Le Huong Ly THAI NGUYEN UNIVERSITY OF AGRICULTURAL AND FORESTRY ADVANED EDUCATION PROGRAM BACHELOR THESIS STUDY ON A HIGHLY SENSITIVE METHOD FOR DETERMINATION OF PESTICIDE RESIDUES IN TEA BY GAS[.]
INTRODUCTION
Overview
Vietnam, as a developing country, relies heavily on its agricultural sector, which often involves the use of plant protection chemicals to combat pests and diseases While pesticides can offer significant benefits for various agricultural products, it is concerning that approximately 200 out of 5,000 pesticides pose direct risks to human health Ensuring that farmers apply these chemicals safely and at the correct dosage is crucial, yet many consumers remain confused about the safety of pesticide-treated products due to widespread rumors The effectiveness of pesticide use also varies based on the knowledge and practices of individuals in different regions Therefore, it is essential to implement strict regulations on the use of safe and effective plant protection products to protect human health and the environment, a pressing issue that demands attention.
Controlling pesticide residues in food, especially tea, is an urgent priority As a widely cultivated crop, tea farmers frequently use pesticides to manage pests and enhance productivity However, these residues pose significant pollution risks Protecting consumer health is paramount, necessitating effective measures to minimize pesticide levels in food products, particularly tea Additionally, it is essential to monitor and regulate the presence of unsafe tea in both domestic and export markets.
Due to economic pressures, there is a high reliance on pesticide use to control insect populations, often resulting in the application of these chemicals just days before tea leaves are harvested, posing health risks Various methods exist for detecting pesticide residues in vegetables and tea, including traditional techniques and the QUECHERS method; however, these methods struggle to identify very low concentrations of pesticides This study proposes a highly sensitive approach for determining pesticide residues in tea using gas chromatography-mass spectrometry (GC/MS), focusing on three banned substances: Chlorpyrifos, α-Endosulfan, and Bifenthrin, as identified by the Ministry of Health.
Implementing effective food toxin analysis methods can significantly benefit farmers by enhancing their knowledge and economic outcomes While scientists have developed analytical techniques tailored to various crops, the effectiveness of these methods can vary due to regional characteristics and the diverse care practices for different crop types.
The objective of my research to evaluate a sensitive and accurate method based on: limit of detection (LOD), limit of quantification (LOQ), linearity, repeatability (Sr), recovery (%R), and reproducibility
Why choose gas spectrometry (GC/MS) method to analyze pesticide residues in tea?
How to confirm validity of the method?
Due to constraints of time and sample responsiveness, the sample size is limited So, certain factors could not be studied in depth.
DOCUMENT REVIEW
Introduction about tea
The product of my optimal method research is tea
The tea tree, scientifically known as Camellia sinensis, is a plant valued for its leaves and buds, which are essential for tea production The term "sinensis" translates to "Chinese" in Latin, reflecting its origins Historically, it has also been referred to by the names Thea bohea and Thea viridis.
The growth cycle of tea trees comprises three key stages Initially, during the young tree stage, tea plants take 3 to 4 years to mature This is followed by the peak productivity phase, lasting 20 to 30 years, influenced by variety, soil, and climate As tea trees age, they produce fewer buds and lower yields, necessitating rejuvenation to extend their productive life by about 5 years Typically, tea can be harvested twice in spring, while summer and autumn allow for monthly harvests In winter, growth slows, limiting harvests to once every three months.
2.1.2 The chemistry composition of tea
Tea contains a complex array of chemical compounds, with thousands of components found in its leaves and buds During processing, these compounds undergo breakdown, interact to form complexes, and create new substances Key chemical components of tea include polyphenols, amino acids, enzymes, pigments, carbohydrates, methylxanthines, minerals, and volatile compounds.
Tea is one of the healthiest beverages globally, rich in antioxidants that offer numerous health benefits Regular consumption of tea can enhance overall health by improving memory, stabilizing blood pressure, regulating blood sugar levels, and aiding in weight loss Additionally, tea supports liver health, promotes recovery, prevents diseases, and contributes to healthy skin Notably, drinking tea can also help prevent cardiovascular diseases and lower the risk of stroke.
