Determination of testosterone esters in serum by liquid ...liu.diva-portal.org/smash/get/diva2:331730/FULLTEXT01.pdf · Determination of testosterone esters in serum by liquid chromatography

1

Department of Physics, Chemistry and Biology

Final Thesis

Determination of testosterone esters in serum by liquid

chromatography – tandem mass spectrometry

(LC-MS-MS)

Erica Törnvall

Final Thesis performed at

National Board of Forensic Medicine

2010-06-03

LITH-IFM-EX--10/2263--SE


Linköping University

581 83 Linköping, Sweden

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Determination of testosterone esters in serum by liquid

chromatography – tandem mass spectrometry

(LC-MS-MS)

Erica Törnvall

Final Thesis performed at

National Board of Forensic Medicine

2010-06-03

Supervisors

Yvonne Lood

Martin Josefsson

Examiner

Roger Sävenhed

3

Datum

Date

2010-06-03

Avdelning, institution Division, Department

Chemistry


Linköping University

URL för elektronisk version

ISBN

ISRN: LITH-IFM-EX--10/2263--SE _________________________________________________________________

Serietitel och serienummer ISSN

Title of series, numbering ______________________________

Språk Language

Svenska/Swedish Engelska/English

________________

Rapporttyp Report category

Licentiatavhandling Examensarbete

C-uppsats

D-uppsats Övrig rapport

_____________

Titel

Title

Determination of testosterone esters in serum by liquid chromatography – tandem mass

spectrometry (LC-MS-MS)

Författare Author

Erica Törnvall

Nyckelord Keyword

LC-MS-MS, MRM, testosterone, testosterone esters

Sammanfattning Abstract

Anabolic androgenic steroids are testosterone and its derivates. Testosterone is the most important naturally existing sex

hormone for men and is used for its anabolic effects providing increased muscle mass. Testosterone is taken orally or by

intramuscular injection in its ester form and are available illegally in different forms of esters. Anabolic androgenic steroids

are today analyzed only in urine. To differentiate between the human natural testosterone and exogenous supply the quote

natural testosterone and epitestosterone is used. Detection of testosterone esters in serum is an unmistakable proof of

exogenous supply of testosterone. The aim of this thesis was to find a method for determining testosterone esters in serum

and to study an extraction method possible for quantification of testosterone esters in serum.

The technique used to separate and identify the Testosterone esters was Liquid Chromatography Tandem Mass Spectrometry

Electro Spray Ionisation. Parameters for chromatography and mass detection were optimized for nine testosterone esters and

evaluated according to selectivity, resolution and intensity. A method that could be used for determination of testosterone

esters in serum was found. The MS-method was set and at least three possible transitions for each testosterone ester were

found. The best choice of column proved to be the C18 column where all the esters were separated and seven of them were

base-line separated. The C18 column along with methanol and ammonium acetate buffer, 5 mM, pH 5 showed the highest

sensitivity for Multiple Reaction Monitoring-detection. A gradient profile for a total runtime of 5.6 minutes was established.

Two alternative extraction procedures were tested, with tert-butylmethylether or diethyl ether/ethyl acetate and both seemed

to work satisfactory. Analysis of serum proved to work well and no severe interference occurred. Results from the linearity

tests indicate that future quantification method in serum will be possible.

4

Abstract

Anabolic androgenic steroids are testosterone and its derivates. Testosterone is the most

important naturally existing sex hormone for men and is used for its anabolic effects

providing increased muscle mass. Testosterone is taken orally or by intramuscular injection in

its ester form and are available illegally in different forms of esters. Anabolic androgenic

steroids are today analyzed only in urine. To differentiate between the human natural

testosterone and exogenous supply the quote natural testosterone and epitestosterone is used.

Detection of testosterone esters in serum is an unmistakable proof of exogenous supply of

testosterone. The aim of this thesis was to find a method for determining testosterone esters

in serum and to study an extraction method possible for quantification of testosterone esters in

serum.

The technique used to separate and identify the testosterone esters was Liquid

Chromatography Tandem Mass Spectrometry Electro Spray Ionisation. Parameters for

chromatography and mass detection were optimized for nine testosterone esters and evaluated

according to selectivity, resolution and intensity. A method that could be used for

determination of testosterone esters in serum was found. The MS-method was set and at least

three possible transitions for each testosterone ester were found. The best choice of column

proved to be the C18 column where all the esters were separated and seven of them were

base-line separated. The C18 column along with methanol and ammonium acetate buffer, 5

mM, pH 5 showed the highest sensitivity for Multiple Reaction Monitoring-detection. A

gradient profile for a total runtime of 5.6 minutes was established. Two alternative extraction

procedures were tested, with tert-butylmethylether or diethyl ether/ethyl acetate and both

seemed to work satisfactory. Analysis of serum proved to work well and no severe

interference occurred. Results from the linearity tests indicate that future quantification

method in serum will be possible.

5

Abbreviations

MeOH Methanol

ACN Acetonitrile

LC Liquid chromatography

MS Mass spectrometry

TIC Total ion chromatogram

AAS Anabolic androgenic steroids

MRM Multiple reaction monitoring

CID Collision induced dissociation

C18 Octadecyl

Rt Retention time

ESI Electrospray ionization

WADA World Anti-Doping Agency

T/E Testosterone glucuronide/Epitestosterone glucuronide

TA Testosterone acetate

TB Testosterone benzoate

TC Testosterone cypionate

TD Testosterone decanoate

TE Testosterone enanthate

TP Testosterone propionate

TPh Testosterone phenylpropionate

TI Testosterone isocaproate

TU Testosterone undecanoate

6

Table of contents

Abstract

Abbreviations

1. Introduction 8

1.1. Anabolic Androgenic Steroids 8

1.2. Testosterone and Testosterone Esters 8

1.3. Methods of Analysis

9

2. Methodology 10

2.1. Liquid Chromatography 10

2.2. Tandem Quadrupole Mass Spectrometry 11

2.3. Multiple Reaction Monitoring 11

2.4. Aim

12

3. Experimental 13

3.1. Chemicals and Reagents 13

3.2. Solutions 13

3.3. Instrumentation 13

3.4. Sample Preparation 14

3.4.1. Infusion Study 14

3.4.2. Mixed Standards 14

3.4.3. Sample Preparation by LLE 14

3.5. Optimization

3.5.1. MS-Specificity

3.5.2. Chromatographic Selectivity

3.5.3. Linearity and Sensitivity

14

14

15

15

4. Results and Discussion 16

4.1. Tandem-MS Detection 16

4.2. Gradient Profile 17

4.3. Mobile Phase Composition 20

4.4. Stationary Phase 22

4.5. Final Gradient Profile 24

4.6. Matrix Test 26

4.7. Linearity 30

4.8. Final Method

32

5. Conclusion 34

6. Acknowledgement 35

References 36

7

Appendix A. Names and Structures of Testosterone Compounds 37

Appendix B. Transitions in the MS-method Tested with Reference Solutions 38

Appendix C. Transitions Based on the Serum Analysis 42

Appendix D. Linearity Study 45

D.1. Chromatograms from the Linearity Study

D.2. Linearity Evaluated by Using Transition 97

45

47

Appendix E. Transitions Used in the Final Study 49

E.1. Final Transition Method

E.2. Tests of Final Transitions

49

50

8

1. Introduction

1.1. Anabolic Androgenic Steroids

Anabolic androgenic steroids (AAS) are testosterone and its derivates. All AAS have both

anabolic properties such as increased muscle hypertrophy and androgenic such as

masculinisation [2].

