Tran Thi Yen THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY TRAN THI YEN STUDY ON A HIGHLY SENSITIVE METHOD FOR ANALYZING CARBARYL RESIDUES IN WHITE RADISH BY LIQUID CHROMATOGRAPHY TAND[.]
INTRODUCTION
Research rationale
Radish is a highly nutritious agricultural product that offers numerous health benefits, serving as both food and medicinal ingredient The two most common types of radish are white and red radish, each valued for their unique flavors and nutritional properties These versatile vegetables are widely used in various culinary dishes and traditional remedies.
Radish in Vietnam not only has traditional medicinal uses such as treating coughs, sore throats, acne, improving water supply, and aiding digestion, but also holds significant economic value due to its utility and demand for high-quality produce To meet this growing demand, exporters are adopting measures like intensive crop cultivation, seed improvement, and the use of plant protection chemicals While pesticides effectively prevent pests and increase crop yields, they also pose environmental and health risks, including pollution, poisoning to humans and livestock, increased production costs, and residual contamination affecting food safety To ensure consumer health and safety, we propose a project titled “Study on a Highly Sensitive Method for Analyzing Carbaryl Residues in White Radish Using Liquid Chromatography,” aiming to accurately detect harmful pesticide residues in radish.
Chromatography tandem Mass Spectrometry (LC-MS /MS)”.
Research’s objective
The objective for this study is evaluate the sensitive and accurate method based on: Linearity, Repeatability, Recovery, Reproducibility, Limit of detection
(LOD), Limit of quantification (LOQ)
- Why choose Liquid Chromatography tandem mass spectrometry method to analyze pesticide residues in white radish?
- How to confirm validity of the method?
Commented [A1]: 15/3/2016 món ngon vị thuốc từ củ cải
Commented [A2]: Sở tài nguyên và môi trường Vĩnh Phúc
(2008) Thuốc bảo vệ thực vật và những tác động của chúng
Due to constraints of time and sample responsiveness, the sample size is limited So, certain factors could not be studied in depth.
Limitations
Due to constraints of time and sample responsiveness, the sample size is limited So, certain factors could not be studied in depth.
LITERATURE REVIEW
Radishes
Radish (Raphanus sativus L) is an important annual vegetable belonging to the Cruciferae family, widely recognized in many countries for its culinary uses Its enlarged root and hypocotyl are primarily consumed as salted vegetables, fresh grated radish, garnish, or in salads, showcasing its versatility Additionally, young leaves and seedlings, known as ‘kaiware,’ are also enjoyed in various dishes Scientifically classified under Plantae, Angiosperms, Magnoliopsida, Brassicales, radish is valued for its nutritional benefits and cultural significance worldwide.
Radish is a globally cultivated vegetable known for its spicy flavor and versatile culinary uses Its tubers can grow up to 40 cm long, with leaves clustered below the ground, and it is harvested approximately 45-55 days after planting, making it a convenient year-round crop Radish is widely consumed across various regions and can be combined with other ingredients to create delicious dishes According to traditional sources like the Hai Thuong book, radish has a sweet yet slightly bitter taste, is non-toxic, and is believed to help relieve sputum, promote circulation, and treat certain ailments.
2.1.2 Chemical composition and nutritional value
Radish is a nutrient-rich vegetable that provides 1.4 grams of protein, 3.7 grams of carbohydrates, and 1.5 grams of dietary fiber per 100 grams It is a good source of essential minerals, including 40 mg of calcium, 41 mg of phosphorus, and 1.1 mg of iron Additionally, radish contains vital vitamins such as 0.06 mg of vitamin B1, 0.06 mg of vitamin B2, 0.5 mg of vitamin PP, and 30 mg of vitamin C, making it a healthy addition to a balanced diet.
