University of Amsterdam

Faculty of Science

MSc Chemistry

Analytical Sciences

Graduation research thesis

Determination of ethanol metabolites in hair

by LC-MS(/MS) and GC-MS(/MS)

by

Liz Leenders

Supervisors:

dr. W. F. Duvivier

dhr. dr. W. T. Kok

2

List of abbreviations

ORS Outer root sheath

IRS Inner root sheath

SoHT Society of Hair Testing

EtG Ethyl glucuronide

FAEEs Fatty acid ethyl esters

FA Fatty acids

TG Triglycerides

LP Lipoproteins

PL Phospholipids

SPE Solid-phase extraction

MS(/MS) (Tandem) mass spectrometry

GC Gas chromatography

LC Liquid chromatography

m/z Mass to charge

CID Collision induced dissociation

SRM Selected reaction monitoring

MRM Multiple reaction monitoring

EI Electron ionization

LOD Limit of detection

LOQ Limit of quantitation

HS-SPME Headspace solid-phase micro extraction

BSTFA N,N-bis(trimethyl-silyl)trifluoro-acetamide

PFPA Pentafluoropropionic anhydride

HFBA Heptafluorobutyric anhydride

PBA Phenylboronic acid

PCI/NCI Positive/negative chemical ionization

ESI Electrospray ionization

HILIC Hydrophilic interaction liquid chromatography

MMS Matrix-matched standards

MMRS Matrix-matched recovery standards

CCα Decision limit

CCβ Detection capability

3

Abstract Hair analysis is increasingly used over the past years in the detection of drugs in both forensic as clinical

science. It is a sufficient and sensitive method to monitor drugs or alcohol use over a long period of time. When

compared to body fluids, compounds in hair can be detected over a longer timeframe, and segmented hair

analysis could give information about drug and alcohol abuse in a retrospective way. The problem in the case of

ethanol is the fact that ethanol itself cannot be detected in hair due to its high volatility, only its metabolites

are incorporated into hair.

In this research project, a method is developed for the detection of two ethanol metabolites in hair with LC-

and GC-MS(/MS). A method for the analysis of one of the metabolites, EtG, was developed and optimized. The

optimized method consisted of finely cutting 50 mg of hair, extraction with Milli-Q water in an ultrasonic bath

for 4 hours followed by overnight incubation, clean-up by SPE, evaporation and reconstitution for LC-MS/MS.

The method was fully validated with a mean trueness of 99.5%, recovery of 90.55%, CCα of 31.83 µg/L and CCβ

of 63.65 µg/L.

The method was further tested on 2 reference and 42 real hair samples. The method was able to measure

known, large concentrations accurately and is able to detect chronic excessive alcohol consumption (30

pg/mg), but the sensitivity is too low for the detection of regular alcohol consumption (7 pg/mg).

Also, the initial method development for the analysis of both EtG as well as FAEEs with GC-MS(/MS) was

executed. Derivatization of EtG was possible with PBA, and EtG-PBA could be detected in GC-MS/MS, however

the intensity was extremely low. For the analysis of FAEEs, ethyl palmitate is extracted from the hair matrix

with hexane and DMSO, followed by overnight incubation, than clean-up by SPE, evaporation and

reconstitution. These steps are similar to the optimized method for ethyl glucuronide for LC-MS/MS, so a

combined sample pre-treatment could be possible in the future. The method was able to analyse ethyl

palmitate with GC-MS/MS.

Samenvatting Haar analyse wordt steeds vaker gebruikt voor het detecteren van drugs in zowel forensische als klinische

scheikunde. Het is een geschikte en gevoelige methode om drugs en alcohol gebruik over een lange periode in

kaart te brengen. In haar zijn moleculen langer te detecteren dan in andere humane materialen, en door een

haar gesegmenteerd te analyseren kan informatie gewonnen worden op een retrospectieve manier. Het

probleem in het geval van ethanol, is het feit dat ethanol niet te detecteren is in haar omdat het een hele hoge

vluchtigheid heeft. Metabolieten van ethanol echter zijn wel te detecteren in haar.

In dit onderzoeksproject is een methode ontwikkeld om ethanol metabolieten in haar te detecteren aan de

hand van LC- en GC-MS(/MS). Een methode voor de analyse van een van de metabolieten, EtG, was ontwikkeld

en geoptimaliseerd tot het volgende: 50 mg haar wordt heel fijn geknipt, EtG wordt geëxtraheerd met water in

een ultrasoon bad voor 4 uur gevolgd door incubatie gedurende de nacht, de monsters worden behandeld met

SPE, gevolgd door verdamping en her-oplossing voor LC-MS/MS. De methode is vervolgens volledig

gevalideerd met een jusitheid van 99.5%, een terugvinding van 90.55%, een CCα van 31.83 µg/L en een CCβ van

63.65 µg/L.

De methode is getest aan de hand van 2 referentie en 42 echte haarmonsters. De methode was in staat om

bekende, hoge concentraties van EtG nauwkeurig te meten, chronisch alcohol gebruik te detecteren (30

pg/mg), maar had helaas een te lage gevoeligheid om regelmatig alcohol gebruik te detecteren (7 pg/mg).

Vervolgens is er geprobeerd om een methode te ontwikkelen voor het detecteren van zowel EtG als FAEEs met

GC-MS(/MS). Het derivatiseren van EtG bleek mogelijk met PBA, en een EtG-PBA complex kom gedetecteerd

worden met GC-MS/MS, echter met een extreem lage intensiteit. Voor de analyse van FAEEs is een methode

gebruikt waarin ethyl palmitaat geëxtraheerd wordt uit het haar door middel van hexaan en DMSO gevolgd

door incubatie gedurende de nacht, de monsters worden behandeld met SPE, gevolgd door verdamping en

her-oplossing voor GC-MS/MS. Deze stappen tonen veel overeenkomsten met de geoptimaliseerde methode

voor de detectie van EtG met LC-MS/MS, dus een gecombineerde monstervoorbewerking zou een mogelijkheid

kunnen zijn in de toekomst. De methode was in staat om ethyl palmitaat te detecteren met GC-MS/MS.

4

Preface The main goal of this master research project is to develop a method to detect ethanol metabolites

in human hair. Hair analysis is a relatively simple, sufficient and sensitive method to monitor alcohol

use over a long period of time. In the last few years, research in the field of hair analysis is rising, also

within RIKILT Institute of Food Safety.

Two ethanol metabolites are studied, and a method to detect these metabolites with both LC-

MS/MS and GC-MS/MS was developed. Optimization and validation of the developed method should

provide us with more insight in the possibilities of hair analysis for detection of alcohol (ab-)use.

Chapter 1 of this thesis is an introduction into hair and hair analysis, ethanol metabolites and the

techniques available for hair analysis. Also, the objectives of this project are described in this chapter.

In Chapter 2, the initial method development for the analysis of one ethanol metabolite is given.

Chapter 3 describes the optimization and validation of one of the methods developed. In Chapter 4

the initial method development of the other ethanol metabolite is described. Chapter 5 gives

recommendations for future research, and general conclusions are stated in the last chapter,

Chapter 6.

All the research described in this thesis is carried out at RIKILT Institute of Food Safety in

Wageningen, under daily supervision of dr. W.F. Duvivier.

5

Table of Contents List of abbreviations ................................................................................................................................ 2

Abstract ................................................................................................................................................... 3

Preface ..................................................................................................................................................... 4

1. Introduction ......................................................................................................................................... 6

1.1 Hair and hair analysis .................................................................................................................... 6

1.2 Detection of alcohol (ab)-use ...................................................................................................... 11

1.3 Analysis techniques ..................................................................................................................... 13

1.4 Aim of this project ....................................................................................................................... 18

2. Initial method development for the analysis of EtG in hair by GC-MS/MS and LC-MS/MS .............. 19

2.1 Introduction ................................................................................................................................. 19

2.2 Materials and methods ............................................................................................................... 19

2.3 Results and discussion ................................................................................................................. 22

2.3.1 Method development for LC-MS/MS ................................................................................... 22

2.3.2 Method development for GC-MS/MS .................................................................................. 27

2.4 Practical obstacles ....................................................................................................................... 29

3. Optimization and validation of LC-MS/MS method for EtG in hair ................................................... 32

3.1 Introduction ................................................................................................................................. 32

3.2 Materials and methods ............................................................................................................... 33

3.3 Results and discussion ................................................................................................................. 35

3.3.1 Optimization ......................................................................................................................... 35

3.3.2 Validation ............................................................................................................................. 38

3.3.3 Reference and real hair samples .......................................................................................... 42

4. Initial method development for the analysis of FAEEs in hair with GC-MS/MS ............................... 46

4.1 Introduction ................................................................................................................................. 46

4.2 Materials and methods ............................................................................................................... 46

4.3 Results ......................................................................................................................................... 47

5. Recommendations for future research ............................................................................................. 48

5.1 Sensitivity LC-MS/MS .................................................................................................................. 48

5.2 Derivatization of EtG for GC-MS/MS and sensitivity ................................................................... 48

5.3 Sensitivity FAEEs with GC-MS/MS ............................................................................................... 48

5.4 Combining sample pre-treatment steps ..................................................................................... 49

6. General conclusions .......................................................................................................................... 50

References ............................................................................................................................................. 52

Attachments .......................................................................................................................................... 56

6

1. Introduction The use of drugs is a part of human culture for decades, and attempts to ban drugs have failed

multiple times in history. Drugs are used on a social or recreational base, but also in an abusing way 1. This uncontrolled usage could have physical impact on a human being, essential organs could be

damaged, and it could play a role in crimes. So, it is important to know the effects of drugs on an

individual, and his or hers general usage of drugs, from both a forensic and a clinical point of view 1.

Hair, blood, urine and oral fluid are all human materials used for toxicological analysis. Traditional

biomarkers are mainly determined in blood (serum or plasma). Hair as a matrix for monitoring

chronic consumption is a powerful evidential tool that has been relied upon in many criminal cases

during the last decade 2. Hair analysis in forensic science is increasingly important, since the

difference between hair and the other materials is its substantially longer detection window in the

order of weeks to several months, compared to that of body fluids (hours to days). Also, the high

stability of hair samples and the fact that it can be stored at room temperature for a long time,

allows hair analysis to be performed even centuries after growth. This makes it possible to

investigate chronic or past consumption of drugs in a retrospective way 1. Hair as a testing matrix

could provide a historical overview of the individual’s exposure to drugs following chronic use, but it

is also possible to detect drugs after a single exposure, which is a great advantage above other

human materials 2. Hair collection is a simple and non-invasive procedure, and the potential for

manipulation of the results is low. Furthermore, there is little to no biosafety risk in handling the

samples, in terms of transmission of diseases 1.

1.1 Hair and hair analysis

The entire skin of the human body is covered in hair, except the outer parts of the lips, feet soles and

hand palms, and some surfaces of the external genitalia 3. Hair is used worldwide for diagnostic

purposes as testing drugs 4, and for the determination of metal concentrations related to sex and

age, for instance in poisoning cases 5. Hair can also provide information about the identity or lifestyle

of its owner, moreover it can be used in diagnosing health disorders 6.

1.1.1 Structure of the human hair

Hairs are derivatives of the epidermis, so the outermost layer of the human skin. Hairs are basically

thin and flexible tubes for 90% made of dead and keratinized cells. Inside the skin hair follicles are

alive, they grow into the dermis and hypodermic fat 7. Macroscopic, human hairs vary among ethnic

groups and individuals in length, colour, diameter and shape 4. In the human hair two different

structures could be separated; hair follicles inside the skin and the ones in the hair shaft visible on

the skin. These structures are shown in Figure 1.

As can be seen in Figure 1B, a hair shaft is made up of a medulla in the centre, surrounded by the

cortex and cuticle cells. The cortex represents most of the hair fibre composition and is an important

part in describing the strength, but also the colour and the texture of human hair. It consists for

roughly 50 to 60 percent of macro fibrils, which consist of cylinders of micro fibrils fixed into a matrix 8.

The cuticle forms the outer part of the hair shaft, its purpose is to cover the hair completely from

root to tip to protect the cortex, and to protect the inner regions of the hair 8. It consists of flat

overlapping cells 6, each cell has a thickness of approximately 0.3 – 0.5 µm. The visible length of the

cuticle is roughly 50 µm and it is made up of several substructures, for instance the epicuticle,

exocuticle and endocuticle, and the cell membrane complex, as shown in Figure 1B. The medulla is

only present in really thick hair, and is made up of empty space 9.

7

Figure 1: A: Formation of hair in a follicle from matrix cells on the basement membrane to the mature hair shaft. B: Structure and constituents of the human hair shaft. Reproduced from Ref. 10.

In Figure 1A, it is shown that hair growth is induced from the follicle. The follicle is the most

important growth structure of the hair. It is a skin organ with a bulb shape, which has two histological

structures; the outer root sheath (ORS) and the inner root sheath (IRS).

The first structure, the ORS, is a part of the epidermis that can be described as some sort of room for

stem cells, such as keratinocyte and melanocyte cells. It surrounds the IRS 7. A part of the ORS, on the

dermal site, consists of two orthogonal layers of collagen fibre, this layer is also known as the dermal

sheet 11.

The IRS consists of three layers, of which one is the cuticle layer. The cuticle layer connects to the

cuticle of the hair shaft, to make sure the hair shaft stays in the skin and the follicle will be

connected. The IRS separates the hair shaft from the ORS, and it produces hormones and keratins to

give strength to the growing hair shaft 7.

The hair bulb can be described as the part of the follicle that produces the hair; the cells in the

bottom of the hair divide just above the papilla 12. This division induces upward movements of the

cells, they are transported to the keratogenous zone in which they are synthesized and keratinized 13.

During the keratinization part, the proteins that are formed bind together, which results in long

fibres 14.

1.1.2 Hair growth cycle

A hair is developed in a dynamic and cyclic process. Many hormones and cytokines coordinate the

growth cycle of a hair. The duration of this cycle is not only dependent on the place of the body the

hair is growing, but also on other factors as age of the individual, its diet, or environmental

alterations such as day length 8. Hormones and cytokines are the particles in the human hair that

instruct the follicle to change, and each hair on the human body could be in a different stage of the

growth cycle at a time 15.

In the hair growth cycle, three phases could be noted: the anagen or growth phase, the catagen or

transitional phase, and the telogen or resting phase (Figure 2). In these phases, the hair follicle

undergoes stages of fast growth and formation of the hair shaft, followed by stages in which the hair

follicle growth is inactive 16,17.

8

Figure 2: Different phases of hair cycle. Reproduced from Ref. 3

In the anagen phase, the hair follicle is in the active growth phase. The follicle grows until it reaches

its typical onion shape, and a hair fibre is produced, as shown in Figure 2. This phase could be divided

into six small stages; the first five stages of the anagen phase are called the proanagen phase, in

which the dermal papilla grows downwards into the skin and differentiates to the hair shaft and the

IRS. Cells in the new hair shaft are divided rapidly, causing growth of the hair. In the sixth phase, the

metanagen phase, the epithelial hair bulb, which surrounds the dermal papilla, is formed, and the

hair root embeds deeply into the dermis and sub cutis 3. The new hair shaft now appears on the

surface of the skin. The anagen phase could last for 3-6 years, and 80-90% of scalp hair is in this

phase 7.

After the anagen phase comes to an end, the catagen phase starts. This phase is also called the

regression phase, and in the beginning of this phase the hair shaft production is completed and the

cell division stops. The hair follicle undergoes regression and is fully keratinized, this results in a

reduction of the follicle of about one-sixth of the diameter. During the catagen phase, a keratinized

structure of the hair looking like a brush, which is called the club hair, is formed on the base of the

hair shaft 3. The catagen phase last for 2-3 weeks, and only 1-2% of scalp hair is in this phase 7.

After the catagen phase the telogen phase starts. In this phase, the hair goes into rest and stops

growing completely. This period can last from a few weeks for eyelashes to a few months for scalp

hair 3. The root of the hair is embedded in the follicle, and approximately 10-15% of all hairs are in

the telogen phase at any given moment 7. At the end of this stage, the hair can be considered dead

and falls out, which is called the exogen phase; a few weeks later, the hair follicle starts again in the

anagen phase 14.

1.1.3 Collection of a scalp hair sample

In hair analysis, scalp hair is preferred since scalp hair has the fastest growth rate, and its follicles

have the highest percentage in the anagen, growing, phase. Therefore, in this paragraph some

features of scalp hair will be summarized, as well as the correct procedure for sampling.

The growth rate of scalp hair is reported as 1 cm per month, continuously for 3-5 years while the

hairs are in the anagen phase 18. After the anagen phase, the hair growth stops and is followed by a

short catagen phase and a telogen phase of approximately 200 months. In this phase, the old hair is

shed, and after the telogen phase the new hair starts to grow from the same follicle during the next

anagen phase.

A scalp hair fibre is approximately 60 – 80 µm in diameter. It consists of a layer of flat and imbricated

scales that point from root to tip of the hair 8.

To avoid errors in the analytical method, proper sampling techniques are required. A scalp hair

9

sample must be cut from the posterior vertex region of the head (Figure 3); this is the region with

less variation in the growth rate, and it must be cut as close as possible to the scalp 19. Locks of hair

with the thickness of a pencil must be collected, with a diameter of about 0.3 – 0.5 cm, and the root

end must be clearly indicated 19. Two locks of hair must be collected to allow initial testing, followed

by confirmatory or re-testing of the sample if necessary 3. To obtain a timeline of drug use, the hair

can be cut into segments starting from the hair root, usually 1 – 3 cm pieces, and these segments are

analysed individually, as shown in Figure 3.

Figure 3: Left: Vertex region of the scalp. Right: Retrospective timeline of drugs in hair. Reproduced from Ref. 1

1.1.4 Hair analysis

As a consequence of the advantages of hair analysis mentioned in the first part of this chapter, it is

routinely used in forensic toxicological investigations, and it could be used in both post-mortem as

alive cases. It is for instance used in drug-related crimes and deaths or child protection cases, and to

monitor drug misuse in workplace drug testing or drug rehabilitation programs. The use of hair

analysis is not limited to human samples, it is also used in veterinary control 1. More and more

laboratories offer hair testing, and the Society of Hair Testing (SoHT) recommends official guidelines

for drug testing in hair 20. Instrument sensitivity and method validity are continuously improving, but

the main challenge is interpretation of the analytical results in evidence cases 1. This challenge is due

to many factors that affect the amount of drugs present in hair, its degradation over time and the

different routes of incorporation.

Researchers were able to detect drugs in hair centuries after growth, for instance arsenic in hair

collected from Napoleon Bonaparte 21 and cocaine in the hair of Peruvian mummies 22. However, the

concentrations of drugs in hair decrease over time because of natural wash out 23, and the stability is

dependent on the physicochemical properties of the hair 24.

