Development of a multi method for the determination of organic wood preservatives in different wood types

Introduction

Introduction

Since the early 20th century, the production of chemicals has significantly advanced, enhancing human comfort and longevity Out of 11 million known chemicals, around 100,000 are produced on a large scale, with approximately 30,000 to 70,000 actively used in the European Union These chemicals serve various purposes, particularly in agriculture and industry, with about 300 million tons of synthetic compounds utilized annually in consumer products In agriculture alone, roughly 140 million tons of fertilizers and several million tons of pesticides are applied each year Additionally, many of these compounds contribute directly to improving human and animal health, ultimately impacting different environmental compartments based on their applications.

Wood is a vital resource globally, serving as a raw material for industries, construction, and fuel In Germany, forests cover approximately 10.6 million hectares, representing 36% of the country's land area, with an annual wood harvest of about 45 million m³, primarily for the building industry Despite being susceptible to damage from fungi and termites, wood remains popular in residential construction and decking To protect wood from decay, especially when exposed to moisture or soil, it is essential to use preservatives, which can be categorized into water-borne, organic solvent-based, creosote, and specialized gas treatments containing biocides like fungicides and insecticides The push for environmentally friendly biocides is a crucial aspect of modern wood preservation, although the high cost of organic options poses challenges, as they can be 10-30 times more expensive than inorganic alternatives Consequently, many organic systems utilize combinations of biocides to enhance efficacy and reduce costs, leveraging their long-lasting effects and versatility to provide comprehensive protection against wood-destroying organisms.

Organic biocides play a crucial role in protecting wood from pest organisms, with various preservatives used in Germany over the years, including PCP, lindane, and tebuconazole Historically, PCP was the most common wood preservative against fungi, but due to regulatory bans on harmful substances like pentachlorophenol and tributyltin (Regulation (EC) No 850/2004), safer alternatives are now favored Compounds such as fenoxycarb are effective at low concentrations (0.015% to 0.05%), while others require higher levels for efficacy The effectiveness of wood treatment can be further improved by incorporating pyrethroid insecticides like permethrin.

The selection of fungicides is based on their specific properties i.e., the ability to control the growth of a particular fungi and their biodegradability (Miyauchi et al., 2005; Schultz et al.,

A mixture of preservatives with complementary chemical and biological properties is typically used in treated wood, with active ingredient concentrations specified by EN 351-1 (2007) to ensure adequate protection Quality control through chemical analysis is crucial for verifying proper treatment, yet accurately determining organic active ingredients poses challenges due to their low concentrations and integration within the wood matrix The increasing chemical diversity in wood treatment products complicates method development, as existing studies on the quantitative analysis of mixed organic preservatives are limited Common preservatives like tebuconazole, propiconazole, and permethrin can be analyzed rapidly using pyrolysis-gas chromatography-mass spectrometry Standard methods, such as those from the American Wood-Preservers’ Association, involve extracting preservatives with methanol followed by chromatography techniques To safeguard consumer interests and the environment while ensuring wood product quality, reliable and efficient analytical methods for simultaneous detection of various organic preservatives are essential Given the diverse chemical properties and wood matrices, developing a sensitive multi-method for analyzing a wide range of organic wood preservatives is a primary objective of this research.

Laboratory practices for extracting organic wood preservatives primarily utilize methanol, leading to extracts that contain numerous co-extracted matrix components from wood (Butte et al., 1992; AWPA, 1997; AWPA, 2006; AWPA, 2008; Schoknecht et al., 2007 and 2009; Rasmussen et al., 2009 and 2010) These co-extractives can interfere with the quantitative determination of active ingredients, diminishing detection sensitivity (Kollmann et al., 1968) Current methods often adjust HPLC techniques to account for these interferences, but clean-up steps are essential for enhancing analytical reproducibility Gel permeation chromatography (GPC) serves as an effective post-extraction clean-up procedure, eliminating matrix components like lipids, pigments, proteins, and polymers that may hinder analyte detection GPC can separate compounds based on molecular size without causing decomposition, making it suitable for both polar and non-polar analytes Its efficiency in separating multi-pesticide residues from plant matrices has been well-documented (Balinova A., 1998; Cervera M.I et al., 2010), and this study also tested GPC for removing co-extractants from wood matrices.

Recent studies have explored various solid phase extraction (SPE) methods for the multi-residue analysis of pesticides in samples including plants, vegetables, foods, and environmental materials (Ramesh et al., 1998; Didier et al., 2004; Teruhisa et al., 2004 and 2005; Herrera et al., 2005; Cervera et al., 2010) These pesticides share similarities with the active ingredients found in wood preservatives A specific SPE technique has been utilized for the cleanup of wood extracts, with Miyauchi et al (2005) employing Oasis MCX cartridges for the HPLC determination of tebuconazole and cyproconazole in treated wood Additionally, the same SPE cartridges were used to quantitatively analyze benzalkonium chloride and its homologues in treated wood (Miyauchi T and Mori M., 2007) Consequently, solid phase extraction (SPE) has been identified as an effective method for the cleanup and enrichment of organic active agents from wood matrices.

Objectives and research activities

Base on reviewing the literature dealing with organic wood preservatives, several important points have to be taken in consideration:

1 Although several studies concerning analytical methods have been already published, the accurate quantitative determination of organic active ingredients in complex wood matrices implies special problems as the concentrations of the compounds are often relatively low and, moreover, they are fixed into the wood matrix Therefore, it requires the application of analytical tools capable of providing the comprehensive in favored enhancing chemical diversity of the wood treating products

2 Concerning the concentrations of organic biocides use as wood preservatives in wood samples only few studies addressing the quantitative determination of a mixture of organic wood preservatives with differing chemical functionalities have been carried out so far This is attributed to the lack of sophisticated analytical method feasible for the simultaneous determination of the important organic wood preservatives in different wood matrices

3 So far in laboratory practice, organic wood preservatives are mainly extracted with methanol from wood and the extracts are analysed without further clean up steps with HPLC/UV-Vis (AWPA, 1997) Since wood contains a large number of compounds that can be extracted with organic solvents, the samples extracted using the above methods include numerous co- extracted components such as wood extractives These different wood types may contain very different pattern and amounts of matrix compounds that may be disturb the analysis of the wood preservatives Consequently, these extractives might interfere with the quantitative determination of organic preservatives

4 Using of only one method is not enough in the identification and quantification these biocides, since the reliable and effectiveness are the most common problem Owing to the different chemical properties, the amounts of such active ingredients in treated wood, and the different matrices of different wood types, much more sophisticated procedures are necessary So far, it can be very troublesome to determine whether a sample of wood contains preservatives or not, predominantly because of lack of analytical multi methods Up until now no multi method for the determination of the different class of biocides in wood preservatives has been described Therefore, combination of at least two techniques may provide complementary information that enabled the more complicated identification process

To safeguard consumer interests, protect the environment, and ensure wood product quality, it is essential to establish reliable, rapid, and cost-effective analytical methods for the simultaneous determination of various organic wood preservatives Developing a comprehensive multi-method that encompasses all significant organic agents can minimize analytical efforts Therefore, this thesis focuses on creating and validating a sensitive multi-analytical method for the simultaneous identification and quantification of organic wood protectants in wood.

This research focuses on developing methods to simultaneously analyze eight selected biocides used in organic wood preservatives, highlighting their environmental importance A significant challenge is analyzing these biocides, which have varying polarities, in complex wood matrices such as cellulose, hemicellulose, and lignin at low concentrations To enhance analyte enrichment, sample preparation procedures, including extraction and cleanup, were optimized to eliminate interfering species, enabling accurate analysis of target compounds The study employed advanced analytical instruments like HPLC/DAD, GC/MS, and LC/MS/MS to achieve precise qualitative and quantitative results, with method detection limits (MDLs) and method quantification limits (MQLs) established through fortification experiments Analytical quality assurance was ensured through repeatability experiments to evaluate accuracy and day-to-day variation Finally, the proposed methods were validated by analyzing real wood samples collected from Dr Matthias Wobst in Braunschweig, Germany, for the target compounds under investigation.

Literature reviews…

The German wood industry

Wood is a vital resource globally, serving as a key raw material for industries, construction, and fuel Following agriculture, forestry is the second largest land use in Germany and plays a crucial role as a natural habitat Germany is one of Europe's most densely wooded countries, with forests covering approximately 11.08 million hectares, which represents 31 percent of the nation's total land area However, woodland coverage varies significantly across regions, from just 10% in Schleswig-Holstein to much higher percentages in other areas.