Strength training is widely used to increase performance in sports with high physical

demands. The use of drugs to further enhance the performance happens via forbidden

substances, methods and manipulations. AAS are wide spread among athletes and the youth

today in gyms. The effects of these drugs on physical performance are documented [1]. AAS

increase the muscle hypertrophy induced by strength training further for athletes involved in

doping. The number of nuclei per muscle fibre increases [2]. Those who have withdrawn from

anabolic steroid usage and training for several years still have a remaining high number of

myonuclei [1].

AAS are used in medical purpose as substitution treatment for men with no natural production

of testosterone. Testosterone is predominantly administrated as intramuscular injection but is

also available as gel and plaster. In forensic investigation AAS are involved in violent

behaviour, depression and criminality and could cause more serious harm such as sudden

cardiac death, damaged liver function and disturbances in the lipid metabolism [1, 2]. In

Sweden it is restricted by law the use of synthetic AAS, testosterone and its derivates, growth

hormone and chemical substances increasing production and secretion of testosterone and its

derivates or growth hormone [3].

1.2. Testosterone and Testosterone Esters

Testosterone is produced in the Leydig cells in the testicles and even in females by the ovaries

in small quantities. Testosterone is naturally secreted to urine [4]. Men produce 6-10 mg

testosterone daily of which approximately 1% is excreted in urine [1]. Because of the short

half-life of only one hour exogenous intake of pure testosterone do not have any effect, since

only 2% of oral intake reaches the muscles. To slow down the metabolism and receive better

effect the testosterone molecule has been modified at its 17-position. This modification

creates stronger anabolic effect and weaker androgen effect as well [5].

O

CH3

CH3 O

R

Figure 1. Structure of Testosterone with an R-

group at its 17h position.

9

Testosterone esters have varying half-life. Testosterone cypionate and testosterone enanthate

injected intra muscularly have a half-life for 4.5 days, testosterone propionate 0.8 days and

testosterone undecanoate 21-34 days. The esters are preferably administrated by intra

muscular injection to directly reach the target cells and delay the liver metabolism [6].

1.3. Methods of Analysis

For forensic laboratories as for doping laboratories it is important to be able to determine

intake of AAS. To differentiate between exogenous and endogenous testosterone the

measured urinary ratio testosterone glucuronide/ epitestosterone glucuronide (T/E) indicates

the use of exogenous intake of testosterone. The naturally occurring epitestosterone is

constant since it is not affected by intake of testosterone. A T/E ratio above 6 indicates

testosterone doping and levels above 4.0 being considered suspicious according to World

Anti-Doping Agency (WADA) and the corresponding ratio used by Department of Forensic

Toxicology is 12 [4]. The chosen value is due to statistical reasons and studies indicate that

the urinary T/E ratios vary between individuals influenced by genetic factors [2, 4].

A study of samples from Swedish and Korean people predicted different effects of

testosterone intake on the T/E ration in the two ethnic groups. The Swedish people had 16-

times higher excretion of testosterone than the Koreans. Recent findings indicate that the gene

UGT2B17 influences the testosterone pattern. All individuals homozygous for the UGT2B17

gene have negligible or no excretion of TE. The genotype is seven times more common in

Asians than in European people [2]. It is a common polymorphism with an allele frequency of

29% in Swedes and 78% in Koreans. The sensitivity and specificity of the T/E test could be

markedly improved by using genotype-based cut off ratios [4]. Studies have showed that even

after a single dose of 360 mg testosterone, 40% of the subjects homozygous for the UGT2B17

deletion never reaches the T/E cut off ratio of 4.0. East Asians such as Japanese, Chinese and

Koreans, have considerably lower T/E ratios than Europeans increasing the risk of false

negative test results, challenging the accuracy of the test [4].

Determination of intact testosterone esters in serum is an unmistakeable proof of exogenous

intake and would avoid the problems related with the varying T/E ratios between individuals

in urine analysis [7].

10

2. Methodology

2.1. Liquid Chromatography

In order to avoid the time-consuming sample processing and derivatization needed for gas

chromatography often used as a method for steroid analysis a simple and rapid liquid

chromatography-tandem mass spectrometry (LC-MS/MS) method with electrospray

ionization (ESI) has been tested. The method has proven to be an alternative choice in steroid

analysis [8, 9].

High-performance liquid chromatography (HPLC) uses high pressure to force solvents,

mobile phase, through columns containing very fine particles, stationary phase, that give high

resolution separations. The HPLC system consists of a solvent delivery system, a sample

injection valve, a high pressure column, a detector, and a computer to display results and

control the system [10].

The compounds in a sample are separated due to their difference in size and affinity for the

stationary phase [10]. The chromatography is most commonly reversed-phase in which the

stationary phase is less polar than the mobile phase. Typically mobile phases for reverse-

phase chromatography are based on acetonitrile or methanol in combination with aqueous

buffer. C18 is the most non-polar and common column and other non-polar alternatives are

C8 and phenyl column. Drugs of interest are mostly less polar and are therefore better

retained by the reversed phase [11]. The chromatogram provides both qualitative and

quantitative information. Each compound in the mixture has its own elution time under a

given set of conditions. Both the area and the height of each peak are proportional to the

amount of the corresponding substance [11].

The growing demand for high-throughput separations in many fields, including forensics,

clinical chemistry and doping require faster separations. The need for enhanced productivity

and a large number of analyses require mandatory rapid analytical procedures. Ultra

performance liquid chromatography (UPLC) provides faster analyses with the same resolution

as HPLC (figure 2) due to the decrease in particle size and column length and increase in

pressure making the mobile phase flow rate faster. The theoretical plate is higher for UHPLC

than HPLC (fig. 2) [12].

11

Figure 2. Changes in efficiency due to linear velocity depicted as a van Deemter plot. Columns packed with 1.7

μm diameter particles perform better independent of flow rate. (waters.com)

2.2. Tandem Quadrupole Mass Spectrometry

Mass spectrometry embraces detection of analytes by ionizing molecules and then sorting the

fragments according to their mass-to-charge (m/z) ratios. Electrospray ionization (ESI) is a

very mild ionization technique especially developed for LC-MS which provide little or no

fragmentation, making it keep the precursor ion intact. ESI produces charged ions directly

from an aqueous/organic solvent system by creating a stream of charged droplets in the

presence of a strong electric field. In the quadrupole (Q1) for tandem-MS the ions transferred

into the vacuum travel through their respective regions, but only the ions with the selected

m/z are detected. Tandem mass spectrometry have two quadrupoles and a collision cell in

between which allows selection of a specific m/z in Q1 to further fragmentation in the

collision cell (Q2) for selection of another specific m/z in the second quadrupole (Q3) is

called multiple reaction monitoring, MRM [13].