Table 2.1: Nutritional value per 100g radish
Encyclopedia of Food Sciences and Nutrition (Second Edition), 2003
Commented [A4]: Seaman, Abby (2013-11-13) "Turnips and Radishes" Integrated crop and pest management guidelines for commercial vegetable production Retrieved 2020-05-20
Commented [A5]: 15/3/2016 món ngon vị thuốc từ củ cải
Radish's spicy flavor offers natural antibacterial, anti-inflammatory, and analgesic benefits During winter, applying radish wrapped in a cloth bag directly to muscular or joint pain can help alleviate discomfort This traditional remedy leverages radish's active compounds to provide soothing relief for sore muscles and joint aches.
- Good for liver, prevent cardiovascular disease
Radish contains the bioactive compound Betaine, which supports healthy liver function by aiding in effective fat breakdown, promoting weight loss, and preventing fatigue and nausea Betaine, also found in beets, plays a crucial role in reducing plasma homocysteine levels, a significant risk factor for cardiovascular disease Incorporating radish into your diet can enhance liver health and contribute to improved heart health.
A study from Wake Forest University reveals that the high nitrate content in radishes can boost blood flow to the brain, leading to improved mental performance Consuming radishes may enhance brain function, support long-term brain health, and offer protective benefits for brain cells over the years This natural vegetable's nitrate levels make it a valuable addition for cognitive health and longevity.
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The natural Vitamin B12 in Radish helps promote absorption of iron, involved in the synthesis of Hemoglobin, so the increased amount of Oxygen
Hemoglobin that good for the body, prevent anemia
Radish leaves are rich in vitamins A and C, with vitamin C content surpassing that of many other vegetables, making them effective in preventing skin aging and the formation of dark spots These nutrients help maintain a bright, soft, and youthful complexion Additionally, radish paste can be used as a natural facial cleanser, promoting healthy and clear skin.
Radishes are a good source for Anthocyanins that keep our hearts functioning properly, reducing the risk of cardiovascular diseases Addition, Radish are high on
Vitamin C, Folic acid, and Flavonoids also.
Plant protection products
Plant protection drugs, also known as pesticides or plant protection products, are toxic substances of natural or synthetic origin used in agriculture to prevent pests and protect crops in fields and gardens These chemical compounds play a crucial role in safeguarding agricultural produce from pests, ensuring healthy plant growth and optimizing crop yields Proper management and understanding of these products are essential for effective pest control and minimizing environmental impact.
Plant protection drugs can be divided into 4 main groups:
- Chlorine group: Are Chlorine derivatives of some organic compounds such as
Diphenyletane, Cyclodiene, Benzene and Hexane This group includes Organic compounds that very stable in the natural environment and have a long half-life
Representatives of this group are Aldrin, Dieldrin, Heptachlo, Lindan, Methoxychlor
The organophosphorus group primarily consists of ester compounds, which are organic derivatives of phosphoric acid These pesticides have a shorter half-life compared to organic chlorine compounds and are more widely used in agricultural applications They affect insect nervous systems by inhibiting the enzyme cholinesterase, leading to disrupted nerve function and ultimately insect death.
The Vinh Phuc Department of Natural Resources and Environment (2008) highlights the harmful effects of certain pesticides on human health, including muscle weakness, dizziness, and even death These pesticides, such as Parathion, Malathion, Diclorvos, and Chlorpyrifos, pose significant risks due to their toxic nature Proper awareness and regulation are essential to minimize their health impacts and ensure environmental safety.
Carbamate compounds are organic derivatives of carbamic acid that include both environmentally less stable chemicals and highly toxic substances to humans and animals When applied, carbamates target the enzyme cholinesterase in the nervous system, disrupting normal nerve function Their toxic mechanism is similar to that of organophosphates, impairing nervous system activity and posing significant health risks.
