Undergraduate Thesis 45 Hp

Master thesis 45 hp

March 2014

Analysis of most common endogenous steroids in plasma

Wenjun Zhao

Degree project in analytical chemistry, Master of Science (2 years) Supervisor: Dr. Kumari Ubhayasekera Analytical Chemistry, Department of Chemistry BMC and Science for Life Laboratory (SciLife), Uppsala University Box 599, 75124 Uppsala, Sweden.

ISRN UU MASTERUTB-EX--(M2014: ) Examiner: Christer Elvingson

Contents Abstract ...... 3 List of Abbreviations ...... 4 1. Introduction ...... 6 1.1 Endogenous steroid hormones and their metabolites ...... 8 1.2 Analytical methodology ...... 9 1.2.1 Sample preparation ...... 9 1.2.2 Analytical techniques for steroid analysis ...... 10 1.3 Scope of this study ...... 13 2. Materials and methods ...... 14 2.1 Chemicals and reagents ...... 14 2.2 Standard solutions ...... 14 2.3 Optimization of sample preparation ...... 15 2.4 TLC analysis ...... 16 2.5 Sample- free plasma preparation ...... 16 2.5.1 Using ISOLUTE SI 6 ml SPE column ...... 16 2.5.2 Using charcoal method...... 16 2.6 Steroids extraction using ISOLUTE SLE + 96-well plates ...... 17 2.7 Method validation ...... 17 2.8 Instrumentation ...... 18 3. Results and discussion ...... 19 3.1 Identification of each steroid...... 20 3.2 Optimized derivatization conditions ...... 27 3.3 Retention times and specificity ...... 31 3.4 Steroid-free experiments ...... 32 3.5 Method validation ...... 33 3.5.1 Calibration curve for each steroid ...... 33 3.5.2 Limit of detection (LOD), limit of quantification (LOQ) and precision...... 35 3.5.3 Normal sample analysis ...... 36 3.5.4 Recovery test ...... 36 4. Conclusion ...... 38 5. Future work ...... 38

6. Acknowledgments ...... 39 7. Popular scientific summary ...... 40 8. References ...... 42

Abstract Steroid hormones are biologically active low molecular weight lipid compounds which are synthesized from cholesterol. These steroids are important in controlling human body functions and regulating the nervous and immune systems. Therefore, a sensitive, stable and rapid method for the analysis and quantification of steroids in the human plasma is necessary in pharmacokinetic studies. This work is establishing a novel GC-MS method for the simultaneous determination of as many as 9 analytes covering 3 classes of endogenous steroids.

The methoxylation and silylation conditions for steroids were optimized in this work. The linearity of the GC-MS method was evaluated at the steroids concentration range of 2 – 50 µg/ml. Correlation coefficient (R2) of cholesterol and estradiol ≥ 0.99; both dehydroepiandrosterone and cortisone’s parameters ≥ 0.93. The recoveries for all the steroids determined at low, medium and high concentration levels varied from 52.70 – 162.78 %. The concentration of cholesterol in normal plasma is 34.82 ± 1.35µg/ml.

Keywords Steroids, methoxylation, silylation, GC-MS, quantification, human plasma

List of Abbreviations

AS Androsterone BSA N,O-Bis(trimethylsilyl)acetamide BSTFA N,O-Bis(trimethylsilyl)trifluoroacetamide BSTFA+TMCS N,O-Bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane

C2Cl3F3 1,2,2-trichloro-1,2,2-trifluoroethane

CAN Concentration of the analyte

CIS Concentration of the internal standard CHOL Cholesterol DHEA Dehydroepiandrosterone DTE Dithioerythritol E Cortisone E1 Estrone E2 Estradiol E3 Estriol ECN Etiocholanolone EI Electron impact GC Gas chromatography GC–MS/MS Gas chromatography mass spectrometry HDL High-density lipoprotein HFBA Heptafluorobutyric Acid HPLC High performance liquid chromatography IS Internal standard LC–MS/MS Liquid chromatography tandem mass spectrometry LOD Limit of detection LOQ Limit of quantification MS Mass spectrometry MSTFA N-Methyl-N-(trimethylsilyl)trifluoroacetamide m/z Mass-to-charge ratio

Rt Retention time SD Standard deviation S/N Signal-to-noise ratio

SIM Selected ion monitoring SPE Solid-phase extraction T Testosterone TBDMCI tert-Butyldimethylsilylchlorosilane TEA Triethylaluminium TMIS Trimethyliodosilane TMS Trimethylsilyl TMSI N-trimethylsilylimidazole TMCS Trimethylchlorosilane

1. Introduction

Steroid hormones are biologically active low molecular lipid compounds which are synthesized from cholesterol (figure 1.1). All the steroid hormones have a tetracyclic base structure which consists of four fused rings: three cyclohexane rings and one cyclopentane [1]; the chemical structure and IUPAC names of the steroids of interest are listed in table 1.1.

These steroids are important in controlling human body functions and regulating the nervous and immune systems [2, 3]. In recent years, steroids have became useful biomarkers for the diagnosis of several pathological conditions [4]. Therefore it is important to improve and continue developing new analysis methods. The most important requirements for the techniques of steroid measurement are reliability, specificity and sensitivity. The tandem mass spectrometry (MS/MS) technique can satisfy these requirements, hence mass spectrometry (MS) coupled with gas chromatography (GC) or high performance liquid chromatography (HPLC) is commonly used in steroid hormone detection during modern times [34].

Figure 1.1: Biosynthesis of steroid hormones in the cholesterol pathway (http://en.wikipedia.org/wiki/File:Steroidogenesis.svg)

Table 1.1. List of the names, abbreviations and chemical structures of the investigated steroids. IUPAC Common name Abbreviation Chemical structure (3β)-Cholest-5-en-3-ol Cholesterol CHOL

3-Hydroxyestra-1,3,5(10)-trien- Estrone E1 17-one

(17β)-Estra-1,3,5(10)-triene- Estradiol E2 3,17-diol

(16α,17β)-Estra-1,3,5(10)- Estriol E3 triene-3,16,17-triol

(3β)-3-Hydroxyandrost-5-en- Dehydroepiandroster DHEA 17-one one

(3α,5α)-3-Hydroxyandrostan- Androsterone AS 17-one

(3α,5β)-3-Hydroxyandrostan- Etiocholanolone ECN 17-one

(17β)-17-Hydroxyandrost-4-en- Testosterone T 3-one

11,17,21-Trihydroxypregn-4- Cortisone E ene-3,20-dione

The aim of this work is to develop and optimize a GC–MS/MS analytical method to serve as a diagnostic test for steroids/biomarkers of endocrine and metabolic diseases.

