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ISSN: 2329-6798
Modern Chemistry & Applications
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Gas Chromatography-Mass Spectrometric Method for Simultaneous Separation and Determination of Several Pops with Health Hazards Effects

Nagwa ABO EL-Maali* and Asmaa Yehia Wahman
Department of Chemistry, Faculty of Science, Assiut University, Assiut, Egypt
Corresponding Author : Nagwa ABO EL-Maali
Department of Chemistry
Faculty of Science, Assiut University
71516-Assiut, Egypt
Tel: 0020-882-080-799
E-mail: [email protected]
Received July 22, 2015; Accepted September 22, 2015; Published September 28, 2015
Citation: EL-Maali NABO, Wahman AY (2015) Gas Chromatography-Mass Spectrometric Method for Simultaneous Separation and Determination of Several Pops with Health Hazards Effects. Mod Chem appl 3:167. doi:10.4172/2329-6798.1000167
Copyright: © 2015 EL-Maali NABO, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

The hazards effect of persistent organic pollutants, POPs, on the human health has lead us to modify the ASTM method D-5175 to enhance both sensitivity and selectivity of their simultaneous separation. As their separation is difficult- due to the similarity in their chemical and physical properties- that lead to co-elution in extraction, we proposed a validated method for their simultaneous determination using liquid/liquid microextraction followed by GC/MS in the SIM mode. The method is advantageous since the time needed for the chromatographic analysis of all analytes is less than 17 min. Method Detection Limits (MDLs) and Limit of Detection (LODs) reached sub- ppb levels and in many cases are lower than those achieved in the standard test method ASTM D-5175 for many analytes. Besides, three pesticides namely: Hexachlorocyclopentadiene, p,p’-DDE and trifluralin have been added to the method with good accuracy and precision. Application to several environmental samples has been successfully assessed and supported by proficiency testing samples provided from Absolute Standards®, Inc.

Keywords
GC-MS; POPs; Validation; Application to environmental matrices
Introduction
The importance of the Persistent Organic Pollutants (POPs) with their health effects has lead to looking for accurate and reliable methods for their determinations [1-6] using chromatographic techniques in many matrices viz. ground water [2], human serum [3,4] water and drinking water [5,6], fruits and vegetables [1,7] and tap water [8].
Organochlorines (OCs) are a lipophilic class of chemicals that include OC pesticides and other persistent organic pollutants, such as Polychlorinated Biphenyls (PCBs). It is well known that environmental and/ or dietary exposure to OCs results in the bioaccumulation of these chemicals in the human body especially, in adipose tissue, serum and breast milk [9,10].
Despite of the long-term adverse effects on humans, animals and environment [11], recent studies in East-Asian countries have reported elevated concentration of OCPs in various environmental media suggesting that same OCPs are still being used [12]. OCexposure has been linked with a number of children diseases such as asthma, abnormalities of the productive tract, diabetes, and growth and neurobehavioral disorders [13,14]. In Spain [15,16], the level of chemical contamination by OCs of the population of the canary Island has been evaluated although they’re banned in Spain in the late 1970s. In US, PCBs exposures are encountered by the general public by eating contaminated food or living near a previously operating PCB factory hazardous waste site [17], although they are banned in the United States in 1977. PCBs have been classified as probable human carcinogenic and are listed in the top 10% of EPA’s most toxic chemical [18]. Of the 209 PCBs congeners four non-ortho and eight mono-ortho congeners are currently recognized by the World Health Organization (WHO) as ‘’dioxin like’’ in their toxic effects [19]. Routine analysis of OCs in environmental in different matrices has been achieved through GC/MS and different extraction techniques [20-23].
Therefore, the aim of the present work is to validate and enhance the sensitivity of the ASTM method D5175 for the determination of OCPs viz. Alachlor, Aldrin, Dieldrin, Endrin, Heptachlor, Heptachlorepoxide, Hexachlorobenzene, lindane, Methoxychlor , PCBs congeners namely: PCB 28, 52,118,138, and 180 cited in Table 1 in the presence of the new analytes viz. Hexachlorocyclopentadiene, p,p’-DDE and the organofluorine pesticides trifluralin in other matrices viz. waste water and transformer oils with new levels lower than those cited in the literature.