Representing for this group as Carbofuran, Carbaryl, Carbosulfan, Isoprocarb,
- Pyrethroids: These are natural-derived insecticides, mixtures of different esters with complex structures separated from flowers of certain daisy varieties
Representatives of this group include Cypermethrin, Permethrin, Fenvalarate,
Deltamethrin, etc In addition, there are some other groups such as: Inorganic insecticides, biological insecticides derived from Bacteria, Fungi, Viruses
(Fungicides, Bacteria ), Inorganic compounds (compounds of Copper, Mercury)
2.2.3 List of pesticides allowed to be used and banned in Vietnam.[4]
According to Circular No 10/2019/TT Ministry of Agriculture and Rural
Development about promulgating the list of pesticides allowed to be used and banned in Vietnam
Commented [A8]: Theo thông tư số 10/2019/TT
BNNPTNT ban hành danh mục thuốc BVTV được phép sử dụng, cấm sử dụng tại Việt Nam
Table 2.2: List number of common name and trade name for pesticides allowed to be used and banned in Vietnam
Type of pesticides Number of Common name Number of Trade name
List of pesticides banned in Vietnam
Pesticides and forest product preservative
- List of pesticides allowed to be used in Vietnam
2.2.4 Effect of pesticide to health [4]
Most pesticides are toxic to humans and animals at difference levels
- Acute toxins: Below the lethal dose, they are not capable of causing death, gradually resolved and excreted
Chronic toxins, such as certain pesticides, can accumulate in the body over time due to their durability and resistance to resolution and excretion These plant protection drugs penetrate the human and animal bodies primarily through three main routes: respiration, digestion, and direct contact Exposure to pesticides can lead to both acute and chronic poisoning, depending on the level and duration of contact, posing significant health risks.
Acute intoxication manifests through symptoms like vomiting, headache, dizziness, and sweating If the poisoning is severe, toxins are metabolized by the liver, leading to neurological issues such as insomnia, poor memory, blurred vision, and hearing loss It can also cause body weakness, reproductive problems in women, including miscarriage and premature birth, and may result in congenital malformations or cancer Additional symptoms include fatigue, skin itching, hoarseness, nausea, increased salivation, watery eyes, and in extreme cases, can be fatal.
- Chronic poisoning: Stimulates cancer cells to grow, cause birth defects, deformities, impaired memory and concentration, serious weakness, affecting the nervous system and brain
Pesticide poisoning symptoms often include digestive issues like nausea and severe diarrhea, along with neurological signs such as drowsiness, coma, convulsions, and muscle paralysis In severe cases, it can lead to hypotension, respiratory failure, and ultimately death The Poison Control Center at Bach Mai Hospital emphasizes that these symptoms are critical indicators of plant protection drug poisoning that require immediate medical attention.
Cholinergic syndrome, often caused by poisoning from organic phosphorus pesticides or carbamates, can lead to urinary problems, vomiting, shortness of breath, chest pain, sweating, body tremors, and seizures This condition affects both adults and children, highlighting the importance of prompt diagnosis and treatment to prevent severe complications.
2.2.5 Current situation of pesticide residues in vegetables
- Determined 790 samples of 52 units of safe vegetables business in the city,
Over the past 11 months, Ho Chi Minh City Plant Protection Department detected pesticide residues in 26 samples, representing a contamination rate of 3.29% The investigation revealed that vegetables such as straw mushrooms from Tra Vinh, celery, lettuce, broccoli (cauliflower), and amaranth contained high levels of pesticide residues, highlighting concerns over food safety and the need for stricter pesticide regulation and inspection.
(3.94%) Including 5 green onion samples of Da Lat, 2 samples of morning glory and choysum of Ho Chi Minh City, which over maximum residue level [5]
- 31/67 people in Hanoi were tested with blood levels of pesticides The results were published by the Institute of Occupational Health and Environment
(Ministry of Health) when conducting random tests at a class of the Center for
Community learning initiatives in the communes and towns across four suburban districts of Hanoi—Soc Son, Dong Anh, Me Linh, and Hoai Duc—aim to raise awareness about pesticide safety and health risks Currently, 31 individuals are identified as at risk due to detectable pesticide residues in their blood, highlighting the urgent need for educational programs to promote safe handling and storage practices Among these, one person faces a higher level of danger, surpassing general risks, which underscores the importance of targeted interventions to protect vulnerable community members.