1.1.5 Challenges in hair analysis

Contamination is the most important issue in hair analysis; the fact that analytical results could be

interpreted as false negatives or false positives should be avoided 1. Alcohol markers are present in

several commercially available hair products. Traces of these compounds were detected in hair

samples of people who frequently used the products 25. Decontamination of the hair sample before

analysis is thus of great importance in order to avoid false positives.

Weather conditions, such as exposure to sunlight, could reduce the concentration of several drugs in

hair on a daily basis 26. Also, hair treatment could cause a reduction of the concentrations.

Shampooing was shown to not significantly affect the concentration of drugs in hair 27,28, but blow-

drying, curling or straightening can damage or destroy the cuticle, which provides routes for

contamination, and it can be responsible for removal of incorporated drugs. Harsher cosmetic

treatments could cause even more damage 19,27,29; all literature studies agree on the fact that

bleaching, dyeing or perming the hair have a destructive effect on the concentrations of drugs in hair.

10

Bleaching created the largest decrease in drug concentrations. These factors influencing the

concentration, or the incorporation of drugs into the growing hair, need to be fully understand by the

analyst, and the results of the hair analysis needs to be fully interpret in order to make the correct

conclusion in criminal cases 1.

1.1.6 Mechanisms of drug incorporation

Up to the present day, the exact mechanism for the incorporation of drugs into hair is not fully

understood, as well as the factors that could influence their stability. Drugs inside the body are

believed to incorporate into the human hair in times of increased activity in metabolism and cell

division during the anagen growth phase 31. However, there are three different ways for the

incorporation of drugs in hair. These three routes are shown in Figure 4, the first one is directly from

the blood supply, the second route is from sebum or sweat bathing the hair, and the last one is from

external contamination 32,33. It is unclear which route contributes to what extent to the drugs

incorporation in hair, but what is known is the fact that it varies from drug to drug 34.

Figure 4: Incorporation routes into the hair follicle. Reproduced from Ref. 1

Scientists proposed several models for the incorporation of drugs, trying to explain the different

drugs profiles in human hair. The first model, which is the most simplistic one, describes the diffusion

of drugs as a passive process, in which the diffusion is directly from the blood supply to the hair

follicle. The diffusion process is passive, which means that the concentration of the drugs in hair are

expected to be correlated to the drug concentrations in blood at the time of the analysis 35. However,

the metabolic profiles in hair and blood are different; in blood, detection of the parent drug is less

common when compared to its primary metabolites, whilst in hair the parent drug is more

commonly detected.

To explain the incorporation of drugs in growing hair in an endogenous way, the ‘biochemical

concept’ was described by Baumgartner et al. 36. In this model, the ratio of the parent drug to its

metabolites in hair is explained. Other subjects in this model are an explanation of the

physiochemical properties of drugs and their dependency on the incorporation, incorporation of a

drug in hair that is not pigmented and the dependency of drugs on hair pigmentation. Using this

model, it became clear that the affinity and binding capacity of hair is different for each drug,

including a different binding mechanism to the hair matrix 37. Another factor influencing the

11

incorporation or binding of the drugs to human hair is the lipophilicity; molecules that are neutral

and lipophilic can penetrate the cell membrane easier than others, which results in a higher

incorporation rate 38.

Examining all existing drugs is beyond the scope of this thesis, so in this thesis the focus will be on

ethanol metabolites incorporated into hair. Different analytical techniques and approaches will be

tested and compared, and factors influencing the incorporation of the metabolites in hair are

examined.

1.2 Detection of alcohol (ab)-use 1.2.1 Ethanol metabolites in hair

For decades, the clinical and forensic research field has been searching for suitable markers for

alcohol consumption. Due to its high volatility, alcohol detection in hair is difficult. However,

metabolites of ethanol are detectable in hair 39.

An overview of possible ethanol markers in hair is given in Scheme 1. Ethanol markers could be direct

or indirect. The direct markers are the markers still containing the carbon atoms of ethanol, the

indirect ones are a product of a pathological change caused by ethanol in the metabolism. Overall,

direct markers are preferable, because of the fact that indirect markers could also be formed via

other pathologic pathways. The direct markers in hair are metabolites of ethanol which still contain

the C2H5-group, or products which were formed during a reaction of acetaldehyde with physiologic

molecules 40.

In this thesis, the focus will therefore be on direct markers. The ethanol markers most studied in

literature are ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs). They have the advantage

that they contain the ethyl group of ethanol which is a great indication that alcohol was responsible

for the positive result 1. These markers will be discussed below in more detail including mechanisms

of drug incorporation in hair. The probability to find these ethanol markers in hair will be discussed

regarding their formation and concentrations in hair, and some of their chemical properties will be

discussed.

Scheme 1: Possible markers of chronically elevated alcohol consumption in hair. Reproduced from Ref 39.

12

1.2.2 Ethyl glucuronide (EtG)

In 1952, EtG was described by Kamil et al. as a minor metabolite of ethanol 41. Investigation of EtG as

important marker of alcohol consumption was mainly performed by Skopp et al. 42. EtG is a direct

metabolite of ethanol, and is a water-soluble and non-volatile compound with high stability 43. EtG is

more sensitive and specific than most ethanol metabolites, because of the fact that EtG is only

detectable in human hair after ethanol intake 44. In Scheme 2, the formation of EtG is shown. Ethanol

is conjugated with glucuronic acid via Uridine 5'-diphospho-glucuronosyl transferase (UDP-

glucuronosyl transferase). Glucuronic acid is located in the endoplasmic reticulum, in liver cells and in

small amounts in for instance the lungs. Glucuronic acid is formed by an enzymatic hydroxylation in

the presence of UDP-glucuronosyl transferase, and is necessary in the glucuronidation step of

ethanol 45. The molecular formula of EtG is C8H14O7, its monoisotopic molecular weight is 222.074

g/mol. The melting point or decomposition temperature is approximately 150 °C 1.

Scheme 2: Formation of ethyl glucuronide by conjugation of UDP-glucuronic acid and ethanol.

The incorporation rate of EtG in hair is low, because the molecule is an acid; EtG is a polar molecule

with a pKa of 3.21. As a consequence, EtG is only detectable in hair in very small amounts and thus

requires very powerful analytical techniques for detection 46. In order to establish universal

interpretation of analytical results for EtG in hair, the SoHT has set some cut-off values for

international use in 2010. A value of 30 pg/mg or more suggests chronic excessive consumption of

alcohol, and a second cut-off value was established in the consensus of 2012 and has not change

since; a value of ≥ 7 mg/mg strongly suggest repeated alcohol consumption. Another rule to

remember is the fact that a positive result for EtG in hair overrules a negative result for FAEEs 18.

1.2.3 Fatty acid ethyl esters (FAEEs)

In Scheme 3, the formation of FAEEs is shown. As can be seen, ethanol undergoes esterification with

free endogenous fatty acids (FA), triglycerides (TG), lipoproteins (LP) and phospholipids (PL) to form

FAEEs in the presence of two enzymes, FAEE synthase and acyl-co-enzyme A(CoA)/ethanol O-acyl-

transferase. FAEE synthase can be found in organs most likely to be damaged by alcohol

consumption; the pancreas and the liver 47. The FAEEs most likely to be formed are ethyl palmitate

and oleate 48, however, all esters are stable and can be found throughout the body 49.

In 1956 the process of the biotransformation of ethanol was described, in which FAEEs were shown

to be products of this reaction 50. However, until 2001 nobody described FAEEs as possible markers

for ethanol in hair, Pragst et al. were the first to do so 51.

13

Scheme 3: Formation of fatty acid ethyl esters (FAEEs). FA: fatty acids. TG: triglycerides. LP: lipoproteins. PL: phospholipids.

Preliminary studies revealed the fact that FAEEs can be found in human hair samples without

exposure to alcohol, which results in false positive results. The exact reason is unclear, but it can be

due to some physiological and pathological processes 1. Because ethanol is a by-product of some

physiological metabolism reactions in the human body, it is possible that some FAEEs could be

detected inside the human body without actual alcohol intake 43.

In order to interpret the results correctly, the SoHT also established cut-off values for FAEEs. The cut-

off values were based on the sum of the four major FAEEs, which are ethyl myristate, palmitate,

stearate and oleate. In 2016, the last consensus of the SoHT, a new cut-off value was established; the

sum of the four FAEEs is no longer necessary, only the FAEE most likely to be formed, which is ethyl

palmitate, is measured in the new consensus. Repeated alcohol consumption could be assumed with

a cut-off concentration of 0.12 ng/mg of ethyl palmitate in the first 0-3 cm hair, or 0.15 ng/mg in the

first 0-6 cm hair. A cut-off concentration of 0.35 ng/mg of ethyl palmitate in the 0-3 cm hair segment,

and 0.45 ng/mg in the 0-6 cm segment indicates chronic excessive alcohol consumption 52.

1.3 Analysis techniques After hair sample collection, the samples need further pre-treatment in terms of extraction of the

targeted compounds from the matrix. Hair samples are cut into small fragments of typically 1 to 3

mm, in order to obtain a larger surface area. Attentively, the samples could be pulverized.

Compounds can be extracted from the hair matrix with organic solvents or aqueous solutions. Which

specific solvent to use is dependent on the physicochemical properties of the compound 1. After

extraction, the sample needs a clean-up and concentration step like solid phase extraction (SPE),

after which the sample can be analysed. Analytical methods for the determination of EtG and FAEEs

in hair were described in several papers 53-56, and often these methods only differ in used

instruments or chromatographic parameters measured. Mass spectrometry (MS) has been widely

accepted as the detection method of choice, and is therefore used in most hair analysis methods.

Before compounds can be detected using MS, they most often are separated using gas

chromatography (GC) or liquid chromatography (LC). Analytical techniques used in hair analysis

should contain some important properties; they must be suitable for explicit identification and

quantitation of the drugs. Chromatographic techniques are powerful screening methods, by their

ability to cover a wide range of analytes in one run 1. Due to the fact that hair contains low

concentrations of drugs and the sample sizes are small, developing a general method for drug

screening in hair is difficult. This is mainly because every compound or drug class needs a specific

14

sample preparation. Therefore, the procedures described for drugs screening are typically developed

for the analysis of a limited number of drugs at the same time 57,58.

1.3.1 Mass spectrometry (MS)

In hair analysis, the mass spectrometric analyser most commonly used is the quadrupole analyser.

Movements of molecules in oscillating fields are stable, and a quadrupole analyser makes use of this

fact to separate ions according to their mass to charge (m/z) ratios. Quadrupole analysers are made

up of four rods of circular section, as shown in Figure 5. The rods must be perfectly parallel 59.

Figure 5: Schematic overview of a quadrupole analyser. Reproduced from Ref 60.

A positive ion entering the space between the rods will be drawn towards a negative rod, and the

other way around. If the potential changes from sign before the ion comes in contact with the rod

and discharges itself, the ion will change direction. An ion without resonance will not reach the

detector in the end, an ion with resonance does 59.

Figure 6: Diagram of a triple quadrupole instrument. Reproduced from Ref. 61

In hair analysis, a triple quadrupole spectrometer is often used. In this spectrometer, three

quadrupoles are next to each other, as shown in Figure 6. The first and last quadrupole (Quad 1 and

Quad 3) are mass analysers. The quadrupole in the centre, Quad 2, is used as a collision cell in which

the dissociation takes place. In Quad 1 the precursor ion is selected and afterwards dissociated into

product ions in Quad 2, followed by a mass scan in Quad 3 61.

In tandem mass spectrometry (MS/MS), a product ion scan could be used to determine the fragment

ions of a compound. The product ion scan, also called daughter scan, selects a precursor or parent

ion of a specific m/z ratio, and afterwards the product or daughter ions are determined based on

collision-induced dissociation (CID) 59, as shown in Figure 7A.

15

Figure 7: Product ion scan (A) and selective reaction monitoring (B) in MS/MS. Reproduced from Ref. 59

Usually, for sensitive and targeted analysis, the detection mode selected reaction monitoring (SRM)

is used. In this mode, ions with a specific m/z ratio are selected and isolated through the collision

cell, as shown in Figure 7B. After CID only fragment ions with a certain m/z ratio are lead into the

detector. This way, the ions selected by the first mass analyser are only detected if they produce this

fragment ion with a certain m/z ratio, by a selected reaction. This means that no time is wasted on

data that is not relevant for the analysis, so the sensitivity comes to a maximum 59.

SRM can also be performed by selecting multiple fragment ions before analysis, this technique is

known as multiple reaction monitoring (MRM).

1.3.2 Gas chromatography – mass spectrometry (GC-MS)

One of the methods frequently used in hair analysis is gas chromatography coupled to mass

spectrometry (GC-MS). GC makes use of a capillary column, this is one of the reasons it is a method

with a high resolution 73. A GC-MS electron ionization (EI) mass spectrum has a really high specificity;

especially if SRM mode is used in the measurement. High specificity can also be achieved by using

internal standards that are deuterated 73. All these features combined make GC-MS a very specific

and sensitive analysis technique for the detection of many drugs and its metabolites. If the

chromatogram is divided into several time windows with different SRM masses, a large number of

compounds can be measured at the same time in one run. Usually, the limit of detection (LOD) of GC-

EI/MS is around 0.03 ng/mg for most drugs 1.

If GC-MS/MS is used, the sensitivity and specificity will be increased when compared to GC-MS.

These advantages lead to the fact that GC-MS/MS is used more often nowadays in hair analysis 62-65.

The sensitivity of the method can also be increased by making use of headspace solid-phase micro

extraction (HS-SPME), in which a derivatized sample is cleaned up by headspace extraction instead of

regular SPE, increasing the analysed amount of sample by accumulation on the fibre 66.

1.3.3 Analysis of ethyl glucuronide using GC-MS

To perform GC-MS, it is essential that the compound of interest is volatile enough and stable at very

high temperatures. If a compound contains free amino (NH2), hydroxyl (OH), or carboxyl (COOH)

groups, these groups are able to form hydrogen bonds inside the compound so that the volatility

goes down and it does not interact with, for example, the column packing 67. These chemical

16

properties are reasons why it is necessary to derivatize the compound before performing GC-MS.

Derivatization reactions used for GC-MS fall into three general reaction types namely; (i) alkylation of

which the general process is esterification, (ii) acylation and (iii) silylation 68. Acylation and silylation

are the two processes used in derivatization of EtG samples.

Silylation

N,N-bis(trimethyl-silyl)trifluoro-acetamide (BSTFA) is a derivatization agent used often for

derivatization of EtG samples. BSTFA is a silyl reagent, which reacts with both hydroxyl as carboxyl

groups in a compound, in order to form trimethylsilyl ethers and trimethylsilyl esters respectively.

BSTFA contains a trifluoroacetyl group, which makes a reaction with BSTFA fast and complete. The

derivatives formed with BSTFA have high volatility and can be easily separated 69. In Scheme 4, the

derivatization of EtG with BSTFA is shown.

Scheme 4: Derivatization of EtG with BSTFA.

Due to the high volatility of derivatives formed with BSTFA, they will elute early, and the derivatives

of BSTFA result in a low detector noise and pollution, since BSTFA is able to derivatize all OH-groups

of EtG 70. No water must come in contact with the samples, as this will lead to hydrolysis of BSTFA,

which leads to the fact that none of the targeted analytes will undergo derivatization.

Acylation

Another type of derivatization used often for EtG samples is acylation. It is a type of reaction in which

an acyl group is introduced to an organic compound. In case of a carboxyl group, the reaction

involves the introduction of the acyl group and loss of the OH - group 71. The two most often used

acylation derivatization agents are fluorinated anhydrides such as pentafluoropropionic anhydride

(PFPA) and heptafluorobutyric anhydride (HFBA). Fluorinated anhydrides such as PFPA and HFBA

could react with amines, alcohols and phenols, producing stable derivatives with high volatility 72.

Scheme 5 and 6 show the derivatization of EtG with PFPA and HFBA, respectively.

Scheme 5: Derivatization of EtG with PFPA.

17

Scheme 6: Derivatisation of EtG with HFBA.

Using GC-MS in chemical ionization mode, both positive and negative (GC-PCI/MS and GC-NCI/MS),

could increase the sensitivity of the method to an LOD between 0.2 and 15 pg/mg for compounds as

benzodiazepines 73, or derivatized,perfluorinated carboxylic acid anhydrides compounds such as EtG 74. Overall, for EtG, NCI delivers better results, but the consequence is the fact that the specificity will

be less due to missing fragmentation.

1.3.4 Analysis of fatty acid ethyl esters using GC-MS

FAEEs also need some sample pre-treatment before performing GC-MS, in the forms of SPE or

headspace solid-phase micro-extraction (HS-SPME). GC-EI/MS is the method most often used in the

analysis of FAEEs in hair, leading to an LOD as low as 0.01 ng/mg for the sum of all four FAEEs, though

nowadays only ethyl palmitate is used. In Scheme 5, the formation of fragment ions of FAEEs is

shown. Molecular ions are used as qualifiers, the quantifiers are the fragment ions from McLafferty

rearrangement (m/z 88) or from β-cleavage (m/z 101) 1, as shown in Scheme 7.

Scheme 7: Formation of fragment ions of fatty acid ethyl esters (FAEEs).

1.3.5 Liquid chromatography – mass spectrometry (LC-MS) Since GC-MS is dependent on derivatization of the compound to avoid issues with volatility and

stability, a promising alternative is liquid chromatography – mass spectrometry (LC-MS) 1. However,

LC-MS is still not used as often as GC-MS because of the fact that it is a very expensive technique. LC-

MS has a lower chromatographic resolution compared to GC-MS for several compound classes. This

is the reason why LC is only possible if coupled to tandem MS/MS in hair analysis, in order to reach a

high sensitivity 1. Liquid chromatography is normally used for compounds that are not volatile and

are not suitable for gas chromatography 59.

18

1.3.6 Analysis of ethyl glucuronide using LC-MS

Several publications describe the use of LC-MS/MS in hair analysis, including the detection of several

drugs as methadone and metabolites 75, benzodiazepines 73, neuroleptics 76, sildenafil 77 and EtG78.

EtG is an anion, so the most often used ionization technique is electrospray ionization (ESI) in

negative ion mode. The transitions most often used are m/z 221 – 75 as quantifier and m/z 221 – 85

as qualifier. If reversed phase columns are used, EtG is eluted in the first part of the chromatogram,

with large interfering signals and matrix effects 79. Further optimization of the chromatography

showed that a high water content in the mobile phase was necessary to retain EtG, as well as post

column addition of acetonitrile to enhance a low ESI yield 80. It was found that these difficulties can

be solved by making use of other columns, such as hydrophilic interaction liquid chromatography

(HILIC) columns 79,81, or silica columns with a mobile phase containing a high content of acetonitrile 82. Many papers perform hair analysis by directly injecting the hair extract on the column, but in

order to avoid false negative results caused by ion suppression, a clean-up by SPE is recommended 1.