Figure 3. Multiple Reaction Monitoring

2.3. Multiple Reaction Monitoring

MRM provides a function for selecting specific m/z and ignores all other fragments. In this

mode selected transitions between the precursor ion and a single fragment are monitored. The

selected precursor ions are selected in Q1 of tandem mass spectrometers, fragmented in Q2 by

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collision with an inert gas and the fragments are analysed in the Q3. Unlike the scan function

were each m/z is scanned shortly, MRM provides prolonged scan time for each transition.

Sensitivity is therefore increased and the signal to noise ratio increased, whereas the spectra

specific for the selected precursor contains less chemical noise or interferences.

Fragmentation energy in the collision cell and cone voltage is optimized to obtain

reproducible spectra for a large group of compounds [14].

2.4. Aim

To prove intake of testosterone esters in a suspected user, it is desirable to be able to identify

the intact esters. The testosterone esters chosen in this study are due to their presence on the

market and the most frequent seizure by the police. The primary aim of this thesis was to find

a method for liquid chromatography tandem mass spectrometry to determine testosterone

esters in serum. The parameters were optimized according to selectivity, sensitivity and

resolution and included selection of transition, column, gradient and mobile phase. The

secondary aim was to study an extraction procedure possible for quantification of testosterone

esters in serum.

13

3. Experimental

3.1. Chemicals and Reagents

Methanol (MeOH), acetonitrile (ACN), acetic acid, formic acid, ammonium acetate and

ammonia were purchased from Merck/VWR International.

The reference substances testosterone acetate (TA), testosterone benzoate (TB), testosterone

cypionate (TC), testosterone decanoate (TD), testosterone undecanoate (TU), testosterone

enanthate (TE), testosterone propionate (TP), testosterone phenylpropionate (TPh) and

testosterone isocaproate (TI) were purchased from Steraloids Inc.

Ammonium formiate was purchased from Fluka/Sigma.

Tert-butylmethylether and diethyl ether and ethyl acetate were purchased from Merck.

Milli-Q H2O was produced in house.

Negative serum was obtained from the blood center at the University Hospital in Linköping.

3.2. Solutions

All standard solutions were dissolved in ACN at 200 µg/mL and 10 µg/mL.

Mixed standard solutions containing TA, TB, TC, TD, TE, TP, TPh, TI and TU was prepared

in ACN at 1 µg/mL and 0.5 µg/mL, 0.1 µg/mL, 0.05 µg/mL, 0.01 µg/mL, 0.005 µg/mL and

0.001 µg/mL. Mobile phases A ammonium formiate buffer, 5 mM pH 3, were prepared from

1 M stock solutions of formic acid and ammonium formiate, and ammonium acetic buffer, 5

mM pH 5, pH 7.8 were prepared from 1 M stock solutions of Acetic acid and ammonium

acetate. Mobile phase B was ACN with 0.05% formic acid and MeOH with 0.05% formic

acid.

3.3. Instrumentation

An electrospray liquid chromatography tandem-mass spectrometry system (ESI-LC-MS-MS)

for gradient chromatography was used. The instrumentation consisted of an Acquity, Ultra

Performance Liquid Chromatographic system (UPLC), equipped with a solvent manager, a

sample manager and a column manager for handling of four columns (Waters, Milford, MA).

Mass detection was performed on a Quattro Premier XE tandem-MS (Waters, Milford, MA)

operating in positive ion mode. The following instrument conditions were used; capillary

voltage, 0.9 kV, extractor voltage 3V, RF lens voltage 0.1 V, multiplier voltage 680 V, source

temperature 120 C, oven temperature 60C, desolvation gas temperature 400C, cone gas

flow 50 L/hr, desolvation gas flow 1100 L/hr, collision gas flow 0.54 L/hr, ion energy (1) 0.5

V, ion energy (2) 0.2 V at LM and HM resolution (1) of 13 and (2) of 15, collision entrance

and exit potential of 1 V. Cone voltage and collision energy were optimized individually for

each testosterone ester, appendix 5. Instrument control was performed using MassLynx 4.1

and integration, processing and calculation were performed using the TargetLynx software.

Infusion experiments for multiple reaction monitoring (MRM) optimizations and ion

suppression studies were performed with an integrated Hamilton syringe pump at a flow rate

of 10l/min.

14

High-resolution liquid chromatographic separation was performed on an AQUITY BEH C18

and an AQUITY BEH phenyl UPLC-column both with the dimensions 502.1 mm i.d. with

1.7 µm particles (Waters, Milford, MA). An injection volume of 2 µL was used followed of a

strong wash of 500 µL ACN, isopropanol, MeOH and water 25:25:25:25 (v/v/v/v) with 0.2%

HFo and a weak wash of 900 µL MeOH: 0.1 M HFo 50:50 (v/v). The mobile phases consisted

of 5 mM ammonium formiate buffer pH 3.0 or ammonium acetate buffer pH 5.0 or 7.8 for

phase A and MeOH with 0.05% HFo or ACN with 0.05% HFo for phase B. Different gradient

profiles were tested. A flow-rate of 0.6 ml/min at 60°C was used. Reference chromatograms

and the retention times are shown in Results section and the Appendices. A MRM method

was prepared including the three or four most intense transitions for each analyte (appendix

5).

3.4. Sample Preparation

3.4.1. Infusion Study

To find suitable transitions for the MS-method, solutions of each ester had to be infused and

the distinctly appearing signals examined. The solution infused was made of 50 µL standard

solution (10 µg/mL), 450 µL MeOH and 500 µL 20 mM ammonium formiate buffer pH 3.

The sample was infused into the LC-MS/MS at a flow rate of 10 mL/min and instrument

parameters were optimized individually.

3.4.2. Mixed Standards

Reference mixtures of equal amounts of testosterone esters in each sample were injected to

the LC at each chromatography. 100 µL of 10 µg/mL of each ester was mixed to a final

concentration of 1 µg/mL in ACN. A 2 µL aliquot was injected into the LC-MS/MS for

studies of different chromatographic conditions.

3.4.3. Sample Preparation by LLE

To 500 µL serum 25 µL ester mixture in ACN (10 µg/mL) was added. Serum was extracted

for 5 min with 3 mL of tert-butylmethylether or 3 mL diethyl ether/ethyl acetate (70/30) [7].

The phases were separated by centrifugation at 4000 rpm for 10 min and the upper organic

phase was transferred to a clean 10-mL conical glass tube and solvents were evaporated to

dryness under nitrogen at room temperature. Residues were reconstituted in 200 µL ACN. A 2

µL aliquot was injected into the LC-MS/MS and interferences from the matrix were studied.

