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[.]
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 TANDEM MASS SPECTROMETRY (LC-MS /MS) BACHELOR THESIS Study Mode: Full-time Major: Food technology Faculty: Advanced Education Program Office Batch: 2016-2020 Thai Nguyen, 2020 Luan van i Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Food Technology Student name Tran Thi Yen Student ID DTN1654290015 Thesis Title Study on a highly sensitive method for analyzing Carbaryl residues in white Radish by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Supervisor(s) Dr Vu Thi Hanh - Thai Nguyen University of Agriculture and Forestry, Vietnam Supervisor’s signature Abstract: Highly sensitivity detection method for analyzing Carbaryl residues in white Radish by liquid chromatography tandem mass spectrometry (LC-MS/MS) Active ingredient Carbaryl extracted with 0.1% Acetic acid in ACN The samples were cleaned by QUENCHERS method Extraction of a 10g sample with 10ml ACN, and then adding 4g MgSO4 and 1g CH3COONa, after centrifugation the extract will be decanted into a tube containing 250g C18 plus 0.9g MgSO4 After a second shaking and centrifugation step, the solution extracted was transferred to autosampler vials for analysis by LC-MS/MS Time for sample analysis is 18 minutes, limit of detection defined were 10ppb, limit of quantitative got 30µg/kg The recovery is about 90-110%, the linear regression coefficient r ^ 2≥0.99 Based on the above survey results, this is a highly sensitive method, easy to carry out, providing fast and accurate analysis results suitable for Carbaryl residue analysis in white Radish Keywords Carbaryl, White radish, LC-MS/MS Number of pages 37 Date of submission 17th August, 2020 Luan van ii ACKNOWLEDGEMENT This work would not have been possible without the support of Thai Nguyen university of Agriculture and Forestry, advanced education program office, lecturers of food technology and biotechnology faculty for their sincere guidance I received to improve my practical as well as laboratory skills First of all, I would especially like to thank Dr Vu Thi Hanh as my teacher and mentor, She had supported to me by showing different method of information collection about the study She helped all time when we needed and she gave right direction toward completion of project Second, I would like to thanks to to Mr Nguyen Xuan Thanh as a person in charge of Laboratory and all members of The center for Technical Services and Professional Training in Agroforestry –Fishery Quality – Thai Nguyen Each member in lab has provided me extensive material and professional guidance and taught me a great deal about both scientific research and life in general Finally, thanks to my parent and my friends, who provide unending inspiration For giving me encouragement, enthusiasm and invaluable assistance Sincerely, Tran Thi Yen Luan van iii LIST OF ABBREVIATIONS ACN Acetonitril ADI Acceptable Daily Intake AOAC Association of Official Analytical CH COONa Sodium Acetate CV Coefficient of variation GC-MS Gas Chromatography Mass Spectrometry HPLC High Performance Liquid Chromatography LD Lethal Dose LC-MS/MS Liquid Chromatography Tandem Mass Spectrometry LOD Limit of Detection LOQ Limit of Quantification EMRL Extraneous Maximum Residue Limit IS Internal Standard m/z Mass to charge ratio MRLs Maximum Residue Limits MRM Multiple Reaction Monitoring MeOH Methanol MgSo Magnesium Sulfate PSA Prostate-Specific Antigen ppm Part per million ppb Part per billion QUECHERS Quick, Easy, Cheap, Effective, Rugged and Safe RSD Relative Standard Deviation S/N Signal to noise ration S Standard deviation for repeatability sR Standard deviation for reproducibility TPP Triphenylphosphate Luan van iv CONTENTS ACKNOWLEDGEMENT ii LIST OF ABBREVIATIONS iii CONTENTS iv LIST OF TABLE vi LIST OF FIGURE vii PART I INTRODUCTION 1.1 Research rationale .1 1.2 Research’s objective 1.3 Research question .1 1.4 Limitations PART II LITERATURE REVIEW 2.1 Radishes 2.1.2 Chemical composition and nutritional value 2.1.3 Benefits of Radish 2.2 Plant protection products 2.2.1 Definition .5 2.2.2 Classification 2.2.3 List of pesticides allowed to be used and banned in Vietnam 2.2.4 Effect of pesticide to health 2.2.5 Current situation of pesticide residues in vegetables .9 2.2.6 Food poisoning situation due to pesticides 10 2.3 Carbaryl 10 2.4 Current methods for determination of Carbaryl in vegetable .13 2.4.1 Traditional methods .13 2.4.2 QUECHERS method 13 2.5 Liquid chromatography tandem mass spectrometry (LC-MS/MS) 14 2.5.1 Instrumentation of HPLC 15 2.5.2 Mass spectrometry detector 18 PART III MATERIALS