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

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

Der Fakultät für Lebenswissenschaften
der Technischen Universität Carolo-Wilhelmina
zu Braunschweig
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr. rer. nat.)
eingereichte
Dissertation

Von Bich Ngoc Nguyen
aus Vietnam


1. Referent: Prof. Dr. mult. Dr. h. c. A. M. Bahadir
2. Referent: Dr. Marit Kolb
eingereicht am: 20 Mai 2016
mündliche Prüfung (Disputation) am: 09 Juni 2016
Druckjahr 2016


Vorveröffentlichungen der Dissertation
Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für
Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden
Beiträgen vorab veröffentlicht.

Braunschweig, Juni 2016
(Nguyen Bich Ngoc)

 


ACKNOWLEDGMENT
This thesis represents the collaborative effort from many people from the staffs at the
Institute

of

Environmental

and

Sustainable

Chemistry,

TU

Braunschweig, Germany to the Ministry of Education and Training of
Vietnam (MOET) and my family. This project would no have been a success if I
did not have the support from everyone I knew and everyone I met along this journey.
There are so many people I want to thank for their support, friendship and love
during my time at the Institute. First of all I would like to express my gratitude to
Professor Dr. mult. Dr. h.c. A. Mufit Bahadir, direct of the Institute for
giving me the opportunity to do my Ph.D. work, supporting my desired topic and for
the interesting supervision on the progress of my research. Most notably I am deeply
indebted to my supervising tutor Dr. h.c Marit Kolb who endless support,

patience and encouragement helped me during the whole time of my work. I also
appreciate her knowledgeable suggestions, her valuable discussions and advises which
contributed to the fulfilment of writing thesis.
For the nice working atmosphere and warm-hearted helps and valuable discussions, I
am gratefully to my colleagues at the Institute of Environmental and
Sustainable Chemistry. In particular, I would like to thank Prof. Dr. Robert
Kreuzig, Dr. Hubertus Wichmann, Dr. Heike Diekmann, Dr. Cornelia
Meier, Dr. Roland Vogt, Dipl. Ing. (FH) Jürgen Hamann, Dipl. Ing.
Bernd Rother, Dipl.-Mineralogin Christiane Schmidt, Mrs. Roswitha
Rumpf, and I would like here to mention special thanks to my good friends Heiko
Stache,

Diana

Ludgen,

Katharina

Heitmann,

Mohammed El-Dosoky, Amjad Al Tarawneh.


 

I

Saptono

Hadi,

In addition I would gratefully acknowledge the support of Dr. Matthias Wobst
from the Institute of Wood Research, Federal Biological Research
Center for Agriculture and Forestry (Braunschweig), during the
experiments in his laboratory.
Special thanks go to the support offered by the Ministry of Education and
Training of Vietnam (MOET) beside the generous scholarship granted during
my stay in Germany.
Last but not least, I am deeply grateful to my beloved family, whose encouragement
and support have continued to be my greatest motivation and for giving me the chance
to realize my dreams. Without them, I would have never achieved lot of things.

Bich Ngoc Nguyen
Braunschweig, Germany, in June 2016


 

II


CONTENTS
CONTENTS
Acknowledgements....................................................................................................................I
List of table……………………………………………………………………………………….…...VI
List of figure………………………………………………………………………………………….VII
List of abbreviations................................................................................................................XI
1. Introduction........................................................................................................................1
1.1. Introduction……..…...…………………………………..………………..…………….…..1

1.2. Objectives and research activities………..………………...…………………..…..…….4
2. Literature reviews…..……………………………………..………………………………….…6
2.1. The German wood industry……..……………………..……………………...…………..6
2.2. Compositions and classifications of wood…………..…………………….....…………12
2.3. The need for wood preservatives..…………………………..………..…………….…..16
2.4. Industrial wood preservative formulations in Germany…………………………….….19
2.5. Selection of the analytes…………………….…………………………..…….…………23
2.6. Environmental impacts of wood preservatives ………...……………………...………34
3. Materials and methods………………….………………………..…………..………………37
3.1. Reference standards and chemicals……………………………..…………...…….…..38
3.2. Sample preparation and extraction………………………………..,…………...………39
3.3. Clean up procedures…………………………………………………………..……….…39
3.3.1. Gel permeation chromatography (GPC) clean up……………………………...39
3.3.2. Clean up with solid phase extraction (SPE)………………………………….….42
3.3.2.1.