Plant protection drugs
2.2.1 Important roles of plant protection drugs
Pesticides are chemical products utilized in agriculture and forestry to prevent and eliminate harmful organisms affecting plants and agro-forestry products, as well as to regulate and stimulate plant growth While these plant protection drugs offer significant benefits to farmers, tea growers are increasingly relying on toxic chemical pesticides to boost production and combat pests and diseases, driven by high market demand and rising tea prices.
2.2.2 Some plant protection drugs used for tea
Here are some typical plant protection drugs used in tea production:
In the Circular No 50/2016/TT-BYT dated December 30, 2016 of the Minister of Health and the appendix [7], this active ingredient has CODE code of
Chlorpyrifos, an organophosphate pesticide with the chemical formula C9H11Cl3NO3PS, has been utilized globally since 1965 for both agricultural and non-agricultural applications In developing countries like Vietnam, it is predominantly employed on small farms for crops such as rice and fruit trees As of 2009, it was the most widely produced pesticide registered for sale by the Ministry of Agriculture and Rural Development The maximum residue limit (MRL) for Chlorpyrifos in fat and tea products is set at 2 mg/kg, and various companies in Vietnam manufacture this pesticide, with formulations containing a minimum of 94% Chlorpyrifos.
Chlorpyrifos, an organophosphate insecticide, inhibits acetylcholinesterase, impacting the nervous systems of insects and posing acute toxicity risks in larger quantities Even small amounts can lead to neurological effects in fetuses and children Exposure can cause mild to moderate corneal damage in the eyes and skin burns or irritation at high concentrations Chlorpyrifos poisoning affects the central nervous system, resulting in symptoms such as slurred speech, loss of reflexes, weakness, fatigue, involuntary muscle spasms, convulsions, and potential paralysis Severe cases may lead to incontinence, mental disorders, abnormal heart rhythms, unconsciousness, seizures, and coma, with respiratory failure or cardiac arrest being fatal outcomes.
Circular No 50/2016/TT-BYT, issued by the Minister of Health on December 30, 2016, outlines the regulations for the active ingredient Endosulfan, which has a CODE of 39 and an ADI index of 0.006 The maximum residue limit (MRL) for Endosulfan in tea products is set at 10 mg/kg This pesticide, classified as an organochlorine compound, poses neurotoxicity risks and appears as earthy brown cream or crystals with a rosin-like odor The chemical formula for α-Endosulfan is C9H6Cl6O3S, with a melting point of 208-210°C, and it is primarily used to control various insect pests Although it is nearly insoluble in water, Endosulfan can persist in soil and water for several years, adhering to clay particles Its mechanism of action involves repeated nerve discharge, which is linked to increased temperature, making it effective against harmful insects that can damage crops, food products, and textiles.
Endosulfan, at a concentration of 260 mg/kg, poses a fatal risk to humans due to its high toxicity, which can lead to both acute and chronic health issues, including reproductive effects and embryonic deformities Despite the ban on α-endosulfan in our country since 2011, farmers continue to use this pesticide indiscriminately, often mixing it with other chemicals or increasing its concentration to enhance its effectiveness.
In the Circular No.50/2016/TT-BYT dated December 30, 2016 of the Minister of Health and the appendix [7], this active ingredient has CODE code of
Bifenthrin, with an ADI index of 0-0.01, is a universal contact insecticide effective against harmful sucking insects like mosquitoes It is crucial to confirm the presence of residues in fat, as the maximum residue limit (MRL) for tea products is set at 30 mg/kg This compound has a toxic taste, which aids in its effectiveness in protecting plants.
Bifenthrin (C23H22ClF3O3) is a highly effective insecticide used to control a variety of pests, including aphids, worms, ants, and spiders, across agricultural and industrial applications Depending on the intended use, Bifenthrin can be formulated in different concentrations, such as Bifenthrin 17.1% combined with Imidacloprid 17.1% for stem borers in corn, or Bifenthrin 50g/l with Imidacloprid 250g/l for pests in rice Classified by the WHO as Type II (moderately hazardous), Bifenthrin poses significant health risks, including high toxicity if inhaled, ingested, or absorbed through the skin Detection methods, such as gas chromatography-mass spectrometry, can identify Bifenthrin in drinking water at a limit of 0.04 µg/L Caution is advised, as burning Bifenthrin can release toxic gases.