AND METHODS .22 Luan van v 3.1 Research object .22 3.2 Research scope .22 3.3 Research content .22 3.4 Chemicals and instruments .22 3.4.1 Chemical 22 3.4.2 Equipment 22 3.5 Methods 22 3.5.1 Preparation of sample and standard solution .22 3.5.2 Evaluate the sensitive and accurate of method 24 PART RESULT AND DISCUSTION 29 4.1 Optimize the conditions for LC-MS/MS 29 4.2 Linearity 30 4.3 Limit of detection and limit of quantification 31 4.4 Recovery and repeatability .33 4.5 Reproducibility .33 PART V CONCLUSION 35 REFEREENCES 36 Luan van vi LIST OF TABLE Table 2.1: Nutritional value per 100g radish Table 2.2: List number of common name and trade name for pesticides allowed to be used and banned in Vietnam .7 Table 2.3: MRL and ADI of Carbaryl in some vegetables 12 Table 3.1: Concentration of the calibration curve 25 Table 3.2: Maximum relative Standard deviation for repeatability depend on concentration range (AOAC) 25 Table 3.3: Recovery that accept at different concentrations (AOAC) 26 Table 3.4: Maximum relative standard deviation for reproducibility follow each concentration range (AOAC) .27 Table 4.1: Optimization of the mobile phase composition in gradient elution reversedphase HPLC 29 Table 4.2: Optimal MRM-MS/MS conversion conditions for Carbaryl analysis 29 Table 4.3 Peak area results were obtained in carbaryl analysis 30 Table 4.4: Limit of Detection and Quantification for Carbaryl analysis 32 Table 4.5: The result of reliability evaluation of LOD value in Carbaryl analysis .32 Table 4.6: Recovery and repeatability of Carbaryl .33 Table 4.7: The result obtained from analysis Carbaryl at concentration of 30 µg / kg between members 34 Luan van vii LIST OF FIGURES Figure 2.1: Structure formula of Carbaryl .11 Figure 2.2: Instrumentation of HPLC 15 Figure 2.3: HPLC and LC-MS/MS equipment 15 Figure 2.4: LC column 50 × mm 17 Figure 2.5: Diagram of a ESI ambient ionization source 19 Figure 3.1: Determine LOD base on S/N ratio .28 Figure 4.1: Standard peak of Carbaryl and TPP in chromatography analysis 30 Figure 4.2: Calibration curves of Carbaryl .31 Luan van PART I INTRODUCTION 1.1 Research rationale As we know that, Radish is one of agricultural products that bring a lot of nutritional value, providing foods, medicine for people etc Currently, there are two main types of Radish that used commonly which are white and red Radish In Vietnam, Radish is also used to treat coughs, sore throats, acne, water supply, digestive aid [1] Radish brings a lot of economic value as well as utility to people, Commented [A1]: 15/3/2016 ngon vị thuốc từ củ cải /vfa.gov.vn/ so the demand for product quality has to be increasingly raised In order to meet that demand, exporters will introduce measures such as intensive crop enhancement, seed improvement and one of the indispensable measures is used plant protection drugs Plant protection drugs are considered to be an effective weapon of human in preventing pests and protecting plants[2] Besides the advantages of protecting, increasing crop yields, pesticides also cause many other harmful effects such as environmental pollution, poisoning to humans and cattle, increasing production costs, and most is leaving residues in agricultural products that affect the quality of agricultural products and consumer health From the benefits that radish brings and to ensure the health and safety for consumers We propose a project: “Study on a highly sensitive method for analyzing Carbaryl residues in white Radish by Liquid Chromatography tandem Mass Spectrometry (LC-MS /MS)” 1.2 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) 1.3 Research question - Why choose Liquid Chromatography tandem mass spectrometry method to analyze pesticide residues in white radish? - How to confirm validity of the method? Luan van Commented [A2]: Sở tài nguyên môi trường Vĩnh Phúc (2008) Thuốc bảo vệ thực vật tác động chúng 1.4 Limitations Due to constraints of time and sample responsiveness, the sample size is limited So, certain factors could not be studied in depth Luan van ... Vietnam Supervisor’s signature Abstract: Highly sensitivity detection method for analyzing Carbaryl residues in white Radish by liquid chromatography tandem mass spectrometry (LC- MS/ MS) Active ingredient... sensitive method for analyzing Carbaryl residues in white Radish by Liquid Chromatography tandem Mass Spectrometry (LC- MS /MS) ” 1.2 Research’s objective The objective for this study is evaluate... conversion conditions for Carbaryl analysis 29 Table 4.3 Peak area results were obtained in carbaryl analysis 30 Table 4.4: Limit of Detection and Quantification for Carbaryl analysis 32 Table