1.4 Aim of this project Hair analysis is increasingly used over the past years in the detection of drugs in both forensic as

clinical science. The detection window of hair is longer when compared to body fluids, and if a hair is

analysed in a segmented way, it could give information about drug and alcohol abuse in a

retrospective way, so it is a sufficient and sensitive method to monitor drugs or alcohol use over a

long period of time. The problem in the case of ethanol is the fact that ethanol itself cannot be

detected in hair due to its high volatility, only its metabolites are incorporated into hair.

In this project, a method is developed for the detection of two ethanol metabolites in hair with LC-

and GC-MS(/MS). A method for the analysis of one of the metabolites, EtG, was developed,

optimized and validated. Validation of the method is important to make sure the method has a

sufficient limit of detection and quantification (LOD and LOQ), accuracy and repeatability. Therefore,

a validation protocol following the quality system of RIKILT is used, in which the used method is

evaluated.

Also, the initial method development for the analysis of both EtG as well as FAEEs with GC-MS(/MS)

was executed. Analysis of both metabolites at the same time is not possible, since the clean-up is

different for each of them. It was tried to combine some steps of the analysis, so that it takes less

time when compared to a separate analysis of each metabolite.

19

2. Initial method development for the analysis of EtG in hair by GC-

MS/MS and LC-MS/MS

2.1 Introduction First step in this project was to develop a method for the detection of EtG with GC-MS/MS and LC-

MS/MS.

EtG is a compound which contains three hydroxyl (OH) and one carboxyl (COOH) groups; these

functional groups are able to form hydrogen bonds inside a compound, and could affect its volatility

and the tendency to interact with, for example, column packing material 82. These chemical

properties are reasons why it is necessary to derivatize the EtG samples before performing GC-

MS/MS. As already mentioned in the introduction, acylation and silylation are the two processes

used in derivatisation of EtG samples. Three derivatization agents could be used for these processes,

namely BSTFA for the silylation and HFBA and PFPA for the acylation. To develop a method for the

analysis of EtG with GC-MS/MS, first the derivatization step of the GC-MS/MS method needs to be

optimized. The three derivatization agents discussed in the introduction were tested under different

conditions, in order to find the optimal derivatization process.

Also, another derivatization agent, phenylboronic acid, was tested. Derivatization with phenylboronic

acid (PBA) is done often in analysis that include glucose-like compounds, since it derivatizes two OH-

groups at once 82. In Scheme 8, the derivatization of EtG with PBA is shown.

Scheme 8: Derivatization of EtG with PBA

Since GC-MS/MS is really dependent on these derivatization steps, another promising and upcoming

technique in the analysis of EtG is LC-MS/MS. To avoid false positives in the analysis, the sample can

be washed before analysis. Since decontamination is not a big issue in case of alcohol use, in this

research the samples are not washed before analysis. To avoid false negatives the samples are

usually cleaned-up by SPE, which is also done in this research 1. Different SPE methods were tested

and compared, as well as the reconstitution solvents and different LC-MS/MS systems.

2.2 Materials and methods 2.2.1 Hair samples

The analysis of EtG in hair was performed on blank hair samples provided by children or abstaining

adults.

2.2.2 Chemicals and reagents

Ethyl glucuronide and ethyl glucuronide-d5 were purchased from Cerilliant (USA). Milli-Q water was

obtained from a Millipore system of Merck (Germany). SPE Oasis MAX 3 cc cartridges were

purchased from Waters (Ireland), Isolute NH2 (aminopropyl) cartridges were purchased from Biotage

20

(Sweden). HFBA, PFPA, PBA, ammonium formate and sodium sulfate were purchased from Sigma-

Aldrich (Germany). BSTFA was purchased from Grace Davison, Discovery Science (USA). Methanol, n-

hexane, ethyl acetate, heptane, isooctane, acetone, HCl and acetonitrile were purchased from Actu-

All chemicals (Netherlands). Formic acid and 25% ammonia solution were purchased from Merck

(Germany).

2.2.3 Sample preparation and clean-up procedure for LC-MS

Approximately 50 mg of the blank hair samples were exactly weighed and cut into pieces (1-2 mm)

with scissors. The hair samples were then spiked with different concentrations of the standard

solution of EtG, which was evaporated to air for 30 min. 20 µL of internal standard solution (EtG-d5,

10 ppm) and 2 mL of Milli-Q water were added, and the extraction was performed by 4h

ultrasonication at 50 °C. After centrifugation for 7 min at 4000 rpm, three different clean-up methods

were performed:

(i) Solid phase extraction using Oasis MAX 3cc cartridges (Waters, Ireland) were conditioned with 2

mL of methanol and 2 mL of Milli-Q water. The extract was transferred on the SPE column and the

columns were dried for 5 min. The samples were washed with 2 mL of Milli-Q/5% ammonia solution

and 2 mL of methanol, and dried for 10 min. EtG was eluted with 2 mL of methanol/2% formic acid in

glass tubes and evaporated to dryness under a nitrogen stream at approximately 50 °C.

(ii) Solid phase extraction using Oasis MAX 3cc cartridges (Waters, Ireland) were conditioned with 2

mL of methanol and 2 mL of Milli-Q water. The extract was transferred on the SPE column and the

columns were dried for 5 min. The samples were washed with 2 mL of n-hexane, and dried for 10

min. EtG was eluted with 2 mL of methanol/2% formic acid in glass tubes and evaporated to dryness

under a nitrogen stream at approximately 50 °C.

(iii) Solid phase extraction using Isolute NH2 (aminopropyl) cartridges (Biotage, Sweden) were

conditioned with 3 mL of methanol, 3 mL of Milli-Q water and 3 mL of acetonitrile. Care was taken to

ensure that the columns did not dry between these conditioning steps. The extract was transferred

on the SPE column and the columns were dried for 5 min. The samples were washed with 3 mL of

Milli-Q/5% ammonia solution, and dried for 15 min under a strong vacuum. EtG was eluted with 3 mL

of methanol/2% ammonia solution in glass tubes and evaporated to dryness under a nitrogen stream

at approximately 50 °C.

Three reconstitution solvents were tested, namely methanol, acetonitrile and mobile phase A (see

below). All residues were reconstituted in 200 µL and 10 µL were injected for measurement by LC-

MS.

2.2.4 Instrumentation for LC-MS/MS

Liquid-chromatography was performed on a Waters Acquity UPLC system, and a Zorbax Eclipse XDB-

C8 column (5 µm, 4.6 x 150 mm) with the mobile phases A = 5mM NH4-formate and 0.01% formic

acid in Milli-Q water and B = acetonitrile, with the following gradient: 0-3 min 100% solvent A, linear

to 90% B in 2 min for 3 min, linear to 100% A in 0.10 min for 2 min. The flow rate was 0.5 mL/min.

The detection was performed on Micromass Quattro Ultima Pt, Waters Xevo TQ-S and AB-SCIEX

QTRAP-6500 mass spectrometer instruments with ESI in negative mode using the following

transitions for EtG: m/z 221 – 95, 221 – 85, 221 – 75, 221 – 57, and for EtG-d5: m/z 226 – 85, 226 –

75.

2.2.5 Method calibration

A standard calibration curve was obtained by preparing EtG solutions in mobile phase A in the

following concentrations: 0, 1, 2.5, 5, 12.5, 25, 50, 125, 250, 500, 1250, 2500, 5000 and 9000 pg/µL

and 100 µL internal standard (EtG-d5, 10 ppm).

21

2.2.6 Derivatization procedure for GC-MS

Two derivatization procedures were tested, one procedure for derivatization with PFPA, HFBA and

BSTFA, and a second procedure for derivatization with phenylboronic acid (PBA).

(i) An EtG solution of 10.000 pg/µL (10 ppm) was prepared in methanol, and 50 µL were evaporated

under a nitrogen stream at 60 °C in MS vials. In order to optimize the derivatization of EtG, several

derivatization agents were tested. Standard solutions of these agents were added to the vials as

mentioned in Table 2. The derivatization time and temperature varied according to Table 2. 2 µL of

some of the samples were injected into the GC-MS/MS after the derivatization, others were

evaporated under a nitrogen stream at 60 °C and reconstituted as mentioned in the table, before

injection of 2 µL into the GC-MS/MS.

# Derivatization agent Derivatization temp./time

Reconstitution Reconstitution solvent

1 50 µL HFBA/acetone (1:4) 1 h, 60°C + 50 µL isooctane 2 50 µL BSTFA/acetone (1:4) 1 h, 60°C + 50 µL isooctane 3 20 µL ethyl acetate/BSTFA (1:1) 20 min, 80°C - - 4 50 µL BSTFA/methanol (1:4) 30 min, 80°C + 50 µL isooctane 5 100 µL PFPA 30 min, 80°C + 50 µL heptane 6 100 µL PFPA/methanol (1:5) 30 min, 80°C + 50 µL heptane 7 100 µL PFPA/methanol (1:5) 30 min, 80°C + 50 µL ethyl acetate 8 20 µL ethyl acetate/HFBA (1:1) 20 min, 80°C - - 9 20 µL ethyl acetate/PFPA (1:1) 20 min, 80°C - -

10 20 µL ethyl acetate/HFBA (1:1) 1 h, 80°C - - 11 20 µL ethyl acetate/PFPA (1:1) 1 h, 80°C - - 12 20 µL ethyl acetate/BSTFA (1:1) 1 h, 80°C - - 13 20 µL ethyl acetate/HFBA (1:1) 20 min, 80°C + 50 µL isooctane 14 20 µL ethyl acetate/PFPA (1:1) 20 min, 80°C + 50 µL isooctane 15 20 µL ethyl acetate/BSTFA (1:1) 20 min, 80°C + 50 µL isooctane

(ii) A PBA solution was prepared by weighing 1g of PBA in a 5 mL vial, 4 mL of an acetone/Milli-Q

water mixture (19/1, v/v) was added and the mixture was shaken vigorously. A sodium sulfate

solution was prepared by weighing 0.25g of sodium sulfate in a 5 mL vial, 5 mL of Milli-Q water was

added and the mixture was placed in an ultrasonic bath to ensure the reagent was completely

dissolved. EtG solutions of 10.000 pg/µL (10 ppm) were prepared in methanol, and 500 µL was put in

a 5 mL vial. 2 mL of the sodium sulfate solution was added, followed by addition of 250 µL of the PBA

solution. The mixture was vortexed for 10s and incubated in an ultrasonic bath at room temperature

for 5min. 250 µL of a HCL (37%)/Milli-Q water solution (1/9, v/v) was added. The PBA derivatives of

EtG were extracted by addition of 1 mL of n-heptane, vortexing the mixture for 10s and transfer of

the upper layer to an empty glass test tube. The extraction was repeated with 1 mL of n-heptane and

the two extracts were combined and evaporated to dryness under a nitrogen stream at 50 °C. The

residue was dissolved in 400 µL of n-heptane and the supernatant was transferred to an empty GC-

MS vial, 2µL were injected in GC-MS/MS in both full scan as well as SIM mode.

2.2.7 Instrumentation for GC-MS/MS

A Varian CP-3800 gas chromatography – 1200L Quadrupole MS/MS system operating in EI mode was

used for analysis. Compounds were separated on a fused silica column (Agilent, DB-35MS) with a

(35%-phenyl)-methylpolysiloxane stationary phase (30 m length x 0.25 mm I.D. x 0.25 µm film

thickness). The carrier gas was helium with a constant flow of 1 mL/min. The injector temperature

was 250 °C, the initial column oven temperature of 75 °C was kept for 1 min and subsequently

22

increased at a rate of 20°C/min to 300 °C. The GC-MS/MS analysis was performed in full scan mode,

as well as SIM mode for derivatives with BSTFA and PBA. The precursor ions of EtG (261) and EtG-d5

(266) were selected for BSTFA, before fragmentation in the collision cell at an energy of 10.0 eV,

giving product ions of 143 for both EtG and EtG-d5. The precursor ions of EtG (308) and EtG-d5 (313)

were selected for PBA.

2.2.8 Data processing

Data was processed using the software MassLynx V4.1 and MS Workstation V6.9.2.

2.3 Results and discussion

2.3.1 Method development for LC-MS/MS Comparison of clean-up methods

To begin with, three different clean-up methods described in literature 83-85 were tested, as explained

in Chapter 2.2. In the method in which Isolute NH2 (aminopropyl) cartridges were used, no EtG was

detected in all chromatograms. The SPE cartridges are anion exchange cartridges, which should work

for an acidic compound as EtG, so the problem has to originate elsewhere. A possibility is that all

sample is already removed from the column by the washing solvent, which was a Milli-Q/5%

ammonia solution. Unfortunately, the washing solvents were not collected in glass tubes, so this

needs some further research later on in the project.

Clean-up method 1 and 2 made use of the same SPE cartridges, only the washing steps were

different. The suspicious part about method 1 is the fact that one of the washing solvents is

methanol, in which many compounds dissolve easily. Therefore, during the analysis, also the washing

step with methanol was collected in glass tubes, evaporated under a nitrogen stream and

reconstituted. In Figure 8, the chromatograms of a 5000 pg/µL sample treated with method 1, both

of the washing step with methanol as of the reconstitution, are shown. The intensities of the peaks

are in the right corner.

Figure 8: Chromatograms of EtG samples in a concentration of 5000 pg/µL with clean-up method 1. A: washing step with methanol. B: reconstituted samples. Chromatograms were recorded with a Micromass Quattro Ultima Pt mass-

spectrometer.

23

As can be seen in the figure, the clean-up method does not work properly. In the washing-step

(Figure 8A) EtG is detected, the amount of EtG that gets lost in the analysis is even bigger than the

amount detected after reconstitution (Figure 8B), which was the case for all concentrations

measured.

In Figure 9 the chromatograms of the clean-up method 2 are shown, with the intensity of the signal

in the right corner. As can be seen in this Figure, this method shows slightly better results, but the

intensities still don’t show a relation as expected. For example, the intensity of the peak for the 2000

pg/µL sample (Figure 9D) should be twice as high as the 1000 pg/µL sample (Figure 9C), but the

intensities are almost the same; even the signal for the 1000 pg/µL signal is slightly higher. Note that

the chromatograms of 5000 and 10000 pg/µL (Figure 9E and 9F) are recorded on a different day,

which could be the reason why the chromatograms are slightly offset. Still it is unusual, since EtG

normally elutes after 2.2 min, not after 2.5 min. Therefore, it can be said the method does not work

properly, since there is a variance in peak elution times and intensities of the peaks in the

chromatogram.

Figure 9: Chromatograms of EtG samples in different concentrations with clean-up method 2. Chromatograms were recorded with a Micromass Quattro Ultima Pt mass-spectrometer.

Overall, none of the clean-up methods worked perfectly. Method 3 was not able to detect EtG

signals, the Oasis MAX 3cc SPE cartridges did show some EtG signals in the chromatograms, but the

results were not as expected. Overall, method 2 in total showed the best results, but all methods

need further improvement in order to work sufficiently. Also, the intensities of the peaks are really

low. For this reason, after these experiments, the MS/MS-spectrometer was replaced by two other,

more sensitive machines, namely Waters Xevo TQ-S and SCIEX QTRAP-6500.

24

Comparison of reconstitution solvents

The EtG samples were reconstituted in methanol, acetonitrile and mobile phase A in order to

compare peak shapes and intensity of the peaks. In Figure 10, the chromatograms of these samples

are shown, with the intensities of the peaks in the right corner.

Figure 10: Chromatograms of EtG samples reconstituted in 200 µL of respectively A: acetonitrile, B: mobile phase A and C: methanol. Chromatograms were recorded with a Waters Xevo TQ-S mass spectrometer.

As can be seen in the figure, acetonitrile (Figure 10A) is not sufficient as reconstitution solvent, since

no peak appeared in the chromatogram. This is not as expected, since acetonitrile is also a polar

solvent such as methanol and water (mobile phase A). A solution could be the fact that the samples

in acetronitrile needed a longer time to reconstitute as in methanol and water (mobile phase A),

since the reconstitution times were all equal it could be that EtG did not have time to dissolve in the

acetonitrile. Both methanol (Figure 10C) and mobile phase A (Figure 10B) are sufficient as

reconstitution solvents, however the peak shape of the sample reconstituted in methanol is tailing.

The peak of the sample in mobile phase A (Figure 10B) is sharper, and the intensity of the peak is

slightly higher, though in the same order of magnitude. Since this peak looked really sufficient, it was

decided to continue with mobile phase A as reconstitution solvent.

Different amounts of reconstitution solvents were also tested, 100, 200 and 400 µL, but the

intensities and shapes of the peaks remained the same. Based on these observations, mobile phase A

was chosen as reconstitution solvent, in an amount of 200 µL, to make sure several measurements

could be performed with LC-MS/MS.

Calibration

In Figure 11A, the calibration curves for EtG are shown. As can be seen, the coefficient of

determination R2 for the method with Waters Xevo TQ-S is 0.9755, the R2 for the method with SCIEX

QTRAP-6500 is 0.9916, which is almost equal to the perfect situation, which is an R2 of 1.

25

Figure 11: Calibration curves of EtG corrected with EtG-d5, recorded with blue: SCIEX QTRAP-6500 mass spectrometer, orange: Xevo TQ-S mass spectrometer. A: calibration curve 0 to 9 ppm. B: calibration curve zoomed in, 0 to 0.0125 ppm.

In Figure 11B, the same calibration curves are shown, but zoomed in on the smaller concentrations of

EtG. As can be seen, the values for R2 become a little smaller, since the first measurements (0 to

0.0025 ppm) give quite similar results, especially when a Xevo TQ-S mass spectrometer is used.

However, since the R2 value and peak areas are still higher with the SCIEX QTRAP-6500 mass

spectrometer, it was chosen to use this machine in further experiments.

Control of evaporation-step in sample preparation and clean-up

Since all clean-up methods with SPE did not work properly, this experiment was done without SPE.

The same sample preparation steps as described in Chapter 2.2.3 were followed. After

centrifugation, 100 µL of the supernatant was collected into vials for LC-MS/MS. The rest of the

supernatant was transferred to glass tubes and evaporated under a nitrogen stream at 40 °C, and

reconstituted in 200 µL of mobile phase A. The resulting peak areas are shown in Figure 12.

Figure 12: Peak areas of EtG for 100µl supernatant (blue) and evaporation and reconstitution in 200 µl mobile phase A (orange).

As can be seen in the Figure, the peak areas for the method in which 100 µL of the supernatant was

collected and immediately measured are much smaller than for the method with evaporation and

reconstitution in 200 µL of mobile phase A. This could be due to the fact that the samples in 100µL

supernatant, in which the hair matrix is still present in the tubes while collecting the sample, are less

concentrated than the reconstituted samples. This leads to the fact that evaporation after clean-up is

a necessary step in the analysis.

