Development and validation of modified QuEChERS method coupled with GC-MS / MS for 123 pesticide residues in food

In this study, a gas chromatography-tandem mass spectrometry (GCMS/MS) instrument, which has been widely used in recent years and has high separation power, selectivity and ability to identify pesticides has been used. It is aimed that the main criterion of this analytical method, in which the QuEChERS methodology is used, is applicable to fast, easy, cheap, environmentally friendly and different matrices. At the same time with this method, 123 pesticide residues and their degradation products were quantitatively assayed by GC-MS/MS as well as method validations in tomatoe, lemon, lettuce, almonds, raisins, honey, green pepper, milk and flour. Tomatoe was selected as potential reference matrixes for the target. The steps of concentration and solvent exchange were performed in the resultant extracts for the purpose of improving analytical performance in terms of recovery, precision, linearity, of reducing the amount of co-extracts. Multiple reaction monitoring (MRM) was used to identify and quantify the pesticides. The samples were extracted with 1% acetic acid in acetonitrile, anhydrous magnesium acetate, anhydrous magnesium sulfate and clearing agent. For all pesticides, good linear calibrations with coefficients (R2) ≥0.99 for nearly all of the analytes were obtained. Limit of quantitation of most of the pesticides were in the range of 5-10 ng/g, and recovery of the method validation accuracy parameter was done at two different concentrations 10 ng/g and 50 ng/g were 88.6 99.7% and CV 1.60 – 14.0%. ARTICLE HISTORY Received: 25 December 2017 Revised: 19 March 2018 Accepted: 23 March 2018


INTRODUCTION
Every kind of chemical compound used to protect agricultural products from disease, harmful and foreign weeds is called pesticide.A pesticide is known to be any compound or mixture of compounds that prevents, removes or protects from the spread of any unwanted organism (pest).The usage of pesticides in agriculture after World War II, the world has multiplied to increase food production.Since then, the development of different types of pesticides belonging to various groups has become important [1].
As a result of producers' unconscious and excessive use of pesticides, resistant populations are formed, natural enemies are adversely affected and harmful effects occur in terms of environment and human health.Regular use of pesticides is detrimental to the ecosystem.Many international organizations and countries are extremely worried about pesticide residues.The pesticide maximum residue limit (MRL) determination [2,3] is being undertaken to protect public health and ensure food safety.Numerous studies have been carried out naturally on the simultaneous determination of analytical techniques, especially multiple residues, in the detection of pesticide residues [4,5].This study is quite up to date on these aspects.
Various extraction methods have been stated in the literature including hydrolysis with chloric or sulfuric acid, soxhlet extraction, liquid-liquid extraction (LLE), extraction with organic solvent directly from the solid matrix, and more recently solid-phase microextraction (SPME) [6][7][8][9][10].Purification might be needed to remove co-eluted matrix material and reduce analytical background noise.To date, purification techniques usually included elution of sample extracts with hexane and dichloromethane on chromatographic columns packed with acidified silica gel, deactivated alumina or florisil [11,12].Recently, an attempt was made to replace these laborintensive clean-up steps by LLE or purification on solid-phase extraction (SPE) cartridges [10].Different extraction and measurement methods have been used by various scientists for the detection of multiple classes of pesticides in many food, vegetables and fruit varieties [13,14].
In 2003, Anastassiades et al. [15] developed a fast, easy, inexpensive, effective, robust and secure (QuEChERS) method to overcome the critical deficiencies and practical limitations of existing methods.Then, Lehotay et al. 2010 developed a sample preparation method with the citrate-buffered QuEChERS procedure [16].The main criterion for choosing any methodology is that analytical method is applicable to fast, easy, inexpensive and different matrices.
Developing a new methodology requires the resolution of a large number of problems.Like extraction solvent selection.Solvents used to identify pesticide residues in food matrices [17][18][19], and these solvents provide high analytical recovery.Acetone can be mixed with water, but it is not possible to separate water from the solvent without using apolar solvents.On the other hand, it is the part where ethyl acetate is mixed with water.It causes unnecessary addition of apolar solvents to separate water, but most of the highly polarized pesticides are not separated.The acetonitrile extracts of foods contain less interfering substances than ethyl acetate and acetone extracts and acetonitrile can be separated from the water quite easily (salt precipitation), which is the preferred extraction solvent of acetonitrile methodologies.The aim of the study is to develop and validate the modified QuEChERS method for 123 pesticide residue in food using GC-MS/MS.
The samples used in the study (tomatoes, lemons, lettuce, almonds, raisins, honey, green pepper, milk and flour) were supplied without pesticides from Fethiye, Datça, Marmaris.Fruits and vegetables were purchased from producers of organic farming.This study has been selected as representative matrices by considering the directive of the European Union.