Methodological principles………………………………………………...42

3.3.2.2.

The stationary phases of five different cartridges……………….…..…44
III


CONTENTS
3.3.3. The QuEChERS approach…………………………………………………….….46
3.4. High-performance liquid chromatography (HPLC) Instrumentation…..……………..48
3.4.1. Liquid chromatography with variable wavelength detector…………….………48
3.4.2. Liquid chromatography with tandem mass spectrometry (LC/MS/MS)…....…49
3.5. Gas chromatography and mass spectrometry (GC/MS) Instrumentation……..…....57

3.5.1. GC/MS principles……………………………………………………………….….57
3.5.2. GC/MS operation system…………………………………………………….……59
3.6. Guidelines for method development…………………………………………………….59
3.7. Method of validation……………………………………………………………..…….….61
3.7.1. Principal aspects…………………………………………..……………………….61
3.7.2. Efficiency extraction experiments……………………………………………..….62
3.7.3. Accuracy and precision……………………………………………………………63
3.7.4. Method detection limits (MDL) and method quantification limits (MQL)...……64
3.7.5. Evaluation of matrix effects……………………………………………………….64
4. Results and discussions…………………….………..……………………………………...66
4.1. Characterization of methanolic extract of pinewood (Pinus sylvestris)…………......66
4.2. Optimization of the suitable HPLC-DAD conditions………………………..…….……69
4.3. Optimization the LC and MS/MS conditions……………………………………..….....72
4.4. GC/M analysis……………………………………….……………………….……...…….78
4.5. Interference of wood extractives………………………………………….……….....….79
4.6. Clean up with GPC……………………………………………………...…….…………..82
4.7. Clean up by QuEChERS approach……………………………..………....……………84
4.8. Optimization of the clean up procedure with solid-phase extraction……...….…...…85

IV


CONTENTS
4.8.1. Sample pre-treatment………………………………………………………….…..90
4.8.2. Washing procedure…………………………………………………………….…..92
4.8.3. Elution of analytes……………………………………………………...……...…..93
4.9. Comparison of extraction methods………………......……………...………………….95
4.10. Evaluation of matrix effects…………………………………………………………..…99
4.11. Quantification and method validation………………………………………………...104
5. Summary……………………...……………………………………………………………….107

6. References………………………………………………….………………………………....110

V


LIST OF TABLE
LIST OF TABLE
Table 2.1

Proportion of woodland areas in federal states of Germany………………....

6

Table 2.2

Wood species harvested in Germany…………………………………………..

9

Table 2.3

Consumption of wood preservatives per user sector employed in
Germany……………………………………………………………………………

Table 2.4

10

Total wood balance in Germany in million m3 converted into round wood
equivalents………………………………………………………………………..

11

Table 2.5

Chemical compositions of some wood species.……………………………….

12

Table 2.6

Characteristics of hardwoods, softwoods and tropical hardwoods modified
from Rowell, 2004………………………………………………………………..

Table 2.7

15

Hazard classes of biological attack by environmental service conditions
according to EN 335-1……………………………………………………………

18

Table 2.8

An overview of wood preservation portfolio…………………………………….

25

Table 2.9

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

27

Table 3.1

Parameters of GPC clean up system…………………………………………...

41

Table 3.2

Physicochemical properties of SPE cartridges………………………………...

44

Table 3.3

Comparison of electrospray (ESI) with atmospheric pressure chemical
ionization (APCI)…………………………………………………………………..