However, these pesticides were used with very high concentration in tea produced Therefore, determination of pesticides with high sensitive and accerated in tea is very important
According to Circular No 50/2016/TT-BYT issued by the Minister of Health on December 30, 2016, the document outlines the maximum allowable limits of pesticide residues in food, as detailed in the accompanying appendix.
"Maximum Residue Level (abbreviated as MRL) is the maximum content of a pesticide residue in food (unit: mg/kg of food)."
“Pesticide Residue is food residues due to the use of a pesticide
Residues of pesticides may come from unknown, unavoidable sources (such as from the environment) or from the use of chemicals
Residues of plant protection drugs include plant protection drug derivatives such as conversion products, metabolites, reaction products and impurities that are considered to have significant toxicity.”
Extraneous Maximum Residue Limit (EMRL) refers to the highest allowable concentration of pesticide residues or environmental pollutants in food, which includes residues from prior agricultural chemicals This limit specifically excludes residues from pesticides or contaminants that are directly or indirectly associated with food, measured in milligrams per kilogram (mg/kg) of food.
“Acceptable Daily Intake (ADI) is the daily intake of a chemical throughout life without harm to human health (unit: mg/kg weight)."
"Plant protection drug code (abbreviated as Code) is a plant protection drug code of the International Food Standardization Committee (CODEX)."
Some methods for determination pesticide in tea
2.3.1 Traditional method: Quantitative analysis method (Ministry of
The residual analysis method is designed to efficiently analyze multiple pesticides in a sample This approach is quick and straightforward, requiring only a minimal amount of solvent while effectively extracting all pesticides present in the sample matrix.
The residual analysis method, established by the US Food and Drug Administration (FDA), primarily focused on the organochlorine group, particularly chlorine pesticides and other non-polar substances In this method, acetonitrile is used to extract these substances from fat-free food samples, followed by dilution with water, allowing the pesticides to be assigned to a non-polar solvent like petroleum ether However, this approach has limitations, as it can lead to the partial loss of average polar insecticides, such as organic phosphorus insecticides, prompting scientists to seek alternative methods to improve identification and analysis beyond the Mills method.
The liquid-liquid extraction method is utilized to determine pesticide levels in tea by separating specific substances or groups of compounds This process involves transferring analytes from a first solvent to a second insoluble solvent, allowing for the recovery of the solvent and the analyte Each analyte requires a different solvent volume based on its properties to minimize loss Key factors influencing the extraction process include the analyte's solubility in the solvent, temperature, and the presence of other solutes Additionally, this method aids in removing impurities from the sample, and can be enhanced by physical factors such as mechanical shaking, high-speed stirring, or ultrasonic waves For samples with higher impurity levels, it may be beneficial to combine this technique with other cleaning processes.
- Extract the compound of interest from the original solution
- Raw high division initially contains too many kinds of compounds from non-polar to very polar into segments with different polarities
Significant changes in recent years have been the introduction of the QUECHERS (short, easy, cheap, effective, rugged, and safe method) The
The QUECHERS method, developed by the United States Department of Agriculture in 2003 and first reported by Anastassisides and Lehotay, is an effective technique for analyzing pesticide residues in various sample matrices, requiring only 70-100% water for dried samples This method simplifies the extraction and cleaning of pesticides from fruits and vegetables through a two-step process involving organic solvents and saline solutions, followed by solid phase dispersion (d-SPE) for purification In 2005, Lehotay's evaluation confirmed its efficacy with 207 out of 235 pesticides in vegetable matrices, although recovery of pH-sensitive substances was a concern To address this, he modified the method using an acetate buffer at pH 4.8-5.0, leading to its adoption as the official AOAC 2007.01 method in 2007 Concurrently, Anastassiades developed a variant using citrate buffer at pH 5, which became the European method CEN 15662 The QUECHERS method is praised for its speed, simplicity, cost-effectiveness, minimal solvent use, and versatility across a wide range of pesticides In Vietnam, researchers are adapting the QUECHERS method to enhance pesticide residue analysis in agriculture, optimizing the process to meet local conditions.