40 % in Rhineland-Palatinate and Hesse, the most thickly wooded Lọnder (federal states) In

Table 2.1 the detailed overview on forest and forest cover in Germany is given

Table 2.1: Proportion of woodland areas in federal states of Germany

Federal states Forest Area Forest Cover (%)

Source: Second National Forest Inventory (2001-2002), Federal Ministry of Consumer Protection, Food and Agriculture, Bonn, Germany

Over the past four decades, Germany's forests have expanded by approximately 1 million hectares, with the proportion of stands over 80 years old increasing from 25% to 33% of total forest area The country's timber stocks are notable, averaging 320 m³ per hectare, and the annual timber increment reaches around 100 million m³ in accessible forests, equating to about 9.5 m³ per hectare This positions Germany as a leader in forestry compared to other European nations.

The distribution of forests in Germany is influenced by specific natural conditions and historical developments According to the latest Federal Forest Inventory (2001-2002) and EUWood (2010), around 73% of German forests are composed of mixed stands Spruce and pine are prevalent conifer species across all federal states, particularly in the construction carpentry industry, with spruce representing the largest share at 28%.

%), followed by pine covering 23 % Other wood species including beech (15 %) and oak (10

The popularity of wood species varies by country and industry, with pine and spruce being utilized by approximately 30% of furniture companies, while beech and oak account for an average of 40% Notably, wooden boards are the most commonly used material in the furniture sector, with around 67% of companies opting for this type The distribution of tree species is influenced by natural features, site conditions, and historical developments In Germany, for instance, pine trees thrive in the northern regions, deciduous trees are prevalent in lower mountain ranges and coastal areas, and southern Germany is abundant in spruce trees (Mantau et al., 2010).

Figure 2.1 Tree species composition in Germany (Second National Forest Inventory

2001-2002, Federal Ministry of Consumer Protection, Food and Agriculture, Bonn, Germany)

Germany, despite being a highly industrialized nation, boasts some of the largest forest volumes in Europe, with an average growth rate of nearly 12 m³ per hectare annually The German forest industry has consistently harvested less wood than the annual growth, with the Federal Statistical Office reporting a total of 42.4 million m³ of wood harvested Notably, around 82% of this harvested wood is coniferous timber, while approximately 18% consists of deciduous wood.

Table 2.2: Wood species harvested in Germany

European beech, other deciduous wood 7,641

Source: Federal Statistical Office, 2002, Wiesbaden and German Pulp and Paper Association, Bonn, Germany, FAO’s Yearbook of forest products, 2003 and EUWood, 2010

Forests face significant risks from both abiotic and biotic hazards, with abiotic threats including strong winds, fire, snow, ice, and frost Among biotic hazards, insect damage is the most severe, particularly affecting pure stands of spruce and pine, while natural oak forests are also vulnerable Consequently, forest protection is crucial in Germany, as highlighted by the assessment guidelines from the Ministerial Conference on the Protection of Forests in Europe (MCPFE).

In Germany, approximately 75 to 80% of the forest area, totaling over 9 million hectares, is protected or serves protective functions This makes estimating the consumption rate of wood preservatives crucial for predicting their environmental impact and effects on the ecosystem However, calculating the exact consumption rate in any region is challenging due to the various ways wood protection products are sold, whether over the counter or via prescriptions, as well as their availability under different commercial names According to OECD (2000), literature has indicated the average consumption of wood preservatives per user sector in Germany.

Table 2.3 Consumption of wood preservatives per user sector employed in Germany

Pressure treatment (water and solvent based, without tar oil) ca 2.000 – 2.200 7,0

Treatment in dipping/immersion plants ca 4.700 – 4.900 16,1

Tar oil pressure and hot/cold dipping ca 5.000 – 6.000 18,3

(injections, brushing) undercoating, varnishing, impregnation ca 12.400 – 12.600 41,8

Professional market undercoating, varnishing, painting ca 1.750 5,9

Do-it-yourself market undercoating, varnishing, painting ca 1.750 5,9

Professional curative treatment (injections, brushing) ca 1.400 – 1.600 5,0

Germany ranks highly among global wood-producing countries, leveraging its own resources to meet the demand for wood and wood products Despite ongoing recycling efforts and not fully exhausting its annual wood increment, the country has maintained a strong wood balance In 2009, Germany's total wood consumption reached approximately 94 million m³, translating to a per-capita consumption of about 1.15 m³ of roundwood equivalent.

Hence, Germany occupies a middle position by international ranking Given a 71 % waste paper utilization rate, Germany holds a top position gauged by international ranking (BMVLV,

Table 2.4 Total wood balance in Germany 2009 in million m 3 converted into round wood equivalents

Harvested 48.1 Increase in stock disposal 0.0

Waste paper from domestic production

Source: Public Relations Division, 2011, German forests, Forest-based industry in Germany, Federal Ministry of Food, Agriculture and Consumer Protection, Berlin, Germany

Forests play a vital role in Germany's economy, environment, and society, providing essential economic benefits and ecological services Consequently, a primary objective of forest policy is to maintain and expand forest areas while ensuring sustainable management practices that protect their size and services.

Germany aims to enhance global recognition of forests' vital roles in alleviating poverty, ensuring food security, supporting rural livelihoods, and promoting environmental conservation and climate protection To achieve this, sustainable forest management must be prioritized, considering all existing and potential forest products and services Effective coordination of international initiatives is essential to combat deforestation and forest degradation while promoting sustainable practices, thereby maximizing their impact.

Compositions and classifications of wood

This section outlines the chemical compositions and classifications of wood, offering essential background on the chemical parameters that influence wood structure Understanding these aspects is crucial for explaining the effects of wood decay fungi and the process of impregnating wood with chemical preservatives.

The chemical composition of wood varies significantly due to factors such as tree species, age, geographical location, and specific parts of the tree Despite these variations, all wood species share basic similarities, while notable differences exist among individual species The primary chemical constituents of wood consist of carbohydrates (65-75%), lignin (18-35%), and minor amounts of wood extractives (4-10%) For detailed chemical compositions of various wood types, refer to Table 2.5.

Table 2.5 Chemical compositions of some wood species (Sjửstrửm, 1993)

Wood primarily consists of carbohydrates, including cellulose and hemicelluloses, both of which are polysaccharides made up of long-chain sugars These carbohydrates are biodegradable, as microorganisms can decompose them into shorter fragments and wood sugars The cellulose content in dry wood typically ranges from 40% to 45%, while hemicelluloses account for 25% to 35% of the total weight (Sjửstrửm, 1993; Stenius, 2000).

Cellulose, a linear polysaccharide polymer composed of β(1→4) linked D-glucose units, is the primary component of the rigid cell walls in wood, contributing significantly to its strength With several hundred to over ten thousand glucose units, cellulose exhibits high tensile strength and remains insoluble in most solvents, making it essential for wood's structural integrity.

Figure 2.2 Structure of cellulose (chain conformation)

Hemicelluloses are a group of compounds related to cellulose, characterized by a lower molecular weight, with approximately 150 repeating units compared to cellulose's 5,000 to 10,000 They play a crucial role as a structural component in wood, particularly in hardwood, where glucuronoxylan is the predominant polysaccharide Hemicelluloses are primarily responsible for moisture sorption, although accessible cellulose, non-crystalline cellulose, lignin, and crystalline cellulose surfaces also significantly contribute Notably, lignin exhibits lower water sorption compared to hemicelluloses and cellulose Furthermore, hemicelluloses are soluble in alkali and can be easily hydrolyzed by acid.

Figure 2.3 Chemical structure of lignin which is responsible for many of wood properties

Lignin is a complex, high molecular weight polymer made up of irregularly bonded phenolic units known as phenypropanes It, along with hemicelluloses, forms the structural matrix of plant cell walls and acts as a natural adhesive that binds cells together The chemical composition of lignin varies between hardwood and softwood, highlighting the differences in their structural properties.