51

Table 3.4

Scan modes selected for the present study……………………………………

54

Table 3.5

Used scan types in the present work using triple quadrupole mode………...

56

Table 4.1

Parameters for characterization of pinewood sample, of 1 g wood

VI


LIST OF TABLE
extracted with methanol…………………………………………………………..
Table 4.2

67

MS/MS parameters for eight biocides by the multiple reaction monitoring
(MRM) method of LC/MS/MS analysis with ESI (+) mode……………………

75

Table 4.3

Recovery rates (%) of the eight biocides in pinewood extractives…………..

80

Table 4.4

Fractionation of eight active agents and wood matrix at GPC clean up
procedure……………………………………………………………………….….

82

Table 4.5

Commercial sorbent composition of hydrophilic polymeric sorbent…………

89

Table 4.6

The effect of the extraction methods and solvents on target compounds
recoveries from spiked pine sapwood (c = 50 mg/kg, n = 5)…………………

Table 4.7

97

Matrix effects of pinewood samples analysed by SPE using Oasis HLB
cartridges and LC-MS/MS (n = 3)……………………………………………….

100

Table 4.8

Matrix effect, extraction efficiency and process efficiency in pinewood……..

103

Table 4.9

Method detection limit (MDL) and method quantification limit (MQL) of
biocides performed by HPLC/DAD……………………………………………...

Table 4.10

105

Linearity, dynamic range, recoveries, variability, method detection limit
(MDL) and method quantification limits (MQL) of eight selected biocides in
pinewood matrices performed by LC/MS/MS………………………………….

VII

106


LIST OF FIGURE
LIST OF FIGURE
Figure 2.1

Tree species composition in Germany ………………....................................

8

Figure 2.2

Structure of cellulose …………………………………………………………….

13

Figure 2.3

Chemical structure of lignin which is responsible for many of wood
properties…………………………………………………………………………..

13

Figure 2.4

Examples of wood destroying fungi and insects……………………………….

17

Figure 3.1

Analytical scheme of wood analysis…………………………………………….

37

Figure 3.2

Schematic of GPC set up………………………………………………………...

40

Figure 3.3

The SPE procedure……………………………………………………………….

43

Figure 3.4

Sorbents of OASIS cartridges (A) hydrophilic-lipophilic balance copolymer
(B) mixed-mode cation exchange sorbent……………………………………..

45

Figure 3.5

The QuEChERS approach……………………………………………………….

47

Figure 3.6

The HPLC/DAD Instrumentation………………………………………………...

49

Figure 3.7

LC/MS/MS/ESI QTRAP Instrument……………………………………………..

50

Figure 3.8

Schematic view of the GC/MS 6890 GC-MSD 5975C...................................

58

Figure 4.1

UV/VIS-spectrum of pinewood extract in methanol by using DR LANGE
Spectrophotometer CADAS 100………………………………………………...

Figure 4.2

67

IR spectra of the acetonitrile (a) and methanol (b) extract of pinewood
Pinus sylvestris……………………………………………………………………

68

Figure 4.3

The absorption spectra of eight biocides selected of wood preservatives….

70

Figure 4.4

HPLC/DAD chromatogram (λ = 200 nm) obtained for the separation of
eight biocides (c=25 ng/µL) of wood preservatives with Ultrasphere ODS

VIII


LIST OF FIGURE
column……………………………………………………………………………
Figure 4.5

Total ion chromatogram (TIC of +MRM) of eight biocides (c=100 pg/µL) of
wood preservatives with LC (+) ESI/MS/MS analysis…………………………

Figure 4.6

84

Recovery rates (%) of the biocides after SPE clean up procedure with a
standard mixture of eight biocides in methanol (c = 50 ng/µL)………………

Figure 4.11

83

HPLC/DAD chromatograms of the QuEChERS eluates of un-spiked and
spiked pinewood matrix samples………………………………………………..

