Bioanalytical strategies for the quantification of xenobiotics in biological fluids and tissues 2

Chapter Chapter Determination of estrogens in ovarian cyst fluid samples by porous membrane protected micro-solid-phaseextraction combined with gas chromatography-mass spectrometry 21 Chapter 2.1 Preface to Chapter To compare the levels of estrogens in benign and malignant ovarian tumor cyst fluids, a cost effective and environmentally friendly extraction technique using porous membrane protected µ-SPE is described. A sorbent (ethylsilane (C2) modified silica) (20 mg) was packed in a porous polypropylene envelope (2 cm × 1.5 cm) whose edges were heat sealed to secure the contents. The µ-SPE device was conditioned with acetone and placed in a stirred (1:5) diluted cyst fluid sample solution (10 mL) to extract estrogens for 60 min. After extraction, the analytes were desorbed and simultaneously derivatized with a 5:1 mixture of acetone and N,Obis(trimethylsilyl)-trifluoroacetamide. The extract (2 µL) was analyzed by gas chromatography–mass spectrometry. Various extraction, desorption and derivatization conditions were optimized for µ-SPE. With this simple technique, low limits of detection of between and 22 ng L−1 and linear range from the detection limits up to 50 µg L−1 were achieved. The optimized method was used to extract estrogens from cyst fluid samples obtained from patients with malignant and benign ovarian tumors. The results showed a pattern of higher levels of estrogen accumulation in benign as compared to malignant samples in the samples tested. This implies that estrogens might play a role in the malignancy associated with epithelial ovarian cancer along with other compounding factors. 22 Chapter 2.2 Introduction Estrogens are a group of steroid hormones which primarily function is to regulate the reproductive systems of both female and male animals and humans. Over the past several decades, estrogens have received much attention due to their association with many types of human gynaecological cancer [1]. In 2002, estrogens were first listed as known human carcinogens by the U.S. Department of Health and Human Services in its Report on Carcinogens (10th edition), based on sufficient evidence from human epidemiology studies [2]. These studies showed that use of estrogen replacement therapy by postmenopausal women is associated with a consistent increase in the risk of uterine endometrial cancer and a less consistent increase in the risk of breast and ovarian cancer. Some evidence suggests that use of oral contraceptives may also increase the risk of breast cancer [2]. The exposure to estrogens comes from both natural hormones that are secreted by the ovaries (e.g. 17β-estradiol and its metabolite estrone) and synthetic forms (e.g. 17αethynylestradiol and diethylstilbestrol) that are widely found as the ingredient of medication for estrogen replacement therapy, oral contraceptives, and many cosmetics [3, 4]. The presence of estrogens in human body fluids such as follicular cyst fluid and nipple aspirate fluid has been demonstrated by many studies [5-8]. Therefore, determining the level of estrogens in these body fluids would be very important for the study of their roles in the carcinogenesis of ovarian and breast cancer. The role of estrogen in the progression of gynaecological cancers such as ovarian cancer is well documented [9]. A high correlation was reported between the presence of certain types of estrogen receptors (ER) and the prevalence of 23 Chapter gynaecological cancers [10]. Determination of estrogens in tumor specimens and accumulating fluids in the cyst (cyst fluid) could reveal information on the cancer. Based on this information (estrogen-positive or -negative) the nature of therapy to be administered to patients, and the prognosis of the cancer may be determined following assessment of genes responsible or ER positive and negative status [11,12]. The challenges in determining the quantity of estrogen arises from the fact that (i) the amount of cyst fluid sample available is very small; and these (ii) samples are characterized by their complexity. Therefore, high preconcentration with efficient sample clean up are required for cyst fluid sample analysis. Techniques for extraction of estrogens in aqueous samples include the established SPE [13], SPME [14] and more recently, polymer-coated hollow fibre microextraction [15] and