Extractives are a diverse group of compounds found in wood, typically constituting a small percentage of its total composition, though they can sometimes be more significant These compounds do not play a crucial role in the structural integrity of wood, as they do not contribute to the cell wall structure However, extractives are essential for providing natural durability and decay resistance in most wood species, often being extracted using solvents like water or organic solvents They include substances such as fatty acids, resin acids, waxes, and terpenes, which impart distinctive colors and odors to wood The variability of extractive content is influenced by wood species and different parts of the same tree Some extractives enhance wood's resistance to fungal decay and insect damage, while others possess medicinal properties.

Extractives can be categorized into two classes based on the solvents used in their extraction: lipophilic and hydrophilic compounds Lipophilic extractives, which account for 90-99% of resin acids, diterpenyl alcohols, fatty acids, sterols, steryl esters, and triglycerides, are extracted using non-polar organic solvents like hexane and dichloromethane In contrast, hydrophilic components such as lignans, oligolignans, and phenolic stilbenes are more effectively extracted with polar solvents, including acetone, methanol, or ethanol The use of water in conjunction with polar solvents can enhance the extraction of more polar compounds, such as lignan glycosides and polyphenols.

Certain wood species contain toxic extracts that enhance the durability of their heartwood against decay, though dark-colored heartwood isn't always a sign of durability Sapwood, lacking these protective extracts, is generally non-durable, even in species with durable heartwood Durability can vary significantly not only among different species but also within individual trees, leading to inconsistent service life and occasional rapid decay in lumber Wood is categorized into two zones: sapwood, which comprises living cells responsible for metabolite synthesis and storage, and heartwood, which consists of non-metabolically active cells that serve as a long-term storage for protective extractives These extractives contribute to essential wood characteristics such as stability and resistance to fungi and moisture Consequently, sapwood is often less valued in the wood industry due to its inferior properties compared to heartwood.

Softwoods have long fibers while hardwoods (including exotic hardwoods) have short fibers The extractive content is similar for both soft and hardwoods but elevated in exotic hardwoods

Table 2.6 Characteristics of hardwoods, softwoods and tropical hardwoods modified from Rowell, 2004

Fibres Long (1.4 – 4.4 mm) Short (0.2 – 2.4) Short (0.2 – 2.4)

Cell type One (tracheids) Various Various

Extractive content Up to about 10 % 1 – 10 % Up to 30 %

Non polar High Low Low

This thesis investigates seven commonly used wood species to encompass all tree classes Softwoods are represented by pine (Pinus sylvestris) and spruce (Picea abies), while hardwoods are exemplified by birch (Betula pendula) Additionally, medium density fiberboard with an oak surface (MDF) is included to represent artificial wood.

The need for wood preservatives

Wood serves various structural purposes in Germany, such as in external joinery like window frames, timber for buildings, railway sleepers, and marine applications Being a natural organic material, wood is susceptible to degradation by organisms, especially in environments conducive to their growth, such as when it is frequently exposed to moisture or in direct contact with the ground.

Wood often requires enhanced durability to meet diverse application needs, particularly in scenarios where a service life of 25 years or more is essential This durability is crucial to prevent structural damage and ensure long-term performance.

Wood is highly susceptible to damage from biological organisms such as insects, fungi, and marine borers, particularly under unfavorable conditions Fungi, in particular, can cause significant decay when the wood's moisture content exceeds 20% for extended periods These organisms can compromise the structural integrity of wood by feeding on its components, breaking down cellulose, hemicellulose, and lignin through powerful enzymes As a result, the wood deteriorates, leading to what is commonly referred to as "rotten" or "decayed" wood, making it one of the most damaging threats to timber.

Fungi are the primary agents of wood decay, thriving both inside and on the surface of wood, with growth colors ranging from white to dark brown The extent of decay is influenced by the duration of favorable conditions for fungal growth, as it halts when moisture levels drop below the fungi's needs Additionally, extreme temperatures can significantly slow the decay process, but it can resume once conditions become suitable again Early signs of decay are more noticeable on freshly exposed surfaces of unseasoned wood compared to weathered and discolored wood.

Wood decay fungi are classified into three main types: brown rot, white rot, and soft rot, with stain fungi also impacting wood by causing discoloration, particularly in newly felled pine sapwood While sap stain fungi do not weaken the wood structurally, they diminish its market value Without proper treatment and prevention, the risk of wood-boring insects, such as beetles, borers, and termites, increases, posing further threats to wood integrity.

To enhance the durability of wood for outdoor applications and extend its service life, it is essential to treat the wood with chemical preservatives that protect against wood-destroying organisms.

Wood-destroying fungi: brown rot, white rot, soft rot

Wood-staining fungi: sap stain fungi and mold

Figure 2.4 Examples of wood destroying fungi and insects

Wood preservatives are designed to alter the conditions that promote fungal growth, effectively protecting wood from decay and biological attacks, thereby extending its service life The use of these chemical preservatives is guided by the hazard classes (HC) outlined in European wood treatment standards (EN 335-1), which categorize different moisture conditions and recommend specific treatment techniques to enhance wood durability.

Table 2.7 Hazard classes of biological attack by environmental service conditions according to EN 335-1 (European standard EN 335-1, 2006) with recommended treatments to extend the service life of wood

Service conditions Biological agents Recommended treatments

1 Without soil contact covered (dry)

2 Without soil contact covered (risk of wetting)

Insects, decay fungi Superficial treatments

Insects, decay fungi (plus termites), disfiguring (blue stain) fungi

4 With soil contact As HC-3 plus soft rot fungi Impregnation

5 Salt water contact As HC-3 plus soft rot fungi Impregnation

(1): Methyl bromide fumigation, quick-dip, spraying or brushing

Compared to hazard classes systems according to EN 335-1 (European standard EN 335-1,

2006), further quality grades dealing with wood preservatives for construction timber are assigned by the German institute for building technology (Deutsches Institut für Bautechnik, DIBt):

Iv: Preventively effective against insects

P Effective against fungi (rot protection) This grade is only assigned when preventive effectiveness against insects has been demonstrated

W: For wood exposed to weathering, however, which is not in permanent ground contact or permanent contact with water

E: Wood exposed under extreme conditions (ground contact, running water, etc.)

(P): effective against fungi (for wood composites)

Ib: Remedially effective against insects

M: Schwammsperrmittel (preservatives for barrier treatment)

Wood preservatives are essential for protecting timber from decay and insect damage by impregnating it with toxic substances that hinder fungi from using the wood as a food source The effectiveness of these treatments relies on several factors, including the chemical formulation, wood species, moisture content, preservative retention, and treatment method Commercial wood preservation methods are primarily categorized into pressure and non-pressure treatments, with pressure treatment being the most prevalent, accounting for about 90% of treated wood due to its deeper and more uniform chemical penetration Non-pressure methods, such as thermal processes and cold soaking, offer varying degrees of effectiveness, with cold soaking providing an average service life of 18 to 27 years, but generally falling short of the durability provided by pressure treatments For wood products exposed to high risks of biological attacks, pressure treatments are typically necessary, offering superior retention and penetration, and ensuring service lives exceeding 30 years.

Industrial wood preservative formulations in Germany

Wood preservation in Germany is crucial for extending the lifespan of wood products Modern wood preservatives are highly effective against wood-destroying organisms and typically contain toxic biocides such as fungicides and insecticides These preservatives can be applied in either organic solvent-based or water-based formulations, and they are categorized into three main types: creosote, water-borne preservatives, and organic solvent-borne preservatives (Cichowlaz, 2005).

Creosote is a brownish or black oil derived from the distillation of coal tar, consisting of hundreds of organic compounds Known for its effectiveness as a preservative, creosote offers high toxicity to wood-destroying organisms and is easy to apply It imparts a brown to black hue to treated wood and is commonly used for preserving railway ties, marine piles, and building piles (Thomasson et al., 2006).

Creosote is an effective wood preservative known for its ability to protect structural timbers in outdoor settings; however, it poses significant drawbacks, including skin irritation, making it unsuitable for indoor applications where human contact is likely (Cichowlaz, 2005; Thomasson et al., 2006) To reduce costs, creosote is frequently mixed with coal tar or heavy petroleum oil, which not only lowers toxicity to fungi but also enhances the service life of the treated wood by minimizing weathering and moisture uptake (Shupe et al., 2008).

