JOURNAL TRANSCRIPT
Laboratory manual of standardized methods for analysis of pesticide and antibiotic residues in aquaculture products
Ilda G. Borlongan Joyce Ng Poh Chuan
SOUTHEAST ASIAN FISHERIES DEVELOPMENT CENTER Aquaculture and Marine Fisheries Research Department
GOVERNMENT OF JAPAN TRUST FUND
On the Cover
Laboratory manual of standardized methods for the analysis of pesticide and antibiotic residues in aquaculture products
ISBN:
9718511660
Published by: Southeast Asian Fisheries Development Center Aquaculture Department Tigbauan, Iloilo, Philippines
Copyright @ 2004 Southeast Asian Fisheries Development Center Aquaculture Department Tigbauan, Iloilo, Philippines
ALL RIGHTS RESERVED No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without the permission in writing from the publisher.
For inquiries:
Fax E-mail AQD website
SEAFDEC Aquaculture Department 5021 Tigbauan, Iloilo, Philippines 63-33-335-1008
[email protected] http://w ww.seafdec.org.ph
PREFACE Aquaculture production in Southeast Asia has grown rapidly and is now a major contributor to food supply worldwide. The use of chemicals in aquaculture systems for various purposes is widely recognized. While aquaculturists acknowledge that some operations are reliant on chemical usage, they also realize the potential danger associated with chemical misuse. The increasing use of chemicals in aquaculture has led to widespread public concern because pesticide and antibiotic residues could eventually end up in aquaculture products. Because of their potential adverse effect on human health, governments worldwide set limits on allowable levels of chemical residues in food and animal feeds and monitor these levels. The monitoring and enforcement actions are, however, dependent on the technical capability to detect residues. Realizing this need, the Government of Japan through the Trust Fund’s Fish Disease Project funded a project to standardize methodologies for detection of pesticide and antibiotic residues in aquaculture products. Two separate studies were implemented by the Aquaculture Department of the Southeast Asian Fisheries Development Center (SEA FDEC/AQD, Philippines) and the Marine Fisheries Research Department (SEAFDEC/MFRD, Singapore) on pesticide and antibiotic residues, respectively. The results of these studies are the basis for this manual, which will benefit all those involved in the monitoring and enforcement aspects of chemical residue limits in aquaculture products in the region.
Dr. Kazuya Nagasawa Fish Disease Project Leader Government of Japan Trust Fund
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ACKNOWLEDGEMENTS
The Government of Japan (GOJ) - Trust Fund through SEA FDEC Aquaculture Department provided financial support for publication of this manual. Dr. Yasuo Inui, the first Fish Disease Project Leader of the GOJ - Trust Fund Project laid down the ground-work for the smooth conduct of the project. Ms. Lai-Kim Tan-Low of MFRD was the study leader of the antibiotic residue project. We thank Dr. Relicardo M. Coloso and Ms. Milagros Castaños for reviewing the manuscript and Engr. Nelson Golez for the cover design.
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CONTENTS
Preface
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Acknowledgements
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Table of Contents
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Chapter 1: DETECTION OF ANTIBIOTIC RESIDUES IN AQUACULTURE PRODUCTS
1
Introduction
2
Determination of Oxolinic Acid by HPLC-f luorescence Method
3
Principle Apparatus Reagents Sampling Procedure Procedure Calculation Method Validation References
3 3 4 5 6 10 10 12
Determination of Oxytetracycline, Tetracycline and Chlortetracycline by HPLC-f luorescence Method Principle Apparatus Reagents Sampling Procedure Procedure Calculation Method Validation References
13 13 13 14 16 16 19 19 21
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Chapter 2: DETECTION OF PESTICIDE RESIDUES IN AQUACULTURE PRODUCTS
23
Introduction
24
Preparation of Samples
25
Multi-residue Method
27
Principle Reagents Extraction Clean-up Petroleum Ether-Acetonitrile Partitioning Florisil Column Clean-up Magnesia Column Clean-up Detection and Quantitation by Gas Chromatography Gas Chromatographic Conditions Procedure
27 27 28 33 33 33 36 37 38 38
Determination of Pesticide Residues in Non-fatty Samples
39
Principle Reagents Extraction Calculation of Equivalent Sample Weight Determination of Polychlorinated Biphenyl Residues Extraction Clean up (Florisil Column)
39 39 39 40 41 41 41
Determination of Carbamate Residues Principle Reagents Extraction and Clean-up
42 42 42 42
Method Validation
45
Limits of Detection
45
References
46
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CHAPTER 1
Detection of Antibiotic Residues in Aquaculture Products
Prepared by
Joyce Ng Poh Chuan Senior Research Officer Marine Fisheries Research Department Southeast Asian Fisheries Development Center Singapore