stir-bar sorptive extraction [16]. All these techniques normally require extensive sample clean up from complex samples such as cyst fluid. Therefore the aim of this study is to develop a better and alternative procedure for extracting estrogens from cyst fluids, that involves no or little additional clean up. A novel, low cost and environmentally friendly extraction technique, called porous membrane-protected µ-SPE, was used for the extraction of various target analytes from complex samples without additional sample clean up [17-20]. The µSPE device consists of sorbent enclosed within a ca. cm × 1.5 cm membrane envelope and is ideally suited to the extraction from a limited amount of sample. The judicious choice of sorbent materials, and therefore to some extent, the selectivity of µ-SPE can be fine-tuned. With the protection afforded by the porous polypropylene membrane, the elimination of substances such as particulates, proteins and humic substances, which can interfere with the extraction, is easily accomplished without additional clean up steps [21-23]. 24 Chapter The objective of the study is to develop a µ-SPE technique for the determination of estrogens in benign and malignant human ovarian cyst fluid. This is the first instance where the µ-SPE technique is applied to human cyst fluid samples. The information regarding the levels of estrogen in tumor ovarian cyst fluids might play an important role in disease diagnostics. This work also investigates the feasibility of applying the simple µ-SPE technique to a complex biological matrix. 2.3 Experimental 2.3.1 Chemicals Diethylstilbestrol, estrone, 17β-estradiol, 17α-ethynylestradiol (Figure 2.1) were purchased from Aldrich (Milwaukee, WI, USA). The HPLC-grade solvents and N, O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) were purchased from Merck (Darmstadt, Germany). Chemical standard solutions were diluted with acetone. Accurel polypropylene flat sheet membrane (200 µm wall thickness, 0.2 µm pore size) was purchased from Membrana (Wuppertal, Germany). The ethylsilane (C2) modified silica, octylsilane (C8) modified silica and octadecylsilane (C18) modified silica, activated carbon, Carbograph, Haye-Sep A and Haye-Sep B sorbents were purchased from Alltech (Carnforth, Lancashire, UK). The ultrasonicator was bought from Midmark (Versailles, OH, USA). 25 Chapter Figure 2.1 Chemical structures of the estrogens studied: (a) diethylstilbestrol, (b) estrone, (c) 17β-estradiol, (d) 17α-ethynylestradiol. 2.3.2 Human cyst fluid samples Cyst fluid obtained from benign and malignant ovarian tumor samples were collected following approval from the Domain Specific Review Board, National Health Group, Singapore. Twenty cyst fluid samples were collected from patients who were diagnosed to have benign and malignant cysts. Small volumes of cyst fluid were collected from patients and in initial studies raw cyst fluid samples without dilution were used for µ-SPE, but this resulted in poor precision and significant matrix interference. However, sample dilution with ultrapure water to a 1:1 ratio improved the extraction precision and extraction efficiency. It is probable that the dilution reduced the extent of interferences by the protein (clogging on the membrane) and the low viscosity of the matrix that allowed more efficient extraction. Standard safety precautions were put in place during the handling of body fluids. All body fluids and solvents used in this project were decontaminated according to standard biohazard disposal protocols. 26 Chapter 2.3.3 GC-MS Analyses were carried out using a Shimadzu (Kyoto, Japan) QP2010 GC–MS system equipped with a Shimadzu AOC-20i autosampler and a DB-5 (J & W Scientific, Folsom, CA, USA) fused silica capillary column (30 m × 0.32 mm internal diameter, 0.25 µm film thickness). Helium (purity 99.9999%) was used as the carrier gas at a flow rate of 2.0 mL min-1. Samples (2 µL) were injected in splitless mode. The injection temperature was set at 300◦C and the interface temperature kept at 280◦C. The GC temperature program used was as follows: initial temperature 90◦C held for min, then increased by 30◦Cmin-1 to 280◦C, and held for min. The standard mixtures and extracts were analyzed in selected ion monitoring mode with a detector voltage of 1.5 kV. 2.3.4 Preparation of µ-SPE device The preparation of the µ-SPE device has been described previously [19]. The µ-SPE