Experimental
Chemicals and reagents
Organochlorine pesticides: Alachlor, aldrin, dieldrin, endrin, heptachlor, heptachlorepoxide, hexachlorobenzene, hexachlorocyclopentadiene, lindane, methoxychlor, p,p’-DDE and the organofluorine one- trifluralin -with purity higher than 96.0% and PCBs 28, 52, 118, 138 and 180 with purity higher the 96.0% were acquired from Sigma, reference standards are acquired from AccuuStandards®, Inc Lot # 209111013 and AbsoluteStandards®, Inc Lot # 032409. Proficiency testing samples are from AbsoluteStandards®, Inc Lot # 091608. Sodium chloride, Sigma, sodium thiosulfate, Merck. Methanol, Hexane and acetone (HPLC grade) were from Sigma. Ultrapure water used was from Milli-Q system model: Milli-Q Gradient A10, Elix 3UV and Tank 60L, Serial NO: F7AN24007K F7BN90274I, USA.
Preparation of standards
Standard solution, Stock: These solutions prepared from pure standard materials of each PCBs and Pesticides (1000 μg/ml).
• By accurately weighting about 1.0 mg of pure material. Dissolve the material in 1 ml of methanol absolute in 1.5 ml vials; the weight is used without correction to calculate the concentration of the stock.
• Store standard solution in freezer and protect from light. Stock standard solution should be checked frequently for assign degradation or evaporation, especially prior to preparation calibration standard from them.
• Store standard solution must be replaced if comparison with checked standard indicates a problem.
Standard solution, secondary dilution: Use the stock standard solution to prepare secondary dilution standard solution in methanol and check frequently for singe of degradation evaporation especially just before preparing calibration standard.
Sample preparation and collection
• When sampling from a water tap, open the tap and allow the system to flush until the water temperature has stabilized (usually about 10 min). Adjust the flow to about 500 ml/min and collect samples from the flowing stream.
• When sampling drinking, surface, well , and waste water a sampling water apparatus model Easy-Load® Masterflex® USE 15, 24 TUBING Model:7518-12 part No: 4,813,855 Assembled in USA is used.
Sample preservation and storage
In 1 L empty bottle, add 8 mL of 1 M sodium thiosulfate just prior to sample collection. The samples must be chilled to 4°C at the time of collection and maintained at that temperature until the analyst is prepared for the extraction process. Store samples and extracts at 4°C until analysis has been completed. Extract all samples as soon as possible after collection. Results of holding time studies suggest that all analytes were stable for 14 days when stored under these conditions.
Instrumentation
GC separation was performed using Gas Chromatograph from Agilent Technologies Model 7890A equipped with temperature programming capability, splitless injector, capillary column, and Mass Quadrupole Spectrometry detector Model 5975B. A computer data system is MSD Chem Station E.0201.1177 used for measuring peak areas and heights.
Gas chromatograph parameters
The analytical columns used were DB-1701P (30 m × 0.25 mm × 0.25 μm), Agilent Part No.122-7732 as a primary column and DB-5ms (30 m × 0.25 mm × 0.25 μm), Agilent Part No.122-5532 as a secondary one, the oven temperature was set at 60°C for 0.50 min, increased to 140°C at 120°C/min, 260°C at 11°C/min then to 260°C for 5.5 min. The volume of the injected sample was 1 μL in split less mode. The injector temperature was set at 250°C. Helium (99.999%, purity) was used as carrier constant flow, 1 mLmin-1.
Mass spectrometer parameter
The mass spectrometer was operated in electron impact (70 eV of ion energy), with 4.0 min solvent delay, SIM acquisition mode, mass quadruple and mass source kept at 150°C and 230°C.
Data analysis
Analysis of data is done using Microsoft® Office Excel 2003 (11.5612.5606) Part of Microsoft Office Professional Edition 2003, Product ID: 73931-640-0000106-75603.
