MS. - Mustapha Hached

It is an ancient fortress and garrison town in the province of ... professor (Dr. Gover Somsen), a number of co-workers and an advisory board which gather .... It is composed of a pump, an injector, a column, a detector and a data recorder. Although ... 18 Marvin C. McMASTER, “LC/MC a pracfical user's guide”, Wiley, 2005. 19 ...
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Margaux Jochym IUT A de Lille Dept Chemistry 2008-2010

Avans Hogeschool

University of Applied Science Lovensdijkstraat 61 4818 AJ Breda, The Netherland

Traineeship from 1st of April to 30th of June

Analysis of three hormones in serum by LC/MS. Method to be used in green iguanas.

IUT supervisor: Mr Ahmed Mazzah

Avans supervisor: Dr Ben de Rooij

Acknowledgements

First of all, I would like to thank Dr. Ben De Rooij for giving me the opportunity to work in this laboratory of analytcal chemistry and for giving me this interestng project. I am grateful to his explanatons and his help. Thanks for being my supervisor and for all of your advice. I am very indebted to Edward Knaven for his availability, his kindness, his invaluable help and his research to solve my various problems. Thanks for giving me responsibilites. Your explanatons about the LC/MS and our daily discussions about my results were very helpful. 2

I am thankful to Linda Silvertand for improving my SPE method. Thanks to Fred and Paul for giving me some advice for my report. I would like to thank the Internatonal Ofce of Lille and Breda, Mr Caillier and Mr Mazzah for ofering me the possibility to carry out my project in the Netherland. Finally, thanks to everybody who supported me during these 3 months.

3

Table of contents: Acknowledgements .................................................................................................................... 1 Introducton................................................................................................................................ 4 I.Presentaton.............................................................................................................................. 5 A.About Avans Hogeschool...................................................................................................... 5 B.The research group............................................................................................................... 5 C.The project............................................................................................................................ 6 1.Problem.............................................................................................................................. 6 2.The purpose........................................................................................................................ 6 D.Green iguana......................................................................................................................... 6 1.Reproducton and behaviour............................................................................................. 6 2.Steroid hormones in female iguanas................................................................................. 6 3.Preovulatory follicle stasis (POFS)...................................................................................... 7 E.Theories................................................................................................................................. 8 1.SPE (Solid Phase Extracton)............................................................................................... 8 2.HPLC (High performance liquid chromatography)............................................................. 9 3.LC/MS Ion trap................................................................................................................. 10 II.Materials and methods used................................................................................................. 11 A.Chemical products and prepared solutons........................................................................ 11 B.Material............................................................................................................................... 12 1.SPE colum......................................................................................................................... 12 2.HPLC................................................................................................................................. 12 3.LC/MS............................................................................................................................... 14 C.Protocol............................................................................................................................... 14 1.SPE method...................................................................................................................... 14 2.Improvement of the analyse method.............................................................................. 16 3.Cleared serum.................................................................................................................. 17 4

4.Sham serum..................................................................................................................... 18 5.Analysis method............................................................................................................... 18 6.Validaton of the method................................................................................................. 18 III. Results.................................................................................................................................. 19 A.Determinaton of the 3 peaks by UV detecton.................................................................. 19 B.Determinaton of the hormones and their fragments by LC/MS.......................................19 C.Improvement of the SPE method....................................................................................... 21 D.Determinaton of low concentratons in serum................................................................. 21 E.Cleared serum step............................................................................................................. 21 F.Sham serum......................................................................................................................... 22 G.Test of the method in diferent serum................................................................................ 23 H.Improvement of the analysis method................................................................................ 24 I.First step of the method validaton...................................................................................... 25 Discussion:.................................................................................................................................27 Conclusion:................................................................................................................................ 28 Appendix:.................................................................................................................................. 29 Appendix 1: Preparaton of the Work Solutons.................................................................... 29 Appendix 2: Determinaton of the volume of injecton......................................................... 31 Appendix 3: Analysis of the volume of serum....................................................................... 31 Appendix 4: Analysis in low concentratons...........................................................................32 Appendix 5: Determinaton of the volume of the serum which could be cleared in one column....................................................................................................................................33 Appendix 6: Artfcial serum and sample preparaton for the calibraton curve................... 34 Appendix 7: Method validaton............................................................................................. 36 Appendix 8: Real concentratons in soluton and in samples................................................38 Appendix 9: Accuracy, standard derivaton (SD) and relatve standard derivaton (RSD) formulas................................................................................................................................. 42

5

Introduction

The green iguana is found from Mexico to tropical South America. These iguanas live in trees and usually only come down to lay their eggs. However, due to the destructon of habitat (mostly the deforestaton in the Amazon) and other factors such as the indiscriminate huntng for their meat and skin, along with iguanas trafcking to sell them as pets, iguanas are in some danger of extncton. To fnd a soluton for this, some associatons are allowed to raise iguanas in captvity in order to increase the number of species. Nevertheless for iguanas living in captvity, reproducton is rare. This could be due to an abnormal concentraton of steroid hormones during the POFS (PreOvulatory Follicle Stasis) which impedes ovulaton. Thus, researchers want to determine the level of concentraton which disturbed the POFS. That is why a method has to be found to detect low concentratons of steroid hormones. This report begins by laying out the theoretcal dimensions of the research (chapter 1). Chapter 2 describes the diferent equipment and the method used. The last chapter reports the results of the project.

6

I. Presentation A. About Avans Hogeschool Avans University of Applied Science was founded in January 2004, following a merger of Hogeschool Brabant and Hogeschool ’s-Hertogenbosch. About 20.000 students study 45 courses and 1.800 employees work in Avans. It is composed of 19 schools, 4 support units and a learning and Innovaton Centre, located in three cites in the southern part of the Netherlands1: • Tilburg •

’s-Hertogenbosch



Breda.

Many diferent courses are ofered in Breda, in partcular in the sectors of business, management, Informaton Technology and research. Breda is a dynamic and welcoming city. It is an ancient fortress and garrison town in the province of Noord-Brabant. The municipality has about 173,000 inhabitants and by populaton is one of the 10 largest municipalites of the Netherland. 1htp://www.avans.nl

7

B. The research group2 A research group in Separaton Sciences was found in 2003 by the University of Professional educaton in Tilburg and the Netherland. This group consists of an associate professor (Dr. Gover Somsen), a number of co-workers and an advisory board which gather experts from several scientfc felds. In 2008, the name of the Research Group is changed into Research Group Analysis Techniques in Life Science. At this moment, cooperaton is launched between the Research Group Analysis Techniques in Life Science of Avans Hogeschool and the Research Group Biomedical Analysis of the Department of Pharmaceutcal Sciences of University Utrecht. The research group is concentratng on three major goals: • Research •

Educaton



Internatonalizaton

Their principal priorites are to initate research program, execute some contract research and realize cooperatve programs with diferent companies and insttutes. For a consistent development of research and educaton programs, a relaton between teacher, student and their knowledge is very important. These research programs are mainly focussed on liquid chromatography (HPLC) and Capillary electrophoresis (CE) as separaton techniques but there is also mass spectrometry as detecton and identfcaton techniques.