device consists of sorbent materials enclosed within a polypropylene sheet membrane envelope. To prepare the device, the longer edge of a polypropylene sheet was folded over to a width of ~2 cm. The edge of the fold-over flap was then heat sealed using an electrical sealer to the main sheet. The fold-over section was then trimmed off from the main membrane sheet. The former was then cut (at ~1.5 cm intervals) into individual (2 cm × 1.5 cm) pieces. One of the two open ends of each piece was then heat-sealed. A glass Pasteur pipet and a glass funnel were used to introduce sorbent (20 mg) into the resulting membrane envelope via the remaining open end that was then heat-sealed to secure the contents. Before use, each µ-SPE device was conditioned (ultrasonication for 10 with 5mL of acetone) and stored in the same solvent. 27 Chapter 2.3.5 µ-SPE procedure For extraction, the µ-SPE device after drying in air for few minutes was placed in 10 mL of sample solution. The sample solution was agitated at 105 rad s-1 for 60 to facilitate extraction. The device tumbled freely within the sample during extraction. After extraction, the device was taken out of the sample solution, dried thoroughly with lint free tissue and placed in a 500 µL autosampler vial for desorption. 100 µL of acetone and BSTFA mixture (5:1 ratio) was added and ultrasonicated for min. After desorption, the µ-SPE was removed from the desorption vial and the extract (~ 80 µL) was kept in a water bath at 60◦C for 20 min. Keeping the extract in warm condition before analysis will facilitate the derivatization process especially for biological matrices. Finally, µL of derivatized extract was injected into the GC-MS for analysis. 2.4 Results and discussion µ-SPE is an equilibrium based extraction procedure involving the dynamic partitioning of analytes between the sorbent material and the sample solution [19]. The analytical factors that influence extraction efficiency such as the type of sorbent, amount of sorbent, extraction time and desorption time, sample pH and ionic strength were evaluated by a stepwise univariate approach. C2 1.50E+06 C8 C18 Peak area 1.00E+06 Activated Carbon Carbograph HAYE-SEP A 5.00E+05 HAYE-SEP B 0.00E+00 DES Estrone Estradiol Ethynylestradiol 28 Chapter Figure 2.2 Suitability of various sorbents for µ-SPE from spiked samples. Samples were spiked at levels of 10 µg L-1 of each analyte. µ-SPE conditions: samples were extracted for 30 with 5-min desorption by ultrasonication using 150 µL of acetone, and 20 derivatization at 60◦C; 15 mg of sorbent was used. Initially, the selection of a suitable sorbent was considered. Various sorbents including ethylsilane (C2) modified silica, octylsilane (C8) modified silica and octadecylsilane (C18) modified silica, activated carbon, Carbograph, Haye-Sep A and Haye-Sep B were evaluated (Figure 2.2). Estrogens are polar compounds and appeared to have greater interaction with the relatively polar C2 sorbent compared with the others under acidic (pH 2) condition. After selecting C2 as a suitable sorbent, the amount of sorbent material was varied from to 20 mg. It was found that with increasing sorbent amount, the extraction efficiency increased, as denoted by higher peak areas during GC-MS analysis (Figure 2.3). Placing >20 mg of sorbent in to an envelope made the device too large to fit into the autosampler vial. As a result, desorption was not efficient since the device could not be immersed completely in the solvent. Thus, 20 mg of sorbent was the maximum amount used in all experiments. 6.00E+06 mg 4.00E+06 Peak Area 10 mg 15 mg 20 mg 2.00E+06 0.00E+00 DES Estrone Estradiol Ethynylestradiol Figure 2.3 Effect of sorbent mass on µ-SPE from spiked samples. Samples were spiked at levels of 10 µg L-1 of each analyte. µ-SPE conditions: samples were 29 Chapter extracted for 30 with 5-min desorption by ultrasonication using 150 µL of acetone and 20 derivatization at 60◦C. The effect of extraction time was investigated since mass transfer is a timedependent process. Extractions of between 10 and 60 were studied in order to determine the adsorption profile of the estrogens (Figure 2.4). To facilitate mass transfer and to decrease equilibration time, the sample was stirred at 105 rad s-1 continuously at room temperature. During extraction, the mass transfer of analyte from the sample solution to the sorbent determines the extraction efficiency [18]. A longer extraction time (60 min) gave better analyte enrichment; probably more time was required for the analyte to diffuse through the porous membrane, and onto the sorbent material. Since the total time was considerable (88 comprising of 60 for extraction and 28 for desorption and derivatization), we did not further extend the extraction time, and 60 was selected. 