Extraction procedure
Stored samples are removed from the fridge and allow to equilibrate to room temperature. To 35 ml of each sample, add 6 g NaCl in the separating funnel. Recap and dissolve the NaCl by inverting and shaking several times (approximately 30 sec). Remove the cap, add 2 ml of n-hexane recap and shake vigorously by hand for 2 min, inverting the separating funnel while shaking. Stand the separating funnels upright and allows the water and hexane phases to separate. Transfer 0.5 ml of hexane layer into an auto sampler vial; inject 1 μl portions into the gas chromatograph for analysis.
For transformer oil samples, solid phase extraction procedure using AccuBOND II FLORISIL Cartridges provided from Agilent Part No. 188-2460 was applied using the following method: weigh 0.2 gm of the transformer oil sample, pass through the FLORISIL Cartridges followed by flush five times with 2 mL aliquots of n-Hexane, the eluent was collected in a 10 mL volumetric flask then completed to the mark and mix thoroughly prior to the GC/MS analysis.
Results and Discussion
GC-MS separation
To confirm the retention times of the POPs – under investigation- OCPs, OFP and PCBs, a mass range of 50-500 m/z was scanned. Thereafter the SIM mode was applied to monitor the mixture. The selected ions (m/z) used for confirmation and quantification are cited in Table 2. POPs- under investigation- are eluted from the column in the following order: Hexachlorocyclopentadiene, Hexachlorobenzene, Trifluralin, lindane, PCB 28, Heptachlor, PCB 52, Aldrin, Alachlor, Heptachlorepoxide, p,p’-DDE, Dieldrin, PCB 118, PCB 138, Endrin, PCB180 and Methoxychlor (Figure 1). It is worthy to mention that the time needed for the chromatographic analysis is less than 17 min.
Optimization of the extraction procedure
In order to achieve the highest recoveries for the compounds under investigation, different organic solvents have been tried for this purpose, among them hexane and dichloromethane. With hexane only 2 ml for 2 min gave rise to best recovery all the analytes under investigation while using dichloromethane it needs 6.3 ml for 6 min is required.
Method validation
Method validation is performed to provide evidence that the method is fit for the purpose for which it is used. Since the key challenges in the validated methods is that only well-characterized reference materials with well documented purities should be used during method validation activities, all steps are validated using reference materials, this includes specificity, accuracy, linearity, precision, range, detection limit, quantitation limit and robustness.
In order to assess these parameters, the method was therefore tested for Linearity, range Table 3. The analytical method demonstrated initial and extended validation as being capable of providing mean recovery values at each spiking level within the range 70-120%, spiked recovery experiments are performed (Figure 2), In order to check the precision of the proposed method, a minimum of 5 replicates is performed Table 4 summarizes these data.
Method Detection Limit (MDL) and Limit of Detection (LOD) - for the analytes under investigation - are cited in Table 5. Under normal conditions, reproducibility of data is tested in order to be sure that the method is robust. By changing the pH of the extract and the oven temperature, laboratory reproducibility as RSD% was found to be ≤ 20%, for all compounds indicating that the method is robust.
Quality control
Validation are supported and extended by method performance verification during analysis through analytical quality control AQC. AQC data are used to validate the extension of the method to new analytes viz Hexachlorocyclopentadiene, p,p’-DDE and trifluralin, new matrices viz. waste water and transformer oils and also to new concentration levels. Minimum quality control requirements are checked for POPs determination these include.
Analysis of laboratory reagent blanks (LRB)
All glassware and reagent interferences are taken under control by checking the extract and the reagent for any source of contamination within the retention time window of all analytes under investigation.
Initial demonstration of capability
This has been checked at different spiking levels which have been selected at a concentration level about ten times the estimate detection limit or at the maximum contaminant level for each analyte. For all aliquots analyzed, the recovery value for each analyte falls in the range 70-120%.
Analysis of laboratory fortified blanks (LFB)
Table 6 illustrates the spiking concentration of each analyte in the LFB sample with the calculated accuracy as percent recovery (%R). The recovery of all analytes under investigation fall inside the control limits (X ± 3S); where X is the mean percent recovery and S is the standard deviation of the percent recovery.