1.1 Endogenous steroid hormones and their metabolites The steroids that will be measured in this study are divided into the following catogories: estrogen; androgen; and glucocorticoid, with their respective metabolites. Steroid hormones are synthesized from cholesterol (CHOL) which can be transformed into other steroids (figure1.1).

Steroids with their metabolites show great clinical effectiveness as biomarkers for disease detection. Fouad (et al) found that the concentration levels of steroids could indicate some disorders such as defective steroidogenesis and other related clinical diseases [5];

Other impacts of steroids on the human body are list in table 1.2.

Table 1.2. List of the steroids and their metabolites and some effects in the human body. Steroid and Metabolite Impact Cholesterol It can control membrane fluidity over the range of physiological temperatures and functions in intracellular transport, cell signaling and nerve conduction [6]. Estrone It is a known carcinogen for human females [7]. Estradiol It could protect women from heart disease by raising the level of HDL and protect bone density in both men and women [8]. Estriol In pregnant women with multiple sclerosis, it reduces the disease's symptoms [9]. Dehydroepiandrosterone It works as a metabolic intermediate in the biosynthesis of the androgen and estrogen sex steroids [10]. Androsterone It would influence human behavior and low levels could be the major correlate of decreased sexual desire [11]. Etiocholanolone It would cause fever, immune stimulation and leukocytosis [12]. Testosterone Development of male reproductive tissues such as the testis and prostate as well as promoting secondary sexual characteristics [13]. Cortisone It can elevate blood pressure and prepare the body for a fight or flight response [14].

Synthesis of steroids occurs in the mitochondria and the smooth endoplasmic reticulum of cells in the adrenal cortex, the gonads and the placenta [15]. The adrenal cortex is mainly responsible for the production of corticosteroid and androgen hormones which has three zones. Aldosterone and mineralocorticoids are mainly produced in the outmost zone and zona glomerulosa [16]. The middle zone, zona fasciculate, releases glucocorticoids such as cortisol and cortisone into the body. Zona reticularis and the innermost zone is responsible for producing androgens like dehydroepiandrosterone (DHEA) and androstenedione (the precursor to testosterone (T)) [16]. Estrogens (E1, E2 and E3) are produced mainly by the ovaries and placenta with small amounts being produced by the adrenal glands.

1.2 Analytical methodology 1.2.1 Sample preparation  Extraction/Purification

Common biological samples used for analysis of steroids includes serum, plasma, urine and saliva. Sample sizes typically ranges from a few microliters to 1mL. Sample preparation for steroid analysis generally involves extraction and enrichment. Extraction techniques are usually compound specific and depend on the sample type, requiring sensitivity and high throughput. Routine sample preparation usually consist of liquid-liquid extraction (LLE), protein precipitation and solid phase extraction (SPE) [17].

 Derivatization i. Silylation

Most steroids are required to be derivatized before injection into GC-MS. Silylation is one of the most common techniques employed in steroid derivatization. The reaction occurs by substituting the proton in

[18] hydroxyl (-OH), amino (-NH2) and sulfide (-SH) groups with a silyl group (R3Si) .

Some of the most common reagents used for derivatization, include MSTFA[19-26], TMIS[23,24], DTE[24,27], BSTFA[24], trimethylsilylimidazole(TMSI)[24,27-29], trimethylchlorosilane (TMCS)[24] , HFBA[25,30] and TEA[25]. To enhance the derivatization efficiency, these reagents are sometimes mixed in different ratios. For example, Cangialosia et al. [21], mixed MSTFA and TMIS in a ratio of 1000 to 2 (v/v) for derivatization at 60°C for 15 min, and shown a good sensitivity. Other derivatization mixtures have also been used, namely MSTFA, TMIS and DTE (1000:2:5, v/v/w); BSTFA and 1% TMCS; MSTFA and 1% TMCS; BSTFA and 1% TMSI; MSTFA and 1% TMSI; a good linearity of response with a coefficient R2 > 0.994 was obtained; Limits of detection (LODs) ranging from 0.01 to 1.0 ng/mL for all steroid hormones were also obtained; All the derivatives were stable for quite some time (24-48 hours) [24]. The conditions for derivatization were

as follows: MSTFA with 2.0% NH4I and 1.5% dithioerythritol (v/w/w) was heated for 10 min at 90°C and the lowest quantification limits were reported to be from 0.002 to 0.6 μg/L for 1.0 mL of saliva [26].

From the discussion above, it is clear that the optimization of derivatization conditions still remains a big challenge in the determination of steroids.

9 ii. Methoxylation

Steroids containing ketone groups such as cortisone or cortisol sometimes cannot be detected by GC–MS after silylation. Normally, the ketone group of these compounds should be derivatized by methoxylamine or ethoxylamine to form a stable oxime. The methoxylation reagent can be used separately or coupled with a silylation step [31].

1.2.2 Analytical techniques for steroid analysis Currently, the main techniques employed for the analysis of steroids are immunoassays [32, 33], LC–MS [34, 35-37] and GC–MS/MS [30, 34, 38-44]. However, immunoassays are mainly applied in the analysis of clinical disorders influenced by cross-reactions, thus negatively affecting the results. The latest techniques are mainly GC–MS and LC–MS based. Both of these techniques are superior to immunoassays in terms of increased specificity, quantification and linearity.

 GC–MS/MS method The principle of GC–MS is that gas-phase ions are separated according to mass-to-charge (m/z) ratio and then sequentially detected in single and multiple modes. A GC–MS instrument is divided into four major parts, i.e. sample introduction, ion source, mass analyzer and detector. A typical schematic diagram of a GC–MS/MS system is shown in figure 1.2.

Figure 1.2: Schematic diagram of Scion GC-MS/MS and q0 is ion guide of this instrument

GC–MS is normally used as the predominant method in the determination of steroids in human plasma, because it ensures high specificity and sensitivity as well as a low limit of detection (LOD) [45]. According to Thenot and Horning all possible steroids with their metabolites can be detected by GC–MS [46]. In 1960, the analysis of steroids by GC was described for the first time by Van den Heuvel [19] who used cholestane as internal standard (IS), and reported that the compounds with a hydroxyl group eluted before the compounds with a ketone group. Four years later, in 1964, Ryhage reported on coupling GC with MS for the first time [47]. This invention paved the way for the determination of the structures of known and

11 unknown steroids [47]. The mass analyzer of most GC–MS systems can be operated in full scan mode and/or selected ion monitoring mode. This provides the operator with the opportunity to detect most of the compounds present in a sample or to detect only a selected few. A review of the literature on steroid analysis revealed reports on the simultaneous determination of some of the steroids of interest as well as methods for individual detection [27, 30].