Over 221 types of pesticides have been detected in vegetables, with nearly half exceeding the maximum permitted or banned levels Over the past decade, countries like Australia, the US, Japan, and the EU have rejected Vietnamese vegetable exports valued at over US $1 billion, primarily due to excessive use of fertilizers and pesticides According to Dr Hong Minh, Vietnamese agricultural products face export challenges because farmers often apply chemicals beyond safe limits, and many enterprises fail to adhere to established standards such as VIETGAP and Global GAP.
- In recent years, the authorities took 7593 samples of vegetables and found
Analysis of 393 food samples revealed that 5.17% were contaminated with banned substances or contained pesticide residues exceeding permitted limits, indicating a high level of violations that fall short of consumer expectations This ongoing issue raises concerns about food safety and consumer trust Non-compliance by manufacturers with proper plant protection practices significantly increases the risk of unsafe food products reaching consumers, emphasizing the need for stricter enforcement and better adherence to safety regulations.
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Commented [A11]: Trung kiên 30/4/2017 Nông sản xuất khẩu "gặp khó" vì tồn dư thuốc bảo vệ thực vật
Commented [A12]: Diệu thu (09/06/2016) Hơn 5% lượng rau bán trên thị trường nhiễm chất cấm
- In Ho Chi Minh City, in the first six months of 2009, more than 2,200 samples of vegetables and fruits were tested in three wholesale markets (Binh Dien,
In the first eight months of 2009, analysis of nearly 310 vegetable samples from markets, production areas, and collective kitchens in Binh Duong revealed that approximately 80 samples contained residues of plant protection drugs, indicating ongoing concerns about pesticide contamination Additionally, in Hoc Mon and Thu Duc districts, out of 50 positive samples tested, 2.4% showed pesticide residues, reflecting a 1.3% increase compared to the same period in 2008, highlighting the need for stricter monitoring and improved safety measures in vegetable production.
2.2.6 Food poisoning situation due to pesticides
- According to a survey of the Department of Preventive Medicine and
Vietnam's environment is significantly impacted by the widespread use of toxic chemical pesticides, with over 5,000 cases of poisoning requiring hospitalization and more than 300 deaths reported annually The harmful effects of plant protection chemicals on human health and the environment are primarily due to misuse that exceeds recommended standards Proper regulation and responsible application of these pesticides are essential to protect public health and preserve ecological balance.
- On 14/11, Xuan Non Kindergarten's collective kitchen organized cooking for 796 lunches and snacks at 14:00 pm on the same day Lunch menu includes:
Enjoy a delicious afternoon meal featuring savory sausage, beef with wine sauce, and flavorful Duong Chau fried rice, complemented by mixed boiled vegetables and refreshing orange juice For dessert, there is creamy yogurt and tasty cakes to satisfy every palate After lunch, three teachers and nearly 200 students were welcomed and seated at Dong, creating a warm and communal dining experience.
Anh General Hospital and North Thang Long Hospital with the symptoms of abdominal pain, vomiting, diarrhea, headache and fever [11].
Carbaryl
Carbaryl (1-naphthyl methylcarbamate) is a widely used pesticide belonging to the carbamate chemical group It is a white crystalline solid commonly marketed under the brand name Sevin, which is a trademark of Bayer First discovered and introduced to the market by Union Carbide in 1958, carbaryl has become a prominent chemical in pest control Bayer later acquired Aventis, further enhancing its portfolio of agricultural chemicals.
CropScience in 2002, a company that includes the union carbide pesticide activities
Dichlorvos (DDVP) remains the third most widely used insecticide in the United States, serving applications in home gardens, commercial agriculture, forest protection, and rangeland management In 1976 alone, approximately 11 million kilograms were applied to U.S agricultural crops, highlighting its significant role in pest control across various sectors.