1.20E+07 Peak area 8.00E+06 10min 20min 30min 40min 4.00E+06 50min 60min 0.00E+00 DES Estrone Estradiol Ethynylestradiol Figure 2.4 Extraction time profiles of estrogens. Samples were spiked at levels of 10 µg L-1 of each analyte. µ-SPE conditions: samples were desorbed by ultrasonication using 150 µL of acetone for min, 20 derivatization at 60◦C; 15 mg of sorbent was used. 30 Chapter The salting-out effect has been widely used to enhance the extraction efficiency of polar compounds in extraction and microextraction techniques [13-16]. Addition of salt decreases the solubility of polar analytes in aqueous samples [24, 25] and thus, in this case, favours extraction by the sorbent. The effect of salt on extraction efficiency was determined by adding sodium chloride (NaCl) (from to 30% (w/v)) to the sample. The highest peak areas were obtained when 5% NaCl was used. 1.50E+07 Peak Area 0% 1.00E+07 5% 10% 20% 5.00E+06 30% 0.00E+00 DES Estrone Estradiol Ethynylestradiol Figure 2.5 Ionic strength profile of estrogens for different salt concentrattion. Samples were spiked at level of µgL-1 of each analyte. µ-SPE conditions: samples were extracted for 60 with 100 µL of acetone as desorption solvent, 20 derivatization at 60◦C; 15 mg of sorbent was used. Estrogens are ionisable compounds and their extraction behaviour at different sample pH (from to 12) was investigated. Sample pH was adjusted by the addition of 1M hydrochloric acid and 1M sodium hydroxide respectively. At a sample pH of 2, better extraction efficiency was achieved when compared to neutral or basic conditions. Acidic sample pH had previously been used for extracting these compounds [26]. Based on this, a sample pH of was used for further experiments. 31 Chapter pH=2 1.80E+07 Peak Area pH=4 pH=6 1.20E+07 pH=8 pH=10 6.00E+06 pH=12 0.00E+00 DES Estrone Estradiol Ethynylestradiol Figure 2.6 Effect of Sample pH. Samples were spiked at level of µgL-1 of each analyte. µ-SPE conditions: samples were extracted for 60 with 100 µL of acetone as desorption solvent, 20 derivatization at 60◦C; 15 mg of sorbent was used. After extraction, analytes were desorbed in the organic solvent via ultrasonication. To select a suitable desorption solvent, various organic solvents were tested including acetone, methanol, toluene, dichloromethane, and hexane. Since BSTFA reacts with methanol, acetone was found to be the best desorption solvent as the highest peak areas were obtained using it. This could be because estrogens are polar compounds so they are preferentially desorbed by relatively polar solvents rather than by the less polar solvents such as hexane and toluene. 9.00E+06 Acetone Peak area Methanol 6.00E+06 Dichloromethane Toluene Hexane 3.00E+06 0.00E+00 DES Estrone Estradiol Ethynylestradiol 32 Chapter Figure 2.7 Desorption solvent profile of estrogens. Samples were spiked at level of µgL-1 of each analyte. µ-SPE conditions: samples were extracted for 60 with 100 µL of acetone as desorption solvent, 20 derivatization at 60◦C; 15 mg of sorbent was used. The effect of desorption time with an acetone:BSTFA mixture (5:1) was also investigated. After extraction, the analyte-enriched sorbent was ultrasonicated from to 10 and kept at 60◦C for 20 to complete the derivatization. Figure 2.5 shows the profile at different desorption times; an desorption time appears to be optimum for all analytes. After there was a slight decrease in the desorption profile; this could conceivably be due to the analytes being re-adsorbed by the sorbent material. 