälsoinstitutet, in Swedish.

2 Sadhra S, Foulds I.S, Gray C.N, Koh D. and Gardiner K. (1994) Colophony - uses, health effects, airborne measurement and analysis. Ann Occup Hyg 38, 385-396.

3 Färm G. (1997) Contact Allergy to Colophony: Clinical and Experimental Studies with Emphasis on Clinical Relevance ,Thesis, Karolinska institutet, Stockholm, Sweden.

4 Hagström K. (2008) Occupational exposure during production of wood pel-lets in Sweden, Thesis, Örebro University, Örebro, Sweden.

5 www.pelletsindustrin.org, downloaded on jan 12th 2012, in Swedish. 6 Berglund M, Elinder C.-G. and Järup L. (2001) Definitions and key consid-

erations. In Human Exposure Assessment: An introduction WHO/SDE/OEH/01.3. World Health Organisation. pp 15-34.

7 Arbetsmiljöverket (2005) AFS 2005:17 - Hygieniska gränsvärden och åt-gärder mot luftföroreningar, in Swedish.

8 Lee T, Harper M, Slaven J.E, Lee K, Rando R.J. and Maples E.H. (2011) Wood dust sampling: field evaluation of personal samplers when large parti-cles are present. Ann Occup Hyg 55, 180-191.

9 Berglund M, Elinder C.-G. and Järup L. (2001) Methods of assessing expo-sure and dose. In Human Exposure Assessment: An introduction WHO/SDE/OEH/01.3. World Health Organisation. pp 65-100.

10 www.fass.se. Downloaded on feb 16th 2012, in Swedish. 11 Stickel F, Buch S, Lau K, zu Schwabedissen H.M, Berg T, Ridinger M, Riet-

schel M, Schafmayer C, Braun F, Hinrichsen H, Günther R, Arlt A, Seeger M, Müller S, Seitz H.K, Soyka M, Lerch M, Lammert F, Sarrazin C, Kubitz R, Häussinger D, Hellerbrand C, Bröring D, Schreiber S, Kiefer F, Spanagel R, Mann K, Datz C, Krawczak M, Wodarz N, Völzke H. and Hampe J. (2011) Genetic variation in the PNPLA3 gene is associated with alcoholic liver injury in Caucasians. Hepatology 53, 86-95.

12 Jenkins H.L. and Tresise J.A. (1969) An adhesive-tape stripping technique for epidermal histology. J Soc Cosmet Chem 20, 451-466.

13 Mattorano D.A, Kupper L.L. and Nylander-French L.A. (2004) Estimating dermal exposure to jet fuel (naphthalene) using adhesive tape strip samples. Ann Occup Hyg 48, 139-146.

14 Nylander-French L.A. (2000) A tape-stripping method for measuring dermal exposure to multifunctional acrylates. Ann Occup Hyg 44, 645-651.

15 Surakka J, Johnsson S, Rosen G, Lindh T. and Fischer T. (1999) A method for measuring dermal exposure to multifunctional acrylates. J Environ Monit 1, 533-540.

55

16 Kammer R, Tinnerberg H. and Eriksson K. (2011) Evaluation of a tape-stripping technique for measuring dermal exposure to pyrene and ben-zo(a)pyrene. J Environ Monit 13, 2165-2171.

17 Breternitz M, Flach M, Präßler J, Elsner P. and Fluhr J.W. (2007) Acute bar-rier disruption by adhesive tapes is influenced by pressure, time and ana-tomical location: integrity and cohesion assessed by sequential tape strip-ping; a randomized, controlled study. Br J Dermatol 156, 231-240.

18 Lademann J, Jacobi U, Surber C, Weigmann H.-J. and Fluhr J.W. (2009) The tape stripping procedure - evaluation of some critical parameters. Eur J Pharmaceut Biopharm 72, 317-323.

19 Kobayashi H, Aiba S, Yoshino Y. and Tagami H. (2003) Acute cutaneuos barrier disruption activates epidermal p44/42 and p38 mitogen-activated pro-tein kinases in human and hairless guinea pig skin. Exp Dermatol 12, 734-746.

20 Bommannan D, Potts R.O. and Guy R.H. (1990) Examination of strateum corneum barrier function in vivo by infrared spectroscopy. J Invest Dermatol 95, 403-408.

21 Lindemann U, Wilken K, Weighmann H.J, Schaefer H, Sterry W. and Lade-mann J. (2003) Quantification of the horny layer using tape stripping and microscopic techniques. J Biomed Opt 8, 601-607.