3.5. Optimization

3.5.1. MS-Specificity

Suitable testosterone ester transitions were selected for MRM determination by performing

infusion experiments. Molecular ion weight for the precursor was easily found knowing the

theoretical value for the molecular weight while using the scan mode. Finding product ions

15

required several infusion experiments at various collision energies at daughter ion scan mode

for detection of specific and common fragments for the testosterone esters. A preset of

selected transitions was tried out using reference substances. Fragments with the highest

intensities for each testosterone ester were chosen and set in the MRM-method.

3.5.2. Chromatographic Selectivity

Based on that testosterone esters are non-polar a phenyl column and C18 column were tested.

Phenyl columns are not as non-polar as the C18 column and were assumed to give the best

separation, due to testosterone esters long fatty tails that was suspected to get very retained in

the column and difficult to elute.

3.5.3. Linearity and sensitivity

By future interest in possible quantification a study for linearity and sensitivity was made

based on serum analysis. Linearity for concentration of each testosterone ester is based on

transition 97.0 or 97.1 at 1.0 µg/mL, 0.5 µg/mL, 0.1 µg/mL, 0.05 µg/mL, 0.01 µg/mL, 0.005

µg/mL and 0.001 µg/mL.

Mobile phase and pH affect the sensitivity for detection at the interface. Mobile phase B were

ACN and MeOH and A were ammonium formiate pH 3 and ammonium acetate pH 5 and pH

7.8. Combined in different combinations at different gradient profiles of mobile phase A and

B the highest sensitivity at the detection was to be found.

16

4. Results and Discussion

The aim of this study was to find a method for identifying the nine most frequently appearing

testosterone esters at the market in serum and test the quantifying range and linearity with LC-

MS/MS. Focus was on selection of the most suitable column, mobile phase, gradient and

MRM-transitions. The optimisation was made with reference substances and after a suitable

method was selected it was tested on spiked serum samples. Transitions found by infusion

tests were set up in a method by which following reference substances and serum sample tests

were analysed and evaluated. Extraction of serum samples was based on two different

methods.

4.1. Tandem-MS Detection

In LC-MS fragmentation is generated by collision induced dissociation (CID). All

testosterone esters are based on the testosterone molecule and have therefore CID-fragments

in common and also fragments specific for each testosterone ester based on the ester structure.

Regulations for LC-MS analysis recommend at least two transitions for identifications of

drugs. Three or four fragments were chosen for each testosterone ester in this study. Common

product ions from the testosterone structure are 97 and 109 (table 1). Specific product ions

could not be found for all of the esters. As can be seen in table 1, fragment 1 and 2 are in

common for all the testosterone esters and are therefore assumed to origin from the

testosterone structure. By evaluations and comparison of mass spectra it was found that

several other fragments were in common as well (eg. 149.1, 163.2 and 175.2). Although

unique fragments not were available for most testosterone esters, unique transitions could be

selected since all testosterone esters had unique precursor ions [M+1].

Table 1. Fragment ions and their intensities.

Analyte MW [M+1] Fragment 1 Fragment 2 Fragment 3 Fragment 4

TA 330.5 331.2 97.0 1.61e6 109.0 2.25e6 135.1 7.40e4

TB 392.5 393.2 97.1 4.83e5 109.1 1.11e5 105.0 8.94e5

TC 412.6 413.3 97.1 5.51e5 na

125.1 1.89e5 163.2 6.20e4

TD 442.7 443.3 97.1 5.45e5 109.0 4.77e5 119.2 7.75e4 123.1 4.26e4

TE 400.6 401.3 97.0 4.28e5 109.0 2.97e5 113.1 3.02e5

TPh 420.6 421.3 97.1 8.43e5 109.0 7.51e5 163.2 1.24e5 173.1 4.52e4

TP 344.5 345.1 na 109.0 1.83e6 123.1 5.84e4 187.2 4.49e4

TI 386.6 387.5 97.1 2.24e5 109.1 1.29e5 149.1 1.03e4 175.2 2.36e4

TU 456.7 457.3 97.1 8.81e5 109.1 6.84e5 169.2 3.70e5

Fragments were selected based on their sensitivity during chromatography and the highest

intensities were chosen and set in the MS-method. One fragment from the infusion test

showed no signal in the method (fig. 4) and was replaced. TC, seen in figure 4, shows a signal

at 2.01 min which origins from the mobile phase (appendix 2). Final transitions table is found

in appendix 5.

17

Figure 4. Transitions tested for TC showing an incorrect transition for 413.3 > 301.2.

4.2. Gradient Profile

The chromatographic gradient profile affects the elution and separation of the testosterone

esters. A steep gradient profile may elute the testosterone esters earlier in the chromatography.

For a final method the testosterone esters were preferred in a wide range retention times for

better separation and determination of the peaks. Short time of analysis within less than 10

minutes for is preferred for routine analysis. Testosterone esters need therefore to elute in the

chromatography and before the wash-period begins. Fairly high content organic solvent in the

mobile phase was needed to elute the testosterone esters.

Initially different gradient profiles were tested on a phenyl column. The first trials had a total

time of 7 minutes. The mobile phase was ACN pH 3 and the flow rate is 0.6 mL/min. In order

to avoid band broadening it was important to begin at a low start concentration of mobile

phase B. The gradient profiles were set at a start concentration of 10-40% (figure 5. A-D).

The higher start concentration of mobile phase B the wider the range in retention time

between the testosterone esters was seen and all nine testosterone esters were visible but not

baseline separated in all of the gradient profiles tested (fig. 5). Gradient C and D proved to

elute the most suitable for fast analysis at a first retention time of approximately 2 minutes.

413.4 > 301.2

413.4 > 163.2

413.4 > 125.1

413.4 > 97.1

TIC

18

Gradient A Gradient B

Gradient C Gradient D Figure 5. Phenyl column and mobile phases ACN pH 3. Gradient profiles starting at (A)10%, (B) 20%, (C) 35%

and (D) 40% of mobile phase B.

7 minutes analyzing time is a fairly long time for routine analysis and a reduction in time to

5.6 minutes was performed. The slope of gradient C with an initial composition of 35% B-

solvent was set as a template and the maximum concentration of gradient C was never

reached in order to save time and instead enter the wash-period directly after the last

testosterone ester had eluted.

Four gradients were tested with MeOH on the phenyl column at pH 3, starting with the result

made by gradient C as a template (fig. 6 Gradient E). Gradient E elute the testosterone esters

late in the chromatography and the start concentration was increased until a suitable gradient

was found based on elution of the first testosterone ester. The higher the start concentration of

mobile phase B, the longer the range in retention time between the testosterone esters are,

which is sought for. The long range in retention time results in seven base line separated

19

analytes. The change in mobile phase B from ACN to MeOH led to two analytes eluting at the

same retention time, TI and TPh. Gradient H was chosen to be the most suitable gradient

profile for the phenyl column. To determine how to go further in selection of gradient profile

the most suitable column must be selected.

Gradient E Gradient F

Gradient G Gradient H

Figure 6. Change in gradient profile to centre the analytes for a decrease in chromatography to 5.6 minutes.