Pesticide Standards Main Stock Solution
All pesticide standard substances Dr. Ehrenstorfer are certified reference materials are taken from local supplier.The pesticide standards were weighed in a 50 mL volumetric flask as approximately 10 mg with a precision of 0.01 mg.The volume was completed with methanol.The main stock solutions are stored at -18 °C.

Pesticide Mix Solutions
The pesticide was withdrawn with an automatic pipette such that the concentration of each of the standard main stock solutions was 1 mg L -1 (1 ppm).The pesticide mix solutions for GC-MS/MS were prepared separately in methanol.The pesticide mixture solutions were stored at 4 °C.

Pesticide Working Solutions
To determine the GC-MS/MS conditions of the pesticides, the concentrations from the parent stock solutions were set to be about 500 ng mL-1 (ppb).The target ion, qualifier ions, collision energies and retention time (Rt) were determined separately in the device.For the method-device optimization, the acquisition method was created by dividing it into eight time segments.

Method
GC-MS/MS instrument coupled with Agilent Technologies 7890A GC gas chromatography 7000B Triple Quadrupole mass spectrometer was used in the study.Agilent Technologies 7693 autosampler and multi-mode inlet system were used in the system.This method involves extraction of the extract using acetonitrile pre-extraction and separating solid phase extraction.For liquid-liquid separation, salt precipitation is carried out using anhydrous magnesium sulfate, so that the water is separated from the sample.
Most of the methods applied worldwide at the clean-up level use Primary Secondary Amine (PSA) in most cases.PSA is an expensive chemical that significantly increases the cost of analysis.In this study, the sample extracts have been studied using silica-gel, which are cheaper, simple and effective chemicals, with GC-MS/MS to detect multiple pesticide residues.
The most common approach to derive from the matrix effect is to use matrix-match calibration standards [20,21].However, a large number of blank matrices are required to work with matrix-match, which requires extra extraction.In this study, a separate matrix-match calibration curve is used for each matrix.Thus, it was already known how each pesticide will behave when interacting with the matrix.
Approximately 1 kg of the sample is disintegrated and homogenized with the aid of the sample shredder.Fifteen grams of the homogenized sample is transferred to a 50 mL falcon tupe.Add 15 mL 1% acetic acid in acetonitrile.It is vigorously shaken for 1.5 min.After addition of 1.5 g of anhydrous magnesium acetate and 6 g of anhydrous magnesium sulfate, the mixture is shaken vigorously for 1.5 min and 150 μL (5 ng mL -1 ) of Internal Standard Solution is added.Centrifuge at 4000 rpm for 5 min.Then transfer to 8 mL of extraction tubes then add 400 mg of clearing agent (Silica-gel, PSA, C18 or GCB) and 1200 mg of anhydrous magnesium sulfate, shake, centrifuge.The extract is transferred to 2 mL vials and 50 μL 3-phenyl phosphate (TPP) is added, for the analysis using GC-MS/MS.

Instrument and Pesticide Optimization
In this study, the parent ion, daughter ions, cone voltage and collision energies and retention times (Rt) of each pesticide were determined separately in the device.Multiple reaction monitoring (MRMs) were generated for the analysis method.The process method was established in which the ions of each pesticide were written at the time of retention.Retention time, parent and precursor ions, collision voltages for each of the pesticide were identified in order to obtain maximum signal.Standard main stock solutions were prepared in the concentration of 50-500 ng mL -1 and precursor and product ions of each the pesticide were determined in GC-MS/MS using SCAN mode.Ions were examined in the detected peak, and compared with molecular weight of each pesticide.It is examined by looking at the structure of the molecule, whether it is equal to the molecular weight or fragmented from the molecule.In order to detect target ion and breakdown ions in the MRM mode, different impact energy is tried in increments of 5 V between 5 and 30 for each transition of the analytes to detect the collision energy and the collision energy producing the highest area was detected.An example of this work is illustrated below for propham in Figure 1.
The pesticide representative, propham, three transitions were examined.Three different collision energies were tried, 10V, 20V, 30V.First is 179.1 > 92.2 m/z.The highest peak area was detected as 3.209 abundance at 20 V. Second is 93.0 > 66.0 m/z.The highest peak area was detected as 198.532 abundance at 20 V.With this study, for propham; 93 > 66 m/z and 93> 65 m/z transitions and the result of the collision energy optimization study have been determined.

Method Optimization
Tomato was used as the first matrix in the method optimization study.Four parallel runs according to the sample preparation procedure.The specimens were run separately for GC-MS/MS and the spikes at 50 ng g -1 were made in parallel from GC mixtures.Approximately 1 kg sample was crushed and homogenized with a chopper.A 15-g portion of the homogenized sample was weighed in an analytical balance with a 50 mL falcon tube and 150 μL 5 ppm di ethatyl ethyl (DEE) (internal standard) and 750 μL 1 ppm pesticide solution were added.15 mL of acetonitrile containing 1% acetic acid was added.It was vigorously shaken by hand for 1.5 min.The previously weighed 1.5 g anhydrous sodium acetate and 6 g anhydrous.Magnesium sulfate was added, then vigorously shaken again manually for 1.5 min and centrifuged for 5 min at 4000 rpm.Previously, a 15 mL centrifuge tube was prepared by weighing 400 mg of cleaning agent (PSA/Silica gel) and 1200 mg of anhydrous magnesium sulfate, and after centrifugation, 8 mL of the supernatant was transferred, then shaken by hand and centrifuged at 4000 rpm for 5 min. 1 mL of the supernatant was transferred to 2 mL vials and 50 μL TPP was added.The tube was agitated in the mixer.The vial was analyzed in GC-MS/MS.