1.50E+07 Peak area 1.00E+07 Ethynylestradiol Estradiol Estrone DES 5.00E+06 0.00E+00 6min 10 Figure 2.8 Desorption profile of estrogens for different ultrasonication times. Samples were spiked at level of µgL-1 of each analyte. µ-SPE conditions: samples were extracted for 60 with 100 µL of acetone as desorption solvent, 20 derivatization at 60◦C; 15 mg of sorbent was used. To improve the sensitivity and the selectivity of estrogen determination by GC-MS, in general, their derivatization is important [27-29]. It has been reported that excessive or inadequate amounts of BSTFA leads to poor derivatization results [30]. Therefore careful optimization was performed. Different volume ratios of (1:1, 1:2, 33 Chapter 2:1 and 5:1) extract:BSTFA were evaluated. An extract:BSTFA ratio of 5:1 by ultrasonication gave the highest peak areas with no additional peaks. Comparing with our previous method, polymer-coated hollow-fibre microextraction of estrogens [15], the current procedure gave similar results. The optimized extraction conditions used for this study were as follows; C2 sorbent, 20mg sorbent mass, 60 extraction time, 5% Ionic strength, pH 2, acetone as desorption solvent, desorption time and extract and 5:1 as extract:deivatization agent. After each extraction, the µ-SPE device was cleaned with mL of toluene for (ultrasonication) to remove the residual analytes. The same µ-SPE device was again desorbed for with acetone BSTFA solvent mixture (5:1 ratio) to test the carryover effect. No estrogen peaks were detected clearly indicated the µ-SPE was reusable. In this study, we were able to reuse the µ-SPE device up to 20 times without compromising the extraction efficiency. 2.4.1 Linearity, limits of detection and repeatability The linearity of the calibration curve was examined for each target estrogen using an aqueous standard solution of a concentration range of 0.5- 50 µgL-1 of the analyte. Extraction was performed under the optimized conditions as determined above. The results are shown in Table 2.1. Good linearity with correlation coefficients (r) of between 0.996 and 0.999 were obtained. This allowed the quantification of the compounds by the method of external standardization. The limits of detection (LODs) for the analytes at a signal-to-noise ratio of under GC-MS selective ion monitoring, ranged between and 22 ng L-1 (Table 2.1). While determining the LODs, blanks were carried out to re-confirm that no sample carryover occurred. The LODs of the proposed method were comparable with previously reported SPE and SPME methods 34 Chapter [13, 14]. The relative standard deviations (RSDs) of the determinations (n = 3) of the analytes were between and 11%. To assess the performance of µ-SPE, one of the cyst fluid samples (with predetermined (using the present technique) concentrations of 17β-estradiol at 3.4 µgL-1 and 17α-ethynylestradiol at 0.63 µgL-1) were spiked at 10 µgL-1 concentrations of each of the analytes. The extraction results are shown in Table 2.2; for µ-SPE, the relative recoveries, which is defined as the ratio of GC peak areas for the analytes in the spiked cyst fluid extract to the spiked ultrapure water extract, ranged between 86 and 97%. The high relative extraction recoveries of µ-SPE also indicated that matrix effects were negligible at 1:1 dilution. The RSDs (n = 6) were calculated to be between 13 and 18% for cyst fluid samples. The inter-day and intra-day RSDs were also measured; they were less than 18% for all analytes, suggesting that the µ-SPE reproducibility could be further improved by using internal standard. Taking into consideration the complexity of the samples under study, these results are acceptable. Figure 2.9 GC-MS trace of (I) Benign ovarian cyst fluid sample; (II) Malignant ovarian cyst fluid sample. Peak identification: (1a, 1b) diethylstilbestrol isomers, (2) estrone, (3) 17β-estradiol, (4) 17α-ethynylestradiol. Experimental conditions are given in the text. (The desired peaks were extracted from the overlay chromatogram) 35 Chapter 2.4.2 Sample analysis For the current study, cyst fluids from malignant and benign ovarian cancer tumor, under serous, mucinous, clear cells and endometroid subtypes were subjected to µ-SPE-GC-MS to determine the concentration of estrogens. A total of 10 samples collected from patients with malignant stage (7 early and late) and 10 samples from patients with benign stage were analyzed. Before extraction, these samples were diluted with deionized water at 1:1 ratio to address matrix interferences. Extractions were performed under the previously determined conditions. The Mann-Whitney Utest was used to compare the concentrations of estrogens between benign and malignant ovarian cyst fluid samples. All P values are given for two-sided tests and P < 0.05 was considered significant. Analyses were done using SPSS 13.0 for Windows (SPSS, Chicago, IL, USA). 