2htp://www.expertseteam-separatonsciences.nl

8

C. The project 1. Problem For the green iguanas living in captvity, no reproducton is observed. From the point of view of biodiversity, it is important to fnd a cure. Some researchers have shown that preovulatory follicle stasis (POFS) could be the reason for this problem. That is why the Dutch research insttute of birds and exotc animals has asked Avans Hogeschool for help. Analysis of the four hormones, testosterone, estradiol, cortcosterone and progesterone, is an important aspect of the research of POFS3. 2. The purpose A few years ago, these hormones were already analysed by using HPLC with UV detecton and by preparing the sample by Solid Phase Extracton (SPE). However, a problem occurred as the detecton was not sensitve enough and hampered by a high limit of detecton (LOD). Another method was processed by using HPLC with fuorescence detecton. The sample reacted with dansylhydrazine to be fuorescent. Nevertheless, the limit of detecton was too high and the method could not be validated. An analysis by radio-immuno assays (RIAs) was tested and was really promising because the limit of detecton was low 0,08 ng/mL for testosterone. Although, it is an extremely sensitve method, it is very expensive.4 Consequently, the aim of this project is to fnd a new analytcal method with more sensitvity and selectvity. The method has to be reproducible and applicable in serum. In this project, only three hormones (testosterone, cortcosterone, progesterone) will be analyzed. Indeed, it is difcult to detect estradiol because its concentraton is extremely low during the nestng period (less than 0,12 ng/mL5) contrary to the others. The hormones 3 “Abstract for traineeship of IUT students”, B.M. de Rooij, G.M. Dorrestein, G.W. Somsen, 2010

4 “Steroids Hormones and aggression in female Galápagos marine iguanas”, Dustn R. Rubenstain, Martn Wikelski, 2005, p.332

5 “Steroids Hormones and aggression in female Galápagos marine iguanas”, Dustn R. Rubenstain, Martn Wikelski, 2005,

9

will be detected by HPLC combined with mass spectrometry. Samples will be prepared by a SPE method in order to separate steroids from the rest of the matrix such as proteins, fat or salts. To begin with, the method will be tested in soluton of testosterone, cortcosterone, and progesterone. Then, the reproducton in serum will be checked. Moreover, it is important to determine the limit of detecton to see if it could be applicable with serum of iguanas.

D. Green iguana 1. Reproduction and behaviour Green Iguanas reach sexual maturity between three and four years of age. Iguanas breed in the dry season, ensuring that young hatch in the wet season when food is more readily available. Female iguanas can lay 20-70 eggs at a tme6. The period of reproducton can be separated into two stages: the matng and the nestng period. Females deposit eggs approximately 65 days afer matng, usually over the course of three days, into nests excavated to a depth of about 1 to 3 feet into the soil. Incubaton takes 90 to 120 days. Young iguanas are independent from the tme of hatching7.

Immediately afer copulatng, morphology and behaviour of females changes to resemble males. They alter their skin colour, gaining red patches on their fanks and bright green colours along their dorsal spines. Moreover, they become more aggressive. Female aggression can occur towards males (female-male aggression) or towards other females (female-female aggression). Female-male aggression can take place to signal that they have already copulated and to protect their ofspring, whereas, female-female aggression happens p.333

6htp://animal-world.com/encyclo/reptles/lizards_iguanid/GreenIguana.php

7htp://foridagardener.com/criters/iguanas.htm

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when females vie for resources, males, or when they fght to establish social rank and to defend territories. Female-male aggression occurs only during the matng period, unlike female-female aggression which mainly occurs during the nestng period.

2. Steroid hormones in female iguanas Aggression in females may be partly due to steroid hormones. Testosterone and estradiol seem to promote aggression, while cortcosterone seems to suppress it. Progesterone may infuence aggression in some contexts but suppress it in others8. Concentratons of steroid hormones in female iguanas suddenly change between the matng and the nestng period. Testosterone has the more signifcant change and tends to increase during matng and vitellogenesis (the process of yolk formaton). It may stmulate oviductal hypertrophy (the increase in the volume of the oviduct) and uterine development. It could be related to female-male aggression during the matng period and might produce the change in skin colour. Concentraton of estradiol is extremely low during the nestng period and gestaton. It is probably responsible for vitellogenesis and can stmulate atractvity or receptvity of female reptles. Cortcosterone reaches maximal levels during breeding and may be released to energy mobilizaton for vitellogenesis. Finally, progesterone decreased towards the end of the matng period, but then rose at the beginning of the nestng period. It is related to the maintenance of oviduct vasculary, such as the inhibiton of follicular growth and oviductal contractlity9.

8 “Steroids Hormones and aggression in female Galápagos marine iguanas”, Dustn R. Rubenstain, Martn Wikelski, 2005, p.329

9 “Steroids Hormones and aggression in female Galápagos marine iguanas”, Dustn R. Rubenstain, Martn Wikelski, 2005, p.336

11

Structure of the steroid hormones: Steroid hormones are steroids that act as hormones. The steroid hormones are synthesized mainly by endocrine glands especially the gonads (tests and ovary) and the adrenals, and are then released into the blood circulaton. They can be regrouped into 5 groups by the receptor to which they bind: • Glococortcoids (i.e. : Cortcosterone) •

Mineralocortcoids



Androgens (i.e. : Testosterone)



Estrogens (i.e.: Estradiol)



Progestagens (i.e.: Progesterone)

12

The steroid hormones are all derived from cholesterol. They contain the same cyclopentanophenanthrene ring which is composed of three six-member rings and one fvemember ring. The main modifcatons occur on the substtuent of the fve-member ring10.

3. Preovulatory follicle stasis (POFS) The preovulatory follicle stasis is the frst step in the process of ovulaton. Through the infuence of a rise in follicle stmulatng hormone (FSH) a few ovarian follicles are stmulated. These follicles, which were present at birth, are developing in a process known as folliculogenesis (POFS is the last step of this process). As they mature, the follicles secrete increasing amounts of estradiol. Under the infuence of several hormones, all but one of these follicles will stop growing, while one dominant follicle in the ovary will contnue to maturity. Afer this is done, a hole will form in the follicle and the ovum will leave the follicle through this hole. The amount of FSH and LH (Luteinizing Hormone) released by the hypophysis controlled by the hypothamus of the brain, suddenly increased and trigger of ovulaton11. 10htp://www.cs.stedwards.edu/chem/Chemistry/CHEM43/CHEM43/Steroids/Steroids.HTML

13

Afer ovulaton, FSH and LH cause the remaining part of the dominant follicle to transform into the corpus luteum, which produces progesterone. The increased progesterone starts to induce the producton of estrogen. The hormones produced by the corpus luteum also suppress producton of the FSH and LH that the corpus luteum needs to maintain itself. Consequently, it degenerates and this is the beginning of the next cycle.