Water-borne preservatives are chemicals that dissolve in water or in water mixed with ammonia or acidic compounds, facilitating their application in wood treatment (Prestemon, 1994; Thomasson et al., 2006) After treatment, it is essential to re-dry the wood, as water serves as the carrier for these chemicals These preservatives, classified as over oil type formulations, offer advantages such as cleanliness, paintability, and minimal odor There are two main categories: leach-resistant and leachable preservatives Leach-resistant preservatives, like chromated copper arsenate (CCA) and ammoniacal copper arsenate (ACA), chemically bond to the wood, preventing loss upon rewetting and making them suitable for ground contact In contrast, leachable preservatives, such as chromated zinc chloride (CZC) and fluoride chrome arsenate phenol (FCAP), do not bond with the wood and are not recommended for areas with a high risk of decay.

1994) Water-borne preservatives are used to treat lumber, plywood, fence posts, poles, pilings and timbers (EPA, 2008)

Organic solvent-based preservatives contain two to four active ingredients, typically fungicides and sometimes insecticides, dissolved in solvents like white spirit These preservatives are resistant to leaching and do not cause wood swelling during treatment, unlike water-based alternatives that use water-soluble or emulsified compounds However, they leave an oily residue that makes the wood unpaintable and generally provide less protection than creosote, often darkening the wood in the process Due to their fumes, these preservatives are unsuitable for indoor use and are primarily applied to poles, lumber, timbers, cross arms, and fence posts.

Wood protection products, commonly referred to as biocides, are essential for enhancing wood productivity, quality, and longevity Over the past 170 years, numerous active substances have been introduced for wood preservation; however, many have failed to gain acceptance due to issues related to chemical toxicity, inefficiency, or high costs To be legally registered for use, biocides must undergo rigorous testing for efficiency, environmental impact, and toxicity Concerns arise from the potential presence of active ingredients and their breakdown products in the environment, posing risks for human exposure.

Traditional wood protection using biocidal chemicals effectively prevents decay but raises environmental concerns due to the potential for chemical contamination Understanding the quantity of biocides used as wood preservatives is essential for assessing safety and environmental impact Growing awareness of health and ecological issues has prompted significant advancements in the development of eco-friendly wood preservative systems that are cost-effective and exhibit low toxicity to mammals and the environment Consequently, innovative wood protection chemicals and processing techniques have emerged in the 21st century, with some European countries, like Germany, mandating the use of entirely organic wood preservatives for various applications Historically, industrial wood preservatives have been categorized into two distinct groups.

1- Inorganic wood preservatives included formulations based on borate, chromium, copper or quaternary ammonium have high resistance to leaching and very good performance in service (AWPA P8) In the last decade products such as copper formulated with combinations of different levels of arsenate e.g ammoniacal copper arsenate (ACA), ammoniacal copper zinc arsenate (ACZA), or chromated copper arsenate (CCA), without arsenic or chromium, include ammoniacal copper quat (ACQ), copper bis- (dimethyldithiocarbamate) (CDDC), ammoniacal copper citrate (CC), and copper azole-Type

Recent environmental legislation has led to the introduction of alternatives to traditional inorganic wood preservatives, such as CBA-A, which replace harmful ingredients like chromium and arsenate with more eco-friendly boron-based systems Borate preservatives, known for their high solubility and leachability, are best applied above ground in high humidity environments These odorless, non-toxic, and fire-retardant treatments are effective against decay, termites, beetles, and carpenter ants, making them ideal for indoor use.

2- Organic wood preservatives formulations historically used active substances like fungicides and/or insecticides to protect wood from pest organism Only four insecticides are commonly used: these are dieldrin, lindane and synthetic pyrethroids, permethrin and cypermethrin Organic preservatives used in Germany recently and in the past like pentachlorophenol (PCP), lindane, dichlofluanid, triazoles such as tebuconazole, propiconazole (which replaced PCP), permethrin, fenoxycarb, IPBC, flufenoxuron, and tributyltin compounds, etc have very different structures In the past PCP was the most common compound to protect wood against damage by fungi Dieldrin has been withdrawn from the Germany market by its sole manufactures, although stocks remain and are currently being used up Lindane, tributyltin and pentachlorophenol are banned now for use as biocides (Regulation (EC) No 850/2004) Organic wood preservatives use white spirit or petroleum distillate as the carrier for the active substances

The current organic preservative formulations primarily utilize agricultural and pharmaceutical biocides for residential use (Preston, 2003) These active ingredients must effectively preserve solid timber and protect engineered wood products, such as wood-polymer composites In agricultural settings, biocides are designed to target specific fungi or insects and degrade quickly; however, wood preservation requires biocides that can combat a broader range of decay fungi and insects while providing long-lasting protection for the expected service life of treated wood Consequently, only a limited number of agricultural chemicals possess the necessary properties for effective wood preservation (Dickey, 2003; Freeman et al., 2006).

The growing interest in environmentally friendly materials has led to an increased focus on organic wood preservation systems (Green III, 2003) However, replacing hazardous preservatives with organic alternatives in high-risk applications remains challenging due to the complexity of biodegradable biocides that target specific wood-degrading organisms Effective wood preservation requires biocides that not only combat these organisms but also maintain their efficacy within the wood structure over extended periods (Wallace et al., 2008) To address these challenges, many organic systems are now being developed that combine multiple biocides, enhancing their effectiveness in protecting treated wood and wood products (Schultz et al., 2007; Binbuga et al., 2008).

The identification of effective organic preservatives, which combine various biocides, is crucial for providing long-term protection against wood-damaging organisms (Schultz and Nicholas, 2006) The widespread use of these preservatives in wood processing has significantly enhanced the supply of renewable resources, influenced by the variability in wood properties and environmental factors While some preservatives demonstrate superior effectiveness and adaptability to specific applications, the long-term efficacy is also dependent on various treatment methods and the treatability of different wood species (Archer and Lebow, 2006) In applications involving ground contact or frequent moisture exposure, it is essential to treat wood and wood-based composites with biocides to safeguard against destructive organisms (Preston, 2000) Nonetheless, there remains a considerable gap in understanding how to enhance the durability of numerous wood species through these preservative systems.

Selection of the analytes

The market has seen the introduction of numerous wood preservatives; however, many have failed to gain acceptance due to issues like chemical toxicity, low effectiveness, high costs, or corrosiveness (Murphy, 1990) As of September 21, 2011, the Federal Health Office (BGA) and the Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV) reported approximately 1,500 wood preservatives available in Germany, yet less than one-third have been evaluated or approved by independent bodies Due to the absence of a statutory authorization procedure for these products, the European Biocidal Products Directive 98/8/EC was established to ensure high efficacy and minimal impact on human health and the environment, which was subsequently enacted into German law as the Biocidal Law on June 20, 2002.

The Federal Institute for Risk Assessment (BfR) has consistently addressed health risks associated with wood preservatives, emphasizing the importance of safety in their use In Germany, two primary testing methods exist for evaluating wood preservatives: the building inspection authorization by the Deutsches Institut für Bautechnik (DIBt) for load-bearing components, and the voluntary inspection procedure by the Gütegemeinschaft Holzschutzmittel e.V (RAL quality mark) for preventive measures on non-load-bearing wood All active substances in chemical wood preservation undergo rigorous testing through these procedures Biocidal products that successfully meet these standards are recognized as effective, safe, and environmentally compatible when used correctly.

The registration process has been applied to only 23 products so far, with just one product currently available in the market Recently, there has been a growing interest in wood preservation methods that utilize environmentally friendly, purely organic systems.

In recent years, the use of organic biocides derived from agrochemical biocides has gained traction, although only a limited number possess the necessary properties for effective wood preservation Most biocidal substances utilized in wood protection are either insecticides or fungicides, leading to the common practice of using mixtures to enhance efficacy In Germany, frequently employed organic mixtures for wood protection include combinations such as propiconazole/dichlofluanid, propiconazole/fenoxycarb, and propiconazole/IPBC, among others Current commercially available biocide mixtures for wood protection are detailed in Table [insert table reference].

Table 2.8 An overview of wood preservation portfolio (Janssen Preservation & Material

Name Description Spectrum and recommen dation

Controls blue stain and decay fungi

- Class IV in combination with copper

- With emulsifiers in water based systems

Controls blue stain, decay fungi and moulds

- With emulsifiers in water based systems

Controls blue stain, decay fungi and moulds

- Solubility in solvents depending on product composition

Controls blue stain, decay fungi and moulds

- Class IV in combination with copper

- Solubility in solvents depending on product composition

- Class IV in combination with copper

- Industrial - Solubility in solvents depending on product composition

Preventive against wood boring insects

Can be used in both water and solvent based products

Preventive and curative against wood boring insects

Can be used in both water and solvent based products

Can be used in both water and solvent based

This study's experimental component includes eight organic biocides that encompass the full range of ingredients in wood preservatives, as mandated by the swift implementation of the European Biocides Directive into German legislation The selected biocides' structure and physicochemical properties are critical for the method development presented in this research.