Figure 4.10

81

HPLC/DAD chromatograms of the GPC eluates (fraction 4 to 9) of unspiked and spiked pinewood matrix samples…………………………………..

Figure 4.9

79

HPLC/DAD chromatograms of a methanol extracts of un-spiked and
spiked pinewood matrix at concentration of 50 mg/kg………………………..

Figure 4.8

75

Total ion current chromatogram of the GC/MS-EI analysis in SIM mode of
eight selected biocides of wood preservatives (c =10 ng/mL)………………..

Figure 4.7

72

86

HPLC/DAD chromatograms of the standard mixture of the target
compounds un-spiked and spiked pinewood matrix samples (50 mg/kg)
after SPE clean up with Oasis HLB cartridges…………………………………

Figure 4.12

HPLC/DAD chromatograms of un-spiked and spiked pinewood matrix (c =

50 mg/kg) samples after SPE clean up with Strata X cartridges…………….

Figure 4.13

88

90

Influence of the solvent modification before sample loading on the
recovery rates of the eight biocides (c = 50 mg/kg) at SPE clean up with
Oasis HLB cartridges……………………………………………………………..

Figure 4.14

Influence of the methanol/water ratio of the third washing solution on the
recovery rates of the eight biocides (c = 50 mg/kg) at SPE clean up with
IX

91


LIST OF FIGURE
Oasis HLB cartridges……………………………………………………………..
Figure 4.15

Influence of elution solvents on the recovery rates of the eight biocides (c =
50 mg/kg) at SPE clean up with Oasis HLB cartridges……………………….

Figure 4.16

95

Recovery rate (%) ± RSD of all selected biocides in spiked pinewood (c =
50 mg/kg) sonication extraction method using different solvents……………

Figure 4.18

94

Flow chart of the final optimization of the SPE clean up procedure with
Oasis HLB cartridge………………………………………………………………

Figure 4.17

93

99

Signal enhancement of IBPC and fenoxycarb in methanol and in pinewood
matrix…………………………………………….…………………………………

X

102


LIST OF ABBREVIATIONS
LIST OF ABBREVIATIONS
ACE

Acetone

ACN

Acetonitrile

Amu

Atomic mass unit

APCI

Atmospheric pressure chemical ionization

API

Atmospheric pressure ionization

AWPA

American Wood Protection Association

BP

Biocidal product

Bw

Body weight

CAD

Collision affected dissociation

CAS

Chemical Abstracts Service

CE

Collision cell energy

CID

Collision induced dissociation

CUR

Curtain gas

CXP

Collision cell exit potential

Da

Dalton

DAD

Diode array detector

DI-SPME

Direct immersion mode solid phase microextraction

DMS

N,N-dimethylsulfamide

DP

Declustering potential

DT50

Disappearance time in days for 50% of the initially applied substance

EC

European Commission

XI


LIST OF ABBREVIATIONS
ECD

Electron capture detector

EEC

European Economic Community

EFSA

European Food Safety Authority

EI

Electron impact ionization

EP

Entrance potential

EPA

Environmental Protection Agency

EPA-RED

Environmental Protection Agency – Reregistration Eligibility Decision

EPI

Enhanced product ion scan

ESI

Electrospray ionisation

ESI (-)

Electrospray ionisation in negative mode

ESI (+)

