Study on a highly sensitive method for analyzing carbaryl residues in white radish by liquid chromatography tandem mass spectrometry (LC MS MS)

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 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 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 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 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 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 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 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 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? 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 24 - Dilute stock standard solution (10 ppm): Combine 0.1 ml of stock standard into a 10ml volumetric flask Add ACN up to the mark, store in freezer, solution can storage for year - Working standard solution (1ppm) Take 1ml of 10ppm standard solution and dilute to volume with ACN in a 10ml volumetric flask The solution can store at to °C and stable for about months - Internal standard stock solution (TPP)1000ppm Weigh 10 mg TPP into a 10ml volumetric flask Dilute by ACN up to the mark Store in freezer, solution can storage for year - Dilute stock internal standard solution TPP (20ppm) Take 200µl of stock TPP 1000ppm and dilute to volume with ACN in a 10ml volumetric flask Shake well, keep to until cool and store in freezer, solution can be storage for year - Internal standard working solution (TPP) 2ppm Take 1µl TPP 20ppm and dilute to volume with ACN into a 10ml volumetric flask, and then shake well, keep for until cool This solution can store at to 8°C and storage about months - Prepare mobile phase solvent Channel A: 0.1% HCOOH solution in distill water: Absorb 1ml of HCOOH in a 100ml volumetric flask and add distill water, then mix and ultrasound Channel B: MeOH Quality of sufficient purity that is free of interfering compounds in LC-MS/MS prepared in mobile phase solution 3.5.2 Evaluate the sensitive and accurate of method [15] * Linearity - Determine the linear range by survey different concentrations Determine the linearity and trueness, the experiments are carried out on a series of standard solutions prepared on the blank sample matrix Commented [A25]: VIỆN KIỂM NGHIỆM AN TOÀN VỆ SINH THỰC PHẨM QUỐC GIA THẨM ĐỊNH PHƯƠNG PHÁP TRONG PHÂN TÍCH HĨA HỌC VÀ VI SINH VẬT DS Trần cao sơn 25 - Calculating the standard curve by the first regression line ensures that the linearity of the regression line not less than 0.99 Table 3.1: Concentration of the calibration curve Carbaryl (µg/kg) 20 60 100 200 300 TPP (µl) 100 100 100 100 100 1:9ACN/H2O 880 840 800 700 600 * Repeatability Repeated analysis of 10 spiked samples on white radish samples background at concentrations of 100μg/kg; 200µg/kg; 300µg/kg; 30µg/kg After obtained the result, calculate the standard deviation (s ) Using the formula: s : Repeatability standard deviation x : The result obtained on the i sample x: Average value of the test results, x = Σxi/n n: Number of testing samples Table 3.2: Maximum relative Standard deviation for repeatability depend on concentration range (AOAC) [15, 26] Analyte ratio Unit CV% -4 10 10-5 10-6 100 ppm 10 ppm ppm 5.3 7.3 11 10-7 10-8 100 ppb 10 ppb 15 21 10-9 ppb 30 Commented [A26]: AOAC Official Method 2007.01 Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate 26 * Recovery Arranging experiments at concentrations of 30µg/kg, 100µg/kg; 200µg/kg; 300µg/kg for radish samples Carried out this experiment for 10 times at each concentration above on the same test In case if using spike sample, must be test the "blank sample" (Unspike - a sample used as a spike but without add standard solution), parallel with the spike sample The result is subtracted from average blank sample value Calculate the recovery from the results