Bioanalytical strategies for the quantification of xenobiotics in biological fluids and tissues 4

Chapter 4 Screening of PCB congeners from ovarian tumor cyst fluids using GC-MS with compound composer database software for simultaneous analysis... 4.1 Preface to chapter 4 This invest

Chapter 4 Screening of PCB congeners from ovarian tumor cyst fluids using GC-MS with compound composer database software for simultaneous analysis

4.1 Preface to chapter 4

This investigation was performed to profile the level of polychlorinated biphenyls congeners in ovarian tumor cyst fluid samples to reveal the association of these persistent organic pollutants in the disease progression A simple method using porous membrane protected µ-SPE coupled with GC-MS was used for the simultaneous quantization of 209 PCB congeners in a single GC-MS run Each congener was individually detected and the concentration was calculated using the response factor for a group congener with the same number of chlorine atoms This is the first research work of its kind to be carried out and the method showed good linearity of the standard calibration solutions over a concentration range of 0.50 to

100 µg L-1, and good linearity with correlation coefficients of 0.9878–0.9999 were obtained LODs for the analytes at a signal-to-noise ratio of 3 under GC-MS selective ion monitoring, ranged between 6 and 29 ng L−1 The relative recoveries ranged from 81.8 to 102% with RSD values between 7.8 and 16.5% These results further demonstrated that the µ-SPE–GC–MS system is highly effective for analyzing trace PCBs in tumor cyst fluid samples From the 30 benign and malignant samples, 87 PCB congeners were detected, of which, 13 congeners present in more than 60% of the samples Most of the total PCBs mean levels are significantly elevated in malignant samples Each congener is individually detected and the concentration is calculated and the values show the higher levels of most persistent and abundant congeners, namely, CB-153 and CB-110 This investigation is significant in the research on the influence of persistent organic pollutants on the tumor malignancies

4.2 Introduction

PCBs are a class of persistent organic pollutants POPs and lipophilic made compounds widely used in industrial and consumer products for decades before their production was banned in the United States and other developed countries in the late 1970s PCBs remain ubiquitous environmental contaminants because of their extensive use and persistence Furthermore, they are distributed globally via the atmosphere, oceans, and other pathways, PCBs released in one part of the world have contaminated even remote regions far from their source of origin [1, 2] The half-life

human-of PCBs in the blood ranges from < 1 to > 10 years, depending on the congener [3, 4] PCBs can be measured in most of the general population because of their environmental ubiquity and persistence For instance, in a report of a large and statistically representative sampling of 1,800 individuals 12 years of age and older

from the U.S population, 31 of 35 PCB congeners measured were detected in over 60% of serum samples, and 21 congeners were detected in over 95% of samples [5] The general population is exposed primarily through ingestion of contaminated foods (e.g., fish, meat, and dairy products), although occupational, ambient, and indoor sources of exposure may exist as well [6-11] Exposure to PCBs can result in an inter-nal dose to the female reproductive tract, as PCBs have been measured in human follicular fluid [12-14], ovarian tissue [15], placenta, uterine muscle, and amniotic fluid [16], providing evidence of exposure to critical tissues and fluid of reproductive system

PCBs have been associated with a range of adverse health effects A large number of studies have reported that some PCB mixtures possess diverse deleterious

effects including carcinogenicity Many have been shown to disrupt development and functioning of certain endocrine pathways, to alter growth, development, cognitive function, and to exhibit immunotoxicity in experimental animals, biota, and humans [17, 18] In 1999 the Agency for Toxic Substances and Disease Registry (ATSDR) stated in their updated Toxicological Profile for Polychlorinated Biphenyls that,

“Overall, the human studies provide some evidence that PCBs are carcinogenic” [19] Many higher-chlorinated biphenyls, persistent and predominant in foods, are active as promoters in carcinogenesis Lower-chlorinated biphenyls, predominating in indoor and outdoor air, are more readily metabolized and inhalation of such biphenyls may expose humans to reactive, possibly carcinogenic intermediates [20]

Measurements of the PCBs or their metabolites in body tissues and fluids (often called biological monitoring) have been carried out as useful approach for assessing the exposure risk in the epidemiological studies It tries to assess how much

of a contaminant can be absorbed by an exposed target individual and how much of the absorbed quantity is actually available to create a biological effect Exposure data concerning the human reproductive system are essential for risk assessment, to identify relationships between chemical exposure and diseases or development abnormalities and to distinguish between exposed and control groups The data obtained from contaminant profiling of body fluids, especially tumor cyst fluids, may provide supporting evidence pertaining to the tumor etiology to some extent Unfortunately, the analysis of PCBs and their metabolites in biological fluid and tissue samples involves complex, and time-and solvent-consuming extraction, separation and clean-up steps