Electrospray ionisation in positive mode

ET

Ethyl acetate

EU

European Union

FAO

Food and Agriculture Organization

FAL

Federal Research Center for Agriculture

FIA

Flow injection analysis

F.R

Flow rate

Gas 1

Nebulizer gas

Gas 2

Vaporizer gas

GC

Gas chromatography

GMP

Good manufacturing practice

GPC

Gel permeation chromatography

HC

Hazard classes

XII

LIST OF ABBREVIATIONS
HLB

Hydrophobic lipophilic balance

HPLC

High performance liquid chromatography

IDA

Information dependent acquisition

IDL

Instrumental detection limit

IS

Ion spray voltage

ISO

International Organisation for Standardisation

IQL

Instrumental quantitation limit

IUPAC

International Union of Pure and Applied Chemistry

Kd

Sorption or distribution coefficient

Koc

Distribution coefficient based on the organic carbon

Kow

Partition coefficient between octanol and water

LC

Liquid chromatography

LC50

Lethal concentration where 50 % of the tested organisms died

LD50

Lethal dose in which 50 % of the tested organisms died

LLE

Liquid-liquid extraction

m/z

Mass-to-charge

MCX

Mixed-mode cation-exchange

MDL

Method detection limit

ME

Matrix effects

MeOH

Methanol

MRLs

Maximum residue limit

MRM

Multiple reaction monitoring

MS

Mass spectrometry

XIII


LIST OF ABBREVIATIONS
MS/MS

Tandem mass spectrometry

MQL

Method quantification limit

MW

Molecular weight

NPD

Nitrogen phosphorus detector

NOEC

No observed effect concentration

ODS

Octadecylsilane

OECD

Organisation for Economic Co-operation and Development

PE

Polyester

PEC

Predicted environmental concentration

pKa

Dissociation constant

PNEC

Predicted no-effect concentration

Prec

Precursor ion scan

Q

Quadrupole

QqQ

Triple quadrupole

QuEChERS

Quick, Easy, Cheap, Effective, Rugged, and Safe

R

Recoveries

RE

Relative errors

RP

Reversed phase

rpm

Round per minute

RSD

Relative standard deviations

RT

Retention time

SD

Standard deviation

SDB

Styrene-divinylbenzene

XIV


LIST OF ABBREVIATIONS
SPE

Solid phase extraction

UBA

Umweltbundesamt, Federal Environment Agency

UVD

Ultraviolet detector

WHO

World Health Organisation

λ

Wavelength

π

Pi

XV


INTRODUCTION
1.1. Introduction
Since the beginning of the last century until now, many chemicals have been produced in
order to improve human life and make it more comfortable and longer. Within 11 million
known chemicals, 100.000 compounds are being produced on large scale and approximately
30.000 – 70.000 from these chemicals are in daily use in the European Union (EU). These
chemicals are designed and used for different purposes including agriculture and industry.
Every year, approximately 300 million tons of synthetic compounds are used in industrial and
other consumer products. In agriculture, about 140 million tons of fertilizers with several
million tons of pesticides are applied every year. Moreover, a wide variety of these chemical
compounds offer direct improvements in human and animal health. Based on the application
for which these compounds are used, they end up in one of the environmental compartments
(Younes, 1999; Schwarzenbach et al., 2006).
Wood is one of the world’s most important resources, as raw material for industries, for
construction and as fuel. Today, forest covers about 10.6 million hectares, accounting for 36
per cent of Germany’s total land area. It is estimated that about 45 million m3 wood is
harvested per year in Germany, and the main part is used in the building industry (UPMSustainable Forestry, 2010). Although wood and wood products are attacked by many
organisms, principally fungi and termites, they are extensively used in residential
construction, decking, and so on. Consequently, when wood is frequently wetted or placed in

ground contact it should be treated with preservatives to protect it against wood destroying
organisms. Wood preservatives can be divided into water-borne preservatives, organic
solvent-based preservatives, creosote and special types like gases, containing various types
of biocides like fungicides and/or insecticides. The use of environmentally friendly biocides
constitutes an important aspect of modern wood preservatives. However, several problems
exist with the development of more effective totally organic wood preservative systems, one
of which is cost. Many organic biocides have been examined as potential wood
preservatives, but most are about 10-30 fold more expensive per pound than the inorganics
(Schultz et al., 2004). Thus, many organic systems combine two or three biocides to ensure
broad efficacy (Schultz et al., 2007). The main reason for the success of organic biocides in
wood protection is their combination of high efficacy, selectivity as well as long-lasting effect
and versatile application that will provide long-term protection against a wide range of
organisms that damage wood to enhance synergies, and to reduce costs (Schultz and
Nicholas, 2006).
Various organic biocides are used for the protection of wood from pest organisms. Organic
preservatives used in Germany recently and in the past like PCP (pentachlorophenol),
1