26

The peak areas for all samples are according to the concentrations of EtG in the sample, they become

higher with higher concentrations. However, also the blank sample with a concentration of 0 ppm

gave a peak at 2.1min, as can be seen in Figure 13. Since all samples, including the blank one, contain

the internal standard EtG-d5, the internal standard was examined to check for contamination with

EtG.

Figure 13: Chromatogram of blank hair sample with internal standard EtG-d5. Chromatogram was recorded with a SCIEX QTRAP-6500 mass spectrometer.

Testing internal standard EtG-d5 and evaluation of the blank hair samples

In order to examine for contamination, a 10.000 pg/μL (10 ppm) solution of the internal standard in

mobile phase A was analysed.

As can be seen in Figure 14, a chromatogram of a 10.000 pg/μL (10 ppm) internal standard solution

EtG-d5 shows a peak of EtG between 2.1 and 2.2 min. The intensities are quite low, in the same

range as was seen in Figure 13 of a blank hair sample containing only the internal standard solution

EtG-d5, so it is possible that the internal standard EtG-d5 contains a small amount of EtG, leading to

these peaks in the chromatogram.

Figure 14: Chromatograms of 10 ppm EtG-d5 in methanol. A: MRM of 2 channels (EtG-d5). B: MRM of 4 channels (EtG). Chromatograms were recorded with a SCIEX QTRAP-6500 mass spectrometer.

Another possibility is the fact that the blank hair samples are in fact not really blank for EtG. Another

possibility could be the fact that the blank hair samples are not blank for EtG. To test this, the

measurements are repeated with cow hair, which should be blank for EtG. In Figure 15, the

chromatograms for the blank cow hair samples are shown.

27

Figure 15: Chromatogram of a blank cow hair sample. Chromatogram was recorded with a SCIEX QTRAP-6500 mass spectrometer.

As can be seen in the Figure, the blank cow hair sample shows a small peak at 2.1min, which could be

originating from EtG. The intensity of the peak is in the same range as was seen in Figures 13 and 14.

This leads to the fact that the internal standard, EtG-d5, contains a small amount of EtG. However,

because it is such a small amount, it will still be used in further experiments, but it will be kept in

mind while interpreting the results that the results are in fact slightly lower than shown in the

chromatogram.

2.3.2 Method development for GC-MS/MS Since the LC-MS method for detection of EtG in hair did not work sufficiently enough, next step in the

project was to develop a method to analyse EtG in hair with GC-MS/MS.

At first, reaction 1 to 7 in Table 2 were performed and tested. Unfortunately, analysis of the

derivatization products did not show any signals for EtG. Reaction number 3 showed some peaks in

the chromatogram, but the spectra belonging to these peaks were not originating from EtG.

Since some signal was visible in reaction number 3, this reaction was investigated further with

reactions 8 to 15. The three derivatization reagents were tested in a mixture with ethyl acetate, and

the temperature/time varied. The last three reactions (13-15) were evaporated after derivatization

and reconstituted in isooctane. Unfortunately, none of the reactions showed any signal for EtG,

including a repetition of reaction number 3 on a different day.

Figure 16: Chromatogram of boldenone derivatized with HFBA. Chromatogram was recorded with a Varian mass spectrometer.

To check if the derivatization method used works, a control sample was also derivatized at the same

time. This control sample contained boldenone in acetone (1:4), and was derivatized with HFBA. This

control sample did show a peak in the GC-MS chromatogram recorded in full-scan mode as shown in

Figure 16, so there must be another reason why the derivatization was not working properly. The

28

fact that none of the derivatization reactions worked, could be due to the fact that there might be

some water present in the samples or the solvents used; in this case the derivatization agent is

hydrolysed, so no derivatization takes places at all.

Lastly, another derivatization method was tested, method (ii) in Chapter 2.2.6. In Figure 17, the

chromatogram and spectrum of this derivatization, recorded in SIM mode in the range from 305-315

m/z, is shown.

Figure 17: Chromatogram (A) of 10 ppm EtG solution derivatized with PBA, and spectrum (B) of EtG-PBA. Chromatogram was recorded with a Varian mass spectrometer.

As can be seen in this Figure, several peaks show up. The first peak, eluting at 4min., could be

originating from the EtG-PBA complex, since this complex has got an m/z ratio of 308.

When derivatizing with PBA, PBA can also react with itself to form a large complex, as shown in

Figure 18. Its mass, an m/z ratio of 311, shows a peak eluting at 5min.

Figure 18: Chromatogram (A) of 10 ppm EtG solution derivatized with PBA, and spectrum (B) of PBA complex formed during derivatization. Chromatogram was recorded with a Varian mass spectrometer.

In Figure 19A, the full chromatogram is once again shown, with the filtered chromatogram for m/z

311 in Figure 19B, and the filtered chromatogram for m/z 308 in Figure 19C. As can be seen, the

intensity in Figure 19C is extremely low, and the largest signal in the chromatogram originated from

m/z 311, which originates from the PBA complex formed by a reaction of PBA with itself. Since this

signal is much larger than the signal originating from EtG-PBA, it is possible that PBA reacts quickly

with itself, leaving less PBA for derivatization of EtG.

29

Figure 19: (A) Full chromatogram and chromatogram of (B) m/z 311 of the PBA complex and (C) m/z 308 of EtG-PBA.

Due to time limitations, unfortunately the derivatization of EtG was not investigated any further, but

the derivatization of EtG with PBA is promising.

2.4 Practical obstacles 2.4.1 Evaluation of the transitions used in the LC-MS/MS method

In Figure 20, the mass spectrum of EtG with its transitions is shown. In the LC-MS/MS measurements

four transitions for EtG were used, namely m/z 221 – 95, 221 – 85, 221 – 75 and 221 – 57, because

they showed the highest intensity peaks in the mass spectrum, as shown in the daughter ion scan

chromatogram in Attachment 1.

Figure 20: Transitions for ethyl glucuronide. Reproduced from Ref 78.

30

In the EtG measurements, the signal originating from the transition m/z 221 – 95 and 221 – 57

usually does not contribute to the signal as much as the transitions m/z 221 – 85 and 221 – 75, as

shown in Figure 21.

Figure 21: TIC and chromatograms of four transitions of EtG (10 ppm). Chromatograms were recorded with a Waters Xevo TQ-S mass spectrometer.

To check if a peak has sufficient selectivity, some measurements could be done according to

literature 86. Unfortunately, the article describes a method only for positive electrospray ionization

(ESI) mode, while these measurements are performed in negative ion mode. However, to give an

idea of the ‘identification power’ of the method used, the selectivity of the transitions used is

calculated using the method described in the article.

The selectivity of a method could be measured according to the probability that a compound shows

the same characteristics in LC-MS/MS as the compound of interest. This probability is called the

probability of interference, P(I), and can be calculated with the following formula:

𝑃(𝐼) = 𝑃(𝑀𝑝𝑟) ∗ max (𝑃(𝑀𝑝𝑑1), 𝑃(𝑀𝑛𝑙1)) ∗ max (𝑃(𝑀𝑝𝑑2), 𝑃(𝑀𝑛𝑙2)) ∗ 𝑃(𝑅𝑇)

In this formula P(Mpr) is the probability of the precursor ion to occur, P(Mpd) the probability of the

product ions of transition 1 and 2 to occur, P(Mnl) the probability of a neutral loss of transitions 1 and

2 to occur, and P(RT) the probability of a specific retention time to occur in the chromatographic

system. In case of a high-resolution reversed phase LC system, P(RT) = 0.2. Max[P(Mpd), P(Mnl)]

represents the maximum value of either P(Mpd) or P(Mnl) in a worst-case scenario. All parameters can

be determined according to mathematical models present in the paper 86.

P(I) is calculated, and compared to a criterion previously calculated in the paper to determine if a

method is sufficient or insufficient. If P(I) < 2*10-7, the method is sufficiently selective.

31

Table 1: Precursor Ion Mass, Product Ion Mass and Retention Time, and calculation of P(I) of several transitions of EtG

Transitions ethyl glucuronide

Precursor ion (m/z) 221.18 221.18 221.18 221.18 221.18 221.18 221.18 P(Mpr) 6.5*10-4 6.5*10-4 6.5*10-4 6.5*10-4 6.5*10-4 6.5*10-4 6.5*10-4 Product ion 1 (m/z) 75.12 85.13 95.2 85.13 95.2 95.2 203.2 P(Mpd1) 0.015 0.019 0.023 0.019 0.023 0.023 0.019 Neutral loss 1 146.06 136.05 125.98 136.05 125.98 125.98 17.98 P(Mnl1) 0.027 0.030 0.031 0.030 0.031 0.031 0.1 Product ion 2 (m/z) 57.24 57.24 57.24 75.12 75.12 85.13 75.12 P(Mpd2) 0.007 0.007 0.007 0.015 0.015 0.019 0.015 Neutral loss 2 163.94 163.94 163.94 146.06 146.06 136.05 146.06 P(Mnl2) 0.021 0.021 0.021 0.027 0.027 0.030 0.027 P(RT) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 P(I) 7.47*10-8 8.16*10-8 8.60*10-8 1.04*10-7 1.10*10-7 1.20*10-7 3.52*10-7

In Table 1, the calculations for several transitions of ethyl glucuronide related to each other are

given. The transitions m/z 221.18 – 95.2, 221.18 – 85.13, 221.18 – 75.12 and 221.18 – 57.24 are the

transitions used in the EtG sample measurements. They all have a value of P(I) lower than 2*10-7,

which leads to the fact that the method used for the measurements should be sufficiently selective.

As a comparison, the transition m/z 221.18 – 203.2 is calculated related to the most characteristic

transition of EtG. The transition to 203.2 stands for loss of H2O, which is not a characteristic

transition for ethyl glucuronide. The P(I) value for this transition is larger than 2*10-7, so this

transition leads to an insufficient selectivity and can’t be used in the method.

2.4.2 Limits of detection (LODs) with LC-MS/MS

As can be seen in the calibration curves in Figure 11A and B, the peak areas of solutions with a small

concentration of EtG show a similar result. In the case of a Xevo TQ-S mass spectrometer,

chromatograms of solutions with a concentration higher than 0.005 ppm (5 μg/L) of EtG really show

a relation between concentration and signal intensity. For this reason, the LOD of the samples used

for the calibration of EtG with this mass spectrometer was set to 5 µg/L. In the case of a SCIEX

QTRAP-6500 mass spectrometer, solutions with a concentration higher than 0.0025 ppm (2.5 µg/L) of

EtG show a relation between concentration and signal intensity, which is a factor 2 smaller than the

Xevo TQ-S mass spectrometer.

However, these solutions were stock solutions, no hair matrix was present in the sample. If these

concentrations were converted to a theoretical concentration in pg/mg, which is the unit the SoHT is

working with, in combination with the method used (50 mg of hair, reconstituted in 200µL) it would

be a concentration of 20 pg/mg for the Xevo TQ-S and a concentration of 10 pg/mg for the SCIEX

QTRAP-6500. This is higher than the cut-off value of 7 pg/mg given by the SoHT, and it would be even

higher if a real hair matrix is present and all sample preparation and clean-up steps are involved.

32

3. Optimization and validation of LC-MS/MS method for EtG in hair

3.1 Introduction The LC-MS/MS methods tested in Chapter 2 did work, but the SPE methods did not work properly

and the detection limits were too high according to the guidelines of the SoHT. Since the

derivatization steps for GC-MS did not work, it was decided to optimize the LC-MS method including

the sample preparation and clean-up steps to as sensitive as possible, and to validate this optimized

method.

In the extraction step of the procedure, selecting the best extraction solvent plays an important role.

In previous research, methanol, a mixture of methanol and water (1:1), water and a mixture of water

and trifluoroacetic acid (9:1) were tested as extraction solvent; water was found to be the best

suitable solvent for the extraction of EtG 1 and is therefore applied in most procedures, including the

procedure used in this research. The extraction is performed in an ultrasonic bath for several hours

often followed by an incubation overnight, but previous research showed that the extraction with

water and ultra-sonication is enough to complete the extraction 1. This was tested in this

optimization.

The first step performed in the optimization was selection of the right conditions for SPE. Clean-up

by SPE is often done in hair analysis, since it removes impurities and matrix interferences and

concentrates the analytes of interest to increase the sensitivity 1. Optimization of the SPE method is

crucial to increase the sensitivity of the full method as much as possible. After sample treatment and

clean-up with SPE, the extracts will be more concentrated and contain less impurities, so the

detectability will be increased 87. SPE is done in five steps, as shown in Figure 22. All steps can be

optimized, excluding step 3.

Figure 22: SPE is a five-step process. Step 1: Selection of the proper SPE tube or disk, step 2: conditioning the SPE tube or disk, step 3: addition of the sample, step 4: washing of the packing material, step 5: elution of the compounds of interest.

Reproduced from Ref 87.

SPE columns are available in many varieties, so selecting the most appropriate product for the clean-

up of EtG samples is important; the column can be for instance an anion exchange (reversed phase)

cartridge because of the fact that EtG is highly acidic, or a carbon-based cartridge, for example EtG

cartridges especially developed for the detection of EtG in urine 1. Before the sample is added to the

column, the column needs to be conditioned. In the case of a reversed phase column, the

conditioning contains two steps. At first, the sorbent-surface needs to be wetted by an organic

solvent, so that the bonded alkyl phase gets permeated. The silica-surface also needs to be wetted,

this is done with water or an aqueous buffer 87.

33

Another important step in optimizing the SPE method is selecting the right washing solvent. Washing

the sample is a necessary step in SPE, since it removes impurities from the sample. Washing is

performed with a solvent that is stronger than the extraction solvent. At the same time, the washing

solvent must be weaker than the solvent necessary to remove the compound of interest, since you

want that compound to stay on the column 87. In Table 2 characteristics of solvents commonly used

in SPE are shown. Solvents with a different polarity than the elution solvent used in the end of the

SPE, could be useful as washing solvents.

Table 2: Characteristics of solvents commonly used in SPE. Reproduced from Ref. 87

Polarity Solvent Miscible in water? Nonpolar Strong reversed

phase Weak normal phase

Hexane

No Isooctane No Carbon tetrachloride No Chloroform No Dichloromethane No Tetrahydrofuran Yes Diethyl ether No Ethyl acetate Poorly Acetone Yes Acetonitrile Yes Isopropanol Yes Methanol Yes Water Yes

Polar Weak reversed phase

Strong normal phase Acetic acid

Yes

Since the sample matrix is water, and reversed phase SPE columns are used, all solvents besides

acetic acid are stronger than the extraction solvent. Several washing solvents covering the entire

range in Table 2 will be tested to optimize the washing step of the SPE method.

Lastly, the compounds of interest must be eluted. During the elution step, the compound of interest

must be removed from the extract and the column, but any impurities that were not removed during

the washing step must be left behind in the column. Previous research showed that methanol/2%

formic acid is a sufficient elution solvent for EtG 78,79, so this solvent is used in the SPE method.

3.2 Materials and methods 3.2.1 Hair samples

The analysis of EtG in hair was performed on blank hair samples provided by children or abstaining

adults. Reference hair samples were purchased from Medichem (Germany).

3.2.2 Chemicals and reagents

Ethyl glucuronide and ethyl glucuronide-d5 were purchased from Cerilliant (USA). Milli-Q water was

obtained from a Millipore system of Merck (Germany). SPE Oasis MAX 3cc cartridges were purchased

from Waters (Ireland), Isolute NH2 (aminopropyl) cartridges were purchased from Biotage (Sweden),

Strata-X cartridges were purchased from Phenomenex (USA). Ammonium formate was purchased

from Sigma-Aldrich (Germany). Methanol, n-hexane, ethyl acetate and acetonitrile were purchased

from Actu-All chemicals (Netherlands). Formic acid and 25% ammonia solution were purchased from

Merck (Germany).

34

3.2.3 Sample preparation and clean-up procedure for LC-MS/MS

Approximately 50 mg of the blank hair samples were exactly weighed and cut into pieces (1-2 mm)

with scissors. The hair samples were then spiked with different concentrations of the standard

solution of EtG, which was evaporated to air for 30 min. 20 µL of internal standard solution (EtG-d5,

10 ppm) and 2 mL of Milli-Q water were added, and the extraction was performed by ultrasonication.

Different ultrasonication times were tested in order to optimize this step in the procedure. After

centrifugation for 7 min at 4000 rpm, the SPE method was optimized using the following procedures:

(i) Solid phase extraction using Isolute NH2 (aminopropyl) cartridges (Biotage, Sweden) were

conditioned with 2 mL of methanol and 2 mL of Milli-Q water. Care was taken to ensure that the

columns did not dry between these conditioning steps. The extract was transferred on the SPE

column and the columns were dried for 5 min. The samples were washed with 3 mL of n-hexane, and

dried for 5 min under a strong vacuum. EtG was eluted with 3 mL of methanol/2% ammonia solution

in glass tubes and evaporated to dryness under a nitrogen stream at approximately 50 °C.

(ii) Solid phase extraction using Strata X cartridges (Phenomenex, USA) were conditioned with 2 mL

of methanol and 2 mL of Milli-Q water. Care was taken to ensure that the columns did not dry

between these conditioning steps. The extract was transferred on the SPE column and the columns

were dried for 5 min. The samples were washed with 2 mL of n-hexane, and dried for 5 min under a

strong vacuum. EtG was eluted with 2 mL of methanol/2% formic acid solution in glass tubes and

evaporated to dryness under a nitrogen stream at approximately 50 °C.

(iii) Solid phase extraction using Oasis MAX 3cc cartridges (Waters, Ireland) were conditioned with 2

mL of methanol and 2 mL of Milli-Q water. The extract was transferred on the SPE column and the

columns were dried for 5 min. Several washing solvents were tested for this procedure, namely

Milli-Q/5% ammonia solution, hexane, Milli-Q water, acetonitrile and ethyl acetate, 2 mL of the

washing solvent was used, and the column was dried for 10 min. EtG was eluted with 2 mL of

methanol/2% formic acid in glass tubes and evaporated to dryness under a nitrogen stream at

approximately 50 °C.

All residues were reconstituted in 200 µL of mobile phase A and 10 µL were injected for

measurement by LC-MS.

3.2.4 Instrumentation for LC-MS/MS

Liquid-chromatography was performed on a Waters Acquity UPLC system, and a Zorbax Eclipse XDB-

C8 column (5 µm, 4.6 x 150 mm) with the mobile phases A = 5mM NH4-formate in Milli-Q water and

B = acetonitrile, with the following gradient: 0-3 min 100% solvent A, linear to 90% B in 2 min for 3

min, linear to 100% A in 0.10 min for 2 min. The flow rate was 0.5 mL/min. The detection was

performed on an AB-SCIEX QTRAP-6500 mass spectrometer instrument with ESI in negative mode

using the following transitions for EtG: m/z 221 – 95, 221 – 85, 221 – 75, 221 – 57, and for EtG-d5:

m/z 226 – 85, 226 – 75.