Gradient (E) starting at mobile phase B of 45% MeOH, (F) starting at mobile phase B of 50% MeOH, (G)

starting at mobile phase B of 55% MeOH and (H) starting at mobile phase B of 60% MeOH on a phenyl column

with mobile phase A as pH 3.

20

4.3. Mobile Phase Composition

Three different pHs’ were tested on the phenyl column. The phenyl column with pH 3, 5 and

7.8 as mobile phase A and ACN as mobile phase B was tested with a mixture of nine

testosterone esters.

Testosterone esters were separated at pH 3 but only five of them were base line separated,

TA, TP, TB, TU and TD (fig. 7). The intensities vary but were fairly equal besides the peaks

at 1.89 minutes, TA and 3.68 minutes, TU. The mobile phase was more sensitive to TA and

least sensitive to TD (fig. 7, table 2).

Figure 7. Phenyl column with ACN as mobile phase B and mobile phase A of pH 3.

Both pH 5 (fig. 8) and 7.8 (fig. 9) show an increase in intensity for the first peak at 1.89

minutes. The intensities for the other peaks are fairly good. Separation is on the same level as

for pH 3 (fig. 9). Chromatograms show that a change in pH does not have any effect on

selectivity (fig. 7-9).

Comparing pH 3 for MeOH and pH 3, 5 and 7.8 for ACN on the phenyl column shows the

highest sensitivity at pH 5. Sensitivity was the lowest for pH 7.8 and was therefore abandoned

for further analysis.

21

Figure 8. Phenyl column with ACN as mobile phase B and mobile phase A of pH 5.

Figure 9. Phenyl column with ACN as mobile phase B and mobile phase A of pH7.8

Table 2. Intensity based on mobile phase A and B on a phenyl

column. (* optimum conditions)

Analyte ACN pH 3 ACN pH 5 ACN pH 7.8

TA 5.64e6 7.26e6* 5.92e6

TB 4.58e5 7.98e5* 4.71e5

TC 1.37e6* 1.26e6 7.41e5

TD 4.60e5 1.04e6* 6.96e5

TE 5.36e5 1.20e6* 5.37e5

TPh 1.23e6* 1.13e6 7.45e5

TP 2.84e6* 1.89e6 1.06e6

TI 1.44e6 1.80e6* 1.24e6

TU 1.35e6 1.06e6 1.91e6*

22

4.4. Stationary Phase

Selection of stationary phase material affects the selectivity for the testosterone esters. Thus

C18 and as well as a phenyl column were tested with a mobile phase consisting of MeOH and

ammonium acetate pH 5.

Eight peaks were visible and seven were base-line separated on the phenyl column (fig. 10A).

TPh and TI have the same retention time and are hidden in the same peak (fig. 10B). The

order of elution is based on the size and polarity of the esters. TI eluted slightly before TPh

probably due to the π-interaction created between the phenyl-groups in TPh and the stationary

phase.

A B

Figure 10. (A) Phenyl column with MeOH as mobile phase B and mobile phase A pH 5. (B) Transition 97 for all


A change of column from phenyl to C18 for increased retardation of the testosterone esters

were expected and could give a better separation. Nine peaks were shown and seven of them

were base line separated using the C18 column (fig. 11). The order of elution has changed,

TPh eluated before TI due to the increased affinity for TI and the stationary phase after the

change in column. C18 column with MeOH as mobile phase B provides better selectivity for

testosterone esters than a phenyl column with MeOH as mobile phase B of the same

dimensions.

TI

TPh

TU

TD

TPh

TC

TE

TB

TI

TP

TA

23

A B

Figure 11. C18 column with MeOH as mobile phase B and mobile phase A of (A) pH 5 and (B) transition 97.

A change in column to C18 and MeOH as mobile phase B shows nine peaks and almost seven

of the testosterone esters are base line separated (fig. 12). Sensitivity is higher for MeOH than

ACN (table 3). TA is the most sensitive to the method and TD is least sensitive in both pH 3

and 5, to be compared with MeOH were the lowest sensitivity varied depending on pH, TU at

pH 3 and TD at pH 5 (fig. 13). The selectivity is the same for MeOH and ACN, but the

sensitivity is much higher for MeOH (table 3). Compared to the phenyl column (table 2) the

C18 column shows higher intensities for mobile phases MeOH and pH 3 (table 3). MeOH has

the best sensitivity at both pH 3 and pH 5 compared to ACN (table 3). MeOH and pH 5 shows

the highest intensity for each one of the testosterone esters.

A B

Figure 12. C18 column with ACN as mobile phase B and mobile phase A of (A) pH 3 and (B) pH 5.

TI TPh

TU

TD

TPh

TC

TE

TB

TI

TP

TA

24

A B

Figure 13. C18 column with MeOH as mobile phase A and mobile phase B of (A) pH 3 and (B) pH 5.

Table 3. Intensity for transition 97 based on pH of mobile phase A on a C18

column.(*optimum conditions)

Analyte MeOH pH 3 ACN pH 3 MeOH pH 5* ACN pH 5

TA 3.36e7 8.13e6 4.01e7 6.37e6

TB 6.06e6 1.25e6 7.76e6 1.21e6

TC 1.05e7 1.23e6 1.30e7 1.74e6

TD 3.50e6 1.12e6 7.03e6 1.12e6

TE 7.96e6 1.51e6 9.93e6 1.42e6

TPh 1.22e7 1.32e6 1.43e7 1.23e6

TP 1.66e7 1.79e6 1.91e7 1.99e6

TI 1.24e7 1.46e6 1.49e7 2.27e6

TU 4.36e6 1.55e6 1.07e7 2.76e6

4.5. Final Gradient Profile

After selection of the C18 column as the better choice, the gradient profiles for both MeOH

and ACN were tested. 60% of MeOH as start concentration equals the same eluent strength as

45% of ACN [9]. Gradient H (fig. 6) gave room for even earlier elution and the start

concentration for ACN was therefore set at 50%.

The gradient with ACN for 5.60 minutes had the first testosterone ester, TA eluting early in

the chromatography and the last two at maximum concentration of mobile phase B, TD and

TU (fig. 14). All nine testosterone esters were separated and seven of them are base-line

separated.

25

The gradient with MeOH for 5.60 minutes (fig. 15) has its first testosterone ester, TA eluting

at approximately the same time as H (fig. 6) but the last two testosterone esters, TD and TU

elutes later when mobile phase B reached its maximum percentage.

Compared to the gradient for MeOH, the gradient for ACN provides a larger range in

retention times between the testosterone esters, 3.22 versus 3.01 (table 4). The gradients differ

due to the higher percentage needed for ACN at the start level. The two esters eluting after

wash-period has started could result in interference with the serum in the serum analysis

following.

Figure 14. C18 column with mobile phase A at pH 3 and mobile phase B starting at a concentration of 50 %

ACN.

Figure 15. C18 column with mobile phase A at pH 3 and mobile phase B starting at a concentration of 60 %

MeOH.

TA TP TB TPh TI TE TC TD TU


26

Table 4. Retention time based on mobile phase A.

