2. QUICK DETERMINATION OF ATRAZINE RESIDUES IN FOODS M. H. EL-SAEID* AND M. A. SALEH BIOMARKERS & ENVIRONMENTAL TOXICOLOGY LAB. CHEMISTRY DEPT. TEXAS SOUTHERN UNIVERSITY, HOUSTON, TEXAS

3. INTRODUCTION  Atrazine herbicide commonly used in agriculture.  Using cause a residues in fruit and vegetables.  Residues cause health problems.  Residues need a quick extraction.  Quick extraction by Supercritical Fluid Extraction & Microwave Solvent Extraction {SFE & MSE.}.  Residues need a quick determination.  Quick determination by Supercritical Fluid Chromatography & Enzyme-Linked Immunosorbent Assay {SFC & ELISA}.

4. SFE & MSE has Potential advantages than conventional extraction. Methods. Reduced time. Superior recovery. Reduction in solvent usage. No cleanup. Safe. SFE can be coupled directly with SFC. MSE has proven to be safe alternative to conventional organic solvent extraction. High ability as Food extraction techniques.

6. SFC is separation techniques used critical points & carrier gases of CO 2. CO 2 is a safe gas to use. Become increasingly apparent in last several years. Complements GC & HPLC, it can be analyze different molecular weights without drivatization. Increase the analysis speed. Wider range of detectors, polarity & sensitivity. Can be coupled with SFE.

7. Enzyme Linked Immunosorbent Assay {ELISA} is An important environmental analysis method used to identify Atrazine in Water & Foods. The EnviroGard Triazine Plate Kit is a quantitative laboratory test for the detection of Atrazine residues in water and foods uses polyclonal antibodies which bind Atrazine-enzyme conjugate for a limited number of antibody binding sites. Antibodies that bind Atrazine are immobilized to the inside of the wells. EPA, FDA, AOAC and Codex methods. Rapid (less than 45 minutes). Inexpensive, portable & can be used to evaluate many samples at the same time. Simple & sensitive (0.02 to 5.0 ppb). No solvent disposal.

8. AIM OF STUDY oExtract and determined Atrazine by high sensitive technique in short time. oModify & Compare SFE and MSE as extraction methods. oModify & Compare SFC and ELISA technique as a determination methods

9. MATERIALS  Eleven types of food Samples –Fruit juice( Guava, Mango and fruit cocktail) –Frozen Vegetables( Mixed, Molukhia, Artichoke, Colocassia, Beans, and Okra) – Jam(Strawberry) Homogenized and divided in tow portion, one spiked and one as control Both portion were freeze-dried. Stored at -20 °C.  Atrazine standard obtained from Chemservice, Inc.  Envirogard Triazine Plate Kit 72110 was obtained from Strategic Diagnostics Inc.

10. ATRAZINE EXTRACTION METHODS +SFE uhp SFE model 7680T with hp 1050 pump & Hypersil ODS 30  m trap was used. u Extraction of 10g food samples was carried out in 3 steps. Parameters Step# I Step# IIStep# III CO 2 Density (g/ml) 0.250.670.67 CO 2 F.R. (ml/min)12.52.5 Pressure (psi)111734693469 Chamber Temp. (°C)408080 Nozzle Temp. (°C)454545 Modifier (MeOH) %0030 Time(min)5155  Extracted sample was eluted from the ODS trap by 1.5 ml MeOH  ODS trap regenerated by rinsing 2 ml of MeCl 2 followed by 2 ml MeOH

11.  MSE CEM Corp., MSE model MES-1000 was used with fiber optic temp. control. Extraction of 10g food samples was carried out at: –Extraction solvent  60 ml acetone : hexane (3:2) –Microwave power  75 % –Temperature  125 °C –Pressure  85 psi –Time  20 min  The food sample was filtrated, washed by same solvent 2 times and concentrated to 1.5 ml by Rotevap.  No cleanup Need for both extraction methods.