3.2.5 Validation

A validation plan was written and executed according to Commission Decision 2002/657/EC 88, also

described in RIKILT SOP A0400 and F0052. The validation plan for the detection of EtG in human hair

is shown in Attachment 2.

Three validation days were executed. Every day, 8 different blank human hair samples were used;

samples A to H on day 1, samples I to P on day 2, and samples Q to X on day 3. Samples A, I and Q

were used for determination of the linearity by preparing a matrix-matched standards (MMS) series

of 0, 0.25, 0.5, 1.0, 2.0 and 3.0 times the permitted level, and for the determination of the recovery

by spiking the sample with 1.0 times the permitted level after clean-up.

Samples B to H, J to P and R to X were used to determine trueness, within-laboratory reproducibility,

repeatability, CCα/CCβ, and selectivity. The determination of the trueness, within-laboratory

35

reproducibility, repeatability*, and CCα/CCβ was done by spiking 7 different hair samples at 0.5, 1.0

and 1.5 times the permitted level. The selectivity was determined with 7 blank samples, without

spiking.

Samples B, J and R are used for the determination of the repeatability. From each batch, 7 hair

samples are spiked at 1.0 times the permitted level. The matrix effect was determined by comparison

of the matrix-matched recovery standards (MMRS) of samples A, I and Q, and the stock solution at

the same level.

Additional criteria is stated in the validation plan displayed in Attachment 2, including a brief

summary of each of the performance characteristics.

3.2.6 Data processing

Data was processed using the software MassLynx V4.1, Multiquant 3.0.2 and the ‘ResVal’ template

provided by RIKILT.

3.3 Results and discussion

3.3.1 Optimization First step in the optimization of the sample preparation was the optimization of the ultrasonication

time. A blank hair sample was spiked with a solution of 10.000 pg/μL (10 ppm) EtG and placed in an

ultrasonic bath. Every hour, the 2 mL of extraction solvent was transferred to a glass tube, and 2 mL

of fresh extraction solvent was added to the hair sample. In Figure 23, the peak areas of both EtG and

its internal standard EtG-d5 are shown for different times in an ultrasonic bath, varying from 1 to 7

hours. As can be seen, during the first three hours most of the EtG present in the hair matrix is

extracted. After four hours, the amount of EtG that is extracted every hour is an equal, small amount.

Therefore, in order to make the sample preparation step sufficient, but at the same time as quick as

possible, the ultrasonication time was set at four hours.

Figure 23: Peak areas of EtG (blue) and EtG-d5 (orange) in ultrasonication testing.

After finding the optimum ultrasonication time, it was tested if extra overnight incubation is

necessary. This was tested by spiking eight blank hair samples with a solution of 10.000 pg/μL (10

ppm) EtG, and placing them in an ultrasonic bath. Every hour, two samples were removed from the

ultrasonic bath; one sample was transferred to a clean glass tube, the other one was set aside for

overnight incubation with the hair matrix still present in the sample.

36

Figure 24: Peak areas of EtG in overnight incubation testing. Blue represents samples directly transferred to a clean glass tube after ultrasonication, orange represents samples with overnight incubation with hair matrix still present.

As can be seen in Figure 24, the amount of EtG removed from the sample matrix is higher if the

ultrasonication time is longer. However, overnight incubation also increases the amount of EtG

removed from the hair matrix. It was therefore decided to perform the incubation by placing the

samples in an ultrasonic bath for four hours, followed by overnight incubation in order to remove as

much EtG from the hair matrix as possible.

To show the effect of using SPE as sample clean-up step, a hair sample was prepared and extracted in

duplicate, and half of the extract was directly evaporated and reconstituted, while the other half was

submitted to SPE clean-up. The SPE method used was an Oasis MAX 3cc column, the washing solvent

was 2 mL of n-hexane and the elution solvent was 2 mL methanol/2% formic acid. Figure 25 shows

the area of the EtG peaks in the chromatograms.

Figure 25: Peak areas in the chromatograms of a procedure with (blue) and without (orange) SPE.

As can be seen in Figure 25, the peak areas are higher in a procedure which includes clean-up with

SPE, especially when the concentration of EtG becomes higher. In the case of a concentration of 10

ppm, the area with SPE is approximately 2 times higher than without SPE, which means SPE is a

crucial step in order to make the method as sensitive as possible.

First step in the optimization of the clean-up step with SPE was testing different SPE columns, the

four different procedures tested in triplicate are listed in Table 3.

37

Table 3: Conditions for SPE column testing.

# SPE column Washing solvent Elution solvent

1 Oasis MAX 3cc (1) 2mL n-hexane 2mL methanol/2% formic acid 2 Oasis MAX 3cc (1) 2mL n-hexane 2mL methanol/2% formic acid 3 Oasis MAX 3cc (1) 2mL n-hexane 2mL methanol/2% formic acid 4 Oasis MAX 3cc (2) 2mL Milli-Q/5% ammonia 2mL methanol/2% formic acid 5 Oasis MAX 3cc (2) 2mL Milli-Q/5% ammonia 2mL methanol/2% formic acid 6 Oasis MAX 3cc (2) 2mL Milli-Q/5% ammonia 2mL methanol/2% formic acid 7 Isolute NH2 (aminopropyl) 3mL n-hexane 3mL methanol/2% ammonia 8 Isolute NH2 (aminopropyl) 3mL n-hexane 3mL methanol/2% ammonia 9 Isolute NH2 (aminopropyl) 3mL n-hexane 3mL methanol/2% ammonia

10 Strata X33 2mL n-hexane 2mL methanol/2% formic acid 11 Strata X33 2mL n-hexane 2mL methanol/2% formic acid 12 Strata X33 2mL n-hexane 2mL methanol/2% formic acid

As can be seen, three different SPE columns were tested, all with solutions of 10 ppm EtG. The

Isolute NH2 (aminopropyl) column is a anion exchange column, and the Strata X is a reversed phase

column. The Oasis MAX 3cc is a column that contains both of these properties; it is a reversed phase,

strong anion exchange column. This column was tested with two different washing solvents. The

results of both washing and elution step are shown in Figure 25.

Figure 26: Peak areas of washing (A) and elution (B) steps with four different procedures including three different SPE columns.

As can be seen in the Figure 26A, the peak areas of the washing step in procedure Oasis MAX 3cc (2)

are extremely high. This means a lot of sample gets lost during the washing step in the procedure.

For this reason, procedure Oasis MAX 3cc (2) was not followed anymore; the washing solutions were

further tested and evaluated in the next step of the optimization. In Figure 26B, the peak areas of the

elution step are shown. As can be seen, the area is highest for procedure Oasis MAX 3cc (1). Since

EtG is a highly acidic compound with a pKa of 3.21, an anion exchange column is likely to give the

highest results for the elution of EtG, especially in combination with the reversed phase feature if the

column, since the sample matrix is water. The Oasis MAX 3cc column is both a reversed phase, as

well as an anion exchange column, so for this reason the Oasis MAX 3cc columns in combination with

methanol/2%formic acid as elution solvent were chosen to use in further experiments. The washing

step was further investigated, because in the procedure Oasis MAX 3cc (1), still some sample gets

lost during analysis, as can be seen in the Figure.

The next step was optimization of the washing solvent. Five different washing solvents were tested,

namely hexane, acetonitrile, Milli-Q water, ethyl acetate and Milli-Q/5% ammonia solution, in order

to cover the entire solvent range showed in Table 2. All washing solvents were stronger than or equal

to the extraction solvent, Milli-Q water, except for Milli-Q/5% ammonia. Since ammonia is a polar

38

molecule, mixing water with ammonia makes the solution more polar, which leads to the fact that

Milli-Q/5% ammonia should be listed in Table 2 below water.

In Figure 27, the results of the area of the peaks originating from the washing and elution steps are

shown. The linearity (R2) belonging to the five washing solvents are as follows: 0.4066 for hexane,

0.9941 for acetonitrile, 0.8654 for Milli-Q, 0.9930 for ethyl acetate and 0.9749 for Milli-Q/5%

ammonia solution, respectively. As can be seen, the peak areas of the washing steps with the Milli-

Q/5% ammonia solution are extremely high, same as seen in Paragraph 2.3.1 during the initial

method testing and Figure 26A during the selection of the SPE column. The statement made above

could thus be true regarding the polarity of Milli-Q/5% ammonia solution; since the washing solvent

should be stronger than the extraction solvent in reversed phase columns, and a mixture of Milli-Q

with 5% ammonia is more polar than just Milli-Q, EtG can be removed from the column during the

washing step. The areas of the washing steps with the other four procedures are small, so all four

methods work properly. Since acetonitrile (R2 = 0.9941) and ethyl acetate (R2 = 0.9930) gave the most

linear relationship between the area and the concentration of EtG, acetonitrile was chosen as the

washing solvent to use in further experiments because the peak areas with acetonitrile were higher.

Figure 27: Peak areas of the washing and elution steps of five different procedures.

3.3.2 Validation

The validation of EtG in human hair was executed with 24 blank hair samples (A to X) with known

concentrations of EtG added to these samples. The theoretical detection limit calculated based on

the calibration curve, from measurements with the most sensitive machine available in previous

research, was 10 µg/L (10 pg/mg). Since no hair matrix, no sample preparation and no clean-up steps

were involved, the real detection limit will be higher. In order to make sure the validation can be

successfully executed and all samples can be measured, the validation level, or permitted level, for

the validation of EtG was set at 100 µg/L. This means all samples are spiked with concentrations of

0.25, 0.5, 1.0, 2.0 and 3.0 times the permitted level (25, 50, 100, 200 and 300 µg/L) before or after

sample preparation and clean-up. The validation consisted of three validation days, the hair samples

were prepared and cleaned-up according to the optimized method shown before. In Attachment 2

the validation plan is shown, the results of the measured performance characteristics are shown in

Table 4.

39

Table 4: Criteria and measured values for performance characteristics in validation.

Performance characteristics Measured value

Criteria

MMS 1 0.9572 Linearity (R2) MMS 2 0.9327 ≥ 0.990 MMS 3 0.9869 MMRS 1 79.07 Recovery (%) MMRS 2 101.22 80-110 MMRS 3 91.43 50 µg/L 99.2 Trueness (T, %) 100 µg/L 99.2 80-110 150 µg/L 100.2 50 µg/L 22.692 Repeatability* (r) 100 µg/L 25.291 150 µg/L 28.358 50 µg/L 16.3 Relative standard deviation of repeatability * (RSDr

*, %) 100 µg/L 9.1 ≤ 14.7 150 µg/L 6.7 Repeatability (r) 100 µg/L 27.626 Relative standard deviation or repeatability (RSDr , %) 100 µg/L 10.1 ≤ 14.7

50 µg/L 26.531 Within-laboratory reproducibility (RL) 100 µg/L 26.182 150 µg/L 30.189 50 µg/L 19.1 Relative standard deviation of within-laboratory reproducibility (RSDRL , %) 100 µg/L 9.4 ≤ 22.0 150 µg/L 7.2 Decision limit (CCα) 31.83 Detection capability (CCβ) 63.65

Linearity

Linearity was calculated on all three validation days (MMS 1 to MMS 3), samples were spiked at 0,

0.25, 0.5, 1.0, 2.0 and 3.0 times the permitted level. In an analysis, there should be a linear

relationship between the peak areas and the EtG concentration in the sample. With this linear

relationship, the concentrations of unknown samples could be determined. In Figure 28, all

calibration lines are shown, including their R2 values. As can be seen, the R2 values are 0.9572, 0.9327

and 0.9869 respectively. This means the linearity is a small amount below the criterion on all days,

which is ≥ 0.990. Note that MMS 2 is missing the 0 times permitted level data point, since this sample

was not prepared properly due to a pipetting mistake in the lab. All three MMS series have one data

point which is slightly too low or high, and are considered to be outliers. If these data points are

removed from the series the linearity follows the criterion, so the slightly low values for the linearity

are accepted.

40

Figure 28: MMS series of all three validation days.

Recovery

The recovery was also calculated on all three validation days. It was determined by comparing the

sample in the MMS spiked with 1.0 times the permitted level, with a sample originating from the

same batch with addition of the same concentration after sample preparation and clean-up. The

percentage of the recovery can be calculated with the formula:

𝑅𝑒𝑐𝑜𝑣𝑒𝑟𝑦 = (𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝐸𝑡𝐺 𝑀𝑀𝑆 (1.0 ∗ 𝑝𝑒𝑟𝑚𝑖𝑡𝑡𝑒𝑑 𝑙𝑒𝑣𝑒𝑙)

𝑝𝑒𝑎𝑘 𝑎𝑟𝑒𝑎 𝐸𝑡𝐺 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑦 𝑠𝑎𝑚𝑝𝑙𝑒) ∗ 100%

The recovery shows the percentage of added EtG, recovered in the analysis; in other words the

efficiency of the sample preparation. The criterion for an analysis of samples with a concentration

around 100 µg/L is 80-110%. As can be seen in Table 4, the percentages of the recovery are 79.07,

101.22 and 91.43 respectively. Only the first percentage is slightly below the criteria, but since it is

less than 1% and all other percentages are within the criteria, this is accepted.

Trueness / Within-laboratory reproducibility / Repeatability* / Repeatability

Within RIKILT, two measurements for the repeatability are used; repeatability* and repeatability.

The trueness, within-laboratory reproducibility, repeatability*, CCα and CCβ were calculated based on

spiking seven hair samples from seven different batches with concentrations of 0.5, 1.0 and 1.5 times

the permitted level, the repeatability was calculated based on spiking seven hair samples from the

same batch at 1.0 times the permitted level.

The criterion for the trueness for an analysis of samples with a concentration around 100 µg/L is 80-

110%. The percentage of the trueness can be calculated with the formula:

𝑇𝑟𝑢𝑒𝑛𝑒𝑠𝑠 (𝑇) = (𝑀𝑒𝑎𝑛 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑙𝑒𝑣𝑒𝑙

𝑃𝑒𝑟𝑚𝑖𝑡𝑡𝑒𝑑 𝑙𝑒𝑣𝑒𝑙) ∗ 100%

The trueness, or accuracy, of the method shows the percentage or the deviation of the measured

concentration of a sample compared to the spiked concentration. As shown in the Table, the

percentages for the trueness are 99.2, 99.2 and 100.2 % respectively, which means they follow the

criterion.

The repeatability* is the similarity between separate analyses, according to the same conditions, as

the same method, laboratory or test material. It is used for the calculation of the relative standard

deviation of the repeatability*, RSDr*, using the following formulas:

𝑅𝑒𝑝𝑒𝑎𝑡𝑎𝑏𝑖𝑙𝑖𝑡𝑦∗ (𝑟∗) = 2.8 ∗ 𝑠𝑟 (𝑠𝑟 = 𝑖𝑛𝑡𝑟𝑎 − 𝑑𝑎𝑦 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛)

𝑅𝑆𝐷𝑟∗ = (

𝑠𝑟

𝑀𝑒𝑎𝑛 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑙𝑒𝑣𝑒𝑙) ∗ 100%

41

The criterion for the RSDr* for concentrations below 120 µg/kg is a maximum of 14.7%. As can be

seen in the Table, the percentages for the RSDr*

are 16.3, 9.1 and 6.7 % respectively. The last two

follow the criterion, the first one is slightly too high.

The repeatability was calculated with the same formulas as the repeatability*, however, all samples

originated from the same hair sample batch and were spiked at 1.0 times the permitted level. The

criterion for the RSDr is the same as the criterion for the RSDr*, which was a maximum of 14.7%. As

can be seen in the Table, the percentage for the RSDr is 10.1%, which is within the criterion.

The within-laboratory reproducibility is the similarity between separate analyses performed under

different conditions in the same laboratory during a sufficiently long period. It can be used for the

calculation of the relative standard deviation of within-laboratory reproducibility, according to the

formulas:

𝑊𝑖𝑡ℎ𝑖𝑛 − 𝑙𝑎𝑏𝑜𝑟𝑎𝑡𝑜𝑟𝑦 𝑟𝑒𝑝𝑟𝑜𝑑𝑢𝑐𝑖𝑏𝑖𝑙𝑖𝑡𝑦 (𝑅𝐿) = 2.8 ∗ 𝑠𝑅𝐿 (𝑆𝑅𝐿 = 𝑤𝑖𝑡ℎ𝑖𝑛 − 𝑙𝑎𝑏𝑜𝑟𝑎𝑡𝑜𝑟𝑦 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛)

𝑅𝑆𝐷𝑅𝐿 = (𝑠𝑅𝐿

𝑀𝑒𝑎𝑛 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑙𝑒𝑣𝑒𝑙) ∗ 100%

The criterion for the RSDRL for concentrations below 120 µg/kg is a maximum of 22.0%. As can be

seen in the Table, the percentages for the RSDRL are 19.1, 9.4 and 7.2 % respectively. All three values

are within the criterion. Since the RSDRL percentage follows the criterion for all concentrations, the

slightly too high value for the RSDr* for the spiked hair samples with 0.5 times the permitted level is

accepted.

CCα / CCβ

The decision limit (CCα) and the detection capability (CCβ) can be calculated using the following

formulas:

𝑚𝑝𝑒𝑟𝑚𝑖𝑡𝑡𝑒𝑑 𝑙𝑒𝑣𝑒𝑙 = ∑ 𝑦𝑝𝑒𝑟𝑚𝑖𝑡𝑡𝑒𝑑 𝑙𝑒𝑣𝑒𝑙,𝑖

𝑛

𝑠𝑝𝑒𝑟𝑚𝑖𝑡𝑡𝑒𝑑 𝑙𝑒𝑣𝑒𝑙 = √(∑ 𝑦𝑝𝑒𝑟𝑚𝑖𝑡𝑡𝑒𝑑 𝑙𝑒𝑣𝑒𝑙,𝑖 − 𝑚𝑝𝑒𝑟𝑚𝑖𝑡𝑡𝑒𝑑 𝑙𝑒𝑣𝑒𝑙)2

𝑛 − 1

𝐶𝐶𝛼 = 𝑀𝑅𝐿 + 1.64 ∗ 𝑠𝑝𝑒𝑟𝑚𝑖𝑡𝑡𝑒𝑑 𝑙𝑒𝑣𝑒𝑙

𝐶𝐶𝛽 = 𝐶𝐶𝛼 + 1.64 ∗ 𝑠𝑝𝑒𝑟𝑚𝑖𝑡𝑡𝑒𝑑 𝑙𝑒𝑣𝑒𝑙

These formulas are used in the ‘ResVal’ calculation program provided by RIKILT. As can be seen in

Table 4, the found values for CCα and CCβ are 31.83 and 63.65 respectively. This means the limit at

and below which it can be concluded with an error probability of α that the concentration of EtG in

hair is divergent, is 31.83 µg/L, and the smallest concentration of EtG in hair that may be quantified

in a sample with an error probability of β of 63.65 µg/L.