It has been reported by Jun Chen, et al (2009) that DHEA, cortisol (E), estrone (E1), 17β-estradiol (E2), T and androsterone (AS) were analyzed simultaneously and the resulting detection limit (LOD) and limit of quantification (LOQ) range show that the sensitivity is quite good and stable[30]. Martin et al.[27] also found the concentration levels of estrogen and androgen groups in the plasma and serum, down to 0.08–0.16 ng/mL and 0.20 – 0.36 ng/mL, respectively; the accuracy of this method was 50–112% in the range 0.10 to 2.00 ng/mL.

 LC–MS/MS method GC–MS methods are well suited for the analysis of almost all steroids and their metabolites, however, some steroids and their metabolites are preferably analyzed using LC–MS/MS methods; e.g. corticosterone[48], tetrahydrocortisol[49,50], and tetrahydrocorticosterone[51], to name a few. All the results show good sensitivity and reproducibility.

 Comparison of GC–MS and LC–MS/MS techniques for steroid analysis GC–MS offers higher resolution than LC–MS/MS due to the longer analysis running time. For some diastereomeric compounds, GC–MS has better separation than LC–MS/MS, which renders it more useful in the diagnosis of some metabolic disorders like apparent cortisone reductase deficiency (ACRD) [54]. GC– MS is also a good tool for analyzing non-target steroids, especially when used in full scan mode which enables storage of the fragmentation information for evaluation at a later stage. This is very useful in, for example, the field of sports for the randomized anti-doping analysis of athletes’ body fluid samples which can be re-checked for previously unidentified drugs [34]. GC-MS needs less sample volume than LC-MS however GC-MS is more sensitive than LC-MS.

Table 1.3: Advantages and disadvantages of the current methods for analysis of steroids Method Advantage(s) Disadvantage(s) GC–MS Good resolution and can be used for High cost and time consuming sample volatile compounds preparation procedures High sensitivity/ throughput Cannot be used for non-volatile and Small sample volume thermal unstable molecules LC–MS/MS Shorter analysis time and can be used for Not good for volatile compounds non-volatile and high molecular weight compounds Immunoassay Simple, rapid, inexpensive Overestimate steroid concentrations

One of the biggest drawbacks of GC–MS is that it is marked by a time consuming sample preparation step which is often less prominent in LC–MS/MS. In many cases, derivatization of steroids are needed, which usually include methoxylation of ketone groups and silylation of hydroxyl groups, to improve their volatility when using GC–MS. After derivatization, compounds can give a mass fragmentation of the molecular ion, so it is easier for determination the functional groups on the steroid [18].

After a comparison of these two techniques, it is evident that GC–MS is a powerful tool that can be employed for the analysis of steroids, especially unknown steroids, despite being prone to time-consuming sample preparation steps and longer analysis times [54, 55]. The advantages and disadvantages of these methods for the analysis of steroids are summarized in Table 1.3.

1.3 Scope of this study The main objective of this study was to develop a GC–MS method for the complete separation and quantification of several steroids to use as a diagnostic test for steroid/biomarkers of endocrine and metabolic disease. Optimizations of sample preparation and clean-up steps as well as derivatization conditions are needed. After method development and optimization, the method should be evaluated and applied in analyzing biological tissues and fluids.

2. Materials and methods 2.1 Chemicals and reagents Cholesterol (CHOL), estrone (E1), estradiol (E2), estriol (E3), dehydroepiandrosterone (DHEA), androsterone (AS), etiocholanolone (ECN), testosterone (T) and cortisone (E) were purchased from

Steraloids (Newport, RI, USA). 5α-Cholestane(C27H48) was used as internal standard (IS); N,O- bis(trimethylsilyl)acetamide (BSA); N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), BSTFA with 1% trimethylchlorosilane (BSTFA+TMCS), tert-butyldimethylsilylchloride (TBDMCL); 1,1,1,3,3,3- hexamethyldisilane + trimethylchlorosilane + pyridine (HMDS+TMCS+Pyridine (3:1:9)); methoxyamine hydrochloride and anhydrous pyridine (99.8%) were obtained from Sigma-Aldrich (Stockholm, Sweden). All chemicals and chromatographic reagents used were of analytical grade. All other chemicals and reagents were purchased from Merck, Eurolab (Stockholm, Sweden), unless otherwise stated. Ultrapure water was deionized and osmosed with a MilliQ purification system (Millipore, Bedford, MA, USA) was used.

2.2 Standard solutions Stock solutions (1 mg/mL) of all steroids were prepared by weighing off, with analytical precision, each steroid and dissolving it in ethanol. A stock solution of 100 ng/µL in ethanol was prepared for the internal standard. The stock solutions were kept in a freezer at -20 °C within tightly capped glass tubes. In order to obtain the working solutions and calibration standards at different concentrations, the stock solutions were diluted with methanol (MeOH). The concentration of methoxyamine hydrochloride was 20 mg/mL in anhydrous pyridine.

Working solutions and calibration standards were prepared by a general protocol. After pipetting 20 µL from stock solution (1mg/mL) and 100µl from internal standard solution (100ng/µL) into a glass tube, the solvent was evaporated by a gentle flow of nitrogen gas. The methoxylation reagent (30 µL) was added. The glass tube was kept in an oven at 60°C for 45min. After incubation, the solvent was evaporated and 100 µL of silylation reagent was added to the sample. The samples were incubated again at 120°C for 30 min. Then the solution was cooled to the room temperature and the solvent was evaporated under nitrogen gas. Finally 100 µL of C2Cl3F3 was added. The solution was centrifuged at room temperature at 3,000 × g for 5 min. The solution was transferred to a small GC vial and injected into the GC–MS instrument for analysis.

2.3 Optimization of sample preparation  Methoxylation Different methoxylation conditions were investigated. Therefore, one sample from each set was paired and subjected to the same set of incubation conditions for methoxylation as shown in Table 2.1. Methoxyamine hydrochloride in pyridine was used as methoxylation reagent. After methoxylation of the compounds, the silylation reagent was added to each set and incubated.

Table 2.1: Different incubation conditions for methoxylation of standards. Set Name of silylation reagent Incubation conditions Incubation conditions (100µL) for methoxylation (30µL) for silylation (100µL) 60°C for 30 min A BSA 60°C for 45min 120°C for 30min 70°C for 30min 60°C for 30 min B BSTFA 60°C for 45min 120°C for 30min 70°C for 30min

 Silylation Different silylation conditions were investigated. Five different silylation reagents or mixtures, namely BSA, BSTFA, HMDS+TMSC, TBDMCI and BSTFA+TMCS, were investigated by incubating at different temperatures and inclubation times and the conditions are shown in Table 2.2.