Table 2.9 are briefly discussed below

Table 2.9 Structure and physicochemical properties of the biocides from wood preservatives used for the method development

Material insects and termites and II

Preventive and curative against wood boring insects and termites

Can be used in both water and solvent based products

Determining the concentrations of selected biocide compounds in wood matrices is crucial for understanding their environmental impact To achieve this, a validated method must be established to accurately quantify these pollutants This chapter details the experiments conducted to develop a qualitative, quantitative, and confirmatory analytical method for simultaneously detecting eight specific biocides in various wood types The initial step involves extracting these compounds from the wood matrices using various techniques, as illustrated in the general analytical strategy in Figure 3.1.

Figure 3.1 Analytical scheme of wood analysis

The removal or isolation of target compounds from interfering substances is crucial for detecting trace amounts in complex matrices A thorough cleaning process of raw extracts significantly impacts subsequent analytical techniques, such as HPLC, GC/MS, and LC-MS/MS (+ESI), especially when analyzing environmental samples known for their complexity and variability Thus, implementing advanced cleanup methods is essential for accurate analysis.

① Shavings were produced by drilling wholes into the plank of un-treated wood

The shavings were ground in laboratory mill grinder to a particle size of ≤ 1 mm

Analytical scheme of wood analysis

Materials and methods

Reference standards and chemicals

Biocides were chosen for their prevalence and significance as organic preservatives, as well as their compatibility with HPLC analysis Each analyte was sourced from Dr Ehrenstorfer in Augsburg, Germany, ensuring the highest available purity The physicochemical properties of the studied substances are detailed in Table 2.9.

In Chapter 2, individual standard stock solutions of target compounds were prepared at a concentration of 1 mg/mL in methanol, using 10.0 mg of each compound measured on an analytical balance and dissolved in a 10 mL volumetric flask For dichlofluanid, a 1.0% acetic acid in methanol was utilized to prevent rapid hydrolysis to its metabolite, DMSA, at neutral pH These reference stock solutions were stored in brown glass containers at -20 °C for up to three months, with dilution occurring as needed to avoid photo-degradation Weekly preparation of mixed working standard solutions for recovery experiments and calibration curves involved diluting the stock solutions with methanol and storing them at 4 °C.

HPLC grade methanol (MeOH) and acetonitrile (ACN), along with a 25% ammonium hydroxide solution, were sourced from Sigma-Aldrich Chemie GmbH in Steinheim, Germany Additionally, 100% p.a grade acetic acid (HoAc) and formic acid (FA) were obtained from Carl Roth GmbH + Co KG in Karlsruhe, Germany Deionized water was prepared using the SERALPUR PRO 90/PRO 90 C Ultrapure water system, equipped with a 0.2 µm filter, purchased from SERAL Elrich Alhọuser GmbH in Bansbach-Baumbach, Germany.

Sample preparation and extraction

Shavings were produced by drilling wholes into the plank of un-treated pinewood (Pinus sylvestris) with a Forstner bit The shavings were ground in laboratory mill grinder (Retsch,

The initial stages of method development involved extracting ground pinewood particles sized at ≤ 1 mm using methanol through sonication at room temperature, following the AWPA method (2006) Approximately 1 g of the ground wood was placed in an Erlenmeyer flask and treated with 20 mL of methanol for 2 hours using an ultrasound bath Sonorex RK 512 S (35 kHz-HF-Frequency) After sonication, the wood matrix was rinsed twice with 10 mL of methanol to eliminate residual pesticides The resulting extract was then filtered through filter paper to remove woody particles and subsequently evaporated using a rotary evaporator.

35 – 40 o C to nearly dryness Depending on the selected clean up protocol, the extracts were re-dissolved in different volumes of solvent or acidified deionized water or in water with ammonia.

Clean up procedures

3.3.1 Gel permeation chromatography (GPC) clean up

Gel Permeation Chromatography (GPC) is a specialized form of size exclusion chromatography that separates molecules based on their hydrodynamic volume, commonly referred to as molecular size In this technique, samples are passed through a porous stationary phase, where the solvated molecules' size influences their ability to enter the resin's pores GPC effectively separates molecules according to their size while utilizing an organic solvent as the mobile phase This method is predominantly employed in laboratories as an analytical tool to assess the molecular distribution within various samples.

Figure 3.2 Schematic of GPC set up

In environmental testing and food and agriculture, laboratories face significant challenges when analyzing for toxins or synthetic products like pesticides, as separating the matrix from the target analytes can be difficult The analytes, often small molecules, are concealed among a complex mixture of proteins, lipids, waxes, sugars, and other matrix components that can interfere with chromatography Effective sample cleanup involves eluting the sample from the column and collecting only the fractions containing the analytes of interest Gel permeation chromatography (GPC) is utilized for purification, ensuring the separation of co-extractants and higher molecular weight substances present in the samples (Specht and Tillkes, 1980).

The Gilson GPC unit from Abimed in Düsseldorf features an isocratic HPLC pump (model M 305) equipped with a manometer module (M 307), an auto-sampler injector (model 231) with a dilutor (model M 401, 5 mL syringe), and a Rheodyne 7010 sampling loop, along with a fraction collector.

A glass chromatography column measuring 35 cm x 2.5 cm I.D was filled with Bio-Beads® S – X8, a styrol-divinylbenzol copolymer that is 8% cross-linked and has a mesh size of 200 – 400, sourced from Bio-Rad Laboratories The parameters for Gel Permeation Chromatography (GPC) are detailed in Table 3.1.

The eluent was a mixture of ethyl acetate and cyclohexane (50:50, v/v) The flow rate was set at 5 mL/min and the injection volume was 4 mL

Table 3.1 Parameters of GPC clean up system

Instrument The Gilson GPC clean up system (Abimed, Düsseldorf)

Injector Gilson auto-sampler injector model M 231 with dilutor model M 401

(5 mL syringe) and Rheodyne 7010 sampling loop

Pump Isocratic HPLC-pump model M 305 with manometer module M 307

Eluent Ethyl acetate and cyclohexane (50:50, v/v)

Column Glass chromatography column 35 cm x 2.5 cm I.D filled with Bio-

Beads® S – X8 (styrol-divinylbenzol-copolymer, 8 % cross-linked, 200–400 mesh) from Bio-Rad Laboratories

Sample collect Gilson fraction collector model M 201

The initial GPC experiment involved a working standard of eight biocides at a concentration of 1 mg/mL in a 50:50 (v/v) mixture of ethyl acetate and cyclohexane A 4 mL aliquot of the samples was injected into the GPC apparatus, resulting in the collection of 12 fractions at 5-minute intervals Additionally, an un-spiked pinewood matrix was fractionated Prior to GPC injection, all samples were filtered through a 0.45 µm PTFE syringe filter to eliminate fine particles The collected sample fractions were then rotary evaporated to near dryness, with the final traces of solvent removed using a gentle nitrogen stream The residue was subsequently dissolved in the HPLC mobile phase to a final volume of 2 mL for analysis using HPLC/DAD.

The second experiment involved both un-spiked and spiked pinewood matrices, with the spiked samples containing 2 mL of a working standard solution of eight biocides at a concentration of 1 mg/mL Following the initial GPC experiment, the first 75 mL fraction was discarded, and a second 150 mL fraction was collected, followed by a rinsing fraction of 50 mL The sample fractions underwent rotary evaporation to near dryness, and any remaining solvent was eliminated using a gentle nitrogen stream The final residue was then dissolved in the HPLC mobile phase to achieve a total volume of 2 mL for analysis using HPLC/DAD.

3.3.2 Clean up with solid phase extraction (SPE)

Solid Phase Extraction (SPE) is an efficient sample preparation technique used for extracting, concentrating, and cleaning up semivolatile and nonvolatile analytes in liquid samples It effectively addresses challenges associated with Liquid-Liquid Extraction (LLE), such as the need for large volumes of organic solvents, incomplete phase separations, and time-consuming processes SPE offers a diverse range of adsorbents and sizes, making it crucial to select the appropriate sorbent for each sample to ensure effective extraction and cleanup of various target compounds with differing polarities, solubilities, and stabilities in acidic and basic conditions.