22 Chao Y.C. and Nylander-French L.A. (2004) Determination of keratine pro-tein in a tape-stripped skin sample from jet fuel exposed skin. Ann Occup Hyg 48, 65-73.

23 Dreher F, Arens A, Hostynek J.J, Mudumba S, Ademola J. and Maibach H.I. (1998) Colorimetric method for quantifying human Stratum corneum re-moved by adhesive tape-stripping. Acta Derm Venereol (Stockh) 78, 186-189.

24 Weigert F. and Mottram J.C. (1946) The Biochemistry of Benzpyrene. II. The Course of Its Metabolism and the Chemical Nature of the Metabolites. Cancer Res 6, 109-120.

25 Harper K.H. and Calcutt G. (1959) Glucuronide and Sulphate Conjugation in vivo and in vitro. Nature 183, 419-438.

26 Ohno S. and Nakajin S. (2009) Determination of mRNA expression of hu-man UDP-glucuronosyltransferases and application for localization in vari-ous human tissues by Real-Time Reverse Transcriptase-Polymerase Chain Reaction. Drug Metab Dispos 37, 32-40.

27 Riches Z, Stanley E.L, Bloomer J.C. and Coughtrie W.H. (2009) Quantita-tive evaluation of the expression and activity of five major sulfotransferases (SULTs) in human tissues: The SULT "pie". Drug Metab Dispos 37, 2255-2261.

28 Baldwin P.E, Cain J.R, Fletcher R, Jones K. and Warren N. (2007) Dehy-droabietic acid as a biomarker for exposure to colophony. Occup Med (Oxf) 57, 362-366.

29 Kawai T, Miyama Y, Horiguchi S, Sakamoto K, Zhang Z.-W, Higashikawa K. and Ikeda M. (2000) Possible metabolic interaction between hexane and other solvents co-exposed at sub-occupational exposure limit levels. Int Arch Occup Environ Health 73, 449-456.

30 Linhart I, Weidenhoffer Z, Mraz J, Smejkal J. and Mladkova I. (1997) The evidence for conjugated mandelic and phenylglyoxylic acids in the urine of rats dosed with styrene. Toxicol Lett 90, 199-205.

56

31 Eriksson K. and Levin J.-O. (1990) Identification of cis- and trans-verbenol in human urine after occupational exposure to terpenes. Int Arch Occup En-viron Health 62, 379-383.

32 Moon J.Y, Ha Y.W, Moon M.H, Chung B.C. and Choi M.H. (2010) System-atic error in gas chromatography-mass spectrometry–based quantification of hydrolyzed urinary steroids. Cancer Epidemiol Biomarkers Prev 19, 388-397.

33 Sinadinovic J.R. and Velle W. (1968) Hydrolysis of estrogen conjugates in bovine urine. Formation of estratetraenol. Acta Endocrinol (Copenh) 58, 243-250.

34 Carrieri M, Trevisan A. and Bartolucci G.B. (2001) Adjustment to concen-tration-dilution of spot urine samples: correlation between specific gravity and creatinine. Int Arch Occup Environ Health 74, 63-67.

35 James G.D, Sealer J.E, Alderman M, Ljungman S, Mueller F.B, Pecker M.S. and Laragh J.H. (1988) A longitudinal study of urinary creatinine and creati-nine clearance in normal subjects: race, sex and age differences. Am J Hyper-tens 1, 124-131.

36 Miller R.C, Brindle E, Holman D.J, Shofer J, Klein N.A, Soules M.R. and O'Connor K.A. (2004) Comparison of specific gravity and creatinine for normalizing urinary reproductive hormone concentrations. Clin Chem 50, 924-932.

37 Boeninger M.F, Lowry L.K. and Rosenburg J. (1993) Interpretation of urine results used to assess chemical exposure with emphasis on creatinine adjust-ments: a review. Am Ind Hyg Assoc J 54, 615-627.

38 Needleman S.B, Porvaznik M. and Ander D. (1992) Creatinine analysis in single collection urine specimens. J Forensic Sci 37, 1125-1133.

39 Dai X, Kutschke K. and Priest N. (2011) Rapid urinary output normalization method using specific gravity. Health Phys 101, 140-143.

40 Vij H.S. and Howell S. (1998) Improving the specific gravity adjustment method for assessing urinary concentrations of toxic substances. Am Ind Hyg Assoc J 59, 375-378.

41 Jatlov P, McKee S. and O´Malley S.S. (2003) Correction of urine cotinine concentrations for creatinine excretions: is it useful? Clin Chem 49, 1932-1934.