Thus, sex hormones are very important during this period in female iguanas. Abnormal concentratons could cause a problem for the reproducton.

E. Theories 1. SPE (Solid Phase Extraction) Defniton: The Solid Phase Extracton (SPE) is a separaton process that is used to extract compounds (called analytes) from a mixture of impurites. It is usually used to concentrate and remove interferences (such as protein in plasma) before using a chromatographic or other analytcal method to quantfy the amount of analytes. Solid phase extracton can be

11htp://en.wikipedia.org/wiki/Ovulaton

14

used to isolate analytes of interest from a wide variety of matrices, including urine, blood, water, beverages, soil, and animal tssue12. Principle: A sample is introduced into a column or a cartridge containing an adsorbent material (statonary phase). The solvent (mobile phase) fows through the column.

By choosing an appropriate combinaton of

mobile and statonary phases, sample components may pass directly through the column, or they may be selectvely retained. Indeed, diferent methods exist depending on the polarity of the analyte of interest, the electrostatc interactons and the solubility in diferent phases.13. •

Reversed Phase: polar liquid phase, non-polar solid phase

12 htp://www.sigmaaldrich.com, “Guide to Solid Phase Extracton”, p.9

13 htp://www.sigmaaldrich.com, “Guide to Solid Phase Extracton”, p.3

15

• •



Normal Phase: non-polar liquid phase, polar solid phase Ion Exchange: electrostatc interactons between the analyte of interest and the positvely charged groups on the statonary phase Adsorpton: interactons of compounds with unmodifed materials

For this project, hormones such as testosterone, progesterone and cortcosterone from serum need to be separated. Steroids have a non-polar structure, which is why in order to be retained, it is necessary to use reversed phase column.

16

SPE involves four main steps14: • Conditoning: Methanol (or a water miscible organic solvent) is usually used, followed by water in reversed phase. Methanol wets the surface of the sorbent and penetrates bonded alkyl phases, allowing water to wet the silica surface efciently. •

Loading sample: The whole sample is put on the column. Analyte is retained by the solid phase, while most of the interference goes through the cartridge.



Washing: The cartridge is washed with bufer or solvent to remove further impurites.



Elutng: The analyte is eluted with a non-polar solvent or a bufer of the appropriate pH.

2. HPLC (High performance liquid chromatography) HPLC is a kind of chromatography used to separate, identfy and quantfy compounds. HPLC Instrument: It is composed of a pump, an injector, a column, a detector and a data recorder. Although the column is the most important part because it is where the separaton occurs, it is usually the smallest one. For temperature-controlled separatons, it is enclosed in a thermostat. Moreover, a short guard column can be used between the injector and the separaton column as a protecton. Its purpose is to extend the life of the separaton column by protectng it from deterioraton caused by sample contaminants and strongly retained compounds15.

14htp://www.interchim.com/interchim/chroma/SommaireCatChromato/A-49-91-TechnologiesInterchim.pdf

15 Colin F. POOLE, “The essence of chromatography”, Elsevier, 2003

17

Principle: To be separated, the sample mixture is distributed between two phases in the column. One phase is statonary while the other phase passes through the column the statonary phase is either a solid, porous in small partcles form atached in the column wall (frequently silica partcles). The mobile phase is a liquid. The mixture is injected into the mobile phase in front of the statonary phase. The mobile phase passes through the column thanks to a pump. Compounds that tend to reside in the mobile phase move more quickly than those that prefer the statonary phase. Then, the mobile phase enters the detector that detects the diferent molecules separately16. HPLC used two diferent methods depending on the relatve polarity of the solvent and the analyte: • The normal phase •

The reversed phase

The reversed phase chromatography is the term used to describe the state in which the statonary phase is less polar than the mobile phase. It is the most popular of liquid chromatography. In the case of steroid hormones, reversed phase is used because steroids are non-polar compounds. The column is flled with tny silica partcles where long hydrocarbon chains have be atached to make it non-polar (usually a chain of 8 or 18 carbon are used)

16 Veronika R.MEYER, “Practcal High-Performance Liquid Chromatography”, 4th editon, Wiley, 2004

18

Moreover, a polar solvent is used, such as a mixture of water with various polar and water miscible organic solvent17. 3. LC/MS Ion trap18 LC/MS is a liquid chromatography (HPLC) combined with mass spectrometry (MS). It is an analytcal chemistry technique for identfcaton of chemical structures, determinaton of mixtures, and quanttatve elemental analysis. LC/MS system: Essentally, an LC/MS system is an

HPLC pumping system, injector, and column combined to a mass spectrometer. Once the compounds have been separated by liquid chromatography, they need to be transferred into the mass spectrometer for detecton. This includes everything coming out of the column: separated compounds, eluton solvent and impurites.

Ion source: Also called interface, the ion source must be capable of removing most of the volatle solvent without removing the target compounds of interest. To do this, the interface used a combinaton of heaters, reduced pressure and nebulizer gas. 17htp://www.chemguide.co.uk/analysis/chromatography/hplc.html

18 Marvin C. McMASTER, “LC/MC a practcal user’s guide”, Wiley, 2005

19

In the electrospray interface, ionizaton is accomplished by passing the HPLC eluant (solvent + analytes) down a capillary tube along which an electric charge diferental is applied. This capillary tube is put on another tube in which an inert gas called nebulizer gas circulates (mostly Nitrogen N2). The evaporatng liquid is sprayed out of the end of the tube as charged droplets, it is called the nebulizaton.

The droplets decrease rapidly in size because of the evaporaton of the solvent causing by a drying gas (nitrogen N2), it is the desolvaton. When the surface tension of the droplets becomes too high, they explode. This is called “Coulomb explosion”, producing a release of ionized analytes in gas phase. Generate ions are then transferred to a mass analyzer. Formed ions could be positve or negatve ions. They are generated by protonaton [M-H] + or deprotonaton [M-H]-.