In general, there are four types of SPE sorbents based on the retention mechanism of the target compounds:

1) Normal phase materials: This procedure typically involves polar analytes in aqueous matrices and a polar stationary phase such as silica, diol bonded silica, aminopropyl bonded silica and cyanopropyl bonded-endcapped silica The interactions of polar target compounds and polar stationary phase include hydrogen bonding, π-π interactions, dipole-dipole interactions, and dipole-induced dipole interactions

2) Reversed-phase materials: This is used for separation of intermediate polar to nonpolar analytes from aqueous matrices with a nonpolar stationary phase, for example phenyl, octyl or octadecyl bonded, endcapped silica Therefore, the hydrophobic interactions involves such as nonpolar-nonpolar interactions and Van der Waals or dispersion forces

3) Ion exchange materials: This type of SPE can be used for compounds that occur in form of ions in aqueous matrices The retention mechanism is mainly based on the electrostatic attraction of the charged functional group of the target compound and the charged functional group of the bonded silica The negatively charged (anionic) compounds can be isolated on aminopropyl bonded silica or quaternary amine bonded silica with Cl - counterions

4) Adsorption materials: The interaction type of this SPE depends on which stationary phase is used Thereby, hydrophobic and hydrophilic interactions are possible Some examples of the stationary phases are alumina (acidic, basic or neutral), magnesium silicate, nonbonded carbon phase or resin-based

Solid phase extraction (SPE) is a popular chromatographic technique used to isolate an analyte from complex mixtures This method involves a column filled with a stationary phase that retains the target analyte, typically made of carbon-based materials like C8 and C18 The SPE process consists of four main steps: conditioning the sorbent, loading the sample, rinsing, and eluting the sample A block diagram illustrating the general SPE process can be found in Figure 3.3.

To perform solid-phase extraction (SPE), the process begins by activating the sorbent with an organic solvent to wet the stationary phase, allowing the initially collapsed hydrophobic ligands to extend and prepare for analyte interaction Next, the sample loading phase introduces a solution containing the target analyte along with potential contaminants onto the stationary phase, where the analyte molecules are retained Following this, a washing step is implemented to eliminate contaminants using a solvent that matches the sample matrix, ensuring that the analyte remains bound to the stationary phase Finally, an elution solvent with distinct properties from the previous solvents is introduced to release the analyte; for instance, acetonitrile may be used to elute a hydrophobic compound from an aqueous plasma solution.

3.3.2.2 The stationary phases of five different cartridges

At the beginning, the five selected SPE materials: C 18 Polar Plus cartridge (1000 mg, 6 cc, J.T Baker, Phillipsburg, USA), SDB-2 (1000 mg, 6 cc, J.T Baker, Phillipsburg, USA), Strara

In this study, we evaluated X (30 mg, 1 mL, Phenomenex, Torrance, USA), Oasis MCX, and Oasis HLB (30 mg, 1 cc, Waters, Millford, USA) following a solid-phase extraction (SPE) protocol recommended by Waters for method development The reversed phase cartridges selected included Oasis HLB, Strata X, SDB-2, and C18 Polar Plus, each chosen for their distinct properties Additionally, the inclusion of Oasis MCX as an ion exchange cartridge was deemed significant for the analysis.

(1): http://www.waters.com/waters/nav.htm?locale=en_US&cid083845

Table 3.2 Physicochemical properties of SPE cartridges n.a.: not available

2: CIEX – cation ion exchange, RP – reverse phase

Silica C 18 Polar Plus Octadecyl n.a 40 60 RP

Polymer Oasis MCX Copolymer 810 30 80 RP + CIEX

Polymer Oasis HLB Copolymer 810 30 80 RP

Oasis HLB features a copolymer of macroporous poly(N-vinylpyrrolidone-divinylbenzene) (PVP-DVB), which enhances its hydrophilic-lipophilic balance, facilitating hydrophobic and π-π interactions with target compounds (Fontanals et al., 2005) This characteristic makes Oasis HLB ideal for extracting a diverse range of compounds across various polarities and pH levels (Yu and Wu, 2011) In contrast, Oasis MCX serves as a mixed-mode strong cation-exchanger, offering both ion-exchange and hydrophobic retention capabilities, allowing it to effectively adsorb polar, non-polar, neutral, and cationic compounds from water samples (Yu and Wu).

High-performance liquid chromatography (HPLC) Instrumentation…

High-performance liquid chromatography (HPLC) is an analytical technique essential for separating, identifying, and quantifying organic and inorganic compounds The HPLC system includes key components such as mobile phase reservoirs, a pump, an injector or auto-sampler, a sample valve, a column, a detector, and a data analysis system To analyze samples, analytes must be dissolved in HPLC-compatible solvents, which are then introduced into the mobile phase stream via the injector The pump generates high pressure to propel the mobile phase through the stationary phase of the column As analytes traverse the column, they interact with the stationary phase, leading to their separation based on the degree of interaction or affinity between the mobile and stationary phases The separated analytes are subsequently detected and recorded at specific times, known as retention times, which serve as unique identifiers for each analyte under defined conditions.

The selection of the mobile and stationary phases in HPLC depends on the polarity of the compounds being analyzed For non-polar compounds, a hydrophilic stationary phase combined with non-polar solvents is necessary In contrast, this study utilized a reversed phase column with polar solvents to analyze two polar compounds and their eight highly polar metabolites Various detectors, such as ultraviolet variable wavelength (UVD), diode array detector (DAD), and mass spectrometry (MS), can be integrated with HPLC instruments for enhanced analysis.

3.4.1 Liquid chromatography with variable wavelength detector

In preliminary experiments, HPLC/UVD and HPLC/DAD techniques were utilized to investigate the chromatographic behavior of the studied compounds All HPLC runs were conducted using a variable wavelength detector (UV), featuring a column oven, an auto-sampler maintained at ambient temperature, and a binary pump.

The study utilized an Agilent 1100 series HPLC system, featuring a photodiode-array (DAD) detector, binary pump, column thermostat, thermostated autosampler, and vacuum degasser system, all controlled by HP ChemStation This setup enabled the acquisition of absorption spectra for target compounds across a wavelength range of 200 to 400 nm.

Chromatographic separation was conducted using a reversed phase column (Eclipse XDB, 150 mm x 4.6 mm, 5 µm; Agilent Technologies, Santa Clara CA, USA) with a mobile phase flow rate of 1.0 mL/min and a sample injection volume of 10 µL via an auto-sampler The column oven temperatures were set at 20, 25, and 30 °C, allowing for a 5-minute re-equilibration before each LC-UV experiment The HPLC system operated at various wavelengths, with all compounds dissolved in methanol and separated during a single gradient run.

Conventional HPLC struggles with low separation capacity, selectivity, and sensitivity, making it unsuitable for the simultaneous determination of eight specific compounds in complex wood matrices Therefore, liquid chromatography coupled with mass spectrometry (LC-MS/MS) emerges as the optimal solution to address these challenges effectively.

Figure 3.6 The HPLC/DAD Instrumentation (HP 1100 series, Agilent, Waldbronn, Germany)

3.4.2 Liquid chromatography with tandem mass spectrometry (LC/MS/MS)

LC-MS/MS analyses were conducted using an Agilent 1200 series HPLC system, featuring a vacuum degasser, binary pump, and high-performance auto-sampler, in conjunction with a 4000 QTRAP tandem mass spectrometer that includes a turbo ion spray ionization source.

The LC/MS/MS system utilized Analyst software (version 1.5) for control, employing an Ultrasphere ODS column (5 µm, 250 mm x 4.6 mm, Beckman Coulter, USA) The mobile phase was maintained at a flow rate of 1.0 mL/min, with an injection volume of 10 µL, allowing the column effluent to be directly transferred into the ESI interface without any splitting.

High-performance liquid chromatography coupled with mass spectrometry (LC-MS/MS) is an effective method for quantifying and verifying chemical compounds, even at low concentrations or within complex matrices Key components of mass spectrometry include the sample introduction systems like HPLC, the ion source, mass analyzer, and detector, as illustrated in Figure 3.7.