Table 2.1: Different incubation conditions for silylation of standards. Set Name of silylation Incubate condition for the Incubate condition reagent (100µL) methoxylated derivatization(30µL) for the silylation A BSA 60°C for 45min 70°C for 1 hour 120°C for 30 min B BSTFA 60°C for 45min 70°C for 1 hour 120°C for 30 min C HMDS+TMCS 60°C for 45min 70°C for 1 hour 120°C for 30 min D TBDMCL 60°C for 45min 70°C for 1 hour 120°C for 30 min

E BSTFA+TMCS 60°C for 45min 70°C for 1 hour 120°C for 30 min

The working flow diagram of experiment with standards Pipette 20µl from each steroid stock solution and 100µl from internal standard solution evaporate solvent add 30µl methoxylation reagent keep the tube in the oven at different programs evaporate solvent add silylation reagent 100 µl into the mixture solution under the N2 gas flow keep in the oven at different programs evaporate solvent the derivatized standards re-dissolved in 100µl C2Cl3F3 centrifuge for 5min GC-MS/MS analysis

2.4 TLC analysis To check the completion of derivatization, underivatized and derivatized steroids were analyzed by analytical pre-modified silica with aliphatic hydrocarbons. The TLC plates silica gel 60, 20 × 20 cm, 0.25 mm thickness were used (Merck, Eurolab AB, Stockholm, Sweden) to check visually the completeness of derivatization. The derivatized samples along with the standard steroids (underivatized) as TLC-references (Steraloids, Newport, RI, USA), were spotted onto a TLC plate and developed in the solvent system diethyl ether: cyclohexane (90:10, v/v). The TLC plate was dried briefly in air and then sprayed with 10 % phosphomolybdic acid in diethyl ether: ethanol (50:50, v/v). The plate was placed in an oven at 120 ºC for 15 min for color development.

2.5 Sample- free plasma preparation 2.5.1 Using ISOLUTE SI 6 ml SPE column Normal plasma (1mL) was diluted with 1mL hexane: diethyl ether (9:1 v/v) and vortexed at room temperature. The diluted sample was loaded onto an ISOLUTE SI 6 ml SPE cartridge (pre-conditioned with 5ml hexane). After distribution of the aqueous phase in the column, the steroids of interest were eluted with 15ml hexane: diethyl ether (9:1 v/v). After that process, washing with 10ml hexane: diethyl ether (1:1 v/v) again. Finally, elution of pure steroid free plasma with 10ml acetone into silanized glass tubes. The collected elution was dried under the N2 gas after the methoxylation and silylation process. The sample solution was compared with the standard cholesterol solution using a TLC plate to check if the steroids were removed.

2.5.2 Using charcoal method The steroid-free plasma was prepared by activated charcoal as described by Aburuz et al [56]. Two sets of normal plasma (200µl) were prepared, one set without charcoal and one set with 8mg charcoal then vortexed

16 and centrifuged. After the filtering, Methanol: CHCl3 (2:1, v/v) was added twice into these two sets of plasma samples. The sample solution dried under the N2 gas after the methoxylation and silylation process. It was then compared with the standard cholesterol solution using a TLC plate to check if the steroids were removed.

2.6 Steroids extraction using ISOLUTE SLE + 96-well plates Normal plasma (100µl) with 2µg internal standard was diluted with MQ water (100µl). The pre-treated sample (200µl) was loaded into a plate followed by a pulse of vacuum to initiate flow and left for 5 minutes. Then eluted with dichloromethane (1ml) dropped directly into a deep well collection plate which was left for 5 minutes. Then a short pulse of vacuum was applied. The eluent was collected followed by methoxylation and derivatization and finally injected into GC-MS.

2.7 Method validation The proposed method was validated using spiked plasma samples as described in the guidelines by the FDA (http:\\www.fda.gov\cder\guidance\4252fnl.pdf.2001). The method was validated using four different steroids (CHOL, E2, DHEA and E). Stock solution of steroids and IS (Cholestane) were prepared independently at 1mg/mL in ethanol and stored at -20˚C. Prior to the measurements, these solutions were serially diluted with the same solvent and further used for the calibration and quality control (QC).

 Linearity In order to accurately quantify the targeted steroids, the measured response must be correlated with the concentration of the steroid hormones. Therefore, calibration curves need to be constructed. Calibration standards of CHOL, E2, DHEA and E were prepared from 1 mg/mL stock solutions according to the general protocol described in section 2.2. The standards for CHOL, E2 and DHEA were prepared to a final concentration of 2, 4, 10, 15, 25 and 50 µg/mL and E was 50, 75, 100, 150 and 200 µg/mL in triplicate, respectively. Each sample contained 15 µg/mL of the internal standard. The calibration range reflected the steroid concentration levels in the human body and the number of replicates was sufficient to determine certain parameters and perform various statistical analyses (linearity, precision, accuracy, sensitivity, stability, limit of detection, etc.). Samples were run in triplicates. Calibration curves were separately prepared by plotting the peak area ratio of each steroid area of IS against the concentration of each compound. The linearity was determined by linear regression analysis.

The ratio of the integrated peak areas (AAN)/(AIS) and the concentration ratio (CAN/CIS) of the analyte (AN) and internal standard (IS) were calculated. Calibration curves of (AAN)/(AIS) versus (CAN/CIS) were constructed for each compound. A linear relationship over the analyte concentration range was displayed by all four calibration curves.

 Precision and accuracy Quality control samples were used for precision and accuracy determination on intra-day and inter-day basis. The intra-day (n=3) and inter-day precisions (n=6) were determined for the spiked samples.

 Limit of detection and quantification The detection limit (LOD) and the quantification limit (LOQ) were calculated using calibration curves for samples spiked with steroids. LOD was calculated using the formula 3.3 δ/S and LOQ was obtained by the formula 10 δ/S, here δ is the residual standard deviation of the regression line and S is the slope of the regression line.

 Recovery Samples were spiked in triplicates with known amounts of four different steroids at five different concentration levels (CHOL, E2, and DHEA was 500ng, 1000ng, 2000ng, 3000ng and 5000ng; E was 5000ng, 7500ng, 10000ng, 12500ng and 15000ng respectively) and analyzed to determine the recovery of each steroid.

2.8 Instrumentation Analysis of steroids was performed using a triple quad mass spectrometer coupled to the gas chromatograph (SCION TQTM GC-MS/MS System, Billerica, MA, USA). Separation of steroids was performed on a non- polar capillary column (DB-5MS, J&W Scientific, Folsom, CA, USA), 30 m  0.18 mm  0.18 m film thickness. Helium was the carrier gas at a linear velocity (1 mL/min). The injector temperature was 250°C and the samples were injected using the split injection mode (20:1). The oven temperature was 120°C followed by an increase of 50°C /min to 240°C and an isothermal hold of 1 min, then ramping at 1.5°C /min to 250 °C, after that increasing of 10°C /min to 260°C, finally by an increase of 50°C /min to 320°C with a hold of 5 min, total running time was 17.27 min. The ionization mode was electron impact (EI). The mass spectra were recorded at an electron energy of 70 eV and the ion source temperature was 260°C. The transfer line temperature was 300°C. The spectra were scanned in the range of 50-600 m/z. Identification

18 of the steroids was done by comparing the mass spectra of standard samples of the steroids and their retention times. Chromatograms were collected in full scan mode (TIC). Quantification of the steroids was accomplished using cholestane as an internal standard due to its physical and chemical behavior similar to that of target analyte as well as its absence in biological samples. Quantification of steroids (area response) was accomplished with the help of the internal standard.