Figure : Reducton of size of a droplet 20

Ion transport: Afer the ion source, the ions go through a glass capillary, a skimmer and a dual octopole which guide ions to the mass analyzer. During the transport, a high-vacuum environment is needed. Indeed, the ionized analytes ions formed in the interface cannot survive collision with air molecules in the analyzer. Moreover, a focusing lens electrically charged with the same charge type as the ions is placed before the mass analyzer to concentrate them into a beam. Ions then enter the analyzer. Mass analyzer: The heart of a mass spectrometer is the mass analyzer, which separates ions by their mass divided by their charge (their m/z rato). Its purpose is to hold ions, select specifc mass ion as the radio frequency (RF) is scanned, and move the selected ion into the ion detector for countng. The ion trap analyzer consists of three electrodes: two hyperbolic electrodes (also called endcaps) which contain small perforatons in the center to allow the ion to enter and exit the ion trap, and one ring electrode. The ions enter the analyzer and are trapped between these three electrodes by a statc (direct current DC) electronic feld and a radio frequency. The increase of the voltage pushes ions into an unstable orbit, causing it to escape through one of the seven holes in the exit electrode and into the ion detector. For efcient trapping and cooling of the ions generated by the electrospray, helium gas is introduced into the ion trap. Detector:

21

The ion detector is shaped like a horn. Thus, when a molecular ion strikes the face of the detector it causes an electron to be released from the other side. These electrons then strike the coated walls of the detector, releasing multple electrons on each impact. This cascade of electrons allows an amplifcaton of the signal and it is transferred to the data system.

II. Materials and methods used A. Chemical products and prepared solutions Solids:  Cortcosterone minimum 92% (C21H30O4), Sigma  Progesterone 98% (C21H30O2), Agrôs organics  Testosterone ≥ 99 % (C19H28O2), Fluka  Sodium chloride 99,5% (NaCl), Acrôs organics  Potassium dihydrogen phosphate (KH2PO4), Merck  Sodium phosphate dibasic heptahydrate (Na2HPO4, 7 H2O), Acrôs organics  Bovine Serum Albumine-Fracton V, Sigma-Aldrich

Liquids:  Acetonitrile HPLC-S (CH3CN), Biosolve  Methanol absolute HPLC (CH3OH), Biosolve  Milli Q Water (MQ) Stock Solutons: The stock soluton (ST) in testosterone is prepared by weighing 25 mg of testosterone and dissolved in a 25 mL volumetric fask in acetonitrile (ACN). The concentraton is 1 g/L. Stock soluton in cortcosterone and progesterone are prepared in the same way. Work solutons: 22

Six work solutons (WS) are prepared from the stock solutons in ACN/MQ (50/50). These solutons are composed of the three steroids in diferent concentratons. Their preparatons and their concentratons are explained in appendix 1, p.47.

B. Material 1. SPE colum

Characteristcs: • Name: Water Sep Pak Vac C18 •

Statonary phase: Silica-based with chains of 18 carbons



Sorbent mass: 500 mg



Applicatons: drugs and metabolites in serum, plasma or urine19

During the second part of the internship, the Water Sep Pak SPE column were replaced by a new one because of a lack of stock.

19htp://www.waters.com

23

Characteristcs: • Name: Biotage Isolute C18 (EC) •

Statonary phase: Silica-based with chains of 18 carbons



Sorbent mass: 200 mg



Applicatons: acids, basics, and neutral drugs from a biological fuids20

2. HPLC

Characteristcs: •

Name: Agilent Technology 1200 series



Column: Agilent Zorbax Eclipse XDB C18



Thermostat temperature: 40°C



Solvent: ACN/MQ (60/40) + 0.1% of Formic Acid



Pump: Binary SL pump

20htp://www.biotage.com

24



Detector: UV detecton DAD (Diode Array Detector)



Injecton system: Autosampler



Injecton volume: 100 µL (frst 10 µL was used)



Flow: 0,5 mL/min (Formic Acid is used in the solvent to improve the protonaton of the molecules for the MS analysis)

Characteristcs of the column:



Name: Agilent Zorbax Eclipse XDB C18



Statonary phase: Silica bonded to C18

Size: 4.6 * 100 mm •

Partcle size: 3.5 µm

In the second part, the column has to be changed because of impurites, due to serum which is staying in it. The pressure soared to 300 bar and shifed retenton tme. The acquisiton run became too high (30 min instead of 15 min). So the column has been changed for a new one and the percentage of ACN in the mobile phase was put on 75% instead of 60%. Characteristcs of the new column: • Name: Agilent Zorbax Eclipse-XDB C8 •

Statonary phase: Silica bonded to C8



Size: 4.6 * 150 mm



Partcle size: 5 µm 25

3. LC/MS Characteristcs:



Name: Agilent Technology 6320 ion Trap LC/MS



Ion polarity: Positve



Ion Source Type: ESI (ElectroSpray Interface)



Dry Temperature: 350°C



Nebulazer Gas: Nitrogen (N2) 15.00 psi



Drying Gas: Nitrogen (N2) 10.00 L/min



Scan Begin: 250 m/z Scan End: 360 m/z

26

For more selectvity and sensibility, each sequence is segmented into three parts. Each part represents the mass of one compound of interest. •

Segment 1 (from 0 to 6.5 min): the 347 m/z rato is measured (more or less 2). It is the mass of cortcosterone + 1 proton.



Segment 2 (from 6.5 to 9 min): the 289 m/z rato is measured (more or less 2). It is the mass of testosterone + 1 proton.



Segment 3 (from 9 to the end of the run): the 315 m/z rato is measured (more or less 2). It is the mass of progesterone + 1 proton.

C. Protocol 1. SPE method Step 1: Preparaton of the sample: • Put 1,0 mL of sample (water or serum) in a test tube. In the frst experiments 0,5 mL was used. •

Spike Work Soluton (WS): the volume and the WS depend of the concentraton is needed.

Step 2: Conditoning step • Put test tubes in the SPE box (Varian Sample preparaton products) and fx columns to it. •

Put 3 mL of MeOH on the column. 27



Add 3 mL of MQ when the column is empty. Cauton: Keep the column wet for these 2 stages,

thanks to the taps under the column. Step 3: Load step • Load 1 column with 1 prepared sample. Leave the liquid to pass slowly through the column. •

Cauton: Keep the column wet for this stage.

Step 4: Washing step • Wash the column with 3 mL MeOH/MQ (30/70). Dry the column by a centrifuge step: put column in test tubes and

put them in the centrifuge Labofuge 200, Heraeus sepatech (3000 rpm/10 min). Step 5: Eluton step • Put new tubes in the SPE box, and fx dry columns. •

Add 3 mL of ACN, and discharge slowly trough the column. 28

Step 6: Evaporaton step •

Collect ACN in tube and evaporate by a stream of air in 45°C thanks

to the evaporator. Step 7: Reconsttuton step •

When the sample is dried, reconsttute it with 500 µL of ACN/MQ (50/50) and mix.