Gas chromatography - mass spectrometry GC-MS
Gas chromatography (GC) is a vital technique in analytical chemistry for separating and analyzing volatile compounds without altering the samples Unlike other chromatography methods such as column chromatography, HPLC, and TLC, GC operates by separating compounds between a stationary phase and a mobile gas phase, while column chromatography uses a solid stationary phase and a liquid mobile phase Additionally, GC utilizes a column furnace to regulate the gas temperature, a feature not present in traditional column chromatography, where temperature remains constant The concentration of a compound in the gas phase is solely determined by its vapor pressure.
Mass spectrometry (MS) is a powerful analytical technique used to identify and classify chemicals in a sample by measuring the mass-to-charge ratio and the quantity of gas-phase ions.
Gas chromatography-mass spectrometry (GC-MS) is a powerful analytical technique that merges gas chromatography and mass spectrometry to effectively identify various substances within a sample This method is capable of detecting even trace amounts of a substance, making it highly sensitive and valuable for analytical applications.
In gas chromatography, the mobile phase is typically an inert carrier gas like helium or nitrogen, while the stationary phase consists of a microfluid or polymer layer within a column The gas chromatograph, which can function as a gas separator or recorder, facilitates the analysis of gaseous compounds that interact with the stationary phase, leading to their separation based on retention time As compounds exit the column, they are electronically detected and identified Various parameters, such as bearing rate, column length, and temperature, can influence the order and duration of compound separation, making retention time a crucial factor in gas chromatography analysis.
Mass spectrometry is an analytical method that quantifies the mass of charged molecules as they move through an electric field Ionized samples transform into various charged molecules, which are then separated according to their m/z values The data obtained from mass spectrometry is automatically captured and utilized for protein identification through bioinformatics tools.
Operation and general structure of a mass spectrometer: Initially, the sample
Mass spectrometry involves the ionization of samples into charged ions, which are then separated according to their mass-to-charge (m/z) ratios The analysis of these separated ions, facilitated by bioinformatics software, allows for effective database searching and result interpretation It's important to note that mass spectrometers measure the m/z ratio rather than the mass itself.
2.4.2 Structure of GC-MS system
The injection set is a crucial component of gas chromatography, responsible for introducing the sample into the continuous carrier air stream It is an integral part of the column head, ensuring efficient sample delivery for analysis.
Transducers are essential in analytical chemistry, with common types including flame ionization detectors (FID) and thermal conductivity detectors (TCD) The TCD is the most widely used probe, operating on the principle of thermal conductivity changes around a tungsten-rhenium wire, which generates voltage signals as analyte molecules interact with carrier gases In contrast, the FID specifically detects organic compounds that produce ions during pyrolysis, displaying results as peaks on a chromatogram Other notable probes include the catalytic combustion detector (CCD) for measuring combustible hydrocarbons, ion discharge detectors (DID) that utilize high-voltage discharges, and dry electrolytic conductive probes (DELCD) for chlorine compounds Additionally, electronic trap detectors (EDC) employ radioactive sources, while flame photometers (FPD) and atomic emission detectors (AED) analyze spectral lines and create atomic emission spectra, respectively The Nit-Phosphorus detector (NPD) functions as an electronic thermal probe, and mass spectrometry probes (MS), or GC-MS, are recognized for their high sensitivity and efficiency with small sample sizes Other types include ionizing photoelectric detectors (PID) and pulse ionizing detectors (PDD).
(TID)/Vacuum ultraviolet detector (VUV)/Infrared probe (IRD)/Helium ionizers (HID)/Electrolytic conductivity probes (ELCD)
• Operation and general structure of a mass spectrometer:
Mass spectrometry systems are versatile and widely used across various fields, particularly in proteomics, where two primary types—MALDI-TOF and ESI-MS/Ms—are employed While these systems share similarities, they complement each other in the identification of proteins A mass spectrometer consists of three key components: the sample ionization source, the mass analyzer, and the detector The configuration and operating principles of the mass spectrometer vary based on the types of ionization sources and mass analyzers used.