42 Sorahan T, Pang D, Esmen N. and Sadhra S. (2008) Urinary concentrations of toxic substances: An assessment of alternative approaches to adjusting for specific gravity. J Occup Environ Hyg 5, 721-723.

43 Sipponen A. and Laitinen K. (2011) Antimicrobial properties of natural co-niferous rosin in the European Pharmacopoeia challenge test. APMIS 119, 720-724.

44 Martin V.J.J, Yu Z. and Mohn W.W. (1999) Recent advances in understand-ing resin acid biodegradation: microbial diversity and metabolism. Arch Mi-crobiol 172, 131-138.

45 Fengel D. and Wegener G. (1983) Extractives. Wood. Chemistry, Ultrastruc-ture, Reactions. Walter de Gruyter & Co, Berlin. pp 182.

46 Fries A, Ericsson T. and Gref R. (2000) High heritability of wood extractives in Pinus sylvestris progeny tests. Can J Forest Res 30, 1707-1713.

47 Peng G. and Roberts J.C. (2000) Solubility and toxicity of resin acids. Water Res 34, 2779-2785.

48 Hausen B.M, Börries M, Budianto E. and Krohn K. (1993) Contact allergy due to colophony. (IX). Sensitization studies with further products isolated

57

after oxidative degradation of resin acids and colophony. Contact Dermatitis 29, 234-240.

49 Karlberg A.-T. (1988) Contact allergy to colophony: Chemical identification of allergens, sensizatation experiments and clinical experiences. Acta Derm Venereol (Stockh) 139, 1-43.

50 Karlberg A.-T. (1991) Air oxidation increases the allergenic potential of tall-oil rosin. Colophony contact allergens also identified in tall-oil rosin. Am J Contact Dermatitis 2, 43-49.

51 Karlberg A.-T, Bohlinder K, Boman A, Hacksell U, Hermansson J, Jacob-sson S. and Nilsson J.L.G. (1988) Identification of 15-hydroperoxyabietic acid as a contact allergen in Portuguese colophony. J Pharm Pharmacol 40, 42-47.

52 Färm G, Lidén C, Karlberg A.-T. (1994) A clinical and patch test study in a tall-oil rosin factory. Contact Dermatitis. 31:102-107.

53 Mathias C. G, Adams R. M. (1984) Allergic contact dermatitis from rosin used as soldering flux. J Am Acad Dermatol. 10:454-456.

54 Estlander T, Jolanki R, Alanko K. and Kanerva L. (2001) Occupational al-lergic contact dermatitis caused by wood dusts. Contact Dermatitis 44, 213-217.

55 Burge P.S, Harries M.G, O'Brien I. and Pepys J. (1980) Bronchial provoca-tion studies in workers exposed to the fumes of electronic soldering fluxes. Clin Allergy 10, 137-149.

56 Karlberg A.-T, Boman A, Hacksell U, Jacobsson S, Labs J. and Nilsson G. (1988) Contact allergy to dehydroabietic acid derivatives isolated from Por-tuguese colophony. Contact Dermatitis 19, 166-174.

57 Keira T, Aizawa Y, Karube H, Niituya M, Shinohara S, Kuwashima A, Ha-rada H. and Takata T. (1997) Adverse effects of colophony. Ind Health 35, 1-7.

58 Bråred Christensson J, Matura M, Bäcktorp C, Börje A, Nilsson J.L.G. and Karlberg A.-T. (2006) Hydroperoxides form specific antigens in contact al-lergy. Contact Dermatitis 55, 230-237.

59 Karlberg A.-T. and Gäfvert E. (1996) Isolated colophony allergens as screen-ing substances for contact allergy. Contact Dermatitis 35, 201-207.

60 Sadhra S, Gray C.N. and Foulds I.S. (1997) High-performance liquid chro-matography of unmodified rosin and its application in contact dermatology. J Chromatogr B: 700, 101-110.

61 Jones K, Garfitt S.J, Calverley A, Channa K. and Cocker J. (2001) Identifica-tion of a possible biomarker for colophony exposure. Occup Med 51, 507-509.

62 Oikari A, Anäs E, Kruzynski G. and Holmbom B. (1984) Free and conju-gated resin acids in the bile of rainbow trout, Salmo gairdneri. Bull Environ Contam Toxicol 33, 233-240.

63 Asakawa Y, Ishida T, Toyota M. and Takemoto T. (1986) Terpenoid bio-transformation in mammals IV. Biotransformation of (+)-longifolene, (-)-caryophyllene, (-)-caryophyllene oxide, (-)-cyclocolorenone, (+)-nootkatone, (-)-elemol, (-)abietic acid and (+)-dehydroabietic acid in rabbits. Xenobiotica 16, 753-767.

