Training Manual
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Training of Trainers Program on Analysis of Veterinary Drug Residues including Antibiotics TRAINING MANUAL Name: __________________________________ Email: __________________________________ Organized by: FSSAI & GFSP in association with PBTI at Punjab Biotechnology Incubator, Mohali, Punjab February, 2018 MANUAL FOR VETERINARY DRUG RESIDUE ANALYSIS INCLUDING ANTIBIOTICS Food Safety and Standards Authority of India Ministry of Health and Family Welfare, Government of India Manual for veterinary drug residue analysis including antibiotics, 2018. © 2018 by Food Safety and Standards Authority of India, New Delhi. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical or photocopying, recording or otherwise without the prior written permission of the publisher or authors. ABOUT THE MANUAL The current manual borrows its majority of content and structure from the Training of Trainers (ToT) manual prepared by IFSTL partners USFDA, USDA and JIFSAN and with their consent. This manual is complementary to the workshop laboratory manual and lecture notes. The manual is meant to provide future trainers with an opportunity to make notes on various aspects of logistics associated with giving the training to a group. COPYRIGHT NOTICE The contents of the manual are protected by the terms of the Food Safety and Standard Authority of India (FSSAI), Ministry of Health and Family Welfare, Govt. of India. Permission to use whole or parts of texts contained in this manual in printed or electronic form must be obtained and is usually subject to royalty agreements. Proposals for non-commercial reproductions and translations are welcomed and considered on a case-by-case basis. Enquiries should be addressed to FSSAI. LABORATORY SAFETY 1. Laboratory coats should be worn at all times in the laboratory. 2. Use fume hoods whenever handling volatile or hazardous chemicals. 3. Safety goggles should be worn at all times in the laboratory. 4. Appropriate gloves should be worn as needed. 5. Gloves and lab coats should not be worn outside the laboratory. 6. Appropriate closed-toed shoes should be worn in the laboratory. 7. All samples and transfer containers must be labelled. 8. MSDS are available in a clearly marked binder in the laboratory if you wish to consult them. 9. In the event of an emergency please contact one of the supervising instructors immediately. FOREWORD Laboratories are crucial to national veterinary drug residue monitoring programmes. However, one of the main challenges laboratories encounter is obtaining access to relevant methods of analysis. Thus, in addition to training, providing technical advice and transferring technology, the FSSAI has resolved to develop clear and practical manuals to support laboratories at national level. FSSAI with the support of scientific committee has developed a number of analytical methods as standard operating procedures (SOPs), which are now compiled in various manuals. This manual contains SOPs on chromatographic and spectrometric techniques, as well as veterinary drug residue analysis in different food commodities. Some analytical method validation protocols are also included. The publication is primarily aimed at food safety laboratories involved in testing veterinary drug residues, including under organized national residue monitoring programmes. It is expected to enhance laboratory capacity and competence of various laboratories in India. Quality Assurance Division FSSAI Page Chapter Contents No. 1. Introduction....................................................................................... 1 1.1. Background............................................................................... 1 1.2. Objective.................................................................................... 1 1.3. Scope.......................................................................................... 1 1.4. Structure.................................................................................... 1 2. Introduction on Veterinary Drugs................................................ 2 2.1. Antibiotics and their veterinary usage................................. 2 2.2. Antibiotic usage and its impact in food chain..................... 3 2.3. Method criteria......................................................................... 5 3. Sample Preparation/Processing for fishery products................ 7 4. Analytical techniques in Antibiotic residues analysis............. 8 5. Determination of Chloramphenicol- HPLC-MS/MS method. 15 6. Determination of Nitrofuran metabolites- HPLC-MS/MS method............................................................................................... 21 7. Simultaneous determination of multi-residue and multi- class Antibiotic residues using - HPLC-MS/MS method........ 28 8. Method Validation protocol for veterinary drugs including antibiotics........................................................................................... 35 9. Annexure - I……………………………………………………... 46 10. Annexure - II…………………………………………………….. 50 11. Annexure - III…………………………………………………… 55 12. Annexure – IV (I-A)…….…………………................................ 59 Annexure – IV (I-B) ………......................................................... 63 Annexure – IV (II) ………........................................................... 68 Annexure – IV (III) ……….......................................................... 71 13. Annexure –V……………….……………………………………. 74 Chapter 1 1. INTRODUCTION 1.1. BACKGROUND The presence of residues of banned substances/ substances permitted but exceeding the prescribed limits by the regulatory authorities in case of veterinary drugs, pharmaceutical products and pharmaceutically active substances in products of animal origin (like muscle, liver, kidney, fish-flesh, egg, milk, honey etc.) and from various species (like bovine, ovine, porcine, caprine, poultry, rabbit, farmed fish etc) is a matter of concern for public health. As a consequence, national food safety authorities and regulatory authorities have banned the use/ strictly regulated its use in veterinary practice or established legal guidance to ensure proper use of these veterinary drugs, pharmaceutical products and pharmaceutically active substances. The successful implementation of national regulation and surveillance monitoring depends on availability of reliable analytical techniques. Various techniques are available, employed and are in practice like Immunoassay for screening and liquid chromatography with Ultra-Violet/ Fluorescence detection/ Mass spectrometry to determine and identify the commercially available veterinary drugs, pharmaceutical products and pharmaceutically active substances in products of animal origin. 1.2. OBJECTIVE The purpose of this manual is to aid food testing laboratories primarily in the routine monitoring, determination and control of residues of some veterinary drugs in animal products. 1.3. SCOPE This manual publication consists of analytical techniques, methods in form of SOPs, for testing selected veterinary drug residues in some animal products. It covers a number of chromatographic−spectrometric techniques. The information in the manual is also presented as an informative guide in some respect. 1.4. STRUCTURE The preliminary (and larger) part of the manual consists of chromatographic and spectrometric SOPs for analysis of antimicrobial residues such as chloramphenicol, nitrofuran metabolites and tetracyclines, sulfonamides as well as quinolones. The manual then concludes a reference validation protocol. TOT Program on Analysis of Veterinary Drug Residues including Antibiotics Page | 1 Chapter 2 2. Introduction on Veterinary Drugs Veterinary drugs are defined as any substance applied or administered to any food- producing animal, such as meat or milk producing animals, poultry, fish or bees, whether used for therapeutic, prophylactic, or diagnostic purposes, or for modification of physiological functions or behavior. Residues of veterinary drugsare pharmacologically active substances, whether active principles, excipients or degradation products, and their metabolites which remain in foodstuffs obtained from animals to which the veterinary medicinal product in question has been administered.The definition includes the parent compounds and/or their metabolites in any edible portion of the animal product, and includes residues of associated impurities of the veterinary drug concerned. There are various functional classes of veterinary drugs. The functional classes as classified by Codex Alimentarius Commission are: Adrenoceptor agonist, Anthelmintic agent, Antibacterial agent, Antifungal and antiprotozoal agent, Antimicrobial agent, Antiparasitic agent, Antiprotozoal agent, Beta-adrenoreceptor blocking agent, Glucocorticosteroid, Growth promoter, Insecticide, Production aid, Tranquilizing agent and Trypanocide. Veterinary drugs are being increasingly used in farming of livestock, poultry birds and aquaculture. The main purpose of use of these drugs is to prevent outbreak of diseases, prevent mortality during transport, increase freshness or to stimulate growth during farming. As a consequence, veterinary drugs appear in the final product as residues and further continue in the food chain. Consumption of animal products (e.g. fish, shrimp, milk, meat, etc.) containing residues of these compounds for protracted period of time can render undesirable adverse health effects. Major concerns of veterinary