The LC/MS/MS/ESI QTRAP instrument utilizes the Turbo V™ ESI ion source to generate ions, which are then directed into the MS/MS region for analysis This mechanism, illustrated in Figure 3.7, demonstrates the ion formation process essential for mass spectrometry applications, as detailed by Hager in 2002.

Electrospray ionization (ESI) and atmospheric chemical ionization (APCI) are the most widely used atmospheric pressure ionization (API) techniques in LC/MS/MS analyses These methods are known for their sensitivity and gentle ionization processes, generating positive or negative ions from polar compounds as they pass through a needle with the HPLC eluent In ESI, ionization occurs in the liquid phase due to high electric fields applied to the needle, resulting in the formation of a spray of charged droplets assisted by a nebulizer gas.

As the solvent evaporates in a vaporizer gas, the charged ion droplets shrink, leading to increased repulsion forces that cause the droplets to become unstable and dissociate into smaller droplets This process, which can result in the separation of ions or the explosion of droplets, generates gas phase ions In Atmospheric Pressure Chemical Ionization (APCI), gas phase molecules are produced using a heater at the LC/MS/MS interface, followed by chemical ionization in a plasma created by a corona needle discharge Although APCI is limited to smaller molecules compared to Electrospray Ionization (ESI), which is more sensitive and suitable for a wider range of thermolabile substances, it is less affected by sample matrix effects Additionally, Atmospheric Pressure Photoionization, which utilizes a xenon lamp for ionization instead of a corona discharge needle, represents a third type of API but is less commonly used.

Selecting an appropriate ion source depends on the analyte's chemical properties, signal sensitivity, and the application matrix A comparison between Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI) is detailed in Table 3.3.

Table 3.3 Comparison of electrospray (ESI) with atmospheric pressure chemical ionization (APCI) (Bruins, 1994; Thurman et al., 2001)

(API) Soft ionisation mode (softest)

Differences Suitable for low and high masses

,polar and highly polar and thermolabile molecules Solution phase ionisation Compatibile with flow rate From 5 àL to 2 mL

Suitable for small masses, volatile, polar, relatively non polar and thermostable molecules Gas phase ionisation Compatibile with flow rate From 200 àL to 2 mL

Signal suppression or enhancement are common Adducts could be formed, e g., [M+NH4] + , [M+Na] + and [M+K] +

Clusters or dimers could be formed, e g., [M+CH3OHH] + , [M+CH3CNH] + and [M+M+H] +

In a mass spectrometer, the vacuum region is isolated from the spray by a capillary orifice, allowing precursor ions to enter the Q0 region with the aid of a curtain gas that declusters the ions and protects the curtain plate Fragmentation of ions can be controlled by adjusting the declustering potential in the Q0 region, where ions can also be accumulated to enhance their concentration before they move to the Q1 region for scanning Subsequently, ions are introduced to the Q2 region, or collision cell, where further fragmentation occurs through interaction with an inert collision gas, producing characteristic product ions The extent of fragmentation is influenced by the pressure of the collision gas and the collision energy Finally, product ions are scanned in the Q3 region, which can also function as a linear ion trap (LIT) mass spectrometer to improve sensitivity by trapping ions prior to scanning.

LC/MS/MS is widely recognized in the literature as a primary tool for structural elucidation due to its numerous advantages (Ahrer et al 2001, Fatta et al.).

Gas chromatography and mass spectrometry (GC/MS) Instrumentation

The integration of high-resolution capillary columns with fast scanning quadrupole or magnetic sector mass spectrometers, known as gas chromatography-mass spectrometry (GC/MS), offers a superior method for identifying a wide range of wood preservatives Notably, GC/MS analysis of environmental samples provides structural insights into many unknown components linked to chromatographic peaks, including those that may be obscured in the baseline of the chromatogram This technique's ability to detect molecular markers relies on the systematic fragmentation of these markers, producing characteristic key ions that facilitate the identification of specific compounds.

GC/MS, or Gas Chromatography-Mass Spectrometry, is a highly effective analytical instrument that merges the benefits of gas chromatography with mass spectrometry This versatile tool is extensively utilized for the separation, identification, and quantification of complex mixtures A typical GC/MS system comprises an injection port, a column, and a mass spectrometry detector, as illustrated in Figure 3.8, showcasing the key components of a modern capillary column GC/MS system.

Figure 3.8 Schematic view of the GC/MS 6890 GC-MSD 5975C (Agilent, Waldbronn, Germany) chromatographic system

Gas chromatography (GC) is a technique used to analyze substances in the gas phase, where a mixture is introduced via micro syringe into a hot injection port for flash evaporation This process converts liquid substances into gas, but it also subjects them to thermal stress, making GC ideal for analyzing less polar and thermally stable analytes The vaporized substances are then carried by an inert gas, such as helium, through a long, temperature-controlled capillary column lined with a stationary phase Separation occurs based on the substances' polarity and boiling points, as they interact differently with the mobile and stationary phases.

The mass spectrometry (MS) unit utilizes a transfer line to ionize substances, separating the resulting ions based on their mass-to-charge ratio Electron impact ionization is the most widely used technique, providing extensive structural information through full scan mass spectra This method, combined with access to commercial libraries, significantly enhances the screening and identification of unknown compounds, making gas chromatography-mass spectrometry (GC/MS) a powerful analytical tool.

GC/MS units offer significant advantages, including the rapid determination of analyte mass and the ability to identify components in incomplete separations These units are robust, user-friendly, and can analyze samples nearly as fast as they are eluted However, mass spectrometry detectors have drawbacks, such as the risk of thermal degradation of samples before detection and the potential for complete sample fragmentation.

The samples were incubated using a multifix shaker S 300 heating block and analyzed with a Hewlett Packard HP 6890 Series GC System, which included an HP 5972 Series mass selective detector, an HP 6890 Series injector, and HP Chem Station G1701AA version A.03.00.

Ionization was performed using electron impact mode (EI) at 60 eV, with the mass spectrometer operating in full scan mode across a mass range of 50-500 amu for screening purposes For quantification of target compounds, single ion monitoring (SIM) mode was utilized A 1 µL aliquot was injected into the split/splitless injector port set at 250-280 °C A DB-5 MS fused silica capillary column, measuring 30 m in length, 0.25 µm in film thickness, and 0.25 mm in inner diameter, was employed The gas chromatography (GC) oven temperature program commenced at 80 °C, held for 2 minutes, and was then ramped to 250 °C at a rate of 10 °C/min, maintaining this final temperature for 5 minutes The transfer line temperature was kept at 250 °C, with a helium carrier gas flow rate of 1 mL/min.

Guidelines for method development

Reliable residue analytical methods are essential for measuring residue levels in commodities and enforcing legal maximum residue limits (MRLs) These methods can be certified through two specifications: traditional standard methods and a more modern "criteria approach." The criteria approach focuses on quality control principles and validation procedures for both screening and confirmatory methods, offering greater versatility This adaptability allows for the incorporation of technical advancements and a swift response to emerging issues, such as previously unconsidered analyte/matrix combinations Adhering to analytical quality control (AQC) requirements is crucial for ensuring the validity of data used in compliance checks.

To support enforcement actions and assess consumer exposure to pesticides, it is essential to ensure accurate reporting by avoiding false positives and negatives, achieving acceptable trueness and precision, and harmonizing cost-effective analytical quality control (AQC) Laboratories analyzing pesticide residues must comply with recognized accreditation schemes, such as ISO 17025 or Good Laboratory Practices (GLPs) These accredited laboratories demonstrate their competence through regular participation in proficiency testing schemes recognized or organized by national or community reference laboratories.

Quality control procedures for pesticide residue analysis, as outlined in the 2002/657/EC Commission Decision, emphasize that chromatographic methods lacking molecular spectrometric detection are unsuitable as confirmatory methods The decision mandates the use of both on-line and/or off-line chromatographic separation Consequently, laboratories worldwide depend on mass spectrometry (MS) for the unequivocal confirmation of pesticide presence in food products.

The quality control guidelines for pesticide residue analysis emphasize the importance of confirming results through increased sensitivity, which can be achieved by scanning a limited mass range or using selected ion monitoring (SIM) The minimum requirement for data confirmation includes obtaining results from two ions with m/z values greater than 200 or three ions with m/z values greater than 100 Additionally, intensity ratios from characteristic isotopic ions can provide valuable insights It is recommended that ions selected for medium or high-resolution mass spectrometry (MS) or tandem mass spectrometry (MS/MS) be specific to the analyte and not commonly found in many organic compounds.