3. Results and discussion A simple analytical procedure for the simultaneous determination of 9 steroids by a GC-MS/MS method has been developed. Many target analytes are not intrinsically volatile and will decompose during the transition between the liquid and gas phase inside the injection port or during elution from the column at high temperatures. Consequently, several steps, including pre-analytical derivatization, are required for steroid analysis by GC/MS. The derivatization is one of the main steps in the sample preparation. This step is required to improve the volatility and thermal stability of steroids. Derivatization of steroids is generally required before analyzing by GC-MS/MS. This process often time consuming.

In the present study, several strategies have been employed to enhance this process, like incubation temperature, time of incubation and different silylating reagents.

Mass spectrometry is the most recent method that has wide acceptance in the steroid field. GC for separation prior to MS (GC/MS) is used, due to the relative ease of coupling these orthogonal techniques with respect to the vacuum considerations. Given the high resolving capability of a GC equipped with a modern capillary column and the superb precision of MS, GC/MS systems have been a mainstay of analytical chemistry. To facilitate the interpretation and discussion of the results, mass spectrometry identification of steroids is briefly discussed below (Figure 3.1).

Figure 3.1: a Schematic diagram of a simple mass spectrometer with source, quadrupole, and detector, sorting ions by m/z. b Generation of a mass spectrum for a pure compound. The molecular ions fragment, and the charged fragments are separated by the quadrupole and quantitated by the detector, yielding a standard mass spectrum. c Mass spectrum of testosterone.

During the methoxylation reaction a methyl group (–CH3) from the methoxylation reagent, with a molecular mass of 15 Da, binds to the oxygen of a ketone group of the steroid. A trimethylsilyl group (–Si(CH3)3) from the silylation reagent, with a molecular mass of 73 Da, will attach to the oxygen of a hydroxyl group of the steroid.

3.1 Identification of each steroid The MS detection of every steroid showed abundant molecular ions [(M-n×H) +n×73+m×15]+, or [(M- n×H) +n×73]+ (M represents the molecular weight of each steroid, n represents the number of hydroxyl groups and m represents the number of ketone groups) providing evidence of the steroid's molecular weight (Table 1.1).

Figures 3.2-3.12 illustrate the chromatogram and mass spectrum using full scan mode for each steroid.

1) The molecular mass of cholesterol is 386.65 Da (Table 1.1) and the structure has one hydroxyl group. The MS detection of cholesterol showed an abundance singly charged molecular ions [(M- H) +73]+ at m/z of 458 providing evidence of cholesterol. Also fragments of m/z 129, 329 and 368 could be detected. The mass to charge ratio m/z of 129 is a basic fragment formation for identifying

steroids; also [M-H2O] gave the formation of m/z at 368. As we can see from the figure, the intensity of the molecular ion with m/z of 458 was relatively low comparing with the other fragments due to that the EI source is a hard ionization technique with high voltage so that most of analyte would be fragmented and leaving a weak molecular ion peak.

Figure 3.2: Representative cholesterol chromatograms and mass spectrum; cholestane was the internal standard. The molecular ion m/z is 458.

2) The molecular mass of androsterone is 290.44 Da (table 1.1); the structure has one ketone group and one hydroxyl group, respectively. During the methoxylation process, the ketone group cannot be completely converted to a methoxy group, so androsterone would show two peaks after GC separation. The MS detection of androsterone showed one charged molecular ions [(M-H)+73]+ at m/z of 362.3 and another was [(M-H) +73+15]+ at 377.3 providing evidence of androsterone. Figure 3.2 shows androsterone with one TMS group (m/z 362.3) and figure 3.3 shows androsterone with one TMS group and one methoxy group (m/z 377.3).

Figure 3.3: Representative Androsterone-TMS chromatograms and mass spectrum; cholestane was the internal standard. The molecular ion m/z is 362.3.

Figure 3.4: Representative Androsterone-TMS-CH3 chromatograms and mass spectrum; cholestane was the internal standard. The molecular ion m/z is 377.3.

3) The molecular mass of etiocholanolone is 290.44 Da (table 1.1), the structure of etiocholanolone has one ketone group and one hydroxyl group, respectively. The ketone group can be completely converted to a methoxy group. The MS detection of etiocholanolone showed an abundance singly charged molecular ions [(M-H) +73+15]+ at m/z of 377.4 providing evidence of etiocholanolone.

Figure 3.5: Representative etiocholanolone chromatograms and mass spectrum; cholestane was the internal standard. The molecular ion m/z is 377.4.

4) The molecular mass of DHEA is 288.42 Da (table 1.1). The structure of DHEA has one ketone group and one hydroxyl group, respectively. The ketone group can be converted to methoxy group completely. The MS detection of DHEA showed an abundance singly charged molecular ions [(M- H) +73+15]+ at m/z of 375.4 providing evidence of DHEA.

Figure 3.6: Representative DHEA chromatograms and mass spectrum; cholestane was the internal standard. The molecular ion m/z is 375.4.

5) The molecular mass of estrone is 270.37 Da (table 1.1). The structure of estrone has one ketone group and one hydroxyl group, respectively. During the methoxylation process, the ketone group cannot be completely converted to a methoxy group, so the estrone still show two peaks in the chromatogram. The MS detection of estrone showed one charged molecular ion [(M-H) +73]+ at

m/z of 342.3 and another was [(M-H) +73+15]+ at 357.4 providing evidence of estrone. Figure 3.7 shows estrone with one TMS group (m/z 342.3) and figure 3.8 shows estrone with one TMS group and one methoxy group (m/z 357.4).

Figure 3.7: Representative estrone-TMS chromatograms and mass spectrum; cholestane was the internal standard. The molecular ion m/z is 342.3.

Figure 3.8: Representative estrone-TMS-CH3 chromatograms and mass spectrum; cholestane was the internal standard. The molecular ion m/z is 357.4.

6) The molecular mass of estradiol is 272.38 Da (table 1.1). The structure of estradiol has two hydroxyl groups. The MS detection of estradiol showed one charged molecular ions [(M-2H) +2×73]+ at m/z of 416.3 providing evidence of estradiol. Figure 3.9 shows the results for the molecular ion of estradiol.