INTRODUCTION Aquacultured animals are under constant threat from bio-aggressors such as vir uses, bacteria, parasites and fungi. These organisms harm either spontaneously or through aquatic animal husbandry practices, and often both. Indeed, it is generally recognized that disease problems follow the development of techniques for animal production. Consequently, fish culture uses a variety of chemicals that represent potential threats to the health of the cultured animal, indigenous biota, and even humans. Chemicals employed in aquaculture include the following: -
Drugs used to treat disease (chemical therapeutants)
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Chemicals introduced through construction materials
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Hormones used to alter reproductive viability, sex, and growth rates
Of these, chemotherapeutic drugs are the most harmful. Chemotherapeutic treatments are initiated after clinical signs of a disease appear in a population of fish. Chemicals used in construction and hormones are not considered because they are relatively non-toxic. The use of chemical therapeutants obviously leads to the transit of drugs and to their persistence in products intended for human consumption. It also leads to the release of drugs or their metabolites to the aquatic environment. Hence the criticisms raised in the press against the use of chemotherapy in aquaculture, and the restrictive legislation set up in many countries under pressure of public opinion. It sometimes appears that people would believe that drug resistance of bacteria responsible for human infections originates exclusively, or almost exclusively, from consumption of animal products such as those provided by aquaculture. It should be noted that in addition to the chemicals that are deliberately used, fish raised in aquaculture are also susceptible to contamination via pesticides present in feed, agriculture run-off water, and sediments. The magnitude of human exposure to these sources has not yet been fully assessed and should be examined periodically in light of the growth and change in this sector of the seafood industry.
Determination of Oxolinic Acid by HPLC-Fluorescence Method Principle Oxolinic acid (OXA) is a powerful synthetic antibacterial agent used in aquaculture in curing or preventing diseases caused by certain species of Yersinia, Aeromonas and Vibrio. The methods for the detection of the bactericide, oxolinic acid, can be generally divided into two categories, namely biological and physiochemical. The biological method, such as bioassay, lacks sensitivity and specificity. The physiochemical method, based mainly on HPLC with U V or f luorescence detection, is much faster, more specific and more sensitive than the biological method. Apparatus a.
High Performance Liquid Chromatograph (HPLC): WATERS Isocratic pump system, WATERS in-line degasser, 600E Multisolvent Delivery System, 600E System Controller, 717 Plus Autosampler equipped with 470 Scanning Fluorescence Detector capable of monitoring emission at 365 nm and excitation at 337 nm.
b.
Chromatographic column: Reverse phase, TSK-GEL ODS-8OTM (150 x 4.6mm) Operating condition: Flow rate set at 0.5 mL/min. Injection volume: 20 ml. As a part of the system shut-down at the end of the experiment, HPLC grade water is pumped through the column for a minimum of twenty min followed by a twenty min rinse with methanol:water (7:3) at 0.5mL/ min
c.
Filter Unit: GL, Chromatodisc 13P, 0.4 mm
d.
Glass centrifuge bottle (150 ml) and tubes (15 mL)
e.
Separatory f lasks (125 mL)
f.
Florentine f lask (100 mL)
g.
Tissue homogenizer
h.
Centrifuge
i.
Rotary evaporator
j.
Ultrasonic water bath
Reagents a.
Acetonitrile (HPLC grade)
b.
Water (HPLC grade, Diamond-Q)
c.
1-propanol (GR grade)
d.
n-hexane (GR grade)
e.
Sodium sulfate, anhydrous (GR grade)
f.
Methanol (HPLC grade)
g.
Sodium chloride (GR grade)
h.
0.1 M citric acid (HPLC grade): Dissolve 21.01 g of monohydrate salt of citric acid in 1L of HPLC grade water.
i.
Oxolinic acid (Sigma Chemical Company): All standard solutions are stored below 10o C. Stock solution is stable for at least 3 months, but diluted solutions should be kept no longer than 2 weeks. Stock solution (100 ppm): Weigh accurately 10 mg of oxolinic acid and make up to 100 mL in a volumetric f lask using acetonitrile:water (1:1) solution. Intermediate solution (10 ppm): Pipette accurately 5 mL of the 100 ppm stock solution into a 50 mL volumetric f lask and make up volume with acetonitrile:water (1:1) solution. Working solution (1 ppm): Pipette accurately 5 mL of the 10 ppm intermediate solution into a 50 mL volumetric f lask and make up volume with acetonitrile-water (1:1) solution. The working solution should not be used after 1 month of refrigerated storage and fresh working solution should be prepared.
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Mobile Phase: ( Acetonitrile:methanol) - 0.1M citric acid solution (2:2:3), filtered through polyvinylidene f luoride membrane (0.45 mm). The mobile phase should be prepared daily.
Sampling Procedures 1. Place frozen shrimps sample at 5o C overnight. Remove the head and shell.