obtained, using the formula: Rm = X / X Spike Rm: Recovery average X: The average value of the test results X = Σxi-xuns/n xuns: Average value of unspike samples XSpike: The concentration of spike samples Table 3.3: Recovery that accept at different concentrations (AOAC) [15, 26] n Analyzed ratio Unit Recovery [%] 10-4 100 ppm 90-107 10-5 10 ppm 80-110 10-6 ppm 80-110 10-7 100 ppb 80-110 10-8 10 ppb 60-115 10-9 ppb 40-120 * Reproducibility At least two members participated Each people analyzed 10 times spike samples on a blank samples background at the same concentration, on the same or different days Then calculate the repeatability standard deviation between two people Calculate the standard deviation of reproducibility: 27  (ai − bi ) sR = 2k - ai, bi are the i measured value of two members - k is the number of repetitions after eliminating the values of large deviations Table 3.4: Maximum relative standard deviation for reproducibility follow each concentration range (AOAC) [15, 26] Analyzed ratio Unit CV% 10 -4 100 ppm 8.0 10 -5 10 ppm 11.2 10 -6 ppm 15.9 10 -7 100 ppb 22.4 10-8 10 ppb 31.7 10-9 ppb 44.8 * Limit of detection (LOD) and Limit of quantification (LOQ) The calibration curve construction was carried out on a blank basis in the range of Carbaryl concentration of 10-400 (µg/kg) with 100µl TPP of 2ppm concentration The equation for the calibration curve is expressed as y = ax + b LOD and LOQ were calculated by repeated analysis of 5-10 times spike samples at low concentrations and calculated by the formula: LOD = × SD and LOQ = 10 × SD Where SD is the standard deviation when analyzing standardized samples at low concentrations when obtain the signal to noise ratio (S/N ≥3) S is height of signal; N is noise of sample matrix (it can be seen in figure 3.1) 28 Figure 3.1: Determine LOD base on S/N ratio 3.5.3 LC-MS/MS analysis Chromatography separation was performed with column Phenomenex C (3µm pore size, x 150mm) and protection column of the same type Linear gradient elution by Channel A contains 0.1% formic acid in distilled water, channel B is MeOH in 18 at 0.4ml/min was performed at 30ºC An injection volume of 10µl was used An infusion experiment was performed for Carbaryl to optimize the transition conditions for an MRM analysis in the electrospray ionization (ESI) positive ionization mode The condition MRM-MS measurements in the experiment for optimizing the LC separation as follows: drying gas (N2), drying temperature 450ºC and 0,4ml/min gas flow Sample inject were follow: Inject standard solutions, blank samples, and then control samples 29 PART RESULT AND DISCUSTION 4.1 Optimize the conditions for LC-MS/MS Table 4.1: Optimization of the mobile phase composition in gradient elution reversed-phase HPLC Time (Min) 0.50 5.00 6.00 7.00 12.00 14.30 Flow rate (ml/min) 0.4 0.4 0.4 0.4 0.4 0.4 0.4 %A %B 70 70 30 15 5 30 30 70 85 95 95 95 Maximum pressure (PSi) 50 50 50 50 50 50 50 After optimization of the mobile phase composition in gradient elution reversedphase HPLC, the result shown in table 4.1, Chromatographic separasion was performed in phenomenex C18 column (3µm pore size, 2mm x 150mm) Linear gradient elution by 0-100% MeOH containing 0.1% Fomic acid in 18 at 0.4 ml/min was perform at 30ºC with injection volume is 10µl Table 4.2: Optimal MRM-MS/MS conversion conditions for Carbaryl analysis Analyte Mother Daugter peak (m/z) peak (m/z) Carbaryl 201.1 145.1 Retention time (min) 3.49 127.1 TPP 327.1 159.1 3.82 After infusion to optimise MS/MS conditions, table 4.2 shown that Carbaryl gives daugters peak having hight intensity (145.1m/z and 127.1m/z ) and TPP 30 was ionized 159.1(m/z) Figure 4.1 was demonstrated that Carbaryl and TPP were run out at 3.5 min, 3.82 respectively Therefor Carbaryl was ionized successfully Figure 4.1: Standard peak of Carbaryl and TPP in