Figure 3.9: Representative estradiol-2TMS chromatograms and mass spectrum; cholestane was the internal standard. The molecular ion m/z is 416.3.

7) The molecular mass of testosterone is 288.42 Da (table 1.1). The structure of testosterone has one ketone group and one hydroxyl group, respectively. The ketone group can be completely converted to a methoxy group. Testosterone has enantiomers, so after the derivatization, there were two split peaks with exactly the same fragment information. The MS detection of testosterone showed an abundance singly charged molecular ions [(M-H) +73+15]+ at m/z of 375.4 providing evidence of testosterone. This is shown in figure 3.10.

Figure 3.10: Representative testosterone chromatograms and mass spectrum

8) The molecular mass of estriol is 288.38 Da (table 1.1). The structure has three hydroxyl groups and can be silylated completely. The MS detection of estriol showed an abundance singly charged molecular ions [(M-3H)+3×73]+ at m/z of 504.3 providing evidence of estriol. Figure 3.11 shows the information about estriol.

Figure 3.11: Representative estriol-3TMS chromatograms and mass spectrum; cholestane was the internal standard. The molecular ion m/z is 504.3

9) The molecular mass of cortisone is 360.44 Da (table 1.1). The structure of cortisone has three ketone groups and two hydroxyl groups, respectively. The ketone groups can be completely converted to methoxy groups. The MS detection of cortisone showed an abundance singly charged molecular ions [(M-2H) +2×73+3×15]+ at m/z of 549.6 providing evidence of cortisone. The figure 3.12 shows the fragment information about cortisone.

Figure 3.12: Representative cortisone-2TMS-3CH3 chromatograms and mass spectrum; cholestane was the internal standard. The molecular ion m/z is 549.6.

3.2 Optimized derivatization conditions Silylation conditions such as different silylation reagents (BSA, BSTFA, BSTFA+TMCS, TBDMCI and HMDS+TMCS+Pyridine (3:1:9)) with different reaction times and reaction temperatures. I also tried to test different reaction times and reaction temperatures for the methoxylation reaction. To measure the efficiency of derivatization, we should compare the peak area for each steroid. Table 3.1 shows the results from the silylation reaction.

Table 3.1. Peak area of every steroid using different silylation reaction condition (methoxylated reaction condition was the same at 60°C for 45min) BSA BSTFA BSTFA+TMCS HMDS TBDM Steroid +TMCS CL 70°C 120°C 70°C 120°C 70°C 120°C 70°C 120°C 60min 30min 60min 30min 60min 30min 60min 30min AN-TMS 1.115e8 3.837e8 1.745e8 4.420e7 1.868e8 3.072e8 1.112e8 1.868e8 ETIO- 2.320e8 9.171e8 4.371e8 1.069e8 4.084e8 6.937e8 2.393e8 4.084e8 TMS AN-TMS- 1.679e8 8.144e8 2.870e8 2.580e8 2.145e8 6.390e8 1.845e8 2.145e8 CH3 ETIO- 6.147e8 2.611e9 1.050e9 7.680e8 8.279e8 2.051e9 6.571e8 8.279e8 TMS-CH3

DHEA 3.230e8 1.556e9 5.676e8 5.093e8 4.120e8 1.229e9 3.608e8 4.120e8 E1-TMS 1.221e8 5.315e8 2.506e8 7.414e7 2.414e8 4.012e8 1.567e8 2.414e8 E2 4.389e8 1.891e9 8.655e8 5.444e8 6.373e8 1.579e9 5.080e8 6.373e8 E1-TMS- 1.429e8 9.242e8 3.190e8 2.809e8 2.291e8 7.431e8 2.045e8 2.291e8 CH3 T 2.860e8 1.226e9 4.815e8 2.683e8 3.507e8 9.713e8 2.504e8 3.507e8 E3 4.824e8 2.088e9 9.235e8 6.457e8 6.202e8 1.692e9 5.425e8 6.202e8 CHOL 4.384e8 1.517e9 7.778e8 4.763e8 6.181e8 1.475e9 4.736e8 6.181e8 E 2.368e8 9.507e8 5.575e8 3.197e8 4.394e8 1.088e9 5.575e8 4.394e8 *For some unknown errors, I did not collect the data information about HMDS+TMCS at 120°C, 30min and TBDMCL at 70°C, 60min; *e represents the base 10.

Comparing all the derivatization yield efficiency information, the reactions of BSA and BSTFA+TMCS (99:1) at 120°C, 30 min seem approximately the same, and better than the other reagents. BSTFA shows the best result among all the reagents at 70°C, 60min. During 120°C, the efficiency of BSA is much better than at 70°C; BSTFA shows the opposite result. Finally, BSTFA+TMCS (99:1) and BSA were chosen to do the experiment for optimization of the methoxylation reaction. The figure 3.13 illustrates that the peak areas are not much different between BSTFA-TMCS (99:1) and BSA as silylation reagents.

Figure 3.13: the chromatograms (overlaid) for 9 steroids when using BSTFA+TMCS (99:1) and BSA as silylation reagent. The red color is for BSA and blue color is for BSTFA-TMCS (99:1).

After finishing the optimization of the silylation reaction, the silylation condition was set to 120°C, 30min to continue the experiment for the methoxylation reaction. Table 3.2 shows the result for optimization of the methoxylation reaction.

Table 3.2 Peak area of every steroid using different methoxylation reaction conditions (silylation reaction condition was everywhere the same at 120°C for 30min). Steroid BSA BSTFA + TMCS (99:1) 60°C 60°C 70°C 60°C 60°C 70°C 30min 45min 30min 30min 45min 30min AN 7.902e8 5.982e8 1.175e9 3.449e8 3.971e8 3.749e8 ETIO 1.932e9 1.430e9 2.897e9 1.073e9 1.160e9 1.058e9 DHEA 1.409e9 1.005e9 2.151e9 7.054e8 8.127e8 7.573e8 E1 9.175e8 7.332e8 1.556e9 4.801e8 6.018e8 5.110e8 E2 9.695e8 6.978e8 1.822e9 4.375e8 5.594e8 5.588e8 E1 4.701e8 2.541e8 6.159e8 1.992e8 2.064e8 1.740e8 T 3.809e7 2.373e7 11.887e7 2.495e7 2.738e7 2.443e7 E3 1.071e9 7.698e8 1.860e9 5.335e8 5.830e8 5.303e8 CHOL 7.986e8 5.225e8 1.329e9 3.616e8 4.232e8 3.338e8 *For some unknown errors, I did not collect the data information about cortisone. *e represents the base 10.