INTRODUCTION
lindane, dichlofluanid, tebuconazole, propiconazole (which replaced PCP), permethrin,
fenoxycarb, IPBC, flufenoxuron, triazoles and tributyltin compounds, etc. have very different
structures. In the past PCP was the most common compound to protect wood against
damage by fungi. Lindane, tributyltin, pentachlorophenol and chromate preservatives are
banned now for use as biocides (Regulation (EC) No. 850/2004). Some of them as e.g.
fenoxycarb are relative effective and selective and thus applied in very low concentrations
(0.015 % to 0.05 % in case of fenoxycarb) whereas other agents have to be used in higher
concentration levels. The treatment efficiency may be further enhanced with the use of
pyrethroid insecticides, such as permethrin (Schultz et al., 2008).
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.,
2008). Typically, a mixture of preservatives is used that possesses complementary chemical
and biological and thus protective properties. The amounts of such active ingredients in
treated wood are specified to guarantee protection according to EN 351-1 (2007). Therefore,
quality control by means of chemical analysis is an essential tool to ensure a proper
treatment. The accurate quantitative determination of organic active ingredients in timber
implies special problems as the concentrations of the compounds are often relatively low
and, moreover, they are fixed into the wood matrix. However, as mentioned above, in wood
treating products enhanced chemical diversity is favoured. This will complicate method
development, as mixtures of chemical with different chemical properties have to be
determined. Up to now, studies addressing the quantitative determination of a mixture of
organic wood preservatives with differing chemical functionalities are limited. Most common
organic wood preservatives such as tebuconazole, propiconazole, dichlofluanid, IPBC or
permethrin can be simply and rapid analysed by means of pyrolysis–gas chromatography–
mass spectrometry (Horn and Marutzky, 1994). The standard methods of the American
Wood-Preservers’ Association also involve propiconazole and tebuconazole extracted from
wood with methanol followed by gas or liquid chromatography (AWPA, 2006). For
determination of permethrin or fenoxycarb in timber, the biocide is first extracted with
methanol then either liquid chromatography (LC) with UV detection (LC/UV), gas
chromatography (GC) or gas chromatography with mass spectrum (GC/MS) is carried out
(Schoknecht et al., 2007 and 2009). For the purpose of acquiring knowledge about the
varieties and application ranges of the organic wood preservatives used, to safeguard
consumers’ interests, to protect the environment, and to ensure the quality of wood products,
it is necessary to establish reliable, rapid, inexpensive, and effective analytical methods for
simultaneous determination of the different organic preservatives. 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,
2

