DEVELOPPEMENT AND VALIDATION OF A QUECHERS EXTRACTION BASED GAZ CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY ``GC-MS/MS`` METHOD FOR THE DETERMINATION OF 7 POLYCHLORINATED BIPHENYLS RESIDUES IN BIVALVES "OYTERS"

* Fatima Habti 1 , Soumia Belouafa 1 , Ahmed Bennamara 1 , Mustapha Tarhy 2 , Noureddine Fatini 2 , Abdelmjid Bahloul And Abdelmjid Abourriche 1 . 1. Laboratory of Biomolecules and Organic Synthesis. Department of Chemistry. Faculty of Sciences Ben M'Sik. University Hassan II of Casablanca. Avenue Driss El Harti B.P 7955, Sidi Othmane Casablanca, Morocco. 2. Moroccan Laboratory of Agriculture (LABOMAG) . Department of Pesticide Residus.Km 10.500 Route de Zenata, Rue ''J'' n°1 Ain Sebaa, Casablanca, Morocco. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5 (7), 1160-1170 1161 PCBs that are widely distributed in the environment are classified as persistent organic pollutants (POPs), such as dioxins and polycyclic aromatic hydrocarbons (PAHs). There are two types of PCBs according to their mechanism of action: PCBs known as "Dioxin-Like" or PCB-DL. Their mechanism of action on cells is similar to that of dioxins: it binds to the same cell receptor and their toxicity is expressed as toxic equivalent factor in relation to the toxicity of TCDD (2,3,7,8-Tetrachloro-Dibenzo para-Dioxin) more commonly known as Seveso dioxin while PCBs called "Non Dioxin-Like" or PCB-NDL. They are found in larger quantities in river fish than Dioxin-Like PCBs. Of all PCBs, seven molecules are particularly found in contaminated products. They constitute indicator PCBs whose dosage is used to quantify the contamination of the products. [3,6,9] Depending on the nature of the studied material, PCB analysis is carried out according to a more or less complex protocol comprising several steps: extraction of residues, purification of the extract, possible separation into groups of compounds, instrumental analysis by coupled gas chromatography triple quadrupole mass spectrometry and ultimately chemical confnmation. [4,5,10] A variety of GC and HPLC methods have been developed for multi-residue determination of organochlorine pesticides employing a variety of sample preparation and cleanup techniques. In recent years, due to its rapidness, easy to use, effectiveness, reliability and safety, the QuEChERS method, as a new extraction method, has become a new sample pretreatment technology that is extensively adopted at home and abroad and is also widely applied in the gas or liquid chromatographic analysis for various pesticides. [1] [7] [9] In this work, the QuEChERS-GC-MS-MS method is used and validated to quickly determine the polychlorinated biphenyls residues : 2,4,4'-trichlorobiphenyl, 2,4',(-tetrachlorobiphenyl, 2,2',4,5,5'-Pentachlorobiphenyl, 2,3',3,4,4',5-Pentachlorobiphenyl, 2,2',3,4,4',5'-Hexachlorobiphenyl, 2,2',4,4',5,5'-Hexachlorobiphenyl, 2,2',3,4,4',5,5'-Hexachlorobiphenyl) in Moroccan bivalves "OYTERS".

Materials and Methods:-
Equipment:-GC-MS/MS system Agilent Triple Quadrupole Mass Spectrometry 7890Awith Sensor Technologies 7000 MSD were used. Analysis is performed in MRM "Multiple reaction monitoring". The instrument is equipped with injector type Multi mode and an automatic sample changer 7693. The software type is mass hunter allows the acquisition to data processing and reporting. A refrigerated centrifuge SIGMA 4 bucketsand an Ultra-Turrax homogenizer were used for in extraction and purification steps.
The 50ml tubes are used for the deposition of the extract, 2 ml tubes of centrifuge EFF and an analytical balance for weighing the samples.