chromatography analysis 4.2 Linearity Table 4.3 Peak area results were obtained in carbaryl analysis Analyte conc (µg/kg) IS conc (µl) Analyte peak area e IS peak area (e ) Ratio analyte peak/IS peak 20 100 1.87 2.90 0.65 60 100 4.93 2.64 1.87 100 100 8.69 2.79 3.1 200 100 15.6 2.74 5.7 300 100 22.6 2.66 8.5 Standard calibration curve was performed by blank sample matrix (sample does not contain pesticide need to analysis) Prepare standard of Carbaryl at 20300µg/kg concentration On this method, TPP will be used as internal standard TPP have chemical and physical properties that relatively similar with Carbaryl Observed in Figure 4.2, the linear correlation of Carbaryl between 20 and 300µg/kg with a coefficient of correlation (r) = 0.999 Analyte peak/ IS peak 31 y = 0.0277x + 0.2001 R² = 0.9994 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 Analyte conc/IS conc Figure 4.2: Calibration curves of Carbaryl 4.3 Limit of detection and limit of quantification Determine the LOD and LOQ procedure by add standard at low concentration into the samples does not contain the chemical that need to analysis On this experiment, we added standard at 50µg/kg concentration, and then parameter the signal to noise ratio (S/N), if S/N ≥ 3, calculate the standard deviation to determine the LOD and LOQ Considering the result on table showed that LOD is 9.81µg/kg and LOQ is 32.71µg/kg To confirm the LOD and LOQ value for this method, we analyzed 10 times reduplicate on blank samples added standard at LOD threshold (10 ppb) See the results on table 3, it can be seen that Carbaryl gives a mother peak and daughter peaks that have S/N ≥ (The results similar with AOAC at survey concentration) So that, can be confirmed that LOD is 10 ppb are acceptable for this method 32 Table 4.4: Limit of Detection and Quantification for Carbaryl analysis n=10 Signal to noise 50µg/kg ratio (S/N) 100.6 48.50 66.5 49.50 93.1 47.90 87.5 48.80 121.5 52.50 197 51.60 166.2 45.50 155.6 56.60 212.1 51.10 10 163.2 46.20 SD 3,27 CV (%) 6,57 LOD (µg/kg) 9,81 LOQ (µg/kg) 32,71 Table 4.5: The result of reliability evaluation of LOD value in Carbaryl analysis at 10ppb concentration (n) Samples S/N ratio S/N ≥ 3 10 498.7 326 443.2 437.6 367.3 236.8 685.7 366.7 515 546.9 ≥3 ≥3 ≥3 ≥3 ≥3 ≥3 ≥3 ≥3 ≥3 ≥3 33 ion, m/z 202>127.1 202>145.1 4.4 Recovery and repeatability Under the optimal LC-MS/MS conditions in table 4.2, we performed the experiment at difference concentration (30µg/kg, 100µg/kg, 200µg/kg, 300µg/kg) Each experiments performed for 10 times on the same samples does not contain the chemical that need to analysis The recovery (R%) was determined by the reality result of Carbaryl after analysis and initial concentration was added Considering the result in table 4.6 that the recovery of Carbaryl at each concentration were higher than 100% In addition, the coefficient of variance bigger than 10 From the result above, it clear that the optimal LC-MS/MS conditions were accurately and suitable for this method Table 4.6: Recovery and repeatability in Carbaryl analyze Samples 30 µg/kg 10 Average SD R% CV% 27,8 30,15 32,1 31,4 30,45 33,0 32,05 31,95 32,3 29,25 31,05 1,61 103,4 5,19 100µg/kg 200µg/kg 300µg/kg 105 110 105 102 109 105 110 105 103 109 106,3 2,95 106,30 2,77 203 195 212 203 209 200 212 203 195 200 203,56 6,18 101,60 3,04 305 307 312 307 309 300 303 307 310 311 307,10 3,70 102,37 1,20 4.5 Reproducibility In this experiment we analyzed at concentration of 30µg/kg on blank background matrix samples Each people performed this experiment repeat 10 times, compare the results between two members and then calculated the CV and 34 SD for the method Observation the results in table 4, it’s conclude that the CV= 4.28 % and SD = 1.32 (the result are similar with AOAC at the same concentration) [27] Table 4.7: The result obtained from analysis Carbaryl