The mass spectrometer gas chromatography system, like other gas chromatography systems, comprises essential components including gas supply, column furnace, sample injection unit, analytical column, probe, signal recorder, and analytical data print department, with the probe functioning as a mass spectrometer The system is primarily divided into two parts: the gas chromatograph and the mass spectrometer The gas chromatograph utilizes capillary columns, where the separation of molecules is influenced by the column's size (length, diameter, film thickness) and phase properties As the sample progresses through the column, differences in chemical properties and relative affinity for the stationary phase lead to the retention and subsequent elution of molecules at varying times.
Mass spectrometers operate by capturing, ionizing, accelerating, deflecting, and detecting individual ionized molecules, which are broken down into ion fragments analyzed by their mass-charge ratio In a gas chromatography-mass spectrometry (GC-MS) system, the sample solution is injected into the GC inlet, vaporized, and transported through a chromatographic column using a carrier gas, typically helium The compounds in the mixture are separated based on their interactions with the stationary phase of the column and the mobile phase As the compounds elute from the column, they pass through a heated line to the ion source, where they are converted into ions A typical GC-MS setup includes a vacuum pump, an automatic sample pump (Compipal), a Trace GC Ultra gas chromatograph, and a Polaris Q ion mass spectrometer, which consists of four main components.
MATERIALS, EQUIPMENT AND METHODS
Establish a high senensitive and accerable method to determine pesticide in tea
To evaluate the sensitive method, we forcus on: LOD, LOQ, linearity, repeatability, reproducibility, recovery
Chlorpyrifos, Bifenthrin, and α-Endosulfan were sourced from Dr Enrenstorfer, with a purity of 99.99% The chemicals used include Sodium Acetate, Nitrogen, Heli, distilled water, Methanol (MeOH), Acetonitrile (ACN), Acetic acid, Acetone, Formic acid, NaCl, MgSO4, Primary Secondary Amine (PSA), C18 stuffed seeds (35-75 µm), and Graphite Carbon Black (GCB), with all other chemicals utilized without further purification For Chemical Preparation M1, a solution was created by diluting 5g ± 0.1g of MgSO4, 1.5g ± 0.05g of NaCl, and 0.5g ± 0.1g of CH3COONa to a final volume of 50ml Chemical Preparation M2 involved mixing 2.5g ± 0.05g of anhydrous MgSO4, 1g ± 0.1g of PSA, 0.5g ± 0.05g of C18, and 0.2g ± 0.01g of GCB.
The gas chromatography-mass spectrometry (GC-MS) system comprises essential components such as gas supply, column furnace, sample injection unit, analytical column, probe, signal recorder, and data printing department, with the probe functioning as the mass spectrometer This system features two primary parts: the gas chromatograph and the mass spectrometer Molecules are retained within the column and elute at varying times, known as retention times The eluting compounds pass through a heated line to the ion source, where they are converted into ions Key components of the GC-MS system include a vacuum pump, an automatic sample pump (Compipal), the Trace GC Ultra gas chromatograph, and the Polaris Q ion mass spectrometer.
3.4.1 Preparation of sample and standard
To prepare a 1000 ppm pesticide-based stock standard solution, accurately weigh 25 mg ± 0.1 mg of the standard substance into calibration boats, dissolve with acetone, and transfer to a 25 ml volumetric flask Add acetone to approximately 20 ml, mix thoroughly using ultrasound or vortex until fully dissolved, and then fill to the mark with acetone Store the solution in dark glass bottles in a refrigerator at 2-8 ℃ for up to one year, ensuring the standard weight is adjusted according to the manufacturer's certificate For a 10 µg/ml standard solution, draw 100 µg from the 1000 µg/ml stock into a 10 ml volumetric flask, add acetone, and fill to the mark This solution can be stored in dark glass bottles in the refrigerator for six months To prepare calibration curves, draw 200 µl, 400 µl, 800 µl, 1000 µl, and 2000 µl of the 10.0 µg/ml working standard, mix well, and store appropriately.
•Prepare test sample: Measure 2 g ± 0.1 g of sample into a 50ml plastic test tube Add 10ml of super clean water, vortex evenly, leave for 30 minutes before proceeding to the next step
• Prepare blank: The blank sample do not contain and prepared similarly to test sample
To prepare a shallow control sample of 100 µg/kg, follow the same procedure as for the blank sample: mix 400 µl of a standard working solution at 10 µg/ml with 10 ml of ultra-pure water, vortex thoroughly, and allow it to stand for 30 minutes before continuing with the subsequent steps.