The 2002/657/EC European Commission Decision outlines performance requirements for various mass spectrometric detection methods, including full mass spectra, selected ion monitoring (SIM), and tandem mass spectrometry (MS/MSn) techniques like selected reaction monitoring (SRM) It emphasizes the use of identification points (IPs) for substance confirmation, requiring a minimum of three or four IPs for reliable identification The report indicates that any mass spectrometry technique or combination can be utilized to achieve the necessary IPs, with the number of IPs depending on the specific technique employed Additionally, the relative intensities of detected ions in both full scan and SIM modes should align with calibration standards, allowing for greater variations in relative ion intensity when it is smaller.

For mass spectrometric determination, it is essential to record full spectra, ensuring that all diagnostic ions—including the molecular ion, characteristic adducts, fragment ions, and isotopic ions—exceed a relative intensity of 10% in the calibration standard spectrum At least four ions must be present with this intensity, ideally including the molecular ion When utilizing SIM or MS/MSn, the molecular ion should be among the selected diagnostic ions, and these ions must originate from different parts of the molecule Additionally, the signal-to-ion ratio for each diagnostic ion should be greater than 3:1 Laboratories may choose to implement more stringent criteria beyond these minimum performance standards.

Method of validation

Validation is an essential process that involves testing various parameters to ensure that the proposed analytical method meets specified requirements and operates within set specifications under defined conditions.

The main objective of this process is fitness for purpose which defined by IUPAC (1999a) as

The effectiveness of data generated by a measurement process is crucial for users to make accurate technical and administrative decisions for specific purposes Alongside the statistical data obtained from testing validation parameters, factors such as practicality (cost) and suitability (simplicity) must also be evaluated, as outlined by IUPAC.

Method validation is a versatile concept that allows analysts to tailor analytical methodologies for various applications According to Boqué et al (2002), calculating the limit of detection (LOD) is unnecessary for methods assessing compounds expected at high concentrations, while it is crucial for residue analysis Therefore, a one-size-fits-all approach to validation requirements does not exist, as different applications necessitate distinct validation processes.

The validation process encompasses a comprehensive analytical procedure, including instrumental signals, representative sampling, sample preparation, and cleanup procedures, while considering parameters that impact method performance It is essential to cover the entire concentration range of the analyte in test samples Key validation parameters include recovery, accuracy, precision (intra-day repeatability and intermediate precision), method detection limit, method quantitation limit, linearity, and matrix effects Method validation followed ICH guidelines (2005), with linearity assessed through the correlation coefficient (R²) using a calibration curve derived from standard solutions ranging from 5 to 50 ng/µL, analyzed in triplicate The regression equation, Y = ax + b, where Y represents peak area and x denotes biocide concentration, demonstrated strong linearity (R² > 0.9959) The limit of detection (LOD) and limit of quantification (LOQ) were calculated as LOD = 3.3 × (SD/Slope) and LOQ = 10 × (SD/Slope), with quantification performed using the external standard method and samples analyzed at least in triplicate.

To assess precision and repeatability, an intra- and inter-day test was conducted by analyzing various concentrations of eight standard solutions This test involved five repetitions within a single day and additional intervals over three days (1, 3, and 5 days) Precision and repeatability were confirmed through the relative standard deviation (RSD) of both intra- and inter-day measurements, calculated by dividing the standard deviation by the measured amount and multiplying by 100 Furthermore, to evaluate accuracy, a recovery test was performed by adding different concentrations of eight biocides to samples, which were analyzed three times.

Fortification experiments were conducted using un-treated pinewood samples, where HPLC/DAD method was applied at concentrations of 10 and 50 mg/kg by adding 100 to 500 µL of a methanol standard mixture (c = 100 ng/µL) to 1.0 g of the pinewood After the addition of the standard solution, the samples were equilibrated for 10 minutes prior to extraction, following the procedures outlined in chapter 3.2 Recovery rates were assessed by comparing the concentrations of samples spiked before and after cleanup Additionally, matrix blanks of non-spiked un-treated pinewood were analyzed to evaluate potential interference from matrix peaks.

Recoveries (RE) were assessed by comparing concentrations of samples spiked before extraction with those spiked after extraction, utilizing external calibration curves with a correlation coefficient of at least 0.995 (Yang et al., 2004a) Calibration curves were established for concentrations ranging from 10 to 50 mg/kg When samples are spiked after extraction and just before clean-up, any losses during the clean-up process and matrix effects are accounted for, leading to a measure of relative recovery or extraction efficiency Conversely, by comparing concentrations of samples spiked before extraction with those spiked right before measurement (post-clean-up), the matrix effect is mitigated, allowing for the determination of absolute recovery.

% Recovery = Concetration of the samples spiked before extraction

Concentration of the samples spiked after extraction

Samples were analyzed in triplicates to ensure accuracy, and blanks made from excrement were tested to assess background contamination and potential interference with the analytes This was done to confirm the chromatographic selectivity of the method used.

The accuracy and precision of the proposed analytical procedures for analyzing target compounds in various matrices were assessed within a specified concentration range Accuracy was quantified through percent recovery or as the relative error, which reflects the difference between the measured mean value and the true value For each analyte, the accuracy was determined using the percent deviation of the mean calculated concentration from the spiked concentration, as detailed in equation 3.2 (Peters et al., 2007) The concentrations of the analytes in fortified samples were derived using external calibration curves, as indicated in equation 3.1.

% RE = Mean calculated - True concentration

“Precision is the closeness of agreement or degree of scatter between a series of measurements obtained from multiple sampling of the same homogeneous sample” (ICH,

In the validation process, the accuracy of a method hinges on its precision, which is crucial for establishing trust in the results This study defines data precision using relative standard deviations (RSD %) To ensure the reliability of the observed precision, three to four replicates at a minimum of three spiking levels were tested, following the International Conference on Harmonization (ICH) guidelines Acceptance criteria for within-day repeatability and intermediate precision were established, requiring RSD to be ≤ 20 % and recovery to fall within ± 20 % of the nominal values.

These parameters were applied according to Wisconsin Department of Natural Resources

3.7.4 Method detection limits (MDL) and method quantification limits (MQL)

The Method Detection Limit (MDL) is defined as the minimum quantity of an analyte that can be reliably distinguished from the background in a specific matrix, while the Method Quantification Limit (MQL) refers to the smallest amount of an analyte that can be accurately quantified within that matrix (Corley, 2003) This study utilized Solid Phase Extraction (SPE) followed by High-Performance Liquid Chromatography with Diode Array Detection (HPLC/DAD) and Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS) to analyze eight selected biocides in a wood matrix The MDLs and MQLs for both analytical methods were subsequently compared.

The MDLs and MQLs were established based on the analysis of seven extracted samples The spiked concentration utilized for calculating the MDL represents the minimum reproducible concentration of a substance measurable in accordance with U.S EPA standards.

(1993) Standard deviation (SD) for each analyte should be calculated first, thereafter, the MDL and MQL were determined according to the following equation (Corley, 2003):

The student's t value for a 99% confidence level is t(n-1, 1-α=0.99) = 3.143, with n = 7 degrees of freedom The standard deviation (SD) is derived from the replicate analysis with n = 7 Additionally, the minimum detectable limit (MDL) can be assessed through specific inequalities.

Calculated MDL < Fortification level < 10 x Calculated MDL

For an effective Minimum Detection Limit (MDL) study, the calculated MDL should be greater than one-tenth of the fortification level, ensuring precision in measurements It is essential that the MDL does not exceed the fortification level; otherwise, it becomes statistically impossible to distinguish between fortified samples and blanks, indicating poor determination precision Meeting these criteria confirms that the fortification level is suitable for accurate analysis.

In analytical chemistry, IUPAC defined the matrix effect as the combined effect of all components of the sample other than the analyte on the measurement of the quantity

In the study by Guilbault and Hjelm (1989), interference is defined as the effect caused by a specific component (Gosetti et al., 2010) The matrix effect is used to describe the ionization efficiency of analytes in LC/MS/MS (Kruve et al., 2008) For validation, matrix effects (% ME) were calculated using a modified equation by Chambers et al (2007), based on the original equation from Matuszewski et al (2003).

% Matrix effect = Response of post - extracted spiked sample

Results and discussions