RESULTS AND DISCUSSION
In this study, all of the analyzed pesticide analytes could be successfully chromatographically separated using an Agilent Technologies 7890A GC gas chromatography and HP-5MS UI (5% phenyl methylsiloxane) 15 m x 250 µm x 0.25 µm capillary column and oven temperature program in MRM mode.The oven program was established in GC for chromatographic separation of analytes.Multiple reaction monitoring (MRM) was used to identify and quantify the pesticides with a precursor ion and at least two product ions in Agilent Technologies 7000B Triple Quadrupole MS/MS.Firstly, the instrument method optimization was accomplished.The pesticide standard working solutions were prepared separately for each of the analte, and then tested to determine the precursor and product ions, collision energies retention time in GC-MS/MS with scan mode.Ions were evaluated in detected peak by comparing its molecular weight.The collision energies were detected for the each of transision in the range of 5-30 V by increments of 5V.The parameters of GC-MS/MS and data related to method validation were displayed in Table 1.Tomatoes, lemons, lettuce, almonds, raisins and honey were selected as representing food matrix for the validation of the method performance.The pesticide matrix solutions (tomato, lemon, lettuce, almond, raisins and honey) were made ten repetitions for three different days for each level at two different concentrations, with concentrations of 10 ng mL -1 and 50 ng mL - 1 for each analyte.
The specificity parameter of an assay is a measure of the extent to which the method can determine a particular compound in the analyzed matrices without interference from matrix components.The validation procedure should confirm the ability of the method to unequivocally assess the analyte in the presence of other components that may be present (for example, impurities, degradation products and matrix components).The chromatographic separation of all analytes from each other was accomplished successfully by an Agilent Technologies 7890A GC gas chromatography and HP-5MS 5% phenyl methylsiloxane (15 m x 250 µm x 0.25 µm) the capillary column, oven program in MRM mode.
The selectivity is that a method analyzes a given compound without interfering with the matrix components in the matrix.It is accomplished by Agilent Technologies 7890A GC gas chromatography 7000B MS/MS Triple Quadrupole GC-MS/MS system.The identification, validation, calculation of an analyte takes place with at least one precursor ion and at least two product ions in the MRM mode.
The limit of detection (LOD) is the limit lowest residue concentration that result could not submit.The limit of quantitation (LOQ) is the lowest concentration at which the analyte can not only be reliably detected but at which some predefined goals for bias and imprecision are met [22].In the European Commission Regulation (EC/299) [23], maximum residue limits (MRLs) are specified using LOQ values.
With the method validation studies, the LOD -LOQ parameter was used in all studied matrices.In the repeatability study of the analysis, the recovery of the method validation accuracy parameter was done at two different concentrations, 10 ng g -1 and 50 ng g -1 , with percent recovery and % CV values.The recovery results obtained in GC-MS/MS for contaminants for authenticity and precision subparameters were 88.6 -99.7% and CV 1.60 -14.0%.
In the reproducibility study data is evaluated, the recovery obtained for each residue at 10 ng g -1 of the matrix containing GC-MS/MS pesticides is 70.1% -120.0%and CV% 1.80 -31.92%.The recovery achieved for 50 ng g -1 level is 70.7% -120.7% and CV is 2.48 -27.59%.It was found that the recovery obtained for each residue in the concentration range of 10-50 ng g -1 of the matrix containing GC-MS / MS pesticides varied between 91.7-98.9% and CV 2.21-5.67%.Combining the recovery results from all the matrices and pesticides in GC-MS/MS resulted in 95.0% average recovery and 4.91% reproducibility % CVR of laboratory data.

CONCLUSION
This method is fast, both as an analysis method and as a method of reading on devices.The device can be analyzed in about 16 minutes in the European Union and in Russia with about 123 pesticides (with their metabolites) that are required for analysis.
Thus, approximately 123 pesticides were validated and quantified in GC-MS/MS.It covers a wide range of products from selected indications to vegetable origin food and food of animal origin.In addition, the method reduces time for analysis by providing time gain.On the other hand, the analysis cost has been reduced, and significant gains have been achieved on the basis of day-month-year as well as instrument consumables, analytical column, vial, working life of all working parts etc.

Table 1 .
GC-MS/MS instrument and the pesticide method validation parameters