To prepare the calibration graph, the sample is treated similarly to the blank sample For fresh tea samples, a mixture standard of 10 µg/ml (ppm) is added to create calibration curve samples with concentrations of 100, 200, 400, 800, and 1000 ppb, as outlined in the accompanying table.
Table 3.4.1a: Concentration and spiked samples 10ppm (àl) to prepare the sample as a calibration curve
STD1 STD2 STD3 STD4 STD5
Then add 5ml of super clean water, vortex evenly, let stand 30 minutes before proceeding to the next step
For dry tea samples, prepare a standard mixture of 10 mlg/ml (ppm) and use it to create a calibration curve with concentrations of 100, 200, 400, 800, and 1000 ppb, as outlined in the accompanying table.
Table 3.4.1b: Concentration and spiked samples 10ppm (àl), 2ppm (àl) to prepare for tea sample
Then add 10ml of super clean water, vortex evenly, stand 30 minutes before proceeding to the next step
To prepare the samples, add 10 ml of a 1% acetic acid solution in acetonitrile to the test tubes containing blank, test, control, and calibration line samples Cover the tubes and mix for 1 minute using a vortex machine, followed by shaking for 30 minutes on a shaking machine.
Add the whole M1 into the test tubes containing the sample above, cover carefully, shake well with hands, mix 1 minute on vortex machine;
Centrifuge the sample at 4500 rpm for 5 minutes at a temperature of 15℃ on a centrifuge;
STD1 STD2 STD3 STD4 STD5
Suck the supernatant liquid into a 15ml plastic test tube and incubate in the freezer for about 2 hours (or overnight if necessary)
Drain the supernatant liquid into a 15 ml plastic test tube and incubate in the freezer for about 2 hours (or overnight if necessary);
Drain the upper layer fluid into a 15 ml plastic test tube containing M2 shake by hand, then mix well on the vortex machine;
Centrifuge the sample at 4500 rpm for 5 minutes at 15 ℃ by centrifuge;
Draw 4ml of the supernatant liquid into a 10ml graduated glass ampoule (if available), blowing medium to dry at temperatures below 400 °C with a stream of nitrogen on a solvent drier;
Dissolve the residue with 2 ml n-hexane: Acetone (9:1) with fresh tea For dried tea samples dissolved with 1ml of n-hexane: Acetone (9:1);
Centrifuge the sample at 4500 rpm for 5 minutes at 15 ℃ Filter the entire layer through a 0.2 µm filter into a vial (for dry tea, use a vial with a 500 µL insert) and inject it into the GC-MS system to analyze the pesticide content in the sample.
3.4.2 Conditions to run the machine GC/MS
Check the status of the machine before analysis, to ensure a stable signal before injection MS probe tune process with the condition of analysis must meet requirements
Column DB-5MS or equivalent (Length 30m, internal diameter of 0.25mm, film thickness of 0.25mm)
Heat program: 50 ℃ (1 minute) → 125 ℃ (0 minutes) → 10 ℃/minute →
Gas carrier; helium (inline type, flow rate: 1ml/minute)
Standard equipment inspection; (inject at least 3 samples with fixed concentrations);
* Note: In case the sample table has more than 10 samples, 10 samples re- inject 1 standard sample to check the device
Calculate the ratio of ions to the equation:
A1: the area of the secondary ion peak is intense
A2: The area of the secondary ion peak has low intensity
The calibration graph illustrates the relationship between the area of the signal ion used for quantification (Q) and the concentrations of substances that form the calibration curve, established through the linear regression method.
Inside: a: slope of the calibration curve b: toss the angle of the standard line x: concentration of standard substance
RF: the area of the ion signal strength used for quantification (Q)
Calculate the amount of analyte content in the test sample based on the calibration curve with a dilution factor of 0.8, correcting the result obtained with the recovery
The repeatability standard deviation is assessed by conducting tests on identical test levels using the same method, operator, and equipment within a short timeframe in a single laboratory In contrast, the reproducibility standard deviation involves testing homogeneous samples with the same method across different laboratories, operators, and equipment Bias refers to the discrepancy between the expected test results and the accepted value, highlighting total systematic errors A larger systematic difference from the accepted reference value indicates a greater bias, which can be categorized into lab bias and method bias.