According the results above, BSA shows the best yield efficiency for the methoxylation reaction at 60°C, 30min (figure 3.14); all the conditions for BSTFA + TMCS (99:1) gave the approximately the same yield efficiency for the methoxylation reaction (figure 3.15). Comparing all the results, using BSA as silylation reagent at 120°C, 30min and methoxylation condition at 60°C, 30min gives the best derivatization efficiency. We still, however, wanted to extend the time for the methoxylation reaction to keep all the analytes stable, and a methoxylation reaction of 60°C, 45min was chosen as the final methoxylation condition.

Figure 3.14: The chromatograms (overlaid) for 9 steroids when using BSA as silylation reagent with different methoxylated conditions.

Figure 3.15: The chromatograms (overlaid) for 9 steroids when using BSTFA+TMCS (99:1) as silylation reagent with different methoxylated conditions

In this study, that samples reacting with 30µl methoxyamine hydrochloride at 60°C for 45min, then with 100 µL BSA at 120°C for 30 min was the optimized condition.

3.3 Retention times and specificity The retention times of 9 steroids are shown in Table 3.3, and the gas chromatogram of 9 steroids is shown in figure 3.16.

Figure 3.16: Gas chromatography of 9 steroids and internal standard. The 9 Steroids were detected via mass spectrometry after being derivatization. (1) Androsterone-TMS; (2) Androsterone-TMS-CH3; (3)

ETIO-TMS- CH3; (4) DHEA; (5) Estrsdiol; (6) Estrone-TMS-CH3; (7) and (8) Testosterone; (9) Cholestane; (10) Estriol; (11) Cholestrol; (12) Cortisone.

Table 3.3 Analysis parameters for 9 steroids Steroids Retention time (min) Fragments (amu) AN-TMS 6.606 129.0;272.3;347.3;362.3a

a AN-TMS-CH3 7.176 129.1;270.3;360.3;377.3

a ETIO-TMS-CH3 7.313 129.1;270.3;360.3;377.3 DHEA 8.061 129.0; 358.3;360.4a E2 8.748 129.0;232.3;285.3;416.3a

a E1-TMS-CH3 8.847 129.1;231.2;340.3;357.4 T 9.091 and 9.207 129.1;358.3;375.4a E3 11.576 129.1;147.0;270.2;504.3a CHOL 12.726 129.0;329.4;368.4;458.5a E 14.027 129.1;369.3;459.3;549.6a a represents the qualifier and quantifier ion of each compound.

3.4 Steroid-free experiments Thin layer chromatography experiments were used for testing the methods of removed the steroids. Among all the steroids in the human body, cholesterol shows the highest concentration level, so the standard of cholesterol was chosen as the reference steroid. Figure 3.17 shows the extraction result of ISOLUTE SI 6 ml SPE column and Figure 3.18 is the result of the charcoal method.

Figure 3.17: SPE extreaction method Figure 3.18: Activated charcoal method

As we can see, cholesterol was not found in the plasma when we used ISOLUTE SI 6 ml SPE column, and the method with activated charcoal shows that this method cannot remove the steroids.

3.5 Method validation

3.5.1 Calibration curve for each steroid During the experimentation process, various errors occurred at different steps of the assay, so the peak area ratio between the steroids and internal standard instead of the absolute peak area of steroids, was utilized to calculate the concentration of steroids in plasma. The peak area ratios were plotted against the concentration of the steroids (Figure 3.19-3.22). The quality of the linear fit was scrutinized by means of several statistical analysis methods as presented in the following section.

3.00E+00 y = 0.045x - 0.0792 R² = 0.9984 2.50E+00

2.00E+00

1.50E+00

1.00E+00

5.00E-01

0.00E+00

Peakarea of Cholesterol/Peak area of IS 0.00E+00 1.00E+01 2.00E+01 3.00E+01 4.00E+01 5.00E+01 6.00E+01 -5.00E-01 Cholesterol concentration (µg/mL)

Figure 3.19: Calibration curve for cholesterol, showing average (n=6) cholesterol to internal standard peak area ratio versus concentration

3.5 y = 0.0617x - 0.0743 3.0 R² = 0.9900

2.5

2.0

1.5

1.0

0.5 Peakarea of Estradiol/Peak areaof IS 0.0 0.0 10.0 20.0 30.0 40.0 50.0 60.0 Estradiol concentration (g/mL)

Figure 3.20: Calibration curve for estradiol, showing average (n=6) estradiol to internal standard peak area ratio versus concentration

1.80

1.60 y = 0.0322x - 0.1663 R² = 0.9833 1.40

1.20

1.00

0.80

0.60

0.40

0.20 Peakarea of DHEA/Peak areaof IS 0.00 0.0 10.0 20.0 30.0 40.0 50.0 60.0 -0.20 DHEA Concentration (g/mL)

Figure 3.21: Calibration curve for DHEA, showing average (n=6) DHEA to internal standard peak area ratio versus concentration

0.80

0.70

0.60 y = 0.0032x - 0.1275 0.50 R² = 0.9312 0.40

0.30

0.20

0.10 Peak areaPeak of Cortisone/Peak IS of area 0.00 40.0 90.0 140.0 190.0 Cortisone concentration (g/mL)

Figure 3.22: Calibration curve for cortisone, showing average (n=6) cortisone to internal standard peak area ratio versus concentration

3.5.2 Limit of detection (LOD), limit of quantification (LOQ) and precision. LOD and LOQ were determined as the concentrations producing signal to noise ratio of 3 and 10 respectively. The instrument precision was determined by multiple injections of the samples. All the results summarized in Table 3.4.

Table 3.4 Relative standard deviation (RSD) values of standard steroid/internal standard peak area ratio at each concentration and each steroid LOD and LOQ Concentration RSD (%) LOD(µg/ml) LOQ(µg/ml) (µg/ml) n=6 4 25.9 Cholesterol 10 17.0 2.6 8.0 15 8.9 50 8.5 50 15.0 Cortisone 65 31.7 62.4 189 75 11.0 100 21.3 4 9.4

DHEA 10 22.0 9.0 27.2 15 26.6 50 11.2 4 10.4 Estradiol 10 3.6 6.6 20.0 15 9.9 50 4.8

3.5.3 Normal sample analysis

The purpose of the steroid quantification method development is eventually to measure concentration of steroids in normal and patient plasma samples. In this study, we used normal plasma to check what kind of steroids we can find using this method. After extraction by ISOLUTE SLE + 96-well plates, only cholesterol can be identified. The chromatogram is shown below (figure 3.23).

Figure 3.23: normal plasma chromatogram

According the peak area ratio and the fitted equation in figure 3.19, concentration of cholesterol is 34.82 ± 1.35µg/ml

3.5.4 Recovery test The recovery of each steroid (cholesterol, cortisone, DHEA, and cortisone) was estimated as the ratio between the areas under the peaks of the extracted samples. The spiking level for DHEA, Estradiol, and Cholesterol was 500ng, 1000ng, 2000ng, 3000ng and 5000ng; recovery level for Cortisone was 5000ng, 7500ng, 10000ng, 12500ng and 15000ng respectively. All the results are illustrated below (table 3.5).