Step 8: Analysis • Put the sample in an autosampler vial which a limited volume insert is placed (the aim of the insert is to reduce the volume of the vial because the capacity of the vial (about 2 mL) is too high compared the volume of resttuton (500 µL)).

Improvement of the washing step: To be sure that the washing step suppresses most of the interference stayed in the column without washes the steroids, we have to research which percentage of methanol (MeOH) is needed.

29

To do this experiment, only one SPE column is used: • Introduce 1mL of WS3 afer the conditoning step. •

2 mL of water is added. It allows to suppress more proteins



Wash with 0% MeOH, 1mL, collect the eluate



Centrifuge, and add the rest of liquid in the eluate



Put a new test tube in the SPE box



Wash directly with 10% of MeOH, 1mL, collect the eluate



Centrifuge, and add the rest of liquid in the eluate



Repeat the washing and centrifuge steps untl 100% of MeOH

Then it is tested in serum, by putng 1 mL of serum instead of WS3. 2. Improvement of the analyse method To determine low concentratons in serum, some improvements are needed: First of all, by increasing the volume of injecton, the intensity of the response should be beter. To check this, 6 samples made in serum (samples) and 6 others made in ACN/MQ (standards) are prepared. Their concentratons vary from 0 µg/L to 2000 µg/L and are obtained thanks to the Work solutons (WS) (In appendix 2, p.48, preparaton of the samples are detailed). Samples in serum are then loaded on the SPE column and analysed by LC/MS. Standard samples are directly analysed. Secondly, the volume of serum can also be increased. By putng 1 mL in the sample instead of 0.5 mL (preparaton of the samples is explained in appendix 3, p.48). Afer these improvements, an analysis in low concentraton can be tested. For that, standard and serum samples are used with concentratons from 100 µg/L to 0.1 µg/L (preparaton of the samples in appendix 4, p.49).

30

3. Cleared serum Testosterone present naturally in the serum disturbs the detecton of low concentraton. That is why a step to clear the serum of testosterone has to be added into the method in order to make a calibraton curve directly in the matrix. Firstly, it is important to know the higher volume of serum which can be cleaned in only one SPE column. For this, 6 SPE columns are conditoned. A batch of serum (+spike) is prepared and diferent volumes are applied on the diferent columns (from 1 mL to 9 mL). Then, the SPE procedure is made. Steroids should stay on the column and the serum should be cleaned of them (Samples’ preparaton in appendix 5,p.49). Then, the clearness of the serum has to be checked. To do this, 3 samples are prepared: • 1 mL of serum + spike with a concentraton of 100 µg/L •

1 mL of serum (blank serum)



1 mL of cleared serum

All of the samples are put on 3 columns and SPE procedure is made. Cleared serum by a repettve way: Sample: 10 mL of serum Step 1: Conditoning of the 6 columns Step 2: Cleaning step • Load in the frst column the 10 mL of serum •

Collect the eluate and keep 1mL apart (1st degree cleared serum)



Load column 2 with the 9 mL remaining



Collect the eluate and keep 1 mL apart (2nd degree)



Do the same for the 4 other columns by putng respectvely 8 mL, 7 mL, 6 mL and 5 mL and always keep 1 mL of each degree of cleaned serum.

31

Each millilitre collected is then put on a new column and the SPE procedure is made. Then the samples are analysed in the normal way.

4. Sham serum Low concentraton in serum cannot be detected, thus, a calibraton curve in sham serum is made. The artfcial serum is prepared from water, phosphate bufer and a bovine protein: BSA (Bovine Serum Albumin) at pH 7,4 like real serum. (In appendix 6, p. 50, preparaton of artfcial serum is explained). 5. Analysis method Afer a certain number of analyses, the system starts to be dirty by impurites present in the matrix. At this moment, pressure really increases (about 20 bars per samples) and thus shifs the retenton tme. That is why, a soluton to resolve this problem have to be found. The frst method that can be tried is to introduce a gradient mode instead of an isocratc one. It means that during the run, the percentage of solvent change. Here it is necessary to wash the system afer each sample by 90% of ACN. Moreover, a needle wash (in ACN/H2O-50/50) is added afer each injecton.

1

Time (min) 0

% ACN

Flow

75

0,5

32

2 3 4 5 6

13 14 19 20 22

75 90 90 75 75

0,5 0,5 0,5 0,5 0,5

Figure : Table illustrated the gradient method

6. Validation of the method To validate the method, the sensitvity, selectvity, accuracy and precision have to be check. For that, some samples in serum called quality control (QC) in low (QCL), medium (QCM) and high (QCH) concentraton (respectvely 5 µg/L, 25 µg/L, 75 µg/L) as well as blanks have to be analysed. Each sample was prepared 4 tmes. In the same tme a calibraton curve in sham serum is made from 1 to 100 µg/L. Moreover, for the analysis, all of the samples are placed in a specifc order in the autosampler to mix solvent, high and low concentraton, and calibraton curve sample and QC samples (The preparaton of the samples and the order is presented in appendix 7, p. 51).

33

III. Results A. Determination of the 3 peaks by UV detection In a serum sample, the three hormones (testosterone, progesterone and cortcosterone) will be present. That is why, the determinaton of the order in which the three hormones come and with which retenton tme are important. To do this, three solutons of each hormone in 10 mg/L are prepared thanks to my stock solutons and analysed with the HPLC, UV detecton. Three chromatograms are obtained and overlaid.

The frst peak is cortcosterone and it has a retenton tme of about 4.1 min. The second peak is testosterone and its retenton tme is about 6 min. And fnally the progesterone comes with a retenton tme of about 14.7 min.

B. Determination of the hormones and their fragments by LC/MS

34

In lower concentraton, LC/MS have to be used because it is more sensitve and efcient than UV detecton. Moreover, LC/MS allows us to check the desired molecule thanks to its m/s rato and its fragmentatons. To determine the m/s rato of each compound and their fragmentatons, the same solutons used previously were injected into the LC/MS.

Cortcosterone:



Compositon: C21H30O4



Molecular mass: 346.47 g/mol



m/z rato afer ionizaton (M+1): 347



Fragmentaton (m/z) : 329, 311, 293

35

36

Testosterone:



Compositon: C19H28O2



Molecular mass: 288.43 g/mol



m/z rato afer ionizaton (M+1): 289



Fragmentaton (m/z) : 271, 253, 109

37

Progesterone: •

Compositon: C21H30O2



Molecular mass: 314.46 g/mol



m/z rato afer ionizaton (M+1): 315



Fragmentaton (m/z) : 297, 279

38

39

C. Improvement of the SPE method

This result shows that cortcosterone starts to pass through the column at 45% of MeOH. That is why, 40% will be beter for the washing step. Moreover, it shows that at least 3 elutons are needed to collect all of the steroids. Thus, 3mL of MeOH is beter to elute analytes of interest because it is cheaper compared with acetonitrile and easier to evaporate. Then, the efciency of the washing step in the serum is checked In conclusion of this experiment, 40% in MeOH is good to wash more efciency the steroids from impurites.