Analysis by GC-MS/MS:-
The analysis of the molecules is done by MRM "Multiple reaction monitoring". A database of molecules provided by Agilent is composed by: The conditions for chromatographic analyzes are detailed in Table 1: Chemicals:-Acetonitrile is used as solvent for PCBs residues analysis. Also, the mixture of salts "anhydrous sulphate crystallized magnesium, hydrogen citrate, disodium citrate sesquihydrate and dihydratetrisodium" are used to saturate the aqueous phase and thus to achieve separation with the organic phase. This product is calcined at about 550 ° C in an oven to remove all traces of phthalates, eg. FlukaNo. 63136. Adsorbents bulk of PSA (Primary and Secondary Amine) and adsorbent Suplclean LC-18, which are used for cleanup, have been selected for their physicochemical properties. The Preparation of standard solutions is based on the purity of standard organochlorine pesticides ( Table  2).

Preparation of standard solutions:-
The stock solutions of compounds were prepared individually from compounds of high purity, at 1 mg/mL in acetone. From these solutions, a standard multi-substance mixture was prepared in 10 ppm of each compound in acetone. This mixture was used to determine the characteristics of ions of Q1 and Q3 SIM modes and transitions MRM mode.
In order to correctly assess the quantification limits and establish relevant calibration ranges, the mixture solutions were prepared in acetone depending on the compounds response coefficient. A wide range of concentrations was used. The previously prepared standard solutions are added to extracts of blank samples to compensate for the effect of matrix samples. The amount of extracts of blank samples used is equivalent to the extract of the sample concentration ready for analysis. These analytical standard solutions also contain quantities of internal standard equivalent to that contained in the extract of the sample ready for injection.

Sample preparation Method:-
Sample of oysters harvested from Walidia (Morocco) region, was placed in containers containing seawater and stored at -20 ° C until use. After crushing, ground fibers were stored in a refrigerator (-20 °C).

Extraction Method:-
The procedure for sample preparation is described in Figure 1.Weigh 10g  0.03g of the ground and homogenized sample, then put it into a centrifuge tube "FEP" 50 ml. Add, with a micropipette in all samples except the "blank samples fortified and White reactive", an amount of internal standard "or surrogate trace the sulfotep". Then, add a volume of 10ml of the acetonitrile extraction solution, on the Farm tubes and shake vigorously for one minute.
Next, add the mixture of previously prepared buffer salts (4g of anhydrous magnesium sulfate, 1 g of NaCl, 0.5g of Disodium hydrogen citrate sesquihytdrate and 1 g of trisodium citrate dehydrate) to the prepared suspension, on Farm tubes, shake vigorously for one minute without delay and ensure that the solvent reacts well with the entire 1163 sample and that agglomerates formed crystals were sufficiently dispersed.Then, centrifuge tubes at 3000rpm for 2 minutes.

Cleanup
An aliquot of the upper organic phase is transferred to a tube containing "150 mg of MgSO4, 50mg PSA and 100mg LC18" per ml of extract. After a mixing and centrifuging step, the purified extract is analyzed by GC-MSMS.

Figure 1:-Organigramme of sample preparation protocol according to QuEChERS method
Chromatographic Analysis:-The injection of 0.2ppm mixed standard (7 PCBs) gave results presented in Figure 2 and Table 3.  This validation was performed by the accuracy, precision, linearity and limit of quantification (LOQ). Accuracy and precision data were obtained with recovery studies by spiking samples with polychlorinated biphenyls standards at levels of 0.2ppm, 0.1ppm,0.05ppm,0.02ppm,0.01ppm. The spiked samples were analyzed in 5 replicates. Precision of the method was evaluated through the relative standard deviations (%RSD) associated with PCBs measurements during recovery.
Linearity was determined by plotting calibration curve with standard solutions in acetonitrile containing five different concentrations (0.01, 0.02, 0.05, 0.1 and 0.2 ppm). Five injections were made at each of the five concentration levels.

Statistical Analysis:-
The data were statically analyzed by using one way ANOVA. All statistical calculations have been done using Excel.    The calibration function was verified by the Fisher test (Table 5). F ratio is less than the critical value of F corresponding to Fisher variable for a risk of 1%. Then, the results show that the linear range is validated and the regression model is acceptable.