• Limit of Detection – Limit of Quantification:
Theoretical detection limit is calculated by the formula: LOD = 3 x Sr
Theoretical quantitative limit is calculated by the formula: LOQ =X + 10 x Sr
Actual detection limit: Repeat analysis 5 to 10 times of spiked samples at theoretical LOQ concentration Determine the standard deviation (S), LOD and LOQ values of the method are determined as follows:
With t: Student coefficient (one-tailed) with 99% confidence and degrees of freedom n-1; S: Standard deviation determined from n incremental sample analyzes
Linearity: Study the appropriate concentration range for the method
To establish a linear range, it is essential to create graphs that plot peak areas against concentration values The calculation of a standard curve using a first-order regression line must yield a correlation coefficient of at least 0.99 to ensure accuracy and reliability.
Investigation on standard solution → evaluating the linearity of equipment, not assessing the effect of the sample matrix → must determine the correct method during method approval
The background survey serves as a foundation for conducting a trueness survey across various concentrations It is essential to assess both accuracy and precision within the specified concentration ranges The minimum levels of precision must be evaluated at both the highest and lowest concentration points within the analytical range.
Repeatability: Repeat the analysis 10 times of spiked sample (spike sample) on the blank at the concentration of 20àg/kg to calculate the standard deviation
Sr: Repeatability standard deviation xi: The result obtained on the ith sample x: The average value of the test results, is calculated as follows: X = Σxi/n n: Number of testing sample
• Recovery: Experimental arrangement at 4 concentrations of 20àg/kg; 200àg/kg; 400àg/kg; 800àg/kg Test spike samples 10 times at each concentration in the same test batch
In the case of the use of a spike sample, a "blank" shall be carried out
(unspike - a sample used as a spike but without spiking) in parallel with the spike The result is subtracted from the mean of the blank
Calculate the recovery from the results, using the following formula:
X: The average value of the test results
Xuns: average value of unspike sample test results
XSpike: concentration of sample spike
• Reproducibility: At least 2 testers participated Each analyst analyzed 10 spiked samples on a blank background of the same concentration, same sample
Sr x i type, same day or different day Then calculate the repeatability standard deviation between the testers
To calculate the standard deviation that reflects the obtained results, use the formula involving the i-th measured values of the two testers, denoted as \( a_i \) and \( b_i \) Additionally, \( k \) represents the number of repetitions after excluding values with large deviations.
RESULTS AND DISCUSSION
Limit of detection and limit of quantification
Actual detection limits calculated as tables
Published detection limit (1) Chlorpyrifos: LODTT = 10 (àg/kg)
The declared detection limit (2) α-Endosulfan: LODTT = 10 (àg/kg)
Published detection limit (3) Bifenthrin: LODTT = 10 (àg/kg)
Actual quantitative limits calculated in the table
Published quantitative limit (1) Chlorpyrifos: LOQTT = 20 (àg/kg)
Published quantitative limit (2) α-Endosulfan: LOQTT = 20 (àg/kg)
Published quantitative limit (3) Bifenthrin: LOQTT = 20 (àg/kg)
To establish the detection limit (LOD) and quantitative limit (LOQ), we introduced a low concentration standard to a blank background and measured the signal-to-noise ratio (S/N) A ratio of S/N ≥ 3 was used to calculate the standard deviation (SD) for determining LOD and LOQ Our calibration at low concentrations yielded results indicating that Chlorpyrifos has an LOD of 4.5 and an LOQ of 13.5, while α-Endosulfan has an LOD of 5.08 and an LOQ of 15.25 Additionally, Bifenthrin exhibited an LOD of 2.29 and an LOQ of 6.88 To validate these LOD results, we conducted 10 replicates on blanks spiked at the LOD, confirming that S/N ≥ 3, which aligns with the AOAC standards.