Analysis of laboratory fortified sample matrix (LFM)
To assess analytes recovery, a known spike of Aldrin, Alachlor , Heptachlorepoxide, Dieldrin, Methoxychlor, p,p’ - DDE, PCB 28, PCB 52, PCB 118, PCB 138 and PCB 180 is added to waste water matrix as shown in Figure 3.
Analysis of reference materials (QCS) and proficiency testing
In order to assure the correct execution of the whole procedure for each individual sample and the correct injection of each final sample extract in the GC system, the use of one or more quality control (QC- ) standards is utilized. These compounds are added at different steps of the procedure e.g., to the samples prior to extraction as surrogate standard or to the final sample extract just before injection as instrument internal standards. Analysis of QC samples provided from Accu Standard®, and Absolute Standards® Inc., are shown in Figure 4 and Table 7 respectively. In addition sharing in a proficiency testing program provided from Absolute Standards®, Inc., was successfully achieved as shown in Figure 5 and Table 8 indicating that our results meet the performance criteria for the provided QC sample datasheets.
Qualifying results with uncertainty data
Measurement uncertainty is a quantitative indicator of the confidence in the analytical data and describes the range around a reported or experimental result within which the true value can be expected to lie within a defined probability (confidence level). Uncertainty ranges must take into consideration all sources of error. To determine the uncertainty associated with analytical results, the available sufficient data derived from method validation /verification, inter-laboratory studies (e.g., proficiency tests provided from Absolute Standards®, Inc proficiency testing provider) and in-house quality control tests provided from Accuu Standard® are applied to estimate the uncertainties. Uncertainty associated with repeatability of measurements for these true samples in the main elements of the uncertainty budget. The expanded uncertainty is calculated and cited in Table 9 as follows:
image
Where K: is the coverage factor (it has a value of 2 at 95% confidence level); SD: is the standard deviations; n: is the number of measurements.
Since uncertainty tends to be greater at lower levels, especially as the LOQ is approached. It was therefore necessary to generate uncertainty data for a range of concentrations if typical uncertainty is to be provided for a wide range of analytes data.
Real samples analysis
Nile River water (Assiut, Egypt), ground water (Assiut), waste water (Zenar, Assiut), tape water from our laboratory and Transformer oils (Cemex, Assiut) were analyzed using the proposed method. Chromatograms are shown in Figure 6 and data are cited in Table 10.
According to the MCL for water provided in Table 1 and the International permissible concentration of PCB’s in Transformer oil as cited in the Environmental Protection Agency, EPA, USA regulations that is: >50 ppm= Non-PCB transformer, 50-500 ppm = PCB-contaminated transformer and ≥ 500 ppm= Repeat the reclassification process until the transformer can be classified as to non-PCB or a PCB-contaminated status; or remove the transformer from service, it is clear that for water samples a contamination with Aldrin and Dieldrin is noticeable while some of the transformer oils are to be considered as PCB-contaminated transformers.
Conclusion
The validation and application of GC MS method in the Selected Ion Monitoring (SIM) mode for the simultaneous determination of the pesticides and PCBs has been evaluated in this study. The optimal conditions of extraction techniques have been obtained. The established method can be applied to determine the concentration of the pesticides in real water samples and transformer oils. The recoveries in water are from 70% to 120%. Adequate repeatability, good linearity and the low detection limits prove the capability and credibility for the validation of method by analyzing proficiency testing samples provided from AbsolueStandards®, Inc.
Comparing the data produced from our proposed method , using the universal detector MS in the SIM mode, with those from the ASTM D 5175 method obtained with electron capture detector, Table 11 gave evidence that our proposed method may solve many environmental pollution problems for ultra- trace pollutants since it can reach subppb and ppt levels.
Acknowledgements
The authors would like to thank the Management of Supporting Excellence Unit, Egyptian Ministry of Higher Education for supporting this work through the grant provided (Project CEP1-043-ASSU, 2014).
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Table 9 Table 10 Table 11

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