2. Mince the sample rapidly and thoroughly with chopper. Remove unground material from the blade of chopper and mix thoroughly with ground material and mince thoroughly again.
3. Turn the mince into the shape of a burger and divide into four equl portions.
1st quadrant
4. Take the first quadrant of minced meat for testing.
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Procedure 1. Accurately weigh 5 g of the minced sample (edible portion of the tiger prawn, not de-veined) into a 150 ml glass ceentrifuge bottle. Spike test sample at this stage with 2 ml of 1 ppm working solution.
2. Add 25 ml of acetonitrile and 10 g of anhydrous soduim sulfate into the sample and homogenize for 1 min.
3. Centrifuge at room temperature for 5 min at 2,500 rpm.
4. Filter the supernatant through Whatman No.1 filter paper into a 125 ml separatory funnel containing 25 ml acetonitrile - saturated nhexane.
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5. Add another 25 ml of acetonitrile to the homogenate and sonicate for 30 sec. gently mixing the sample with a glass rod during this process.
6. Centrifuge at room temperature for 5 min at 2,500 rpm and filter the supernatant into the earlier 125 ml separatory funnel (Step 4). Rinse the filter paper with acenotrile and allow washing to drain into separatory funnel
7. Shake f lask vigorously for 10 min. Allow to stand for separation ito layers.
8. Slowly drain off the acetonitrile ( lower) layer into a 100 ml f lorentine f lask.
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9. Add approximately 5 ml of 1-propanol (add more if mixture boils too vigorously) and evaporate the acetonitrile layer to dryness using the rotary evaporator at 40o C.
10. Flush f lask with nitrogen gas to remove any trace of propanol.
11. Add accurately 2 ml of acetonitrile:water (3:7) and sonicate until the residue is dissolved.
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12. Transfer the the sonicated solution into a 15ml glass centrifuge tube. Add 100 ml of soduim chloride and 1 ml of acetonitrile- saturated n-hexane and sonicate the mixture. At this stage, an orangelayer develops at the top. This is a sign of lipids moving on to the top n-hexane layer.
13. Centrifuge at room temperature for 5 min at 2,500 rpm.
14. Very carefully, using a pasteur pipette, pipette out the acetonitrile (lower, aqueous) layer into a plastic syringe (fixed with filter Chromatodisc 13P) and filter the extract into a glass vial.
15. The filtered sample is then ready for injection into HPLC. Inject 20 µl sample using a f low rate of 0.5 ml/min with detector excitation set at 337 nm and emission at 365 nm.
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Calculation Concentration of oxolinic acid in sample (ppm) = Std conc.(mg) x Sample peak area x Final vol. of extract x F x Inj. vol.(sample) Std. peak area Sample weight Inj.vol.(std)
where F = dilution factor = 1
Method Validation Method validation was performed in compliance with regulations to ensure that the analytical methodology is accurate, specific, reproducible, and adaptable over the specified range of an analyte. System suitability tests are used to verif y that the resolution and reproducibility of the system are adequate for the analysis to be performed. The system suitability was checked by injecting 2 ppm of oxolinic acid standard for 10 times into the HPLC. Percentage relative standard deviation calculated from the 10 injections is 1.8 % (criteria: %RSD < = 2%). This indicates that the autosampler of HPLC is suitable for use. All the criteria like Number of theoretical plates (N), Tailing factor (T), Precision (%RSD) and Resolution (R) were met. The results are shown in Table 1. The retention time of oxolinic acid is about 6.5 min. Analysis time per injection is only 9 min, which is practical. Thus, this shows that f low-rate, column length, and solvent composition are suitable for this application. Relative standard deviation of retention time between replicates is < 1%. T, was determined by calculating the asymmetrical factor derived from generated chromatogram. Calculated T was found to be about 1.6 (criteria: 0.5 < = T = < 2) and N, was 3306 (general criteria: N > = 2000). The N and T values indicate that the column used, TSK-GEL ODS-80 TM, is suitable for this application.
Table 1. System suitability of the HPLC-f luorescence detector system for oxolinic acid determination.
Parameters Retention Time (min) Area Height W1/2 (min) A (min) B (min) Tailing Factor (min) Resolution (R) Theoretical Plates (N)
Oxolinic acid* 6.481± 0.018 %RSD = 0.28 4697763 ± 84561 %RSD = 1.80 229946 ± 4287 %RSD = 1.86 0.266 ± 0.008 %RSD = 2.90 0.317 ± 0.000 %RSD = 0.00 0.205 ± 0.013 %RSD = 6.15 1.60 ± 0.10 %RSD = 6.00 7.41 ± 0.02 %RSD = 0.28 3306 ± 203 %RSD = 6.13
* Mean ± Standard Deviation Criteria: 1. Number of theoretical plates, N >= 2000 [N=16(tR/W)^2] 2. Tailing Factor, 0.5