Selectivity / Specificity

The selectivity was determined according to the following procedure:

(i) A statistical approach according to Berendsen et al.86

(ii) The chromatograms of the blank hair samples must be free of interferences on the retention time

of EtG.

The transitions m/z 221.18 – 95.2, 221.18 – 85.13, 221.18 – 75.12 and 221.18 – 57.24 are the

transitions used in the EtG sample measurements. As shown in Chapter 2.4.1, they all have a value of

P(I) lower than 2*10-7.

42

When looking at the retention time of EtG of 2.1 min in Figure 29, no interfering signals are observed

which could originate from the hair matrix. These two observations lead to the fact that that the

method used is sufficiently selective.

Figure 29: Chromatogram of a blank hair sample without internal standard EtG-d5. Chromatogram was recorded with a SCIEX QTRAP-6500 mass spectrometer.

Ruggedness

The ruggedness is a measure for the sensitivity of the method in case of small changes in the

method. Three changes were tested during the validation:

(i) Incubation for 5h at 50 0C instead of 4h

(ii) 4 mL washing solvent instead of 2 mL

(iii) 10 min longer evaporation after the residue is dry

All measurements were evaluated in the ResVal sheet, their concentrations were calculated and are

shown in Table 5.

Table 5: Values for measured concentrations and trueness for three changes in the method

Spiked concentration (µg/L)

Measured concentration (µg/L)

Trueness (%)

(i) 100

102.8 95.4

102.8 95.4

(ii) 100

68.7 86.3

68.8 86.3

(iii) 100

75.3 95.6

75.3 95.6

Since the criterion for the trueness is 80-110%, the percentages for the trueness for the first change

are all still within this criterion. One measured concentration for the second change (68.6%) and for

the third change (75.3%) has a too low percentage for the trueness, which means the amount of

sample detected was a bit smaller than before the change. Since the second change was more

washing solvent, this means more sample is washed away during the washing step. While

evaporating longer than necessary, the sample becomes extremely dry which means it is harder to

dissolve it again in the reconstitution solvent. However, the differences were not extremely big and

other percentages for these changes are within the criteria, so it can be said the ruggedness of the

method is sufficient if the protocol is followed.

3.3.3 Reference and real hair samples After the validation, reference hair samples with concentrations of 100 and 200 pg/mg of EtG were

tested to see if the method works properly. In Attachment 3, the certificate and instruction sheets of

the reference hair samples are shown.

In Table 6, the gravimetric and target values as well as the confidence interval and the control range

of the reference hair material is shown. In the last column, the measured value is given. These values

43

are measured according to the calibration curve measured with the SCIEX QTRAP-6500 mass

spectrometer shown in Figure 11. The reference hair material was prepared and cleaned-up in

duplicate according to the optimized method.

Table 6: Reference hair material for ethyl glucuronide in hair. Concentrations 100 and 200 pg/mg.

Unit Gravimetric value

Target value Confidence interval

Control range Measured value

Ethyl glucuronide 100

pg/mg 105.4 107.7 ± 1.9 105.8 – 109.6 60.4 – 155.1 106.3 108.9

Ethyl glucuronide 200

pg/mg 203.6 201.1 ± 2.5 198.6 – 203.6 118.4 – 283.9 199.4 206.1

As can be seen in the Table, the confidence interval for the reference hair material with a

concentration of 100 pg/mg is 105.8 - 109.6. The measured values, 106.3 and 108.9, fall within this

range. The confidence interval for the reference hair material with a concentration of 200 pg/mg is

198.6 – 203.6. The measured value of 199.4 falls within this range, the measured value of 206.1 does

not. Since the value is slightly higher than the confidence interval and the other measured value is

within the interval, it can be assumed that the reference hair material is cleaned-up and measured

properly.

The method was further tested by measuring real hair samples. During the collection of the hair

samples, the individuals were asked to answer a question sheet regarding their drinking habits and

cosmetic hair treatment. This question sheet is shown in Attachment 4. People were requested to

rate their drinking habits according to the following scale:

- Teetotaller: No drinking of alcohol for at least three months before hair sampling

- Limited: No strict abstinence of alcohol use, but only drinking alcohol at for instance a

birthday party or a wedding, so really low doses at once and usage with a mean of maximum

two alcoholic consumptions a month.

- Tolerable: Consuming alcohol up to 6 standard glasses a day (60g alcohol/day).

- Excessive: Alcohol use equal to or more than 6 standard glasses a day (60g alcohol/day).

Further questions according to hair colour, usage of shampoo, hair gel, conditioner, paint etcetera

were also answered by the individuals. Results are shown in Attachment 5.

Hair samples of 42 individuals were tested. The lowest concentrations were measured in the

teetotallers group, followed by the limited group, which is according to the classification.

However, when the concentrations measured in all groups were compared to the cut-off value stated

by the SoHT to discriminate between teetotallers or social drinkers, and excessive alcohol abusers,

which is 30 pg/mg, some positive signals were detected. In Table 7, this comparison between self-

reported drinking behaviour and the measured results for EtG is shown.

Table 7: Comparison of self-reported drinking behaviour with measured results for EtG in hair. N: number of hair samples. Positive / %: number of positive samples with a EtG concentration ≥ 30 pg/mg / percentage.

Self-reported behaviour Ethyl glucuronide

N Positive / %

Teetotaller 7 0 / 0% Limited 4 1 / 25%

Tolerable 29 3 / 10.3% Excessive 2 2 / 100%

44

As can be seen, the self-reported drinking behaviour is not necessarily suitable or trustworthy for

evaluation, since the percentages for the limited and tolerable categories are positive, which means

in these categories people are present with a measured concentration of EtG above the cut-off value

of 30 pg/mg.

Effects of cosmetic hair treatments were also tested on these 42 individuals. People were asked

questions about bleaching or dying hair, and the use of hair sprays, waxes, gels, oils and conditioners.

All individuals used shampoo, so this hair product was not included in the measurements. The hair

samples were analysed under the assumption that the drinking behaviour of an individual is

independent of its hair products use. The measured values for all hair products were compared to

the individuals who reported themselves as no treatment users. Results are shown in Table 8.

Table 8: Measured concentrations of EtG in hair with cosmetic treatment.

Treatment N Mean measured EtG concentration (pg/mg)

Effect

No treatment used 20 30.8 -

Conditioner 10 29.1 No effect

Hair spray 4 31.2 No effect

Bleaching 1 10.1 Decrease

Dying 8 18.2 Decrease

Hair gel,wax,oil 7 30.5 No effect

As can be seen in the Table, bleaching and dying the hair sample leads to a decrease in the EtG

concentration in human hair. The mean measured values are below the LOD of the method, which

means they could vary a bit in practice, but it is clear that there is a decrease in the amount of EtG

detected. In many hair dyes an oxidizing compound such as H2O2 is present, which could oxidize EtG,

and make its concentration in hair lower. These findings were already shown in previous research 19,27,29. Several studies showed that the use of conditioners and hair sprays, gels, waxes and oils had

no effect for the measured concentration of EtG 27,28, so this experiment confirms this.

Lastly, a retrospective timeline was created by dividing a hair sample of an individual reported as

excessive alcohol user, drinking more than 4 days in a row during weekends, and almost every day on

vacations. The hair sample was cut into pieces of 1cm, and each cm was analysed individually, to

show the ability of the method for the creation of a retrospective timeline of alcohol use. The results

are shown in Figure 30.

45

Figure 30: Retrospective timeline of a regular alcohol user.

As can be seen, the hair samples 5,6 and 10 cm from the scalp show a really high alcohol content,

some above the chronic excessive alcohol consumption border of 30 pg/mg. Note that the smaller

corrected areas, so the smaller alcohol contents, might vary since these values are below the

calculated CCα and CCβ, but chronic excessive alcohol consumption could be detected with this

method. The hair was sampled in the beginning of October, and the individual was on vacation

during April and May, which is 5-6 months before sampling, in the cms of hair with the really high

alcohol content. Also, 10 months before sampling, it was December/January, so the high alcohol

content in this cm of hair could be due to partying during Christmas. To conclude, the retrospective

timeline is quite accurate when it comes to drinking behaviour.

46

4. Initial method development for the analysis of FAEEs in hair with

GC-MS/MS

4.1 Introduction Fatty acid ethyl esters are formed via FAEE synthase, as shown in Scheme 3. Since FAEE synthase is

present in organs that are damaged by alcohol consumption, the pancreas and the liver 47, signals of

FAEEs in human hair are a good indication for alcohol (ab-)use. Often the analysis of FAEEs is used in

combination with EtG to detect chronic excessive alcohol consumption in human hair. All esters are

stable and can be found throughout the body, but the esters most likely to be formed are ethyl

palmitate and ethyl oleate 48. This is the reason why the SoHT reconsidered their consensus in 2016,

instead of using all four FAEEs for the detection of chronic excessive alcohol consumption, only ethyl

palmitate,which is most present inside the body and gives the largest signal in a GC-MS/MS

chromatogram, is used for detection 52.

Scheme 9: Beta-cleavage of ethyl palmitate, leading to transition m/z 101.

FAEEs need sample pre-treatment before performing GC-MS/MS to clean-up the sample and to

remove the unwanted compounds. This is usually done in the forms of SPE or HS-SPME. In this

Chapter, the pre-treatment of a hair sample containing ethyl palmitate by SPE is tested, a method

already performed in literature is used 89. In this method, the molecular ion of ethyl palmitate, 284

m/z, is used as quantifier, the transition 101 m/z will be used as quantifier, since it is the β-cleavage

of ethyl palmitate, as shown in Scheme 9.

4.2 Materials and methods 4.2.1 Hair samples

The analysis of ethyl palmitate in hair was performed on blank hair samples provided by children or

abstaining adults.

4.2.2 Chemicals and reagents

Ethyl palmitate and DMSO were purchased from Sigma-Aldrich (Germany). Isolute NH2 (aminopropyl)

cartridges were purchased from Biotage (Sweden). Methanol, n-hexane and dichloromethane were

purchased from Actu-All chemicals (Netherlands).

4.2.3 Sample preparation and clean-up procedure for GC-MS/MS

An ethyl palmitate solution of 10.000 pg/µL (10 ppm) was prepared in methanol. Blank hair samples

were spiked with 50 µL of this solution and evaporated to air for approximately 30 min. To each

spiked hair sample, 4 mL of hexane and 0.5 mL of dimethyl sulfoxide were added and overnight

incubation followed. The incubation layer of hexane was extracted with Isolute NH2 (aminopropyl)

SPE cartridges, that were initially conditioned with 3 mL dichloromethane, followed by 3 mL hexane.

After elution of the incubation solvent, the analytes were extracted by 3 mL of hexane and,

subsequently, 3 mL dichloromethane, and the resulting extract was evaporated under a nitrogen

47

stream at 50 °C. The residue was dissolved in 50 µL of hexane and 2 µL were injected into the GC-

MS/MS.

4.2.4 Instrumentation for GC-MS/MS

A Varian CP-3800 gas chromatography – 1200L Quadrupole MS/MS system operating in EI mode was

used for analysis. Compounds were separated on a fused silica column (Agilent, DB-35MS) with a

(35%-phenyl)-methylpolysiloxane stationary phase (30 m length x 0.25 mm I.D. x 0.25 µm film

thickness). The carrier gas was helium with a constant flow of 1 mL/min. The injector temperature

was 300 °C, the initial column oven temperature of 100 °C was kept for 0.5 min, then increased to

200 °C at a rate of 12 °C/min, to 300 °C at 8 °C/min, and held at 300 °C for 3 min. Identification was

performed in SIM mode with the following transitions for ethyl palmitate: 101 and 284 m/z

(quantifier was 101).

4.2.5 Data processing

Data was processed using MS Workstation V6.9.2.

4.3 Results The method used was a method from literature 89, to test if ethyl palmitate is detectable with GC-

MS/MS. As can be seen in Figure 31, ethyl palmitate does show a signal in GC-MS/MS. In Figure 31A,

the full chromatogram is shown, recorded for the transitions 101 and 284 m/z. Since m/z 101 was

used as quantifier, in Figure 31B the chromatogram for this transition is shown. As can be seen, the

largest peak in the chromatogram, eluting at 12 min., originates from ethyl palmitate.

Figure 31: Chromatograms of ethyl palmitate. A: full chromatogram, and B: chromatogram from transition m/z 101. Chromatograms were recorded with a Varian mass spectrometer.

This result is a promising indication for the method tried to actually work, but the intensity of the

peaks in the chromatogram is still quite low. Due to time limitations, no further investigation was

performed.

48

5. Recommendations for future research Since there were still some parts in this research that did not work according to plan, in this Chapter

some recommendations for future research are given.

5.1 Sensitivity LC-MS/MS The main problem in this project was the fact that the sensitivity was quite low. By switching from

TQ-S to QTRAP-6500 mass spectrometer, the sensitivity already got 2-3 times higher and we were at

least able to detect chronic excessive alcohol consumption. Measuring with a more sensitive machine

could thus in the future be promising to make sure the sensitivity is another 3-4 times higher, in

order to be able to detect the cut-off value of 7 pg/mg.

Another possibility to make the sensitivity of the method higher, is to change some steps in the

analysis. The sample pre-treatment was optimized in this research, but only with the resources

available in the lab. SPE cartridges especially designed for EtG are available, which are UCT Clean

Screen ETG SPE cartridges. They are especially designed for detection in urine, but it might work for

the detection in hair as well, so using these cartridges in the sample pre-treatment could work in the

future.

Until now, the method was tested with a C8 column for LC-MS/MS, with a high acetonitrile amount

in the mobile phase, to overcome interfering signals and to make sure the EtG eluted a bit later in the

chromatogram, which gave nice results for EtG. HILIC columns could also be used and solve the same

difficulties as the C8-column did. An Acquity BEH HILIC column was tried during the initial method

development, but did not change the sensitivity of the method. Using a different HILIC column could

also be tested in future research, or the addition of post-column acetonitrile for solving the same

difficulties.

5.2 Derivatization of EtG for GC-MS/MS and sensitivity The derivatization process of EtG was a difficult step in this research. Several derivatization agents

such as BSTFA, PFPA and HFBA were tested, but none of them showed a reasonable result. All

solvents need to be completely clean of water during the derivatization, otherwise the derivatization

agents will hydrolyse and no derivatization takes place. In this research, only isooctane and acetone

were completely free of water due to an addition of salt to the solvent, ethyl acetate, methanol and

hexane were not. In future experiments, the derivatization processes could be tried again, but with

different solvents that are clear of water.

Derivatization with PBA did show a result in the chromatogram, but the intensity was extremely low.

During derivatization with PBA, also a PBA complex is formed by a reaction of PBA with itself. The

peak in the chromatogram for this complex was way higher than the peak originating from EtG-PBA,

so it could be that all PBA already reacted with itself, so no PBA was left to derivatize EtG. In future

experiments it could be tried to add more PBA to the EtG during the derivatization, just to make sure

there is still some PBA left for derivatization of EtG.

5.3 Sensitivity FAEEs with GC-MS/MS The signal of ethyl palmitate in the GC-MS/MS spectrum was low, so the use of another machine

could be a solution. Since the Varian CP-3800 gas chromatography – 1200L Quadrupole MS/MS

system is an old machine with insufficient sensitivity.

Another possibility to increase the signal is changing the clean-up method. In the analysis in Chapter

4, SPE is used. Another possible clean-up method is HS-SPME. HS-SPME is a technique based on the

fact that the analytes present in the head space, the gas phase that lies upon the sample, could be

absorbed by a fibre 1. This fibre consists of a very thin polymeric film, which is attached to a fused

49

silica core. This polymeric film is than exposed to the head space of the sample 1. Steps in the analysis

by HS-SPME are shown in Figure 32.

Figure 32: Steps in the analysis by HS-SPME. Reproduced from Ref. 90

If analytes used are volatile enough at the temperature at which the extraction is performed, HS-

SPME is an ideal technique since it is non-destructive, and samples at different experimental

conditions could be evaluated. Another advantage is the fact that HS-SPME makes sure the extract is

protected from impurities, since the vial is closed at all-time 90. Especially this last feature is useful,

since the solutions of ethyl palmitate are volatile, and could evaporate quickly.

5.4 Combining sample pre-treatment steps In the goal it was stated that it would be nice of the sample pre-treatment steps for the two ethanol

metabolites could be combined, in order to make a more sufficient overall method for the detection

of both metabolites.

It would be nice to perform the analysis of both EtG and FAEEs with the same technique. The

problem in this case is the fact that FAEEs are usually determined with GC-MS/MS, since they are

highly volatile which could be a problem in LC-MS/MS. EtG is usually determined with LC-MS/MS,

since the derivatization of EtG could be a problem, as experienced in this project.

However, even if two analysis techniques are used, the sample pre-treatment steps could be

combined. The optimized method for EtG includes ultrasonication and overnight incubation, clean-up

with SPE, evaporation and reconstitution. The sample pre-treatment of FAEEs used in Chapter 4 also

includes overnight incubation, clean-up with SPE, evaporation and reconstitution. In future research,

the sample pre-treatment of the two ethanol metabolites could thus be performed at te same time,

which makes the method more sufficient. Also, if the sample pre-treatment for FAEEs is changed

with HS-SPME included, the samples still need overnight incubation and clean-up, but this time with

HS-SPME, so this does not change much in the sample pre-treatment steps and a combination of the

two methods is still possible.

50

6. General conclusions To conclude, a method was developed and optimized for the detection of EtG in human hair with LC-

MS/MS. The fully optimized method was as follows; 50 mg of hair was finely cut, 2 mL of milli-Q

water as extraction solvent was added, 50 μL of a 10 ppm solution of the internal standard was

added. The samples were placed in an ultrasonic bath at 50 °C for 4 hours, followed by overnight

incubation. The next day, the samples were cleaned-up by SPE, previously conditioned with 2 mL of

methanol and 2 mL of Milli-Q water, samples were washed with acetonitrile and eluted with 2 mL

methanol/2% formic acid solution. Finally, the extract was evaporated under a nitrogen stream at 40

degrees and reconstituted in 200μL of mobile phase A for LC-MS/MS.

The method was fully validated according to a permitted level of 100 pg/mg. The recovery and

trueness/accuracy are nicely within the criteria set by RIKILT. The relative standard deviation of the

repeatability for 0.5 times the permitted level (50 pg/mg) is slightly too high, but since the relative

standard deviation of the within-lab reproducibility for 0.5 times the permitted level is within the

criteria, and all other values for 0.5, 1.0 and 1.5 times the permitted level are within, this was

accepted. The only thing that did not match the criteria, is the linearity. The values for all three

validation days were slightly below the criteria of 0.990. In all MMS series, one point was way above

the calibration line, and when these points were removed, the measured value was above the

criteria of 0.990. These three points were therefore considered outliers, and the linearity was also

accepted.