(*Rt last peak – Rt first peak)

Analyte ACN pH 3 MeOH pH 3

TA 1.73 2.04

TP 2.14 2.47

TB 2.72 3.20

TPh 2.84 3.36

TI 3.19 3.61

TE 3.63 4.04

TC 3.73 4.13

TD 4.64 4.94

TU 4.95 5.05

Range* 3.22 3.01

4.6. Matrix Test

Transitions and parameters were optimized for testosterone esters in reference solution. A

change of matrix to serum could show interference with serum and extraction solutions and

also suppress the sensitivity for the testosterone esters at the detection. To find out if serum

gives any interferences it is important to pre-run a serum analysis with a blank of mobile

phase A and blank serum. Two extraction methods are tested, tert-butylmethylether and

(70:30) diethyl ether/ethyl acetate and two serum blanks. The serum analysis involves serum

spiked with the nine testosterone esters at the same concentration before extraction as the

reference solution.

Blank of mobile phase pH 5 showed a system peak at 1.33 and an increase in intensity at 4.06

and 6.14 minutes (fig. 16). The high intensity at 1.33 is a system peak and the high intensity at

6.14 minutes is due to the wash-period where MeOH is at 95%.

Figure 16. Blank of mobile phase pH 5.

27

The blank serum extracted with diethyl ether/ethyl acetate and tert-butylmethylether both

shows interference at 4.1 minutes (fig. 17, 18). Transitions selected for TA showed

interference at 4.15 minutes (fig. 16, 17).

A B

Figure 17. Blank serum extracted with diethyl ether/ethyl acetate. (A) Chromatogram (B) Transition 97 for all


A B

Figure 18. (A) Blank serum extracted with tert-butylmethylether. (B) Transition 97 for all testosterone esters.

TU

TD

TPh

TC

TE

TB

TI

TP

TA

TU

TD

TPh

TC

TE

TB

TI

TP

TA

28

The interference in serum is shown for TA (fig, 17, 18) at transition 97 and 109 (fig. 19)

during a test for all transitions for TA. This could be due to carry over or a similar natural

testosterone hormone with the same precursor and some transitions as TA.

A B

Figure 19. A) Blank serum extracted with diethyl ether/ethyl acetate and B) tert.butylmethylether.

Both extraction methods are equally working (fig. 20). All testosterone esters were separated

and sensitivity for each ester was good enough for determination (appendix 4). The sensitivity

is still higher for TA and least sensitive for TD and TU.

A B

Figure 20. Serum spiked with mixture of the nine testosterone esters extracted with (A) diethyl ether/ethyl

acetate and (B) tert.butylmethylether run on a C18 column.

29

Since TA showed an increase in intensity at 4.1 (fig.19) which was the retention time for TC

an investigation whether it is due to carry over or not, two blanks following one blank matrix

from the diethyl ether/ethyl acetate method were run. Four more transitions for TA were set in

the MS-method to find out if more transitions show interference.

Neither of the blanks showed any transitions at 4.15 minutes at either the first blank run or the

second(fig. 21 A, C) and nor did the transitions (fig. 21B, 21D). Based on this there was no

proof for carry over. The third run, the blank matrix showed an increased intensity at 4.15

minutes (fig 22). Transitions 97 and 109 showed peaks for TA but the other transitions except

for 253.2, which has an increased intensity for the system peak seemed to be working.

Transitions 97 and 109 for TA needed therefore to be replaced.

A B

C D

Figure 21. (A) First blank run and (B) the transitions. (C) Second blank run and (D) the transitions.

30

A B

Figure 22. (A) Blank serum extracted with diethyl ether/ethyl acetate. (B) Transition 97 and 109 for TA.

There is not any difference in retention time between the reference mixture and the extraction

methods (table 5). Intensities for the extraction methods are fairly equal (fig. 24) and for

further analysis both of them can be used.

Table 5. Retention time based on extraction method

Analyte Referens mix

Serum mix

diethylether

Serum mix tert-

butylmetylether

TA 2.07 2.04 2.04

TP 2.51 2.49 2.49

TB 3.26 3.22 3.22

TPh 3.40 3.36 3.36

TI 3.65 3.62 3.62

TE 4.08 4.04 4.04

TC 4.17 4.13 4.13

TD 4.95 4.95 4.94

TU 5.06 5.06 5.05

Range 2.99 3.02 3.01

31

4.7. Linearity

In order to see if quantification analysis was possible it was important that the intensity peak

area was linear dependent on the ester concentration. Linearity was tested based on the results

from the matrix test (4.6). Each signal for every transition was integrated and based on the

total intensity for each ester graphs were created. Concentrations tested ranges from 0.001

µg/mL to 1 µg/mL.

Intensity was linear dependent on the ester concentration. A representative calibration for TI

showed a straight line close to the intensity/concentration-ratio without any outliers (fig. 23).

The linearity has an r-factor of 0.999 showing that quantification is possible (fig. 23A). The

lowest concentration tested, 0.001 µg/mL, here showed for TI in figure 23B shows that

quantification is possible even for low sample concentrations. The signals did not show any

interference and the peaks are close to symmetric. Linearity for the other testosterone esters is

attached in appendix 3 and table 6 shows the raw data.

Table 6. Intensity (peak area) based on testosterone ester concentration for C18 column with mobile phase B

MeOH and A pH 5.

Analyte

1

µg/mL

0.5

µg/mL

0.1

µg/mL

0.05

µg/mL

0.01

µg/mL

0.005

µg/mL

0.001

µg/mL

TA 4.01e7 2.27e7 4.55e6 2.50e6 4.00e5 1.88e5 4.30e4

TB 7.76e6 4.41e6 8.93e5 4.66e5 7.98e4 3.21e4 7.76e3

TC 1.30e7 6.96e6 1.43e6 7.86e5 1.28e5 6.86e4 2.30e4

TD 7.03e6 3.70e6 7.51e5 4.34e5 7.84e4 2.21e4 8.98e3

TE 9.93e6 5.43e6 1.07e6 6.24e5 1.55e5 7.54e4 3.19e4

TPh 1.43e7 8.06e6 1.57e6 8.59e5 1.49e5 6.31e4 1.64e4

TP 1.91e7 1.03e7 1.96e6 1.09e6 1.97e5 8.44e4 1.66e4

TI 1.49e7 8.02e6 1.61e6 8.56e5 1.67e5 6.04e4 1.24e4

TU 1.07e7 6.06e6 9.07e5 4.87e5 1.18e5 3.07e4 1.54e4

A B

Figure 23. Test of linearity for TI from concentrations of 0.001 µg/mL to 1 µg/mL. (A) Linearity based on

transition 97 and (B) integration of peak areas for all transitions at 0.001 µg/mL.

32

The linearity study makes it possible to estimate the limit of detection from the lowest

concentration at 0.001 µg/mL (table 7). Calculations backward based on varying amount of

serum and reconstitution solution (3.4.3.) shows that the factor divided by the lowest

concentration is 2.5 whichever concentration for the testosterone esters is used. The factor

affects how low the concentration in the serum could be. Varying amount of serum and

reconstitution solution changes the factor and an increase in the factor makes it possible to

detect even lower quantities.