Commented [A13]: Công An Ninh Thuận ( 2009), Hơn hai triệu người ăn rau bẩn
Commented [A14]: Nguyễn Thị Thanh Huyền 01/11/2010
Tác hại của thuốc hóa chất bảo vệ thực vật đối với sức khỏe con người và môi trường
Carbaryl have chemical formula is C12H11NO2, molar mass is 201.225 gãmol−1, melting point at 142 °C (288 °F; 415 K), the solubility in water very low
Figure 2.1 structure formula of Carbaryl
Carbaryl, the active ingredient in Carylderm shampoos, is effective for treating head lice infestations, helping to eliminate lice until the problem is resolved While Carbaryl is approved for use on over 100 crops in the United States, it is illegal in countries such as the UK, Austria, Denmark, Sweden, and Iran due to safety and environmental concerns.
The Acceptable Daily Intake (ADI) for carbaryl is set at 0.008 mg/kg of body weight, ensuring consumer safety In toxicity studies, mice exhibit an LD50 ranging from 250 to 850 mg/kg, indicating the dose at which 50% of subjects would likely experience lethal effects The LC50 for mice falls between 0.005 and 0.023 mg/kg, representing the concentration at which half of the test population experiences lethal outcomes Additionally, the Maximum Residue Level (MRL) for carbaryl in beets is maintained at 0.1 mg/kg to minimize potential health risks for consumers.
• Carbaryl is used to control a wide range of pests, including beetles, cockroaches, ants, ticks and mosquitoes
• Carbaryl products can be made in the form of dust, absorbent powders, liquid concentrates, granules
• Carbaryl products are used on fruits, vegetables, rangeland, lawns, ornamental plants, trees, and building foundations
- Effect of carbaryl to health
Early symptoms of carbaryl exposure typically include headache, muscle weakness, nausea, stomach cramps, sweating, and restlessness The impact of carbaryl on human health and the environment varies depending on the level and duration of exposure, emphasizing the importance of proper handling and safety precautions.
Commented [A15]: Robert L Metcalf “côn trùng Control” trong Bách khoa toàn thư Ullmann của Hóa học công nghiệp”Wiley-VCH, Weinheim, 2002 doi : 10,1002 / 14356007.a14_263
Commented [A16]: Dữ liệu nguy hiểm về thuốc trừ sâu
Carbaryl được lưu trữ ngày 11 tháng 5 năm 2010, tại Wayback Machine
Commented [A17]: 2003 NPIC national pesticide inf center present and the length and frequency of exposure The reaction also depends on the health of each person or certain environmental factors
Greater exposure to Carbaryl can lead to mydriasis, tearing, excessive salivation, runny nose, vomiting, diarrhea, muscle twitching, and coordination problems Severe poisoning can lead to convulsions, coma and death
• Carbaryl is practically non-toxic to birds such as ducks, quails, geese and pheasants [20]
• Carbaryl ranges from toxic to extremely toxic to some fish [20]
Carbaryl is highly toxic to beneficial insects, including bees, which are likely to be affected if present at the application site Although bee kill incidents have been reported, only a single incident involving carbaryl use on asparagus has been definitively linked to such effects Concerns about using carbaryl for fruit thinning in orchards have been addressed through field studies, which found that this application does not significantly impact bee mortality or behavior.