Sample preparation and sample amount are critical steps in the analytical procedure of POPs in human biological fluids They play an important role and can influence the results provided by the instrumental techniques in quantitative determination, which is the final step of the analysis Classical methods use relatively large volumes of solvents i.e 10 to 200 mL, and limit the application of these methods to adults only [21] In addition, most of them require fractionation into sub-samples during sample preparation and/or multiple chromatographic injections Recently modern microextraction techniques such as SPME [22], dispersive liquid–liquid microextraction [23], LPME [24] have been developed for PCB analysis from biological samples However, extremely small sample size and meager quantities of analytes present in the samples drive the need for an more efficient extraction technique suitable for complex ovarian cyst fluid samples

Porous membrane-protected µ-SPE is an effective technique for the extraction

of various target analytes from complex samples without additional sample clean up [25-28] Previously, µ-SPE has been successfully employed for the ovarian tumor cyst fluid samples and for the extraction of estrogens by our group [29] The preparation of µ-SPE device has been explained in chapter 2

GC-MS is the most frequently used analytical technique because of its high sensitivity, selectivity, and flexibility, even for monitoring trace amounts of chemicals However, before actual samples can be tested, standards of target substances must be analyzed for the determination of retention times and the preparation of calibration curves, which are often affected by subtle differences in GC-MS conditions [30, 31] The necessity for standards restricts the number of

chemicals that can be simultaneously analyzed by GC-MS; at the present time, that number seems to be on the order of hundreds

To overcome this problem, we employed an analytical approach that can simultaneously determine 209 PCB congeners by means of GC-MS For quantification, exact retention times are essential for correct identification of targets; standard substances must be analyzed for exact retention times; and preparing all standards before sample analysis is costly and time consuming On this basis, new compound composer database software for simultaneous analysis (Shimadzu) by GC-

MS has been employed to overcome some of the limitations of traditional GC-MS analysis The database consists of three databases - mass spectra, retention times, and calibration curves, all of which are essential for both identification and quantification

of target substances As long as the GC-MS conditions remain constant, the database system can be used to predict exact retention times and to obtain reliable quantification results without prior analysis of standards In addition, new substances can be easily added to the database Therefore, any chemical to which the specified

GC conditions are applicable can be analyzed by means of the system Moreover, if similar databases were constructed using different GC conditions, it would theoretically be possible to analyze, without standards, most of the chemicals to which

GC is applicable

In this current study, for the first time, µ-SPE coupled with GC-MS with compound composer database software for simultaneous analysis was used for the simultaneous quantization of 209 PCB congeners from the malignant and benign ovarian tumor cyst fluid samples in a single GC-MS run Each congener is

individually detected and the concentration is calculated using the response factor for

a group congener with the same number of chlorine atoms

4.3.1 Chemicals

HPLC grade solvent n-Hexane was purchased from Tedia Company Sodium chloride & sodium sulfate were ordered from Goodrich Chemical Enterprise (Singapore) Ultrapure water was prepared from a Nanopure water purification system (Barnstead, Dubuque, USA) Surrogate standard solution (1 µg mL-1) containing 13C-Labelled Mono-Deca PCBs, Internal standard solution Perylene-d12 with a concentration of 200 µg mL-1 and PCB standard solution (1 µg mL-1; mono- and di-CB; 2 µg mL-1) were purchased from Cambridge Isotope Laboratories, Inc (Andover,

MA, USA) Accurel polypropylene flat sheet membrane (200 µm wall thickness, 0.2

µm pore size) was purchased from Membrana The ethylsilane (C2) modified silica, octylsilane (C8) modified silica and octadecylsilane (C18) modified silica, activated activated carbon, Carbograph were purchased from Alltech (Carnforth, Lancashire, UK) The Ultrasonicator was bought from Midmark (Versailles, OH, USA)

mL volumetric flask with hexane Perylene-d12 was used as an internal standard All working solutions and stock standard solutions were stored at 4˚C

4.3.3 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 Thirty 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 diluted with ultrapure water to a 1:1 ratio to avoid matrix interferences and to improve the extraction precision and extraction efficiency Moreover for complex body fluids, 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 work were discarded according to standard biohazard disposal protocols

4.3.4 GC-MS Analysis

A Restek-PCB capillary column (60 mm × 0.25 mm i.d., df = 0.25 mm, Restek Coporation, USA) was used Helium was used as a carrier gas with linear flow rate of 32.6 cm s-1 The injection port and interface temperatures were both set at 280˚C The GC-MS system temperature was set at 110˚C (hold for 3 min); 15˚C min-1

to 210˚C; 2˚C min-1 to 310˚C min-1 and 5˚C min-1 to 320˚C (hold for 10 min) 4 µL of the sample was injected into the GC-MS in splitless mode and the total GC-MS analysis time was 55.00 min SIM mode employed for the set of target PCB compounds The method file “PCB_RtxPCB.qgm” was obtained from the United Nations University, Tokyo; and used in this analysis The retention indices of 209 PCBs are registered in the method file Correction of retention time was carried out using n-alkane data

4.3.5 Preparation of µ-SPE device

The preparation of the µ-SPE device has been described previously of Chapter

2 Briefly, the device consisted of sorbent held within an envelope made from polypropylene membrane sheet of dimension 2 cm × 1.5 cm The edges were heat sealed