INTRODUCTION
it can be very troublesome to determine whether a sample of wood contains preservatives or
not, predominantly because of lack of analytical multi methods. So the main objectives of this
work is to develop a sensitive and effective method that can cover a broad range of important
organic wood preserving agents with differing chemical structure with one analytical multi
method to reduce the analytical effort as much as possible.
So far in laboratory practice, organic wood preservatives are mainly extracted with methanol
from wood and the extracts are analysed without any further clean up steps (Butte et al.,
1992; AWPA, 1997; AWPA, 2006; AWPA, 2008; Schoknecht et al., 2007 and 2009;
Rasmussen et al., 2009 and 2010). Since wood contains a large number of compounds that
can be extracted with organic solvents, the extracts contain numerous co-extracted matrix
components (Kollmann et al., 1968). In the published methods so far, depending on the
wood matrix, the co-extractives are only considered by modifications of the HPLC method
(i.e., gradient program) (AWPA, 2006; AWPA, 2008). Consequently, these extractives might
interfere in case of the quantitative determination of organic active ingredients and thus
reduce the detection sensitivity. In order to increase the detection sensitivity and to assure
the analytical reproducibility, clean up steps are generally necessary. Gel permeation
chromatography (GPC) is used as an effective post-extraction clean up procedure and thus
widely used in residue analysis for eliminating matrix components such as lipids, pigments,
proteins, and polymers that may interfere analytes. Compounds can be separated according
to their molecular sizes, without decomposition or irreversible adsorption in the GPC column.
The GPC technique is appropriate for both polar and non-polar analytes so it can be
effectively used to clean up extracts containing a broad range of compounds. Therefore, the
efficiency of Bio-Beads S-X with an organic solvent to separate multi-pesticide residues in
plant or in fruits and vegetables from matrix components has been extensively documented
(Balinova A., 1998; Cervera M.I. et al., 2010). Therefore, GPC is one of the clean up method
that was tested in this study also for removing the co-extractants from the wood matrices.
Recent publications have reviewed various solid phase extraction (SPE) procedures as clean
up procedure for the multi residue analyses of pesticides in different samples such as plants,
vegetables, foods and also environmental samples (Ramesh et al., 1998; Didier et al., 2004;

Teruhisa et al., 2004 and 2005; Herrera et al., 2005; Cervera et al. 2010). These pesticides
are partly identically with the active agents of the wood preservatives. Single SPE method
was also reported for clean up of wood extracts. Miyauchi et al., 2005 applied Oasis MCX
cartridges in analytical method for the determination of tebuconazole and cyproconazole in
treated wood using HPLC with UV detection. Furthermore, the SPE Oasis MCX cartridges
were used for the quantitative determination of benzalkonium chloride and homologues in
treated wood (Miyauchi T. and Mori M., 2007). Therefore, solid phase extraction (SPE) was
3


INTRODUCTION
also chosen to be tested as clean up and enrichment method for organic active agents from
wood matrices.
1.2. 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 coextracted 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.

4


INTRODUCTION
For the purpose of acquiring knowledge about the varieties and scope of the organic wood
preservatives used, to safeguard consumers’ interests, protect the environment, and ensure
the quality of wood products, it is deemed absolutely necessary to establish several reliable,
rapid, inexpensive, and effective analytical methods for simultaneous determination of the
different organic preservatives. Also, to reduce the analytical effort as much as possible it
would be favourable to develop a method that can cover all importance organic agents with
one analytical multi-method. Thus, the main goal of this thesis was the development and
validation of a sensitive multi analytical method for the simultaneous identification and
quantification of organic wood protecting in wood.
In this sense, the specific approaches of this work were determined to be the major aims of
the present work:
Ø Due to the important of organic wood preservatives in the environment, therefore, the

present research activities focus on the method development to simultaneously analyse eight
selected biocides (Table 2.9) used in wood preservatives.
Ø The particular challenge was to analyse the selected biocides of different polarities in wood
sample matrices of different complexity, i.e., cellulose, hemicellulose, lignin, wood
extractives, etc. at low concentrations.
Ø For analytes enrichment, sample preparation procedures, e.g., extraction and clean up
procedures for complete removal of species that interfere with the determinations were check
to allow for analysis of the target compounds at low concentrations.
Ø The analytical instruments used for accurate qualitative and quantitative analysis of organic
preservatives were HPLC/DAD, GC/MS and also LC/MS/MS. The MDLs and MQLs of target
compounds were determined after fortification experiments.
Ø For analytical quality assurance, repeatability experiments were carried out in order to
assess the accuracy and the day-to-day variation.
Ø Finally, to evaluate the performance of the purposed methods by the analysis of real wood
samples taken from Dr. Matthias Wobst (Braunschweig, Germany) were screened for target
compounds under study.