Results:-
In conclusion, the calibration function is validated on the studied domain with a 1% risk because the error of the model is significantly negligible compared to the experimental error observed and the criterion observed is less than the critical value. Then the calibration function is linear over the analyzed range [0,01; 0.2 ppm]. (Figure 3 and 4) 1167 It is necessary to check that all the biases observed on each analyzed standard are acceptable from an acceptable maximum deviation fixed by the exporter. All biases are lower than the fixed EMA for each standard then the calibration function is considered acceptable in the studied field. In conclusion R 2 > 0.995 for all molecules PCBs and C model is less than V critical model Thus the biases observed are lower than the EMA set by the experimenter. The sensitivity of the used apparatus was estimated by determining limit of quantification (LOQ=0.01ppm) ( Table  6). These low LOQ indicate good sensitivity.  Table 6 shows that the two inequalities ( ̿ -2 S LQ > LQ -60% * LQ and ̿ + 2 S LQ < LQ + 60% * LQ] for all PCBs are verified. From the recovery study (Table 7), it is clear that the method is accurate for quantitative estimation of PCBs in bivalves as the statistical parameters are within the acceptance range (from 70% to 120%) according to the most recent EU guidelines (SANCO, 2015). For the study of accuracy an example of PCB 28 which is representative for all the other molecules will be shown. The same results were obtained for all PCBs. The accuracy of the method is investigated on samples of reference value at 0.01ppm, 0.05ppm and 0.16ppm. The used experimental values for the study of accuracy at these three levels of concentrations are derived from the LQ study and the study of yields. The results are interpreted to verify the accuracy of the method against an acceptable deviation around each reference value set by the laboratory (Tables 8 and 9).    (Table 4). The slopes values close to 1 .Therefore, linearity has also been demonstrated for all PCBs in the range (0.01 to 0.2 ppm) because the coefficient determination are more than 0.995 (r 2 ). The calibration function was verified by the Fisher test (Table 5). F ratio is less than the critical value of F corresponding to Fisher variable for a risk of 1%. And also by a comparison of all observed biases on each standard analyzed are acceptable from an acceptable maximum deviation set by the exporter.
All biases are lower than the fixed EMA for each standard, the calibration function is considered acceptable in the field studied.Then, the results show that the linear range is validated and the regression model is acceptable. The sensitivity of the apparatus used was estimated by determining limit of quantification (LOQ=0.01ppm) ( Table 6). These low LOQ indicate good sensitivity. Table 7 shows that the two inequalities ( ̿ -2 S LQ > LQ -60% * LQ and ̿ + 2 S LQ < LQ + 60% * LQ﴿ are verified. Thus the accuracy of LOQ at 0.01ppm is verified. From the recovery study (Table 8), it is clear that the method is accurate for quantitative estimation of 7 PCBs in bivalves as the statistical parameters are within the acceptance range (from 70% to 120%) according to the most recent EU guidelines (SANCO, 2015). The calculated accuracy error (Table 9) is less than 2. Then, it is considered insignificant [17]. Therefore, the uncertainty associated to accuracy of the method is equal to the uncertainty of the reference material used for testing accuracy study. Table 9 shows the value of Standard deviation EN ≤ Criterion=2 is verified. Therefore the method is just and shows that the two inequalities (Z + 2 SLQ <Réf + EMA and Z -2 SLQ >Réf-EMA﴿ are verified. Thus the accuracy at 0.05 and 0.16ppm are acceptable. The table below shows the example of PCB 28, which is a summary table of all the results of validation criteria of the same PCBs.  The data processing provided precise results and was corroded effectively for the validation of polychlorinated biphenyls. The procedure was successfully applied to the reel bivalve sample.
In conclusion, the QuEChERS method remains a method: fast, easy to implement, inexpensive, specific, exact, sensitive, multi-residual, repeatable and reproducible. According to the results of validation obtained the method proves effective for the detection and the dosage of the 7 polychlorinated biphenyls studied in the oysters of Walidia in "MOROCCO".
The QuEChERS method (Quick, Easy, Cheap, Effective, Rugged and Safe) has been readily accepted by many pesticide residue analysts and many quality control laboratories of marine products in particular and also that are intended for export.
This analytical method should be extended and used for the validation of other bivalves and other organic pollutants.