D. Determination of low concentrations in serum

Variaton of the volume of injecton: Variaton of the volume of serum:

40

These experiences proved that by increasing the volume of injecton, the response is ride by 5 and by put more serum in the sample, the intensity is about twice higher (except for progesterone).

Analysis in low concentraton This analysis shows that very low concentratons (from 5 µg/L to 0,1 µg/L) can be detected but a method has to be found to suppress the natural testosterone.

E. Cleared serum step

These results show that almost 9 mL of serum can be cleaned in one column. It is proportonally the same amount of hormones in 1 mL and 9 mL. Analysis shows that blank serum and cleared serum are quite the same concentraton of testosterone. But the area for the cleared serum is slightly lower than in the blank serum. Thus, a method to clean the serum in a repettve way is tried. 41

In fact, the serum will be cleaned 6 tmes and a reducton of the concentraton of testosterone is expected.

This experiment proves that a litle amount of testosterone always stays in the serum (except for the 5th and 6th degree where the results are impossible and probably due to the system). The SPE method is not able to extract and keep all of the testosterone. This amount is maybe due to the proteins present in serum. The afnity between testosterone and these proteins should be too strong to separate them. Thank to the analysis, an estmaton of the concentraton of the testosterone staying in the blood can be made. Calculaton: •

Concentraton of the serum +spike = 10 µg/L



Area of the testosterone peak = 270000



Concentraton of the cleared serum = a



Area of the testosterone peak for cleared serum = 100000



Response of the analysis = Resp

42

In conclusion, the estmate concentraton of testosterone present naturally in the serum is about 6 µg/L. That is why, in the analysis of hormones at low concentraton (chapter III, D), the area of testosterone in serum stays quite the same afer a concentraton of 8 µg/L and does not lower under the area of 100000.

F. Sham serum As a concentraton below 8 µg/L cannot be detected, a calibraton curve in a “sham serum” is made from 1 µg/L to 10 µg/L. This result shows that a calibraton curve in low concentraton can be made. Detecton is possible from a concentraton of 1 µg/L. But artfcial serum also contains some amount of testosterone in a blank sample as the experience proves. A comparison between artfcial and real serum is made by making the same calibraton curve in higher concentraton (from 10 µg/L to 200 µg/L).

Cortcosterone and testosterone curves are quite the same in real and artfcial serum. However, for progesterone, the response is clearly lower in real serum. This phenomenon could be due to ion suppression. This is occurred in the early stages of the ionisaton process in the LC/MS interface when a component eluted from the HPLC column infuences the ionizaton of an analyte (here progesterone). Even if the interfering compound is not detected, its presence in the matrix (e.g. the real serum) stll afects the response of the analyte of interest. A cleaning step in real serum is really necessary to have a calibraton curve in low concentraton and to eliminate the ion suppression phenomenon.

G. Test of the method in different serum 43

The method has to be test with diferent kind of serum. For example, it is tested in serum from bovine, sheep and horse. Two samples of each serum are prepared: one blank and one spike to have a concentraton of 10 µg/L. Serum from bovine:

Figure 41: Chromatogram of blank serum from bovine

Figure : Chromatogram of serum (+ spike 10 µg/L) from bovine Serum from sheep:

44

Figure : Chromatogram of blank serum from sheep

Figure : Chromatogram of serum (+spike 10 µg/L) from sheep Serum from horse:

Figure : Chromatogram of blank serum from horse 45

Figure : Chromatogram of serum (+spike 10 µg/L) from horse This experience proves that the method is applicable in other samples. Indeed, all of the compounds are detected.

H. Improvement of the analysis method A sequence of 36 samples is put on the autosampler and the pressure at the beginning is checked: This result is promising because the pressure stays quite stable. Moreover, the gradient mode allows the system to wash. Efectvely, afer a high concentraton 80 µg/L, solvent is introduced (ACN/W) and no peak is observed.

46

Figure : Chromatogram of sham serum at 80 µg/L

Figure : Chromatogram of solvent (ACN/W) This method allows the pressure to keep stable and to clean the system afer each sample. Besides, it allows the life of the column to extend.

I. First step of the method validation The used method is quite sensitve because the limit of detecton LOD (which means that the signal has to be about 3 tmes higher than the noise, it is the s/n rato) is between 0 and 1 µg/L for testosterone and progesterone and a litle bit higher for cortcosterone.

47

Indeed, with the height of the results, an estmaton can be made. For instance, the rato between the 0 µg/L and 1 µg/L gives the approximate concentraton of 0,3 µg/L in sham As the method is sensitve enough, a validaton could be made. A calibraton curve in artfcial serum and with the real concentraton is created in order to calculate the accuracy and the precision of the method. Moreover, the equatons of the regressions lines allow the determinaton of the measured concentraton (table with the real and measured concentraton is presented in appendix 8, p.52).

Overlaid chromatograms of two Quality Control in low, medium and high concentratons are shown below:

48

Figure : Chromatogram of the QCL (5µg/L)

Figure : Chromatogram of the QCM (25 µg/L)

Figure : Chromatogram of QCH (75 µg/L)

49

The accuracy is the measured concentraton/real concentraton rato: Cortcosterone Testosterone QCL QCM QCH

84,6 % 88,9 % 91,6 %

85,1 % 80,3 % 88,4 %

Progestero ne 59,3 % 64,3 % 57,1 %

The accuracy is correct if it is about 90% (like cortcosterone in high concentraton). It is acceptable for a percentage of between 80% and 90%, but a lower percentage have to be rejected. The accuracy of the method has to be improved, especially for progesterone. This result confrms the phenomenon of ion suppression met before. The precision is obtained by the relatve standard derivaton: Cortcosterone Testosterone QCL QCM QCH

11,2 % 8,3 % 4,9 %

14,7 % 7,5 % 4,6 %

Progestero ne 12,2 % 12,7 % 8,2 %

Lower the percentage is, higher the precision is. In conclusion, in high concentraton, the precision of the measurement is correct for cortcosterone and testosterone and acceptable in medium concentraton. But as the accuracy, a real improvement has to be made in low concentraton and for progesterone. (calculaton of accuracy and relatve standard deviaton is explained in appendix 9, p.53)