5


LITERATURE REVIEWS

2.1. The German wood industry
Wood is one of the world’s most important resources, as raw material for industries, for
construction and as fuel in comparison with other materials. So, after agriculture forestry is
the second largest form of land use and almost the most important natural living space in
Germany. Germany ranks among the densely wooded countries in Europe ((BMVLV, 2011).
According to The Food and Agriculture Organization of The United Nations’ Global Forest
Resources Assessment (U.N FAO, 2005 and 2010) forests cover about 11.08 million
hectares, accounting for 31 percent of Germany’s total land area. In regional terms, the

proportion of woodland cover varies widely, ranging from 10 % in Schleswig-Holstein to over
40 % in Rhineland-Palatinate and Hesse, the most thickly wooded Länder (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 (%)

Baden-Wuerttemberg

1,362,299 ha

38.1

Bavaria

2,558,461 ha

36.3

16,000 ha

18.0

1,055,733 ha

35.2

0 ha

0

3,000 ha

4.0

Hesse

880,251 ha

41.7

Mecklenburg-Western Pomerania

534,962 ha

23.1

1,159,522 ha

23.5

Berlin

Brandenburg

Bremen

Hamburg

Lower Saxony

6


LITERATURE REVIEWS

North Rhine-Westphalia

887,550 ha

26.0

Rhineland-Palatinate

835,558 ha

42.1

Saarland

98,458 ha

38.3

Saxony

511,578 ha

27.8

Saxony-Anhalt

492,128 ha

24.1

Schleswig-Holstein

162,466 ha

10.3

Thuringia

517,903 ha

32.0

11,075,799 ha

31.0

Germany as a whole

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

Forests increased by approx. 1 million hectares in Germany over the past four decades. The
percentage of over 80-year old stands also rose from one quarter to one third of the forest
area. The timber stocks in Germany account for 320 m3 per hectare, with the annual timber
increment totaling around 100 mill rapidly separate lipid classes from acetone
extracts of wood and pulp, Tappi Journal Vol. 77, p. 235 – 240.
Chen H. C., Wang P. L., Ding W. H., (2008), Using liquid chromatography–ion trap mass
spectrometry to determine pharmaceutical residues in Taiwanese rivers and
wastewaters. Chemosphere Vol. 72, p. 863 – 869.
Chin-Kai Meng (2008), Determination of pesticides in water by SPE and LC/MS/MS in both
positive

and

negative

ion

modes,

Application

Note

5989-5320EN,

Agilent

Technologies, Inc., USA.
Chocholousˇ P., Sˇatínský D., Sladkovský R., Pospísˇilová M., Solich P., (2008),
Determination of pesticides fenoxycarb and permethrin by sequential injection

chromatography using miniaturized monolithic column, Talanta Vol. 77, p. 566 – 570.
Chupin L., Motillon C., Charrier-El Bouhtoury F., Pizzi A., Charrier B., (2013),
Characterization of maritime pine (Pinus pinaster) bark tannins extracted under
different conditions by spectroscopic methods, FTIR and HPLC, Industrial Crops and
Products Vol. 49, p. 897 – 903.
Commission of European communities (1999), Commission Directive 1999/51/EC of 26 May
1999 Adapting to Technical Progress for the Fifth Time Annex I to Council Directive
76/769/EEC on the Approximations of the Laws, Regulations, and Administrative
Provisions of the Member States Relating to Restrictions on the Marketing and Use of
Certain Dangerous Substances and Preparations.
Commission of European communities (2002), Commission Decision 2002/657/EC
Implementing Council Directive 96/23/EC, concerning the performance of analytical
methods and the interpretation of result, Analysis, p. 8 – 36.
Commission

of

European

communities

(2002),

Commission

directive

2002/63/EC

establishing Community methods of sampling for the official control of pesticide

residues in and on products of plant and animal origin and repealing Directive
79/700/EEC, Official Journal of the European Communities L187, 16/07/2002, p. 30–
43.
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