Table 7. Estimation of detection limits at changed parameters (3.4.3.).

Parameters Initial conc. and amounts Alternative A Alternative B Alternative C

testosterone

esters

0.001 µg/mL 0.001 µg/mL 0.001 µg/mL 0.001 µg/mL

serum 0.5 mL 1 mL 1 mL

reconstitution

solution (ACN)

0.2 mL 0.1 mL 0.1 mL

Calc. factor 2.5 5 5 10

100% recovery 0.4 ng/mL 0.02 ng/mL 0.02 ng/mL 0.01 ng/mL

80% recovery 0.5 ng/mL 0.025 ng/mL 0.025 ng/mL 0.0125 ng/mL

4.7. Final method

This study has ended up in a suggestion for a method setup based on the best results of

selection of gradient, column, mobile phases, MS-method and serum preparations. The MS-

method was set after the serum runs in favour to receive a cleaner chromatogram. Unique

transitions are found for each ester. The peaks were separated and seven of nine esters are

base-line separated using reference mixture on a C18 column with MeOH and ammonium

acetate 5 mM pH 5 as mobile phases (fig. 24). The chromatography for the spiked serum (fig.

25) provides all nine testosterone esters visible and separated. The selectivity was not as good

as for the reference mixture (fig. 24), due to the decreased concentration from the extraction

and the spiked serum is not as pure as the references showing a less clean chromatogram. TA

was no longer the testosterone ester with the strongest sensitivity for the method after the

change in transitions. TU still proves to be the one least sensitive to MeOH. The sensitivity

for the testosterone esters depends on the amount of transitions and how large the signal for

each one of them was. Signal intensity was high enough for quantification in serum based on

the intensity for the lowest concentration tested (4.7.).

The final transitions are shown in appendix 5.

33

Figure 24. Reference mixture of nine testosterone esters at a concentration of 1µg/mL run on a C18 column with

mobile phase A at pH 5 and MeOH for mobile phase B.

Figure 25. Serum spiked with nine testosterone esters at a concentration of 1 µg/mL extracted with diethyl

ether/ethyl acetate run on a C18 column with mobile phase A at pH 5 and MeOH for mobile phase B.

Figure 26. Blank serum extracted with diethyl ether/ethyl acetate run on a C18 column with mobile phase A at

pH 5 and MeOH for mobile phase B.

TA

TP

TB TPh

TI

TE

TC

TD TU

TA

TP

TB TPh

TI

TE TC

TD TU

34

5. Conclusion

A method that could be used for determination of testosterone esters in serum has been found.

A suggestion for a method set up of testosterone esters has been made in reference solutions

as a primer for quantification in serum.

The MS-method was set and at least three possible transitions for each testosterone ester were

found. The best choice of column proved to be the C18 column where all the esters were

separated and seven of them were base-line separated. The C18 column along with methanol

and ammonium acetate buffer, 5 mM, pH 5 showed the highest sensitivity for Multiple

Reaction Monitoring-detection. A gradient profile for a total runtime of 5.6 minutes was

established. Two alternative extraction procedures were tested, with tert-butylmethylether or

diethyl ether/ethyl acetate and both seemed to work satisfactory. Analysis of serum proved to

work well and no severe interferences occurred. Results from the linearity tests indicate that

future quantification method in serum will be possible.

The suggested method has been proven to work well. Further development should be focused

on validation such as determinations of; limit of detection, precision, calibration and

suppression test before entering routine analysis.

35

6. Acknowledgement

I am grateful for having had the opportunity to write this report. It was of big interest

and I have been exited from the very beginning.

I would like to thank my supervisors Yvonne Lood and Martin Josefsson for all the support

and help. I also would like to thank all the people at the National Board of Forensic Medicine

that I have been in contact with during this final thesis.

36

References

[1] A. Eriksson, Strength training and anabolic steroids – A comparative study of the vastus

lateralis, a thigh muscle and the trapezius, a shoulder muscle, of strength-trained athletes, PhD

thesis, Umeå University, 2006

[2] F. Sjöqvist, M. Garle, A. Rane, Use of doping agents, particularly anabolic steroids, in

sports and society, Lancet 371 (2008) 1872-1879

[3] Svensk författningssamling, Lag (1991:1969) om förbud mot visa dopingsmedel, hämtat

2010-05-20, www.riksdagen.se

[4] J. Jakobsson Schultze, Genetics of androgen disposition – Implications for doping tests,

PhD thesis, Karolinska Institutet, 2007

[5] T. Rosén, Hormondoping in, Endokrinologi, Ed. S. Werner, Liber (2006) p262

[6] H.M. Behre, E. Nieschlag, Testosterone buciclate (20 Aet-1) in hypogonadal men:

pharmacokinetics and pharmacodynamics of the new long-acting androgen ester, J Clin

Endocrinol Metab. 75 (1992) 1204-1210

[7] X. De la Torre, J. Segura, A. Polettini, Detection of testosterone esters in human plasma

by GC/MS and GC/MS/MS in, Recent advances in doping analysis, Ed. M. Donike, H. Geyer,

U. Mareck-Engelke, Sport und Buch Strauß (1995) 59-80

[8] U. Turpeinen, S. Linko, O. Itkonen and E. Hämäläinen, Determination of testosterone in

serum by liquid chromatography-tandem mass spectrometry, Scand J Clin Lab. 68 (2008) 50-

57

[9] S-H. Peng, J. Segura, M. Farré, J.C. Gonzáles. X. De la Torre, Plasma and urinary markers

of oral testosterone undecanoate misuse, Steroids 67 (2002) 39-40

[10] D.C. Harris, Quantitative chemical analysis, sixth edition, W.H. freeman and Company

(2003) chapter 23, 25

[11] V.R. Meyer, Practical High-performance Liquid Chromatography, fourth edition, John

Wiley and sons (2004) chapter 1, 10

[12] D. Guillarme. J. Ruta, S. Rudaz and J-L. Veuthey, New trends in fast and high-resolution

liquid chromatography: a critical comparison of existing approaches, Anal Bioanal Chem. 3

(2009) 1069-1082

[13] P.J. Taylor, Method development and optimization of LC-MS in, Applications of LC-MS

in toxicology, Ed. A. Polettini, Pharmaceutical Press (2006) p23

[14] P. Marquet, Systematic toxicological analysis with LC-MS in, Applications of LC-MS in

toxicology, Ed. A. Polettini, Pharmaceutical Press (2006) p111

37

Appendix A. Names and Structures of Testosterone Compounds

Table I. Names and structures of compounds sorted by retention time from the final results (*not tested)

Transitions chosen are presented in the m/z-column, were the precursor is marked with M

.