Table 4.1a: Limit of Detection and Quantification
Table 4.1b: The result of reliability evaluation of LOD value
Linearity
Calibration curves were established on a blank basis, ensuring that the samples did not contain Chlorpyrifos, α-endosulfan, or Bifenthrin, within a concentration range of 20-400 µg/kg The results, as shown in the accompanying table and graph, indicate that the correlation coefficients (r²) for the calibration graphs of the three substances exceed 0.998 across this concentration range Consequently, the calibration curve is deemed appropriate for the analysis of this group of chemicals within the specified concentration range.
Table 4.2: The result of concentration, peak area and the correlation coefficient of linear value
Figure 4.2.1b: Calibration curve of Chlorpyrifos
Figure 4.2.2b: Calibration curve of α -endosulfan
Figure 4.2.3b: Calibration curve of Bifenthrin
Repeatability - Recovery
Repeatability (R, %) and recovery are crucial for assessing the accuracy of the analysis method for Chlorpyrifos, α-endosulfan, and Bifenthrin in tea We conducted performance tests under optimized GC-MS conditions by analyzing samples ten times, ensuring the top of the sample was blank for three repetitions before adding the standard for analytes The repeatability (R, %) is calculated as the ratio of the actual analytical standard additive to the concentration of the added standard.
The table indicates that the recovery efficiency of the three substances is consistent, with variability in the analysis of the three compounds being less than 10% This demonstrates that the sample handling method and GC-MS optimization conditions are both accurate and reliable for the analysis of these substances.
Achieving the AOAC criterion at a concentration of 20 àg/kg (RSD% ≤
Table 4.3: The result of the repeatability and the recovery
Reproducibility
In our study utilizing GC-MS equipment, we conducted experiments with four different concentrations: 20, 200, 400, and 800 µg/kg, each repeated ten times to ensure method accuracy As indicated in Table 4.4, the reproducibility of all three substances approached 100%, aligning with AOAC standards This confirms that the GC-MS method is suitable for the experimental conditions employed.
Table 4.4: The result of the reproducibility
CONCLUSION
The data indicates that the standard addition value at the detection limit is 10 àg/kg, with a signal-to-noise ratio greater than 3, and consistently shows one parent ion and three daughter ions, in compliance with the criteria of 2002/657/EC The gas chromatography-mass spectrometry (GC-MS) method for determining three active substances yielded a limit of detection (LOD) of 10 àg/kg and a limit of quantification (LOQ) of 30 àg/kg The average recovery rates of the analytes ranged from 100% to 110%, with a linear regression coefficient of r² ≥ 0.998 and a variation of less than 10% These results confirm that the method is fast, accurate, and reliable for analyzing three active substances in tea.
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National Center for Biotechnology Information; p.1.2.3
[11] VN MARD, The registration list of pesticides in Vietnam in 2009 2009
[12] Minister of Agriculture and Rural Development; 2018; Appendix I "List of plant protection drugs licensed to use in Vietnam"; circular No 03/2018 / TT-BNNPTNT; p.57, p.58, p.59
[13] US Department of Health and Human Services (2015), Toxicological Profile for Endosulfan, Agency for Toxic Substances and Disease Registry
The study by NXTong and NTTHuong (2018) evaluates the toxicity of the endosulfan insecticide on the growth of Daphnia magna Conducted by the Natural Science Institute of Science, Technology and Environmental Management at Ho Chi Minh City University of Industry, in collaboration with the Academy of Science and Technology and the Vietnam Academy of Science and Technology, the research highlights significant environmental concerns regarding the impact of endosulfan on aquatic life.
[15] L.L Foersom, P.L DeFur and S Tuberty (2002), "Effects of endosulfan on moulting in juvenile red swamp crayfish, Procambarus clarki", Con Ference paper in Integrative and Comparative Biology, 42, pp.1218-1219
[16] P Palma, V.L Palma, R.M Fernandes, A.M.V.M Soares and I.R Barbosa
(2009), “Endosulfan sulphate interferes with reproduction, embryonic development and sex differentiation in Daphnia magna”, Ecotoxicology and Environmental Safety, 72(2), pp.344-350
[17] Ministry of Agriculture and Rural Development (2011), "List of drugs allowed, restricted and banned for use in Vietnam", Circular No 36/2011 /