- Maximum residue limit of carbaryl [15]
- Maximum Residue Level - MRL is the maximum content of a pesticide residue in a food (unit: 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 body weight)
Table 2.3: MRL and ADI of Carbaryl in some vegetables
Pesticides ADI (mg/kg) Products MRL (mg/kg)
Commented [A18]: A World Compendium: The Pesticide
Manual, 12th ed.; Tomlin, C D S., Ed.; British Crop Protection Council: Farnham, Surrey, UK, 2000; pp 67-68
The Environmental Fate and Effects Division of the Office of Pesticide Programs has conducted a revised risk assessment of carbaryl to support the Reregistration Eligibility Decision (RED) This comprehensive evaluation provides updated insights into carbaryl’s environmental impact and safety profile, essential for regulatory decision-making (Source: EPA, 2003; [link])
Commented [A20]: R David Jones, Ph.D., Senior
Agronomist Thomas Steeger, Ph.D., Senior Biologist March 18, 2003 Revised EFED Risk Assessment of Carbaryl in Support of the Reregistration
Commented [A21]: Theo thông tư số 50-2016 TT/BYT về Quy định giới hạn tối đa dư lượng thuốc bảo vệ thực vật trong thực phẩm.
Current methods for determination of Carbaryl in vegetable
Some traditional methods are used to determine pesticide residues in fruits and vegetables such as: Liquid–Liquid Extraction, Microwave Assisted Extraction,
Solid Phase Extraction, Supercritical Extraction Most of which take a long time
Liquid–liquid extraction, also known as solvent extraction or partitioning, is a technique used to separate compounds or metal complexes based on their solubility in two immiscible liquids This method involves transferring the analyte from one solvent to another, followed by recovery of the solvent to isolate the desired analyte Selecting the appropriate solvent is crucial to prevent analyte loss and to effectively remove impurities, with factors like solubility, temperature, and the presence of other solutes influencing the extraction process Enhancing extraction efficiency can be achieved through auxiliary physical agents such as mechanical shaking, ultrasonic waves, or super-speed agitation For samples with high impurity levels, combining liquid-liquid extraction with additional purification methods ensures better sample cleanliness.
Solid phase extraction (SPE) is a powerful technique used to separate compounds dissolved or suspended in liquids based on their physical and chemical properties, making it essential in analytical laboratories This method is widely employed to concentrate and purify samples for accurate analysis, enabling the isolation of target analytes from diverse matrices such as blood, water, beverages, soil, and animal tissue Despite its low cost and high accuracy, SPE may face challenges like incomplete removal of interferences and variability in results, which are important considerations for optimal analytical performance.
The QUECHERS method it means quick, easy, cheap, effective, rugged and safe It was expanded by Michelangelo Anastassiades in the years 2001 and 2002
The first time, the methodology was developed for the analysis of veterinary drugs
Commented [A22]: Berk, Zeki (2013) "Food Process
Engineering and Technology (Second Edition)" Advances in Molecular Toxicology, 2014 ISBN 978-0-12-415923-5
The article discusses the presence of certain compounds in animal tissues and highlights their significant potential in extracting polar and basic compounds It was subsequently tested for pesticide residue analysis in plant materials such as fruits and vegetables Currently, the QuEChERS technique serves as the foundation for widely accepted testing methods globally, including AOAC 2010 (2001.07) and EN standards.
15662 and is being applied in the large and modern laboratories in our country [25]
This article introduces a simple, rapid, and cost-effective method for detecting pesticide residues in fruits and vegetables The procedure involves extracting 10 grams of the sample with 10 milliliters of acetonitrile, followed by a liquid-liquid partition with added anhydrous magnesium sulfate and sodium chloride to separate the phases To remove residual water and purify the extract efficiently, the method employs a quick dispersion-SPE technique using 150 mg of anhydrous sorbent This streamlined approach offers an effective solution for pesticide residue analysis, adhering to best practices for cost and time efficiency.
A simple mixture of 25 mg MgSO4 and secondary amine (PSA) sorbent is combined with 1 ml of acetonitrile extract for efficient sample preparation In dispersion-SPE using PSA, polar matrix components like organic acids, certain pigments, and sugars are effectively removed from food extracts Subsequently, gas chromatography-mass spectrometry (GC/MS) is employed for accurate quantification and confirmation of pesticides suitable for GC analysis.
A recovery of 85 to 101% (mainly> 95%) and repeatability