Structure Analyte Rt (min) MW (g/mol) m/z

O

CH3

CH3 OH

Testosterone

(4-Androsten-17β-ol-3-one)

*na 288.414 289.3M

[8]

CH3

CH3O

O

CH3

O

Testosterone acetate

(4-Androsten-17β-ol-3-one Acetate)

2.04 330.46 135.1

253.2

289.3

331.2M

CH3

CH3

O

O

O

CH3

Testosterone propionate

(4-Androsten-17β-ol-3-one

Propionate)

2.49 344.49 97.1

109.0

123.1

187.2

345.1M

O

CH3

CH3O

O

Testosterone benzoate


Benzoate)

3.22 392.53 97.1

105.0

109.1

393.2M

CH3

CH3

O

O

O

Testosterone phenylpropionate


Phenylpropionate)

3.36 420.591 97.1

109.0

163.2

173.1

421.3M

CH3

CH3 O

O

O

CH3

CH3

Testosterone isocaproate


Isocaproate)

3.62 386.58 97.1

109.1

175.2

271.2

387.2M

CH3

CH3 O

O

O

CH3

Testosterone enanthate


Enanthate)

4.04 400.59 97.0

109.0

113.1

401.3M

CH3

CH3 O

O

O

Testosterone cypionate


Cypionate)

4.13 412.6 97.1

109.1

125.1

163.2

413.3M

CH3

CH3 O

O

O

CH3

Testosterone decanoate


Decanoate)

4.96 442.67 97.1

109.0

119.2

443.3M

CH3

CH3 O

O

O

CH3

Testosterone undecanoate


Undecanoate)

5.06 456.7 97.1

109.1

169.2

457.3M

38

Appendix B. Transitions in the MS-method Tested with Reference Solutions

Figure I. Blank of ammonium formiate pH 3 showing a peak at 2.01 minutes.

Figure II. Transitions for TA tested on a phenyl column of mobile phase A pH 3 and mobile phase B ACN.

Figure III. Transitions for TB tested on a phenyl column of mobile phase A pH 3 and mobile phase B ACN.

331.2 > 135.1

331.2 > 109

331.2 > 97

TIC

393.2 > 109

393.2 > 105

393.2 > 97.1

TIC

39

Figure IV. Transitions for TC tested on a phenyl column of mobile phase A pH 3 and mobile phase B ACN.

Figure V. Transitions for TD tested on a phenyl column of mobile phase A pH 3 and mobile phase B ACN.

Figure VI. Transitions for TE tested on a phenyl column of mobile phase A pH 3 and mobile phase B ACN.

413.3 > 301.2

413.3 > 163.2

413.3 > 125.1

413.3 > 97.1

TIC

443.3 > 97.1

443.3 > 119.2

443.3 > 109

443.3 > 123.1

TIC

401.3 > 113.1

401.3 > 109

401.3 > 97

TIC

40

Figure VII. Transitions for TP tested on a phenyl column of mobile phase A pH 3 and mobile phase B ACN.

Figure VIII. Transitions for TPh tested on a phenyl column of mobile phase A pH 3 and mobile phase B ACN.

Figure IX. Transitions for TI tested on a phenyl column of mobile phase A pH 3 and mobile phase B ACN.

345.1 > 187.2

345.1 > 123.1

345.1 > 109

TIC

421.3 > 173.1

421.3 > 163.2

421.3 > 109

421.3 > 97.1

TIC

387.2 > 175.2

387.2 > 149.1

387.2 > 109.1

387.2 > 97.1

TIC

41

Figure X. Transitions for TU tested on a phenyl column of mobile phase A pH 3 and mobile phase B ACN.

457.3 > 169.2

457.3 > 109.1

457.3 > 97.1

TIC

42

Appendix C. Transitions Based on the Serum Analysis

Transitions based on the analysis for blank serum and spiked serum.

A

B C

D E

Figure XI. A) Blank serum extracted with diethyl ether/ethyl acetate. B-E) Transitions chosen for testosterone

esters. The testosterone esters do not show any interferences except for TA which shows an increase in signal at

4.15 for transition 97 and 109.

TIC

43

A

B C

D E

Figure XII. A) Blank serum extracted with tert-butylmethylether. B-E) Transitions chosen for testosterone

esters. The testosterone esters do not show any interferences except for TA which shows an increase in signal at

4.14 for transition 97 and 109.

44

A

B C

D E

Figure XIII. (A) Serum spiked with testosterone esters of 1 µg/mL extracted with diethyl ether/ethyl acetate. (B-

E) Transitions chosen for testosterone esters.

TU

TD

TPh

TE TB

TI

TPh TC

TE

TP

TA


45

Appendix D. Linearity Study

D.1. Chromatograms from the Linearity Study

A B

C D

Figure XIV. (A) Blank of mobile phase A pH 5. (C) Reference mixture for the testosterone esters of 1 µg/mL

and transition 97 for the testosterone esters in the (B) blank and (D) the reference mixture.

TU

TD

TPh

TC

TE

TB

TI

TP

TA

TU

TD

TPh

TC

TE

TB

TI

TP

TA

46

A B

C D

Figure XV. Reference mixture for the testosterone esters of (A) 0.005 µg/mL and (C) 0.001 µg/mL. Transition

for 97 for the testosterone esters at the concentration of (B) 0.005 µg/mL and (D) 0.001 µg/mL.

TU

TD

TPh

TC

TE

TB

TI

TP

TA

TU

TD

TPh

TC

TE

TB

TI

TP

TA

47

D.2. Linearity Evaluated by Using Transition 97

A B

C C

Figure XVI. Test of linearity for the reference mixtures for concentrations of 0.001 µg/mL to 1 µg/mL.

Linearity based on transition 97 for (A) TA, (B) TB, (C) TP and (D) TU.

48

A B

C D

Figure XVII. Test of linearity for the reference mixtures from concentrations of 0.001 µg/mL to 1 µg/mL.

Linearity based on transition 97 for A) TC, B) TD, C) TE and (D) TPh.

49

Appendix E. Transitions Used in the Final Study

E.1. Final Transition Method

Table II. Final transition method.

Precursor Product ion Cone (V) Coll (eV) Rt Compound

331.20 135.10 30.00 25.00 2.04 Testosterone acetate



345.10 97.10 35.00 25.00 2.49 Testosterone propionate




387.20 97.10 35.00 25.00 3.62 Testosterone isocaproate




393.20 97.10 35.00 25.00 3.22 Testosterone benzoate



401.30 97.00 35.00 20.00 4.04 Testosterone enanthate



413.30 97.10 40.00 25.00 4.13 Testosterone cypionate




421.30 97.10 30.00 25.00 3.36 Testosterone phenylpropionate




443.30 97.10 25.00 25.00 4.96 Testosterone decanoate



457.30 97.10 35.00 25.00 5.06 Testosterone undecanoate



50

E.2. Tests of Final Transitions

Tests of transitions used in the final study based on the reference mix, spiked serum sample

and blank serum.

A B

C D

Figure XVIII. (A-D) Transitions from the reference mix.

51

A B

C D

Figure XIX. (A-D) Transitions from the spiked serum sample.

52

A B

C D

Figure XX. (A-D) Transitions from the serum blank.

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