The method was further tested on reference hair samples of 100 and 200 pg/mg, and the measured

concentrations were within the confidence interval given by the company, so it can be stated that

the method works accurately.

Also, some real hair samples were tested, and the method was able to detect chronic excessive

alcohol consumption. A retrospective timeline was obtained to show the ability of the method for an

historical overview of alcohol (ab-)use. The sensitivity of the method was unfortunately too low for

the detection of regular alcohol consumption, the limit of detection for the method is around 30

pg/mg, which is the cut-off value of the SoHT for chronic excessive alcohol consumption.

It was also tried to develop a GC-MS/MS method for the detection of both EtG as well as the FAEE

ethyl palmitate. Derivatization of EtG was quite difficult, since BSTFA, PFPA as well as HFBA did not

give any signals for a derivatized EtG in GC-MS/MS. This could be due to the fact that all solvents

used need to be completely clean of water, otherwise the derivatization agents will hydrolyse and no

derivatization takes place. Since for some of the solvents used, no precaution steps were taken, it

could be that the sample was not completely dry.

Derivatization with PBA did work and gave signals in GC-MS/MS at m/z of 308, which is the mass of

the derivatized EtG-PBA. However, the largest signal in the spectrum originated from m/z 311, which

is a PBA complex formed by a reaction of PBA with itself. This signal was much larger than the signal

originating from EtG-PBA, so it is possible that PBA reacts quickly with itself, leaving less PBA for

derivatization of EtG.

Ethyl palmitate was also analysed with GC-MS/MS. The sample pre-treatment steps tried were quite

similar to the optimized method for EtG with LC-MS/MS, which is a promising feature for combining

these steps in future research, in order to make the method as sufficient as possible. A GC-MS/MS

signal was visible for m/z 101, which originates from beta cleavage of ethyl palmitate. Also in this

case the signal was quite low, so further research in the future is necessary before it can be used as

analysis method.

51

Acknowledgements Here, I would like to thank Andries Koops for the opportunity to work on my project in his research

group at RIKILT. Also, very special thanks to Wilco Duvivier, Saskia Sterk and Marco Blokland for their

daily supervision and Wim Kok for helping me over the course of this project. Last but not least, a

thank you note for the entire BU Veterinary Drugs and all other employees at RIKILT for all sociability

and helping me out with anything over the past 9 months.

52

References

1. Kintz, P.; Salomone, A.; Vincenti, M., ‘Hair analysis in clinical and forensic toxicology’, Academic Press (Elsevier),

Oxford, UK, 2015.

2. Kintz, P., ‘Value of the concept of minimal detectable dosage in human hair’, Forensic Sci. Int., 2012, 218, 28-30.

3. Buffoli, B.; Rinaldi, F.; Labanca, M.; Sorbellini, E.; Trink, A.; Guanziroli, E.; Rezzani, R.; Rodella, L.F., ‘The human

hair: from anatomy to physiology’, Int. J. Dermatol., 2014, 53 (3), 331-341.

4. Kelly, R.C.; Mieczkowski, T.; Sweeney, S.A., ‘Hair analysis for drugs of abuse. Hair colour and race differentials or

systematic differences in drug preferences?’, Forensic Sci. Int., 2000, 107, 63-86.

5. Khalique, A.; Kim, K.S.; Lee, G.J., ‘Investigation of aging effects in human hair using atomic force microscopy’, Skin

Res. Technol., 2011, 17, 63-68.

6. Gurden, S.P.; Monteiro, V.F.; Longo, E., ‘Quantitative analysis and classification of AFM images of human hair’, J.

Microsc., 2004, 215, 13-23.

7. Randall, V.A.; Botchkareva, N.V., ‘The biology of hair growth’, In: Ahluwalia, G.S., ‘Cosmetic Application of Laser

and Light-Based System’, William Andrew (Elsevier), Norwich (NY), USA, 2009, p. 3-35.

8. Swift, J.A., ‘Human hair cutticle: biologically conspired to the owners advantage’, J. Cosmet. Sci., 1999, 50, 23-47.

9. Wolfram, L.J., ‘Human hair: a unique physicochemical composite’, J. Am. Acad. Dermatol., 2003, 48, 106-114.

10. Pragst, F.; Balikova, M.A., ‘State of the art in hair analysis for detection of drug and alcohol abuse’, Clin. Chim.

Acta, 2006, 370, 17-49.

11. Rogers, G.E., ‘Electron microscope observations on the glassy layer of the hair follicle’, Exp. Cell Res., 1957, 13,

521-528.

12. Peus, D.; Pittelkow, M.R., ‘Growth factors in hair organ development and the hair growth cycle’, Dermatol. Clin.,

1996, 14, 559-572.

13. Blume-Peytavi, U.; Vogt, A., ‘Human hair follicle: reservoir function and selective targeting’, Br. J. Dermatol., 2011,

165, 13-17.

14. Wosicka, H.; Cal, K., ‘Targeting to the hair follicles: current status and potential’, J. Dermatol. Sci., 2010, 57, 83-89.

15. Paus, R., ‘Principles of hair cycle control’, J. Dermatol., 1998, 25, 793-802.

16. Bull, J.J.; Pelengaris, S.; Hendrix, S., ‘Ectopic expression of c-Myc in the skin affects the hair growth cycle and

causes an enlargement of the sebaceous gland’, Br. J. Dermatol., 2005, 152, 1125-1133.

17. Kloepper, J.E.; Sugawara, K.; Al-Nuaimi, Y., ‘Methods in hair research: how to objectively distinguish between

anagen and catagen in human hair follicle organ culture’, Exp. Dermatol., 2010, 19, 305-312.

18. Cooper, G.A.A.; Kronstrand, R.; Kintz, P., ‘Society of Hair Testing guidelines for drug testing in hair’, Forensic Sci.

Int., 2012, 218, 20-24.

19. Kintz, P., ‘Segmental hair analysis can demonstrate external contamination in post-mortem cases’, Forensic Sci.

Int., 2012, 215, 73-76.

20. Society of Hair Testing, ‘Statement of the society of hair testing concerning the examination of drugs in human

hair’, Forensic Sci. Int., 1997, 84, 3-6.

21. Smith, H.; Forshufvud, S.; Wassen, A., ’Distribution of arsenic in Napoleon’s hair’, Nature, 1962, 194, 725-726.

22. Springfield, A.C.; Cartmell, L.W.; Aufderheide, A.C.; Buikstra, J.; Ho, J., ‘Cocaine and metabolites in the hair of

ancient Peruvian coca leaf chewers’, Forensic Sci. Int., 1993, 63, 269-275.

23. Chatterton, C.; Kintz, P., ‘Hair analysis to demonstrate administration of amitriptyline, temazepam, tramadol and

dihydrocodeine to a child in a case of kidnap and false imprisonment’, J. Forensic Legal Med., 2014, 23, 26-31.

24. Pötsch, L.; Moeller, M.R., ‘On pathways for small molecules into and out of human hair fibers’, J. Forensic Sci.,

1996, 41, 121-125.

25. Skopp, G.; Pötsch, L.; Mauden, M., ‘Stability of cannabinoids in hair exposed to sunlight’, Clin. Chem., 2000, 46,

1846-1848.

26. Arndt, T.; Schröfel, S.; Stemmerich, K., ‘Ethyl glucuronide identified in commercial hair tonics, Forensic Sci. Int.,

2013, 231, 195-198.

27. Favretto, D.; Tucci, M.; Monaldi, A.; Ferrara, S.D.; Miolo, G., ‘A study on photodegradation of methadone, EDDP,

and other drugs of abuse in hair exposed to controlled UVB radiation’, Drug Test Anal., 2014, 6, 78-84.

28. Röhrich, J.; Zorntlein, S.; Pötsch, L.; Skopp, G.; Becker, J., ‘Effect of the shampoo Ultra Clean on drug

concentrations in human hair’, Int. J. Legal Med., 2000, 113, 102-106.

29. Skopp, G.; Pötsch, L.; Moeller, M.R., ‘On cosmetically treated hair – aspects and pitfalls of interpretation’, Forensic

Sci. Int., 1997, 84, 43-52.

30. Dawber, R., ‘Hair: its structure and response to cosmetic preparations’, Clin. Dermatol., 1996, 14, 105-112.

31. Jurado, C.; Kintz, P.; Menedez, M., Repetto, M., ‘Influence of cosmetic treatment of hair on drug testing’, Int. J.

Legal Med., 1997, 110, 159-163.

53

32. Ryder, M.L., ‘Nutritional factors influencing hair and wool growth’, In: Montagna, W.; Ellis, R.A., ‘The biology of

hair growth’, Academic Press (Elsevier), New York, USA, 1958, p. 305-334.

33. Harkey, M.R., ‘Anatomy and physiology of hair’, Forensic Sci. Int., 1993, 63, 9-18.

34. Chittleborough, G.; Steel, B.J., ‘Is human hair a dosimeter for endogenous zinc and other trace elements?’, Sci.

Total Environ., 1980, 15, 25-35.

35. Kronstrand, R.; Scott, K., ‘Drug incorporation into hair’, In: Kintz, P., ‘Analytical and practical aspects of drugs

testing in hair’, CRC Press, Boca Raton (FL), USA, 2007, p. 1-23.

36. Baumgartner, W.A.; Hill, V.A.; Bland, W.H., ‘Hair analysis for drugs of abuse’, J.Forensic Sci., 1993, 34, 1433-1453.

37. Pötsch, L.; Skopp, G.; Moeller, M.R., ‘Biochemical approach on the conservation of drug molecules during hair

formation’, Forensic Sci. Int., 1997, 84, 25-35.

38. Kikura, R.; Nakahara, Y.; Mieczkowski, T.; Tagliaro, F., ‘Hair analysis for drug abuse XV. Disposition of 3,4-

methylenedioxy-methamphetamine (MDMA) and its related compounds into rat hair and application to hair

analysis for MDMA abuse’, Forensic Sci. Int., 1997, 84, 165-177.

39. Pötsch, L., ‘A discourse on human hair fibres and reflections on the conservation of drug molecules’, Int. J. Legal

Med., 1996, 108, 285-293.

40. Pragst, F.; Spiegel, K.; Sporkert, F.; Bohnenkamp, M., ‘Are there possibilities for the detection of chronically

elevated alcohol consumption by hair analysis? A report about the state of investigation’, Forensic Sci. Int., 2000,

107, 201-223.

41. Kamil, I.A.; Smith, J.N.; Williams, R.T., ‘A new aspect of ethanol metabolism: isolation of ethyl glucuronide’,

Biochem., 1952, 51, 32-33.

42. Skopp, G.; Schmitt, G.; Pötsch, L.; Drönner, P.; Aderjan, R.; Mattern, R., ‘Ethyl glucuronide in human hair’, Alcohol

Alcoholism, 2000, 35(3), 283-285.

43. Cabarcos, P.; Álvarez, I.; Taberno, M.J.; Bermejo, A.M., ’Determination of direct alcohol markers: a review’, Anal.

Bioanal. Chem., 2015, 407, 4907-4925.

44. Tarcomnicu, I.; Van Nuijs, A.; Aerts, K.; De Doncher, M.; Covaci, A.; Neels, H., ‘Ethyl glucuronide determination in

meconium and hair by hydrophilic interaction liquid chroamtography-tandem mass spectrometry’, Forensic Sci.

Int., 2010, 196, 121-127.

45. Foti, R.S.; Fisher, M.B., ‘Assessment of UDP-glucuronosyltransferase catalyzed formation of ethyl glucuronide in

human liver microsomes and recombinant UGTs’, Forensic Sci. Int., 2005, 153, 109-116.

46. Kharbouche, H.; Steiner, N.; Morelato, M.; Staub, C.; Boutrel, B.; Mangin, P.; Sporkert, F.; Augsburger, M.,

‘Influence of ethanol dose and pigmentation on the incorporation of ethyl glucuronide into rat hair’, Alcohol,

2010, 44(6), 507-514.

47. Lapasota, E.A.; lange, L.G., ‘Presence of nonoxidative ethanol metabolism in human organs commonly damaged

by ethanol abuse’, Science, 1986, 231, 497-499.

48. Dan, L.; Lapasota, M., ‘Ethyl palmitate and ethyl oleate are the predominant fatty acid ethyl esters in the blood

after ethanol ingestion and their synthesis is differentially influenced by the extracellular concentrations of their

corresponding fatty acids’, Alcohol Clin. Exp. Res., 1997, 21, 286-292.

49. Cabarcos, P.; Álvarez, I.; Bermejo, A.M.; Tabernero, M.J.; López, P.; Fernández, P., ‘Analysis of fatty acid ethyl

esters in hair by headspace solid-phase microextraction and gas chromatography-mass spectrometry’, Anal. Lett.,

2009, 42, 2962-2977.

50. Brodie, B.B., ‘Pathways of drug metabolism’, J. Pharm. Pharmacol., 1956, 8, 1-17.

51. Pragst, F.; Auwärter, V.; Sporkert, F.; Spiegel, K., ‘Analysis of fatty acid ethyl esters in hair as possible markers of

chronically elevated alcohol consumption by headspace solid phase microextraction and gas chromatography-

mass spectrometry’, Forensic Sci. Int., 2001, 121, 76-88.

52. Society of Hair Testing, ‘Consensus for the use of alcohol markers in hair for assessment of both abstinence and

chronic excessive alcohol consumption’, 2016.

53. Paul, R.; Tsanaclis, L.; Kingston, R.; Berry, A.; Guwy, A., ‘Simultaneous determination of GHB and EtG in hair using

GC-MS/MS’, Drug Test. Analysis, 2011, 3, 201-205.

54. Concheiro, M.; Cruz, A.; Mon, M.; de Castro, A.; Quintela, O.; Lorenzo, A.; López-Rivadulla, M., ‘Ethyl glucuronide

determination in urine and hair from alcohol withdrawal patients’, J. Anal. Toxicol., 2009, 33, 155-161.

55. Auwarter, V.; Sporkert, F.; Hartwig, S.; Pragst, F.; Vater, H.; Diefenbacher, A., ’Fatty acid ethyl esters in hair as

markers of alcohol consumption. Segmental hair analysis of alcoholics, social drinkers, and teetotalers’, Clin.

Chem., 2001, 47(12), 2114-2123.

56. Albermann, M.E.; Musshoff, E.; Madea, B., ‘Comparison of ethyl glucuronide (EtG) and fatty acid ethyl esters

(FAEEs) concentrations in hair for testing abstinence’, Anal. Bioanal. Chem., 2011, 400, 175-181.

57. Gaillard, Y.; Pepin, G., ‘Screening and identification of drugs in human hair by high-performance liquid

chromatography – photodiode-array UV detection and gas chromatography – mass spectrometry after solid-

phase extraction. A powerful tool in forensic medicine’, J. Chromatogr. A, 2002, 958, 231-238.

54

58. Pragst, F.; Balikova, M.A.,’State of the art in hair analysis for detection of drug and alcohol abuse’, Clin. Chim.

Acta, 2006, 370, 17-49.

59. De Hoffman, E.; Stroobant, V., ‘Mass spectrometry – principles and applications’, John Wiley & Sons, West Sussex,

UK, 2007.

60. Kicman, A.T.; Parkin, M.C.; Iles, R.K., ’An introduction to mass spectrometry based proteomics – detection and

characterization of gonadotropins and related molecules’, Moll. Cell. Endocrinol., 2007, 260-262, 212-227.

61. https://www.biologie.hu-berlin.de/de/gruppenseiten/oekologie/meth/massspec/mass_sp

Visited July 7, 2017.

62. Uhl, M.,’Determination of drugs in hair using GC-MS/MS’, Forensic Sci. Int., 1997, 84, 281-294.

63. Sachs, H.; Uhl, M.; Hege-Scheuing, G.; Schneider, E., ‘Analysis of fentanyl and sufentanil in hair by GC-MS/MS’, Int.

J. Leg. Med., 1996, 109, 213-215.

64. Pichini, S.; Pacifici, R.; Altieri, I.; Pellegrini, M.; Zuccaro, P., ‘Determination of opiates and cocaine in hair as

trimethylsilyl derivatives using gas chromatography – tandem mass spectrometry’, J. Anal. Toxicol., 1999, 23, 343-

348.

65. Uhl, M., ‘Tandem mass spectrometry: a helpful tool in hair analysis for the forensic expert’, Forensic. Sci. Int.,

2000, 107, 169-179.

66. Agius, R.; Nadulski, T.; Kahl, H.G.; Schräder, J.; Dufaux, B.; Yegles, M., ‘Validation of headspace solid-phase

microextraction-GC-MS/MS for the determination of ethyl glucuronide in hair according to forensic guidelines’,

Forensic Sci. Int., 2010, 196, 3-9.

67. Zaikin, V.G.; Halket, J.M., ‘Derivatization in mass spectrometry – 2. Acylation’, Eur. J. Mass Spectrum., 2003, 9(5),

421-434.

68. Blau, K.; Halket, J., ‘Handbook of derivatives for chromatography’, John Wiley and Sons, Chichester, UK, 1997.

69. Scott, R.P.W., ‘Gas chromatography. Book 2 Chrom-Ed Book Series.’ The Reese-Scott Partnership, Chesire, UK,

2003.

70. Orata, F., ‘Derivatization reactions and reagents for gas chromatography analysis’, In: Advanced gas

chromatography – progress in agricultural, biomedical and industrial applications, Intech, Croatia, 2012, 83-108.

71. Schauer, J.J.; Kleeman, M.J.; Cass, G.R.; Simoneit, B.R.T., ‘Measurement of emissions from air pollution sources’,

Environ. Sc. Technol., 2002, 36, 1169-1180.

72. Lin, D.L.; Wang, S.M.; Wu, C.H.; Chen, B.G.; Liu, R.H., ‘Chemical derivatization for the analysis of drugs by GC-MS –

a conceptual review’, J. Food Drug Anal., 2008, 16(1), 1.

73. Negrusz, A.; Moore, C.M.; Hinkel, K.B., ‘Deposition of 7-aminoflunitrazepam and flunitrazepam in hair after a

single dose of Rohypnol’, J. Forensic Sci., 2001, 46, 1143-1151.

74. Yegles, M.; Labarthe, A.; Auwarter, V., ‘Comparison of ethyl glucuronide and fatty acid ethyl ester concentrations

in hair of alcoholics, social drinkers and teetotallers’ Forensic Sci. Int., 2004, 145, 167-173.

75. Kelly, T.; Doble, P.; Dawson, M., ‘Chiral analysis of methadone and its major metabolites (EDDP and EMDP) by

liquid chromatography – mass spectrometry’, J. Chromatogr. B, 2005, 814, 315-323.

76. Muller, C.; Vogt, A.; Goerke, R.; Kordon, A.; Weinmann, W., ‘Identification of selected psychopharmaceuticals and

their metabolites in hair by LC/ESI-CID/MS and LC-MS/MS’, Forensic Sci. Int., 2000, 113, 415-421.