36 Chapter Table 2.1 Quantitative data of µ-SPE. Estrogens D Diethylstilbestrol Estrone 17β-Estradiol 17α-Ethynylestradiol Linearity range (µg L-1) Coefficient of correlation (r) RSD (%, n = 3) Limits of detection (ng L-1) Limits of quantitation (ng L−1) 0.5–50 0.5–50 0.5–50 0.998 0.998 0.996 11 14 22 27 42 65 0.5–50 0.999 19 60 Table 2.2 Relative recoveries of µ-SPE Estrogens Concentrations detected in benign samples µg L-1 Amount detected in samples spiked with 10 µg L-1 of each estrogens Relative recovery (%) RSD (%) (n =6) Diethylstilbestrol Estrone 17β-Estradiol nd nd 3.4 9.1 ± 1.4 8.6 ± 1.3 12.1 ± 1.5 91 86 90 16 16 13 17α-Ethynylestradiol 0.63 10.3 ± 1.8 97 18 37 Chapter Table 2.3 Concentrations of estrogens detected in malignant and benign ovarian cyst fluid samples (n = 3). *, a Mean concentration in µg L-1 Estrogens Malignant samples (A-E, K-O) A B C D E Benign samples (F-J, P-T) K L M N O Diethylstilbestrol 0.39 nd nd nd nd 0.3 0.2 nd 0.2 nd Estrone 3.02 2.12 1.8 2.3 1.2 2.7 1.8 2.2 17β-Estradiol 6.25 9.15 6.7 4.4 4.5 5.4 5.3 4.9 3.4 8.9 17α-Ethnylestradiol 4.2 3.4 3.3 3.3 2.3 3.2 2.8 1.9 2.5 3.1 nd - not detected * Malignant and benign data were combined based on cyst type, respectively. a Concentrations in raw cyst fluid samples. F G H I 0.2 nd nd nd 3.7 2.5 3.5 2.1 13 11 8.6 13 4.6 4.9 3.9 5.8 J P nd 4.4 8.9 5.9 38 Q R S T nd 0.6 0.1 nd nd 2.2 1.7 2.5 3.35 3.08 8.9 7.3 9.9 12 8.67 3.1 3.6 2.2 3.45 Chapter Estrogens were detected in most of the cyst fluid samples obtained from patients with malignant and benign ovarian tumors (Table 2.3). Estrogen compounds 17βestradiol and 17α-ethynylestradiol were present in higher levels in benign samples. Except for diethylstilbestrol (1.21 times more in malignant cases) all the other three estrogen metabolites were present in higher concentration in benign samples (estrone (0.73 times more in benign samples), 17β-estradiol (0.58 times more in benign samples) and 17α-ethynylestradiol (0.72 times more in benign samples). Figure 2.6 shows a GCMS trace of an extract of malignant and benign cyst fluids. Our studies showed a pattern of higher levels of estrogen accumulation in benign samples as compared to malignant samples in the samples tested. Previous studies have demonstrated the conversion of circulating estrone sulphate to 17β-estradiol by the tumor tissue could be one important reason for raised serum 17β-estradiol levels in postmenopausal women with ‘non estrogen- producing’ ovarian tumors [31,32]. Benign serous cyst fluid samples obtained from ovarian cysts were found to contain high level of 17β-estradiol and 17α-ethynylestradiol compared to malignant samples. These results showed the impact of estrogens levels on malignant transformation of benign cyst fluids to some extent. 2.5 Conclusion The simple porous membrane protected µ-SPE technique was used successfully in conjunction with GC-MS, to determine estrogens in complex ovarian tumor cyst fluid samples. The protection afforded by the porous membrane precluded the need for sample cleanup prior to extraction; in fact, µ-SPE is a single-step cleanup and preconcentration 39 Chapter approach. Using the most suitable extraction conditions, µ-SPE has been shown to be an efficient and effective method for the processing of complex biological samples without the use of large amounts of toxic organic solvents. Based on this preliminary study on 20 samples, our results showed that estrogens might play a role in the malignancy associated with epithelial ovarian cancer along with other compounding factors. Analysis on larger numbers of clinical samples is required for a better understanding of the role of these compounds in the progression of ovarian cancer. From the results obtained using the µ-SPE technique, we infer that we might be able to obtain a clear trend between the levels of the metabolites and the nature of tumor (benign or malignant) if a large number of samples are subjected to this technique. µ-SPE has been demonstrated to be capable of dealing with limited volume of cyst fluid samples. 40 Chapter 2.6 References [1] Gadducci, A. Fanucchi, S. Cosio, A.R. 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Endocrinol. 23 (2007) 25. 42 [...]