50

Discussion: The method which is improved and used during this training period has some conclusive points. Indeed, the detecton can be made in low concentraton untl 0,35 µg/L for testosterone. However, a problem stll occurs, the cleared serum always contains a litle amount of testosterone which disturbed the analysis and the quantfcaton. This amount could be due to proteins present in serum which bonded with the hormone. A method has to be found to suppress totally the natural testosterone. Antbodies might be a future soluton because testosterone is a hormone. That is why it could be compared to an antgen, a chain of proteins. Thus, an antbody ant-testosterone could catch the antgen testosterone and take away from serum. It should work because antbodies’ bonds are strongest than proteins’ bonds. But it is needed a real contributon. Secondly, the problem of ion suppression has to be resolved because it causes a reducton of the intensity and thus a false quantfcaton of the compound. A deuterated internal standard could be added in the preparaton of the sample to fnd a soluton to that. Indeed, this sort of compound will have almost the same compositon of the hormones, just protons will be replaced by deuterium. In this way, the two signals will be quite a similar and really distnguishable by the mass spectrometry (the molecular mass with deuterium will be higher). So, by making the rato between the analyte signal and the internal standard signal the problem of ion suppression could be resolved. Improvements on the accuracy and the repeatability have to be made, especially in low concentratons. For progesterone, the results should change if the phenomenon of ion suppression will be solved. This method is not completely validated because of a lack of tme. Only one experiment for the validaton is made, and it is necessary to do this 3 tmes and one tme more by another person. Afer some improvements, this method could be tried in iguanas’ serum since, as the results showed, the method is applicable in diferent samples. In a future project, the method has to be tested in lower concentraton (about 0,2 µg/L for testosterone) to be really applicable in iguanas. 51

52

Conclusion: This training period in Avans Hogeschool allowed me to discover the working life in a laboratory. It was for me a great experience, really enriching and interestng. It is rewarding to know that it is a real project which could be used in a near future. Even if it was not easy every tme, because it is quite frustratng to do not manage to solve problems, it was opened my mind to the research life. I learned to do an intellectual reasoning to fnd solutons and explanatons on the results. Moreover, I improve my knowledge on HPLC and discover the principle of mass spectrometry and SPE. By having responsibility on this equipment, it gave me selfconfdence. Finally, this internship allows me to improve my English in writen work just as well in oral work.

53

Appendix: Appendix 1: Preparation of the Work Solutions. WS1 in ACN/MQ

Concentraton of testosterone in the soluton Concentraton of progesterone in the soluton Concentraton of cortcosterone in the soluton

100 µg/mL 100 µg/mL 100 µg/mL

Preparaton of WS1:

1ml of testosterone 1g/L 1ml of progesterone 1g/L 1ml of cortcosterone 1g/L Concentraton of testosterone Concentraton of progesterone Concentraton of cortcosterone

in a fask of 10 mL

diluted by ACN/MQ (50/50)

in the soluton in the soluton in the soluton

10 µg/ml 10 µg/ml 10 µg/ml

Preparaton of WS2: WS3 in ACN/MQ

1ml of WS 1 Concentraton of testosterone Concentraton of progesterone Concentraton of cortcosterone

in a fask of 10 mL in the soluton in the soluton in the soluton

diluted by ACN/MQ (50/50) 1 µg/ml 1 µg/ml 1 µg/ml

Preparaton of SS3: WS4 in ACN/MQ

1ml of WS 2 Concentraton of testosterone Concentraton of progesterone Concentraton of cortcosterone

in a fask of 10 mL in the soluton in the soluton in the soluton

diluted by ACN/MQ (50/50) 0,1 µg/ml 0,1 µg/ml 0,1 µg/ml

Preparaton of WS4: WS5 in ACN/MQ

1ml of WS 3 Concentraton of testosterone Concentraton of progesterone Concentraton of cortcosterone

in a fask of 10 mL in the soluton in the soluton in the soluton

diluted by ACN/MQ (50/50) 0,01 µg/ml 0,01 µg/ml 0,01 µg/ml

Preparaton of WS5: WS6 in ACN/MQ

1ml of WS 4 Concentraton of testosterone Concentraton of progesterone Concentraton of cortcosterone

in a fask of 10 mL in the soluton in the soluton in the soluton

diluted by ACN/MQ (50/50) 0,001 µg/ml 0,001 µg/ml 0,001 µg/ml

Preparaton of WS6:

1ml of WS 5

in a fask of 10 mL

diluted by ACN/MQ (50/50)

WS2 in ACN/MQ

54

55

Appendix 2: Determination of the volume of injection Samples in serum:

S1 S2 S3 S4 S5 S6

Compositon 0,5 mL of serum + 10µL of WS1 0,5 mL of serum + 50µL of WS2 0,5 mL of serum + 25µL of WS2 0,5 mL of serum + 12,5µL of WS2 0,5 mL of serum + 50µL of WS3 0,5 mL of serum

E1 E2 E3 E4 E5 E6

Compositon 10µL WS1 + 490µL ACN/MQ 50µL WS2 + 450µL ACN/MQ 25µL WS2 +475µL ACN/MQ 12,5µL WS2 + 487µL ACN/MQ 50µL WS3 + 450µL ACN/MQ 500 ACN/MQ

Concentraton (µg/L) 2000 1000 500 250 100 0

Standard samples: Concentraton (µg/mL) 2000 1000 500 250 100 0

Appendix 3: Analysis of the volume of serum Samples in serum S1 S2 S3 S4 S5 S6

Compositon 1 mL of serum + 20µL of WS1 1 mL of serum + 100µL of WS2 1 mL of serum + 50µL of WS2 1 mL of serum + 25µL of WS2 1 mL of serum + 100µL of WS3 1 mL of serum

Concentraton (µg/mL) 2000 1000 500 250 100 0

Standard samples Compositon

Concentraton (µg/mL)

56

E1 E2 E3 E4 E5 E6

20µL WS1 + 980µL ACN/MQ 100µL WS2 + 900µL ACN/MQ 50µL WS2 + 950µL ACN/MQ 25µL WS2 + 975µL ACN/MQ 100µL WS3 + 900µL ACN/MQ 1000µL ACN/MQ

2000 1000 500 250 100 0

Appendix 4: Analysis in low concentrations Samples in serum S1 S2 S3 S4 S5 S6 S7 S8 S9 S10

Compositon 1,0 mL of serum + 100 µL of WS3 1,0 mL of serum + 50 µL of WS3 1,0 mL of serum + 20 µL of WS3 1,0 mL of serum + 100 µL of WS4 1,0 mL of serum + 50 µL of WS4 1,0 mL of serum + 20 µL of WS4 1,0 mL of serum + 100 µL of WS5 1,0 mL of serum + 50 µL of WS5 1,0 mL of serum + 20 µL of WS5 1,0 mL of serum + 10 µL of WS5