77. Dumestre-Toulet, V.; Cirimele, V.; Gromb, S., ’Last performance with VIAGRA: post mortem identification of

sildenafil and its metabolites in biological specimens including hair sample’, Forensic Sci. Int., 2002, 126, 71-76.

78. Janda, I.; Weinmann, W.; Kuehnle, T.; Lahode, M.; Alt, A., ‘Determination of ethyl glucuronide in human hair by

SPE and LC-MS/MS’, Forensic Sci. Int., 2002, 128, 59-65.

79. Tarcomnicu, I.; van Nuijs, A.L.; Aerts, K.; de Doncker, M.; Covaci, A.; Neels, H., ‘Ethyl glucuronide determination in

meconium and hair by hydrophilic interaction liquid chromatography – tandem mass spectrometry’, Forensic Sci.

Int., 2010, 196, 121-127.

80. Williams, J.; Patsalos, P.N.; Wilson, J.F., ‘Hair analysis as a potential index of therapeutic compliance in the

treatment of epilepsy’, Forensic Sci. Int., 1997, 84, 113-122.

81. Shi, Y.; Shen, B.; Xiang, P.; Yan, H.; Shen, M., ‘Determination of ethyl glucuronide in hair samples of Chinese

people by protein precipitation (PPT) and large volume injection – gas chromatography – tandem mass

spectrometry (LVI-GC-MS/MS)’, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 2010, 878, 3161-3166.

82. Imbert, L.; Gaulier, J.M.; Dulaurant, S.; Morichon, J.; Bevalot, F.; Izac, P., ‘Improved liquid chromatography –

tandem mass spectrometric method for the determination of ethyl glucuronide concentrations in hair:

Applications to forensic cases’, Int. J. Legal. Med., 2014, 128, 53-58.

83. Kintz, P.; Villain, M.; Vallet, E.; Etter, M.; Salquebre, G.; Cirimele, V., ‘Ethyl glucuronide: Unusual distribution

between head hair and pubic hair’, Forensic Sci. Int., 2008, 176, 87-90.

84. Cabarcos, P.; Hassan, H.M.; Tabernero, M.J.; Scott, K.S., ‘Analysis of ethyl glucuronide in hair samples by liquid

chromatography – electrospray ionization – mass spectrometry (LC-ESI-MS/MS), J. Appl. Toxicol., 2013, 33, 638-

643.

55

85. Suesse, S.; Pragst, F.; Mieczkowski, T.; Selavka, C.M.; Elian, A.; Sachs, H.; Hastedt, M.; Rothe, M.; Campbell,

J.,’Practical experiences in application of hair fatty acid ethyl esters and ethyl glucuronide for detection of chronic

alcohol abuse in forensic cases’, Forensic Sci. Int., 2012, 218, 82-91.

86. Berendsen, B.A.; Stolker, L.A.; Nielen, M.W.F., ‘The (un)certainty of selectivity in liquid chromatography tandem

mass spectrometry’, J. Am. Soc. Mass Spectrom., 2013, 24, 154-163.

87. Sigma-Aldrich Co., ‘Bulletin 910: Guide to solid phase extraction’, Supelco, USA, 1998.

88. Commission Decision 2002/657/EC, ‘Implementing council directive 96/23/EC concerning the performance of

analytical methods and the interpretation of results’, Official Journal of the European Communities, 2002.

89. Politi, L.; Mari, F.; Furlanetto, S.; Del Bravo, E.; Bertol, E., ‘Determination of fatty acid ethyl esters in hair by GC-MS

and application in a population of cocaine users’, J. Phar. Biomed. Anal., 2011, 54, 1192-1195.

90. Valduga, E.; Valerio, A.; Treichel, H.; Nascimento Filho, I.; Furigo Junior, A.; Di Luccio, M., ‘Head space solid phase

micro-extraction (HS-SPME) of volatile organic compounds produced by sporidiobolus salmonicolor’, Cienc.

Technol. Alliment., 2010, 30(4), 987-992.

56

Attachments

57

Attachment 1

Daughter ion scan of EtG

58

Attachment 2

Validation plan

1. Performance characteristics to be determined

The performance characteristics to be determined for each type of analysis method is described in SOP A0400,

and are shown in Table 1. The method that needs to be validated is a quantitative confirmation method. The

performance characteristics that need to be validated are represented in blue.

Table 1. Performance characteristics to validate for each type of analysis method

Performance characteristics Qualitative method Quantitative method

Screening Confirmation Screening Confirmation

Relative standard deviation of repeatability (RSDr (%)) - - + +

Standard deviation of repeatability (sr) - - + +

Repeatability (r) (7 x same sample) - - + +

Relative standard deviation of repeatability* (RSDr*(%)) - - 0 0

Standard deviation of repeatability* (sr*) - - 0 0

Repeatability* (r*) (7 different samples) - - 0 0

Relative standard deviation of within-laboratory reproducibility (RSDRL (%)). - - + +

Standard deviation of within-laboratory reproducibility (sRL) - - + +

Within-laboratory reproducibility (RL) - - + +

Decision limit (CC) - + - +

CC on RPA - - + +

Detection capability (CC) + + + +

Trueness (T, %) - - + +

Recovery (%) 0 0 0 0

Matrix effect (%) 0 0 0 0

Selectivity + + + +

Ruggedness + + + +

Linearity - - 0 0

Stability + + + +

Traceability standards + + + +

+ = Mandatory according to 2002/657/EC1.

0 = Not mandatory according to 2002/657/EC, within RIKILT (*) established as desirable

- = Not mandatory

2. Experimental design

In Table 2, the experimental design for the validation is shown.

59

Table 2. Experimental design for validation

Day Linearity

Recovery

(MMS and

MMRS)

Trueness

Within-laboratory reproducibility

Repeatability*

CCα CCß

Repeatability

Selectivity

Ruggedness

Total

1 7 (A) 7*3 (B-H) 7 (B) 7 (B-H) 42

2 7 (I) 7*3 (J-P) 7 (J) 7 (J-P) 42

3 7 (Q) 7*3 (R-X) 7 (R) 7 (R-X) 6 (Q) 48

Every day, 8 different hair samples will be used; 1 for linearity (MMS) and recovery (MMRS), 7 different ones for

trueness, repeatability*, within-laboratory reproducibility, selectivity, CCα, CCß and 7 hair samples, from the same

batch as used for linearity, for repeatability. In Attachment 1 the used hair samples are listed.

Linearity (MMS): 6 samples originating from the same batch, spiked at 0 – 0.25 – 0.5 – 1.0 – 2.0 – 3.0 * PL on 3

different days.

Recovery (MMRS): 1 sample originating from the batch used for linearity, spiked after clean-up at 1.0 * PL on 3

different days.

Trueness: 7 different hair samples spiked at 0.5 – 1.0 – 1.5 * PL on 3 different days.

Within-laboratory reproducibility: 7 different hair samples spiked at 0.5 – 1.0 – 1.5 * PL on 3 different days.

Repeatability*: 7 different hair samples spiked at 0.5 – 1.0 – 1.5 * PL on 3 different days.

Repeatability: 7 hair samples originating from the same batch, spiked 1.0 * PL on 3 different days.

CCα / CCß: 7 different hair samples spiked at 0.5 – 1.0 – 1.5 * PL at 3 different days.

Selectivity: 7 blank hair samples at 3 different days.

Matrix effect: Comparing MMRS and stock solution at the same level at 3 different days.

Stability: Stability is already tested by preliminary investigation.

Ruggedness: Three changes in the clean-up procedure will be compared in duplicate with the standard procedure.

3. Components to be validated

Ethyl glucuronide

4. Overview permitted level (PL)

For ethyl glucuronide in hair, no maximum residual limit (MRL) or reference point of action (RPA) is established by

authorities. Based on guidelines created by the Society of Hair Testing (SoHT), the cut-off value for ethyl

glucuronide in hair is 7 pg/mg of hair for repeated alcohol consumption, and 30 pg/mg for chronic excessive alcohol

consumption. Based on previous research, the permitted level (PL) was set at 100 µg/L (100 pg/mg with the used

procedure), since the cut-off values could not be reached due to sensitivity issues with the mass spectrometer.

Component Permitted level (µg/l)

Ethyl glucuronide 100 *

* In final extract (200 µL). 50 mg of human hair is used, so the concentration will be 100 pg/mg.

60

5. Choice sample material

Samples used are blank human hair samples of both children and abstaining adults. The used hair samples are

listed in Table 3.

Table 3. Blank hair samples

Hair Gender

(M/W)

Child (C) /

Adult (A)

Hair color

A W C Brown

B W A Brown

C W A Blonde

D W C Blonde

E M C Brown

F M C Brown

G W C Blonde

H W C Blonde

I W C Blonde

J W A Blonde

K W C Brown

L M A Brown

M W C Blonde

N W C Blonde

O W C Black

P W C Black

Q M C Blonde

R W C Brown

S M C Blonde

T M C Black

U M C Black

V W C Blonde

W W C Blonde

X M C Blonde

6. Criteria performance characteristics

6.1 Trueness (T) / Repeatability (r) / Within-laboratory reproducibility (RL)

Repeatability r = 2.8 * sr (sr = intra-day standard deviation)

Within-laboratory reproducibility RL = 2.8 * sRL (sRL = within-lab standard deviation)

Trueness T = mean measured level / permitted level * 100%

RSDT RSDT = RSDRL = sRL / mean measured level * 100%

RSDr RSDr = sr / mean measured level * 100%

Table 4. Design for performance characteristics

Level**

7x

Level***

7x

Day 1 0.5 – 1.0 – 1.5 * PL 1.0 * PL

Day 2 0.5 – 1.0 – 1.5 * PL 1.0 * PL

Day 3 0.5 – 1.0 – 1.5 * PL 1.0 * PL

** = 7 different hair samples

*** = 7 hair samples from the same batch

61

In Table 4, the experimental design for the performance characteristics trueness, repeatability and within-laboratory

reproducibility is shown. The values for the trueness, RSDr and RSDRL must follow some criteria during the

validation, these criteria are listed in Table 5.

Table 5. Criteria for trueness, RSDr and RSDRL as described in SOP F0052

Component PL Range Criteria

(µg/l) T (%) RSDr (%)* RSDRL (%)*

Ethyl glucuronide

50 80-110 14,7 22,0

100 100 80-110 14,7 22,0

150 80-110 14,7 22,0

*The RSDRL, and resulting from that the RSDr that is calculated based on Horwitz2, is very large at low

concentrations. Thompson et al.3 prescribed that, if concentrations are below 120 µg/kg, a maximum RSDRL of 22%

may be used. It follows that the maximum RSDr will be 14,7%.

The maximum RSDr of 14,7% applies for the repeatability of 7 hair samples originating from the same batch, spiked

at 1.0 * PL at 3 different days. The repeatability* (7 different hair samples, spiked at 0.5 – 1.0 – 1.5 * PL at 3 different

days) has the same maximum RSDr of 14,7%.

6.2 CCα / CCß

Seven different hair samples will be spiked at 0.5 – 1.0 – 1.5 * PL at 3 different days, and cleaned-up. CCα and CCβ

are calculated with the ResVal sheet according to ISO 118434 and 2002/657/EC.

6.3 Selectivity / Specificity

The selectivity will be determined according to the following procedure:

(i) A statistical approach according to Berendsen et al.5 (ii) The chromatograms of the blank hair samples must be free of interferences on the retention time of ethyl

glucuronide.

6.4 Ruggedness

Information about the ruggedness of the method was obtained during the development of the method. Different

temperature of the ultrasonic bath (40 – 50 0C) and the evaporation temperature (40 – 55 0C) were tested. These

variations did not influence the final outcome. Additional features were tested by spiking 6 hair samples from the

same batch at 1.0 * PL, and using the variations in the method as shown in Table 5 during the sample preparation

and clean-up steps.

Table 5. Variations in the method to test ruggedness

Level Procedure (SOP #) Variations

1.0 * PL Incubation 4h, 50 0C Incubation 5h, 50 0C

1.0 * PL Incubation 4h, 50 °C Incubation 5h, 50 0C

1.0 * PL 2 mL washing solvent 4 mL washing solvent

1.0 * PL 2 mL washing solvent 4 mL washing solvent

1.0 * PL Evaporation till dryness 10 min longer evaporating

1.0 * PL Evaporation till dryness 10 min longer evaporating

62

The mean value as well as the trueness are calculated for every condition. The trueness needs to follow the criteria

for the trueness, as they are determined for the normal trueness.

6.5 Stability

Preliminary investigation showed that the standard stays stable for at least 6 months at -20 0C. Hair samples are

expected to be stable for several months when stored at room temperature in the dark, based on segmented hair

analysis used in literature. Extracts can be kept at -20 0C for at least a week, without losing their stability.

6.6 Linearity (MMS)

To obtain the linearity, matrix-matched standards (MMS) are produced by spiking a hair sample once at every level

prior to the clean-up, according to Table 6.

Table 6. MMS levels and criteria for the validation

Level Criteria

Day 1-3 0 – 0.25 – 0.5 – 1.0 – 2.0 – 3.0 * PL R2 0.990

6.7 Recovery (MMRS)

To obtain the recovery of an analyte during the clean-up, matrix-matched recovery standards (MMRS) are produced

by spiking a hair sample after the clean-up. This sample will be compared to a sample spiked at the same level (1.0

* PL), but before clean-up. The recovery will be determined with a different hair sample on each validation day

(MMRS A, MMRS I and MMRS Q, all prepared on the validation day itself).

6.8 Matrix effect

The matrix effect will be obtained by comparing a sample with spike after clean-up (MMRS) with a standard solution

of the same level. The matrix effect will be obtained on every validation day, all days for a different batch.

6.9 Confirmation

According to the decisions made in 2002/657/EC, at least 95% of the samples must follow the criteria listed in this

validation plan. The mass spectrometric detection shall be carried out by employing the MS-technique multiple

reaction monitoring (MRM). The ratio of the chromatographic retention time of the analyte to that of the internal

standard, i.e. the relative retention time of the analyte, shall correspond to that of the calibration solution at a

tolerance of ± 2.5 % for LC.

The relative intensities of the detected ions, expressed as a percentage of the intensity of the most intense ion or

transition, shall correspond to those of the calibration standard, either from calibration standard solutions or from

spiked samples, at comparable concentrations, measured under the same conditions, within tolerances described

in Table 7.

Table 7. Maximum permitted tolerances for relative ion intensities using LC-MSn

Relative intensity (% of base peak) LC-MSn (relative)

> 50 % ± 20 %

> 20 % to 50 % ± 25 %

> 10 % to 20 % ± 30 %

≤ 10 % ± 50 %

63

6.10 Traceability

The traceability of the standard could be tested by checking if other suppliers also deliver the standard used in order

to compare them. Unfortunately, no second supplier of ethyl glucuronide was found.

Standard Supplier Second supplier

Ethyl glucuronide Sigma-Aldrich E-015 ?

The Society of Hair Testing (SoHT) organizes proficiency tests for ethyl glucuronide two times a year, so

participation in these tests is a possibility.

References

1. Commission decision 2002/657/EC, ’Implementing council directive 96/23/EC concerning the performance of analytical methods and the interpretations of results’, Official Journal of the European Communities, 2002.

2. Horwitz, W.; Kamps, L.R.; Boyer, K.W., ‘Quality assurance in the analysis of foods for trace constituents’ , J. Assoc. Off. Anal. Chem., 1980, 63, 1344-1354.

3. Thompson, M., ‘Recent trends in inter-laboratory precision at ppb and sub-ppb concentrations in relation to fitness for purpose criteria in proficiency testing’, Analyst, 2000, 125, 385-386.

4. ISO 11843, ‘Capability of detection – Part 1: Terms and definitions, Part 2: Methodology in the linear calibration case’, 1997.

5. Berendsen, B.J.A.; Stolker, A.A.M.; Nielen, M.W.F., ‘The (Un)Certainty of Selectivity in Liquid Chromatography Tandem Mass Spectrometry’, J. Am. Soc. Mass Spectrom., 2013, 24, 154-163.

64

Attachment 3

Certificates and instruction sheets of reference hair material of 100 and 200 pg/mg

65

66

Attachment 4

Personal data information for the analysis of hair samples on alcohol consumption

1. Name (optional): .................................................................................... #: .....

2. Gender:

□ Male

□ Female

3. Age: ..........

4. Hair colour:

□ Blond

□ Brown

□ Black

□ Red

□ Grey

5. Hair sample length:

□ 0-3 cm

□ 0-6 cm

□ > 6 cm

6. Hair sample originates from:

□ As close as possible to the scalp

□ End parts of the hair (cutting by hairdresser for instance)

7. Is the hair sample:

- Bleached □ yes □ no if the answer is yes, brand (optional): ................................

- Dyed □ yes □ no if the answer is yes, brand (optional): ................................

8. Use of hair cosmetics:

- Shampoo

□ yes, every day

□ yes, 3-4 times a week

□ yes, 1-2 times a week

□ yes, a couple of times a month/year

□ no

if the answer is yes, brand (optional): ............................

- Conditioner

□ yes, every day

□ yes, 3-4 times a week

□ yes, 1-2 times a week

□ yes, a couple of times a month/year

□ no

if the answer is yes, brand (optional): ............................

67

- Hairspray

□ yes, every day

□ yes, 3-4 times a week

□ yes, 1-2 times a week

□ yes, a couple of times a month/year

□ no

if the answer is yes, brand (optional): ............................

- Hair gel, wax or oil

□ yes, every day

□ yes, 3-4 times a week

□ yes, 1-2 times a week

□ yes, a couple of times a month/year

□ no

if the answer is yes, brand (optional): ............................

9. Over the last six months, how do you evaluate your drinking behaviour?

□ Teetotaller: No drinking of alcohol for at least three months before sampling.

□ Limited: No strict abstinence of alcohol use, but only drinking alcohol at for instance a birthday party

or a wedding, so really low doses at once and usage with a mean of maximum two alcoholic

consumptions a month.

□ Tolerable: Consuming alcohol up to 6 standard glasses a day (60g alcohol/day).

□ Excessive: Alcohol use equal or more than 6 standard glasses a day (60g alcohol/day).

10. What was the last time you consumed alcohol before hair sampling?

□ ............. days

□ ............. weeks

□ ............. months

□ I am a teetotaler for the period: ..........

11. How many glasses of alcohol do you consume in a week on a regular base?

□ 1-2

□ 3-6

□ 7-12

□ > 12

□ N/A

12. Have you been using other drugs in the last six months?

□ yes, namely ....................... , for ............ times

□ no

68

Attachment 5

Results personal data information

The results for questions 2-5 and 7-11 in the personal data information are summarized below in charts. All hair

was cut as close as possible to the scalp, every individuals used shampoo, and none of the individuals used any

other drug in the last six months, so this is not included in the charts.