... associated with epithelial ovarian cancer along with other compounding factors Analysis on larger numbers of clinical samples is required for a better understanding of the role of these compounds in the progression of ovarian cancer From the results obtained using the µ-SPE technique, we infer that we might be able to obtain a clear trend between the levels of the metabolites and the nature of tumor (benign... linearity of the calibration curve was examined for each target estrogen using an aqueous standard solution of a concentration range of 0.5- 50 µgL-1 of the analyte Extraction was performed under the optimized conditions as determined above The results are shown in Table 2. 1 Good linearity with correlation coefficients (r) of between 0.996 and 0.999 were obtained This allowed the quantification of the compounds... Chapter 2 [13, 14] The relative standard deviations (RSDs) of the determinations (n = 3) of the analytes were between 4 and 11% To assess the performance of µ-SPE, one of the cyst fluid samples (with predetermined (using the present technique) concentrations of 17β-estradiol at 3.4 µgL-1 and 17α-ethynylestradiol at 0.63 µgL-1) were spiked at 10 µgL-1 concentrations of each of the analytes The extraction...Chapter 2 The salting-out effect has been widely used to enhance the extraction efficiency of polar compounds in extraction and microextraction techniques [13-16] Addition of salt decreases the solubility of polar analytes in aqueous samples [24 , 25 ] and thus, in this case, favours extraction by the sorbent The effect of salt on extraction efficiency was determined by adding sodium chloride... compounds by the method of external standardization The limits of detection (LODs) for the analytes at a signal-to-noise ratio of 3 under GC-MS selective ion monitoring, ranged between 9 and 22 ng L-1 (Table 2. 1) While determining the LODs, blanks were carried out to re-confirm that no sample carryover occurred The LODs of the proposed method were comparable with previously reported SPE and SPME methods... Figure 2. 5 shows the profile at different desorption times; an 8 min desorption time appears to be optimum for all analytes After 8 min there was a slight decrease in the desorption profile; this could conceivably be due to the analytes being re-adsorbed by the sorbent material 1.50E+07 Peak area 1.00E+07 Ethynylestradiol Estradiol Estrone DES 5.00E+06 0.00E+00 2 min 4 min 6min 8 min 10 min Figure 2. 8... are shown in Table 2. 2; for µ-SPE, the relative recoveries, which is defined as the ratio of GC peak areas for the analytes in the spiked cyst fluid extract to the spiked ultrapure water extract, ranged between 86 and 97% The high relative extraction recoveries of µ-SPE also indicated that matrix effects were negligible at 1:1 dilution The RSDs (n = 6) were calculated to be between 13 and 18% for cyst... be between 13 and 18% for cyst fluid samples The inter-day and intra-day RSDs were also measured; they were less than 18% for all analytes, suggesting that the µ-SPE reproducibility could be further improved by using internal standard Taking into consideration the complexity of the samples under study, these results are acceptable Figure 2. 9 GC-MS trace of (I) Benign ovarian cyst fluid sample; (II)... profile of estrogens for different ultrasonication times Samples were spiked at level of 5 µgL-1 of each analyte µ-SPE conditions: samples were extracted for 60 min with 100 µL of acetone as desorption solvent, 20 min derivatization at 60◦C; 15 mg of sorbent was used To improve the sensitivity and the selectivity of estrogen determination by GC-MS, in general, their derivatization is important [27 -29 ]... been used for extracting these compounds [26 ] Based on this, a sample pH of 2 was used for further experiments 31 Chapter 2 pH =2 1.80E+07 Peak Area pH=4 pH=6 1 .20 E+07 pH=8 pH=10 6.00E+06 pH= 12 0.00E+00 DES Estrone Estradiol Ethynylestradiol Figure 2. 6 Effect of Sample pH Samples were spiked at level of 5 µgL-1 of each analyte µ-SPE conditions: samples were extracted for 60 min with 100 µL of acetone . [5-8]. Therefore, determining the level of estrogens in these body fluids would be very important for the study of their roles in the carcinogenesis of ovarian and breast cancer. The role of estrogen. 0 .2 nd 0 .2 nd 0 .2 nd nd nd nd nd 0.6 0.1 nd nd Estrone 3. 02 2. 12 1.8 2. 3 2 1 .2 2. 7 2 1.8 2. 2 3.7 2. 5 3.5 2. 1 4.4 2. 2 1.7 2. 5 3.35 3.08 17β-Estradiol 6 .25 . section was then trimmed off from the main membrane sheet. The former was then cut (at ~1.5 cm intervals) into individual (2 cm × 1.5 cm) pieces. One of the two open ends of each piece was then heat-sealed.