Concentraton (µg/L) 100 50 20 10 5 2 1 0,5 0,2 0,1

E1 E2 E3 E4 E5 E6 E7 E8 E9 E10

Compositon 400 µL ACN/MQ + 100 µL of WS3 450 µL ACN/MQ + 50 µL of WS3 300 µL ACN/MQ + 200 µL of WS4 400 µL ACN/MQ + 100 µL of WS4 450 µL ACN/MQ + 50 µL of WS4 300 µL ACN/MQ + 200 µL of WS5 400 µL ACN/MQ + 100 µL of WS5 450 µL ACN/MQ + 50 µL of WS5 300 µL ACN/MQ + 200 µL of WS6 400 µL ACN/MQ + 100 µL of WS6

Concentraton (µg/L) 100 50 20 10 5 2 1 0,5 0,2 0,1

Standard samples:

57

Appendix 5: Determination of the volume of the serum which could be cleared in one column Preparaton of sample: • Take 30 mL of serum and spike 3 mL of WS3. Mix gently



SPE step: •

Conditon the column



Load the column with sample: o Column 1: 1 mL o Column 2: 2 mL o Column 3: 3 mL o Column 4: 5 mL o Column 5: 7 mL o Column 6: 9 mL



Wash, centrifuge, elute, evaporate and reconsttute in the normal way

Appendix 6: Artificial serum and sample preparation for the calibration curve Artfcial serum • Prepare a phosphate bufer o Prepare 250 mL of KH2PO4, of 50 mmol/L in milliQ water (pH~4) o Prepare the same amount of a soluton of Na2HPO4, 7 H2O of 50 mmol/L in milliQ water (pH~8) o With the help of a pHmeter, adjust with KH2PO4 to have pH=7.40 •

Add in his bufer, 0.9% of NaCl (= 9g NaCl/ L of bufer) 58



Add 0.1% of BSA



Check pH afer these additons



Keep it in a fridge

Samples

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10

Compositon 1,0 mL of serum (artfcial or cleared) + 1,0 mL of serum (artfcial or cleared) + 1,0 mL of serum (artfcial or cleared) + 1,0 mL of serum (artfcial or cleared) + 1,0 mL of serum (artfcial or cleared) + 1,0 mL of serum (artfcial or cleared) + 1,0 mL of serum (artfcial or cleared) + 1,0 mL of serum (artfcial or cleared) + 1,0 mL of serum (artfcial or cleared) + 1,0 mL of serum (artfcial or cleared)

20 µL of WS2 100 µL of WS3 50 µL of WS3 20 µL of WS3 100 µL of WS4 60 µL of WS4 40 µL of WS4 20 µL of WS5 100 µL of WS5

Concentraton (µg/L) 200 100 50 20 10 6 4 2 1 0

59

Appendix 7: Method validation Preparaton of the Quality Control samples: Compositon 1 mL of cleared serum 1 mL of cleared serum + 50 µL of WS4 1 mL of cleared serum + 25 µL of WS3 1 mL of serum + 75 µL of WS3

Blank QCL QCM QCH

Concentraton (µg/L) 0 5 25 75

Preparaton of the calibraton curve in sham serum: CC Blank CCL

CCH

Compositon Concentraton (µg/L) 1 mL of sham serum 0 1 mL of sham serum + 100 µL of WS5 1 1 mL of sham serum + 20 µL of WS4 2 1 mL of sham serum + 40 µL of WS4 4 1 mL of sham serum + 60 µL of WS4 6 1 mL of sham serum + 100 µL of WS4 10 1 mL of sham serum + 20 µL of WS3 20 1 mL of sham serum + 40 µL of WS3 40 1 mL of sham serum + 60 µL of WS3 60 1 mL of sham serum + 80 µL of WS3 80 1 mL of sham serum + 100 µL of WS3 100

Order of analysis:

1 2 3 4 5 6 7 8 9 10 11

ACN/MQ ACN/MQ CCH 20 CC Blank QCH 75 QCM 25 CC 80 ACN/MQ CCL 1 QCL 5 Blank

13 14 15 16 17 18 19 20 21 22 23

QCM 25 ACN/MQ CCL 4 QCH 75 QCM 25 QCL 5 Blank ACN/MQ CC Blank CCL 10 QCH 75

25 26 27 28 29 30 31 32 33 34 35

QCH 75 ACN/MQ Blank CCH 40 CCL 2 CCL 6 QCL 5 ACN/MQ QCL 5 Blank CCH 100

60

12 CCH 60

24 QCM 25

36 ACH/MQ

61

Appendix 8: Real concentrations in solution and in samples Real concentratons in the stock and work solutons: Cortcosterone Testosterone Mass (g) Real concentraton (g/L) WS1 (µg/L) WS2 (µg/L) WS3 (µg/L) WS4 (µg/L) WS5 (µg/L)

0,0253 1,012 101200 10120 1012 101,2 10,12

0,0254 1,016 101600 10160 1016 101,6 10,16

Progesteron e 0,0251 1,004 100400 10040 1004 100,4 10,04

Real concentratons in the calibraton curve samples: Expected concentraton (µg/L) 1 2 4 6 10 20 40 60 80 100 Cortcosterone 1,012 2,024 4,048 6,072 10,12 20,24 40,48 60,72 80,96 101,2 Real concentratons Testosterone 1,016 2,032 4,064 6,096 10,16 20,32 40,64 60,96 81,28 101,6 (µg/L) Progesterone 1,004 2,008 4,016 6,024 10,04 20,08 40,16 60,24 80,32 100,4

Real concentratons in quality control samples: QCLow 5 µg/L Cortcosterone Testosterone Progesterone

1 2 3 4 3,626906798 4,307497892 4,410004164 4,769857792 3,376555182 4,558747356 4,610966569 4,74025823 3,008254 3,466441 2,832854 2,606439

QCMedium 25 µg/L Cortcosterone Testosterone Progesterone

1 2 3 4 20,17828592 21,72964073 24,00696348 24,02232941 18,68656002 19,51160284 21,83274719 21,55338993 13,521084 16,783692 18,448519 15,8393

QCHigh 75 µg/L Cortcosterone Testosterone

1 2 3 4 66,29985523 69,90576053 66,32257769 73,27987754 63,86561805 66,79842708 67,37040554 71,32109687

62

Progesterone

40,151223

44,543305

39,987746

47,165983

63

Appendix 9: Accuracy, standard derivation (SD) and relative standard derivation (RSD) formulas.

Accuracy formula:

Standard derivaton formula:

Relatve standard derivaton formula:

64