12. ATRAZINE DETERMINATION METHODS  SFC  hp SFC model G1205A attached to an hp 1050 DAD& G1205A modifier pump was used.  SFC parameters was carried out at:  Temp. (°C)  30  Press. (psi)  80-150  Flow (ml/min)  1-2  Modifier (%)  2-3 MeOH  Column  Alltec Hypersil APS 25m, 205mm, ID 4.6mm  W.L. (nm)  210  Time (min)  10

13.  ELISA procedures  (1) Strip format of negative control {NC} (C), 3 calibrators (C1- C3) and (S1 to S88). for food samples extract.  (2) Add 80  l of NC (C), 80  l of each calibrator (C1-C3) and 80  l of each food extract (S1 to S88) to their respective wells.  (3) Add 80  l of Atrazine-Enzyme conjugate to each well.  (4) Rapid circular motion for 1 minute to mix contents of wells.  (5) Cover by parafilm to prevent evaporation and incubate at ambient temperature for 1 hour with orbital mixing at 200 rpm.  (6) Vigorously shake the contents of the wells, five times washing by a micrototer plate washer to flood the wells completely with cool running tab water, then shake to empty.  (7) Add 80  l of substrate to each well (C), (C1 to C3) and (S1 to S88).

14.  (8) Add 40  l of chromogen sol. to each well.  (9) Mix for 1 min., cover, & incubate for 30 min. with orbital mixing at 200 rpm.  (10) Add 40  l of stop sol. to each well arrest the blue color development and turn the reaction solution yellow, mix thoroughly without spilling until all of the blue has converted to yellow.  A Microplate Reader system model MR 5000 with Biolinx assay manag. software was using as microtiter plate reader to interpret the results at wavelength 450 nm.  Auto-zero on air in a blank well to measure and record the Optical Density (OD) of each wells contents as %B o using semi- log curve with data reduction capabilities fit for the standard curve.

15.  After OD reading by microtiter plate reader for all of the 96 wells, average the OD of each set of calibrators and food samples and calculates of the B o.  The %B o calculation is used as a means of equalizing different runs of assay, also %B o relationship of calibrators and samples to the negative control should remain fairly constant. Graph %B o of each calibrator against its Atrazine concentration on the semi-log scale.  Determine the Atrazine concentration of each food sample by finding its %B o value and the corresponding concentration level on the graph.  Interpolation of sample concentration is only valid if the %B o of the sample falls within the range of the

16. % B o ’s set by the calibrators. Average OD of calibrator or sample %B o = --------------------------------------------------- x 100 Average OD of negative control 100- % B o C x = C determined ------------------------ 100 OD= Optical Density. C x = Actual concentration of Atrazine.

17. RESULTS è Minimum Detection Limits (MDL) of Atrazine by SFC/UVD at 220 nm and ELISA technique at 450 nm. Atrazine (ppb)SFCELISA 0.001ND*D** 0.03NDD 0.06NDD 0.09DD 0.1DD 0.5DD 1.0DD ------------------------------------------------------------------------------------------- *= Not Detected **= Detected

18. è Extraction & Determination Time Extraction time(min)/ sample SFEMSE 15.05.0 Determination time (min)/ sample SFCELISA 10.02.1

19. è Atrazine residues (ppb) & RSD in Juice samples Samples SFE MSE SFC ELISA SFC ELISA Guava ND 0.02  03 ND 0.02  03 -------------------------------------------------------------------------------- Mango 0.31  05 0.35  08 0.27  07 0.34  05 -------------------------------------------------------------------------------- cocktail ND ND ND ND --------------------------------------------------------------

20. èAtrazine residues (ppb) & RSD in frozen samples & strawberry jam.

21. èAtrazine recovery % & RSD in Juice samples Samples SFE MSE SFC ELISA SFC ELISA Guava93.3  1.2 98.7  0.8 92.4  1.6 97.4  1.6 ------------------------------------------------------------------------------------------------- Mango 94.4  1.1 97.2  0.6 93.2  1.3 97.1  1.3 ------------------------------------------------------------------------------------------------- cocktail 94.2  1.5 96.8  0.7 94.8  2.7 95.5  2.7 ------------------------------------------------

22. èAtrazine recovery % & RSD in frozen samples & strawberry jam.

23. CONCLUSION u The MDL of Atrazine was 0.06ppb by SFC & 0.01 ppb by ELISA. u Extraction time / sample was less for MSE than SFE. uDeter. Time was 10 min. by SFC & 2.1 min. by ELISA. uELISA is considerably fast with LDL than SFC. uAtrazine risidue ND in cocktail juice by both techniques. uAtrazine risidue ND in guava juice, frozen artichoke, colocassia, & okra by SFC techniques. uAtrazine risidue were detected in 54.5% and 90.9% of investgated food samples using SFC & ELISA respectively. uELISA technique coupled with SFE & MSE is less time consuming, high recovery% and more senstive with lower detection limit of Atrazine residues in foods.