at concentration of 30 µg/kg between members n Member Member 27,8 30,94 30,15 32,35 32,1 30,4 31,4 30,4 30,45 29,2 33,01 29,65 32,05 31,05 31,96 31,65 32,3 30,25 10 29,25 29,7 Average 31,045 30,56 CV(%) 4,27 SD 1,32 35 PART V CONCLUSION From the results above, it shown that the method for determined the active substance Carbaryl by liquid chromatography tandem mass spectrometry LC-MS/ MS with the following results: Limit of detection for this method is 10µg/kg, Limits of quantitation were demonstrated to be ≥30µg/kg The average recoveries of pesticide were in the range of 100 –110% with linear regression coefficient r ≥0.99 and the variation is less than 10% From the above analysis results, it can be confirmed that method is fast, sensitive, accurate and reliable for analyzing the active substance Carbaryl in white radish 36 REFEREENCES I Vietnamese references 15/3/2016 medicinal delicious dish from radish Vinh Phuc resource base and environment (2008) Plant protection drugs and their effects 6/7/2016 the benefits of radish According to Circular No 10/2019 / TT BNNPTNT promulgating the list of pesticides allowed and banned to use in Vietnam Danchi (2006) vegetables with excess pesticides Danchi (31/07/2018) More than half of people tested were infected with pesticides in their blood Trung Kien30/4/2017 Agricultural production for export " difficulty" because of residues of plant protection drugs Dieu Thu (09/06/2016) More than 5% of the vegetables sold on the market are contaminated with banned substances Ninh Thuan Police (2009), More than two million people eat dirty vegetables 10 Nguyễn Thị Thanh Huyền 01/11/2010 The harmful effects of plant protection drugs to human health and the environment 11 Van Hai (17/01/2018) Nearly 200 children in Hanoi suffered from food poisoning 12 Robert L Metcalf “Insect Control” Encyclopedia Ullmann for Chemical industry”Wiley-VCH, Weinheim, 2002 13 The Hazard data in pesticide Carbaryl was archived on May 11, 2010, Wayback Machine 14 Circular No 50-2016 TT/BYT on Regulations on maximum limit of pesticide residues 37 15 Tran Cao Son, National Institute of Food Safety and Hygiene Evaluation of methods in chemical and microbiological analysis II English references 16 S.K Roy, A.K Chakrabarti, in Encyclopedia of Food Sciences and Nutrition (Second Edition), 2003 17 Seaman, Abby (2013-11-13) "Turnips and Radishes" Integrated crop and pest management guidelines for commercial vegetable production Retrieved 2020-05-20 18 2003 NPIC national pesticide information center 19 A World Compendium: The Pesticide Manual, 12th ed.; Tomlin, C D S., Ed.; British Crop Protection Council: Farnham, Surrey, UK, 2000; pp 67-68 20 Oct 2003, U.S Environmental Protection Agency, Office of Pesticide Programs, Environmental Fate and Effects Division.Revised EFED Risk Assessment of Carbaryl in Support of the Reregistration Eligibility Decision 21 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 Eligibility Decision (RED) 22 Berk, Zeki (2013) "Food Process Engineering and Technology (Second Edition)" Advances in Molecular Toxicology 23 Journal of Chromatography a volume 856, Issues 1–2, 24 September 1999, Pages 3-54 24 Quechers Methodology: AOAC Method Q-sep™ Packets cat 26237 and 26238 25 Michelangelo Anastassiades, 2003 26 AOAC Official Method 2007.01 Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate 38 ... 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... Liquid chromatography tandem mass spectrometry (LC- MS/ MS) Mass spectrometry liquid chromatography (LC- MS) is an analytical chemical technique that combines the physical separation of liquid chromatography. .. Therefor Carbaryl was ionized successfully Figure 4.1: Standard peak of Carbaryl and TPP in chromatography analysis 4.2 Linearity Table 4.3 Peak area results were obtained in carbaryl analysis Analyte