64 Matsumoto T, Hayashi N, Ishida T. and Asakawa Y. (1990) Metabolites of (+)-dehydroabietic acid in rabbits. J Pharm Sci 79, 540-547.

65 McMartin D.W, Peru K.M, Headley J.V, Winkler M. and Gillies J.A. (2002) Evaluation of liquid chromatography-negative ion electrospray mass spec-

58

trometry for the determination of selected resin acids in river water. J Chro-matogr A 952, 289-293.

66 Lee B.L, Ong H.Y, Koh D. and Ong C.N. (1994) High-performance liquid chromatographic method for determination of dehydroabietic and abietic ac-ids, the skin sensitizers in bindi adhesive. J Chromatogr A 685, 263-269.

67 Hrobonová K, Lehotay J, Skacáni I. and Cizmárik J. (2005) HPLC determi-nation and MS identification of dehydroabietic acid and abietic acid in pro-polis. J Liq Chromatogr Rel Technol 28, 1725-1735.

68 Lee B.L, Koh D, Ong H.Y. and Ong C.N. (1997) High-performance liquid chromatographic determination of dehydraoabietic and abietic acids in tradi-tional Chinese medications. J Chromatogr A 763, 221-226.

69 Ehrin E. and Karlberg A.T. (1990) Detection of rosin (colophony) compo-nents in technical products using an HPLC technique. Contact Dermatitis 23, 359-366.

70 Holmbom B, Avela E. and Pekkala S. (1974) Capillary gas chromatography-mass spectrometry of resin acids in tall oil rosin. J Am Oil Chem Soc 51, 397-400.

71 McGuire J.M. and Powis P.J. (1998) Gas chromatographic analysis of tall oil fractionation products after methylation with N,N-dimethylformamide di-methylacetal. J Chromatogr Sci 36, 104-108.

72 Latorre A, Rigol A, Lacorte S. and Barceló D. (2003) Comparison of gas chromatography-mass spectrometry and liquid chromatography-mass spec-trometry for the determination of fatty and resin acids in paper mill process waters. J Chromatogr A 991, 205-215.

73 Meriläinen P.S, Krasnov A. and Oikari A. (2007) Time- and concentration-dependent metabolic and genomic responses to exposure to resin acids in brown trout (Salmo trutta m. lacustris). Environ Toxicol Chem 26, 1827-1835.

74 Örså F. and Holmbom B. (1994) A convenient method for the determination of wood extractives in papermaking process waters and effluents. J Pulp Pa-per Sci 20, 361-365.

75 Dethlefs F. and Stan H.-J. (1996) Determination of resin acids in pulp mill EOP bleaching process effluent. Fresenius J Anal Chem 356, 403-410.

76 Smith P.A, Gardner D.R, Drown D.B, Jederberg W.W. and Still K. (1998) Oxidized resin acids in aerosol derived from rosin core solder. Am Ind Hyg Assoc J 59, 889-894.

77 Smith P.A, Son P.S, Callaghan P.M, Jederberg W.W, Kuhlmann K. and Still K.R. (1996) Sampling and analysis of airborne resin acids and solvent-soluble material derived from heated colophony (rosin) flux: a method to quantify exposure to sensitizing compounds liberated during electronics sol-dering. Toxicology 111, 225-238.

78 Smith P.A, Gardner D.R, Drown D.B, Jederberg W.W. and Still K. (1997) detection of resin acid compounds in airborne particulate generated from ros-in used as a soldering flux. Am Ind Hyg Assoc J 58, 868-875.

79 Pengelly M.I, Groves J.A, Foster R.D, Ellwood P.A. and Wagg R.M. (1994) Development of a method for measuring exposure to resin acids in solder fume. Ann Occup Hyg 38, 765-776.

80 Bergauff M, Ward T, Noonan C. and Palmer C.P. (2008) Determination and evaluation of selected organic chemical tracers for wood smoke in airborne particulate matter. Int J Environ Anal Chem 88, 473-486.

81 Humbert L. (2010) Extraction en phase solide (SPE): théorie et applications. Annales de Toxicologie Analytique 22, 61-68. in French.

59

82 Neue U.D, Mallet C.R, Lu Z, Cheng Y.-F. and Mazzeo J.R. (2003) Tech-niques for sample preparation using solid-phase extraction. In Handbook of Analytical Separations. Edited by Wilson I.D. Elsevier Science. pp 73-90.

83 Zief M. and Kakodar S.V. (1994) Solid phase extraction for sample prepara-tion. In Progress in Pharmaceutical and biomedical analysis. pp 127-143.