Table 3.5 Recovery of four steroids Recovery (%) Spiking level (ng) 500 1000 2000 3000 5000 Cholesterol 119.49 - 122.35 126.69 125.27 Cortisone 128.88 125.25 127.26 - - DHEA 99.27 52.70 83.56 - - Estradiol 149.75 115.20 105.89 - 162.78 A dash represents that I did not collect the data information because some unknown errors.

4. Conclusion The aim of my study was to develop a sensitive, stable and rapid method for the analysis of steroids in biological tissues by GC-MS, a more reliable technique than the usual immunoassay technique, which allows also a wide range of steroids to be identified in a single sample. The steroids I tried belonged to the androgens, estrogens, and corticoids sub-groups. The utilization of different derivatization methods during the sample preparation part is also important. The developed GC-MS method is robust and reached the level of sensitivity and reproducibility which is required by clinical testing. Hence, the method can be incorporated into the daily routine testing of steroids.

5. Future work Future work can possibly explore the derivatization method, looking for effective ways to convert a ketone group to a methoxy group. In this project my focus was to explore only 5 silylation reagents but in the future one can also test more reagents. Examples would be looking at MSTFA, TMIS, DTE, (TMSI), HFBA and TEA or mixing different reagents using different ratio to compatible more steroids which did not test in this project.

6. Acknowledgments My gratitude to all of you is beyond any words. First of all I would like to Professor Jonas Bergquist for accepting me to work on this project. You gave me a lot of trusts and helps since the first day we met. Thank you for trust and giving me the opportunity to switch my master program from another department 2 years ago. Your guidance and encouragement from the start to the end of this project is highly appreciated. My sincere thanks is to my supervisor Dr. Kumari Ubhayasekara for the continuous guidance valuable suggestions. You are really like my father as I mentioned before and I’m very lucky to be with you during these months. You are really cared so much on my work, patience to my questions and explained everything to me. My sincere thanks is also to Neil De Kock for your valuable helps, good suggestions and enjoyable discussions during the lab experiments. Also I thank Warunika Aluthgedara, for her kindness, sweet and valuable discussion. I would also like to thank all the staff at the analytical chemistry department for their support and making my time fun here. Finally, I would like to give my deepest thanks to my father, for your many years of enormous support to my study in Sweden. Last but not least, I would like to say thanks to my boyfriend, Eugene, thank you for your love and support, for your understanding everything of me during my study.

7. Popular scientific summary Steroids are lipid compounds which can be synthesized from cholesterol. The steroids are mainly divided into the following catogories: estrogen; androgen; glucocorticoid; progesterone; and mineralocorticoid. The synthesis of these steroids occurs in the mitochondria and the smooth endoplasmic reticulum of cells in the adrenal cortex, the gonads and the placenta. The adrenal cortex is mainly responsible for the production of corticosteroid and androgen hormones which has three zones. Aldosterone and mineralocorticoids are mainly produced in the outmost zone and zona glomerulosa. The middle zone, zona fasciculate, releases glucocorticoids such as cortisol and cortisone into the body. Zona reticularis and the innermost zone is responsible for producing androgens androstenedione (the precursor to testosterone (T)). Estrogens are produced mainly by the ovaries and placenta with small amounts being produced by the adrenal glands.

These steroids with their metabolites are important in controlling human body functions and regulating the nervous and immune systems. For example, estrogens could develop and maintain the female secondary sex characteristics; androgens could develop and maintain the male secondary sex characteristics; glucocorticoids could be acted as an immunosuppressant; progesterones could maintain the pregnancy and regulate the cyclical changes of menstruation; and mineralocorticoids could regulate the blood pressure.

In recent years, steroids have became useful biomarkers for the diagnosis of several pathological conditions and the concentration levels of steroids in the human body could indicate some disorders. Therefore it is important to develop rapid and accurate analysis methods for steroids.

Currently, the main techniques for the analysis of steroids are immunoassays, liquid chromatography or gas chromatography coupled to mass spectrometry. The most important requirements for the techniques of steroid measurement are reliability, specificity and sensitivity. The technique of immunoassays might give negative results due to cross-reactions, so this technique is not reliable enough. The tandem mass spectrometry (MS/MS) technique can give reliable, specific and sensitive results, hence mass spectrometry (MS) coupled with gas chromatography (GC) or high performance liquid chromatography (HPLC) is now being commonly used in steroid hormone detection.

GC–MS/MS offers a higher resolution compared to LC–MS due to the longer analysis running time and can analyse volatile compounds, so GC–MS/MS is a powerful tool that can be employed for the analysis of steroids and is more sensitive than LC–MS. In this study, GC–MS/MS was used for the analysis of steroids.

Normally, biological samples need extraction and an enrichment process for purification before injection into the instrument. Routine sample preparation usually consists of liquid-liquid extraction (LLE), protein precipitation and solid phase extraction (SPE). After comparing different techniques, the method of ISOLUTE SLE + 96-well plates were shown to give high throughput, so this method was used for extraction of steroids.

Most steroids are required to be derivatized for improving their volatility before injection into GC-MS. Optimization of derivatizated conditions was investigated for enhancing the derivatization efficiency. In this study, that samples reacting with 30µl methoxyamine hydrochloride at 60°C for 45min, then with 100 µL BSA at 120°C for 30 min was the optimized condition.

In order to accurately quantify the targeted steroids, the measured response must be correlated with the concentration of the steroid hormones. Therefore, calibration curves need to be constructed. The calibration range reflected the steroid concentration levels in the human body and the number of replicates was sufficient to determine certain parameters and perform various statistical analyses (linearity, precision, accuracy, sensitivity, stability, limit of detection, etc.). The linearity of the GC-MS method was evaluated at a concentration range of 2 – 50 µg/ml. The correlation coefficient (R2) of cholesterol and estradiol ≥ 0.99; both DHEA and cortisone's parameters ≥ 0.93. The recoveries for all the steroids determined at low, medium and high concentration levels varied from 52.70 – 162.78 %. The concentration of cholesterol in normal plasma is 34.82 ± 1.35µg/ml.

The aim of this study was to develop a sensitive, stable and rapid method for the analysis of steroids in biological tissues by GC-MS, which allows also a wide range of steroids to be identified in a single sample. The utilization of different derivatization methods during the sample preparation part is also important. The developed GC-MS method is robust and reached the level of sensitivity and reproducibility which is required by clinical testing. Hence, the method can be incorporated into the daily routine testing of steroids.

Future work can be done on the derivatization method, to find more effective derivatization methods compatible with steroids which were not tested in this project.

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