Before use, each µ-SPE device was conditioned (ultrasonication for 10 min with 5 mL

of methanol) and stored in the same solvent

4.3.6 µ-SPE procedure

For extraction, the µ-SPE device after drying in air for few minutes was placed in 10mL of sample solution The sample solution was agitated at 105 rad s−1for 60 min to facilitate extraction After extraction, the device was taken out of the sample solution, dried thoroughly with lint free tissue and placed in a 500 µL auto sampler vial for desorption 100 µL of acetone and BSTFA mixture (5:1 ratio) was added and ultrasonicated for 8 min After desorption, the µ-SPE was removed from the desorption vial and the extract was kept in a water bath at 60◦C for 20 min Finally, 2 µL of derivatized extract was injected into the GC-MS for analysis

4.4 Results and discussion

The µ-SPE is the equilibrium based extraction procedure involving the dynamic portioning of analytes between the sorbent material and the sample solution [10] To evaluate µ-SPE, consideration was given to factors that influence extraction efficiency such as sample size, extraction time and desorption time, desorption solvents, pH, and ionic strength

4.4.1 Extraction time

Since µ-SPE involves dynamic partitioning of the PCBs between the sorbent material and the sample solution, the extraction efficiency depends on the mass transfer of analyte from the aqueous sample to the solid sorbent phase packed within the µ-SPE device The effect of extraction time was examined in this study as mass transfer is a time-dependent process The sample was continuously stirred at room temperature (25˚C) with a magnetic stirrer to aid the mass transfer process and to decrease the time required for equilibrium to be established The stirring speed was fixed at 105 rad s-1 The adsorption profile of the PCBs in tumor cyst fluid sample on the µ-SPE was determined by extracting the analytes for 10 to 40 min The highest extraction was achieved at 30 min, and after more than 30 min, no considerable improvement in peak area response was observed In fact, for some analytes, extraction decreases beyond 30 min This result is often observed in similar extraction work Therefore, 30 min was chosen as optimum extraction time

4.4.2 Type of sorbent materials and ratio of composition

The selection of a suitable sorbent is an important parameter Various sorbents including ethylsilane (C2) modified silica, octylsilane (C8) modified silica and octadecylsilane (C18) modified silica, activated carbon, Carbograph, (divinylbenzeneethyleneglycoldimethacrylate), and HayeSep B (divinylbenzenepolyethyleneimine) were evaluated for µ-SPE C18 has the highest hydrophobicity followed by C8 and C2 HayeSep A is of intermediate polarity and HayeSep B is of high polarity Different combinations of polar and non-polar (1:1, 10

mg of each) sorbent materials were tested in extracting target analytes (An unpublished previous experiment showed efficient extraction with combination of

polar and non-polar sorbents for PCBs) A total of six different combinations were investigated by weighing an equal ratio of two types of sorbent materials within each

µ-SPE device The combinations tested were: (i) HayeSep A with C18; (ii) HayeSep A with C8; (iii) HayeSep A with C2; (iv) HayeSep B with C18; (v) HayeSep B with C8; (vi) HayeSep B with C2 (Figure 4.1) Based on peak area analysis, HayeSep A-C18was found to be the effective combination for adsorption than others The moderate to high hydrophobicity of the mixture was probably most compatible to the analytes considered

4.4.3 Sorbent mass

After selecting HayeSep A-C18 as a suitable sorbent, the suitable amount of sorbent material (ranging from 5 to 20 mg) was investigated Obviously, it was found that with increasing sorbent amount, the extraction efficiency increased, as denoted by higher peak areas during GC-MS analysis The auto sampler vial cannot accommodate more than 20 mg of sorbent material Thus, 20 mg of sorbent was the maximum amount used in all experiments

Figure 4.1 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 min with 10 min desorption by ultrasonication; 20mg of sorbent was used

4.4.4 Extraction volume

The influence of extraction sample volume (from 10 to 25 mL) on extraction efficiency was investigated Larger extraction efficiencies were observed as sample volumes were increased This phenomenon is due to increasing analyte enrichment with increasing volume of the sample A limit to this enrichment is reached when the analyte fully saturated with the adsorption sites of the sorbent The extraction efficiency was reached at a maximum at 20 mL of sample Hence, 20 mL was selected as the optimal sample volume

4.4.5 Desorption solvent

Selection of a suitable desorption solvent was assessed based on solubilization capability Various organic solvents such as methanol, acetone, toluene, dichloromethane and hexane were tested Polar solvents such as methanol and acetone were not effective in desorbing the target analytes as peak areas from analysis of respective extracts were relatively small Since PCBs are generally non polar compounds, they should be more favorably desorbed by non-polar solvents This proved to be the case as hexane and toluene gave comparatively better results than the other solvents, with the latter showing the most favorable performance

4.4.6 Desorption time and carryover effects

The effect of desorption time over the range of 5 to 20 min was investigated All the PCBs were desorbed completely within 15 min of ultra-sonication Desorption efficiency was declined when shorter periods of time were used Above 15 min, no significant increase in peak area response was observed (Figure 4.2)