84 Euerby M.R. and Petersson P. (2005) Chromatographic classification and comparison of commercially available reversed-phase liquid chroma-tographic columns containing polar embedded groups/amino endcappings using principal component analysis. J Chromatogr A 1088, 1-15.

85 O'Gara J.E, Walsh D.P, Phoebe Jr C.H, Alden B.A, Bouvier E.S.P, Iraneta P.C, Capparella M. and Water T.H. (2001) Embedded-polar-group bonded phases for High Performance Liquid Chromatography. LC GC 19, 632-642.

86 O'Sullivan G.P, Scully N.M. and Glennon J.D. (2010) Polar-embedded and polar-endcapped stationary phases for LC. Anal Lett 43, 1609-1629.

87 Czajkowska T, Hrabovsky I, Buszewski B, Gilpin R.K. and Jaroniec M. (1995) Comparison of the retention of organic acids on alkyl and alkylamide chemically bonded phases. J Chromatogr A 691, 217-224.

88 van den Berg K.J, van der Horst J, Boon J.J, Shibayama N. and de la Rie E.R. (1998) Mass spectrometry as a tool to study ageing processes of diter-penoid resins in work of art: GC- and LC-MS studies. In Advances in Mass Spectrometry. Edited by Karjalainen E.J, Hesso A.E, Jalonen J.E. and Kar-jalainen U.P. Elsevier Science Publishers B. V, Amsterdam. pp 563-573.

89 Fredriksson R. (2011) A water plug injection technique for boosting HPLC column efficiency. Innovations in Pharmaceutical Technology 39, 20-22.

90 de Hoffman E. and Sroobant V. (2007) Electrospray. In Mass spectrometry, Principles and Applications, 3rd edition. John Wiley & Sons Ltd, Chichester. pp 43-55.

91 Trufelli H, Palma P, Famiglini G. and Cappiello A. (2011) An overview of matrix effects in liquid chromatography-mass spectrometry. Mass Spectrom Rev 30, 491-509.

92 Tang L. and Kerbarle P. (1993) Dependence of ion intensity in electrospray mass spectrometry on the concentration of the analytes in the electrosprayed solution. Anal Chem 65, 3654-3668.

93 Kebarle P. and Verkerk U.H. (2010) On the Mechanism of Electrospray Ion-ization Mass Spectrometry. In Electrospray and MALDI Mass Spectrometry. Edited by Cole R.B. WILEY, Hoboken. pp 3-48.

94 Taylor P.J. (2005) Matrix effects: The Achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometry. Clin Biochem 38, 328-334.

95 Mallet C.R, Lu Z. and Mazzeo J.R. (2004) A study of ion suppression effects in electrospray ionization from mobile phase additives and solid-phase ex-tracts. Rapid Commun Mass Spectrom 18, 49-58.

96 Antignac J.-P, de Wasch K, Monteau F, De Brabander H, Andre F. and Le Bizec B. (2005) The ion suppression phenomenon in liquid chromatography-mass spectrometry and its consequences in the field of residue analysis. Anal Chim Acta 529, 129-136.

97 Bonfiglio R, King R.C, Olah T.V. and Merkle K. (1999) The effects of sam-ple preparation methods on variability of the electrospray ionization for model drug compounds. Rapid Commun Mass Spectrom 13, 1175-1185.

98 King C.S, Barton S.P, Nicholls S. and Marks R. (1979) The change in prop-erties of the stratum corneum as a function of depth. Br J Dermatol 100, 165-172.

60

99 Weigand D.A. and Gaylor J.R. (1973) Removal of stratum corneum in vivo: an improvement on the cellophane tape stripping technique. J Invest Derma-tol 60, 84-87.

100 Maicas S. and Mateo J.J. (2005) Hydrolysis of terpenyl glycosides in grape juice and other fruit juices: a review. Appl Microbiol Biotechnol 67, 322-335.

101 Demers P.A, Teschke K, Davies H.W, Kennedy S.M. and Leung V. (2000) Exposure to dust, resin acids, and monoterpenes in softwood lumber mills. AIHAJ 61, 521-528.

102 Fransman W, McLean D, Douwes J, Demers P.A, Leung V. and Pearce N. (2003) Respiratory symptoms and occupational exposures in New Zealand plywood mill workers. Ann Occup Hyg 47, 287-295.

103 http://limitvalue.ifa.dguv.de, GESTIS database of international limit values, Institute for Occupational Safety and Health of the German Social Accident Insurance (IFA), downloaded on feb 27th 2012.