Chapter 1
Basic Considerations for the Analyst for Veterinary Drug Residue Analysis in Animal Tissues

James D. MacNeil1 and Jack F. Kay2

1Department of Chemistry, St. Mary's University, Halifax, Nova Scotia, Canada

2Department of Mathematics & Statistics, University of Strathclyde, Glasgow, Scotland

1.1 Introduction

It is not sufficient to be expert in the techniques applied in an analytical method to produce a meaningful result when applying a method for the analysis of veterinary drug residues, as is the reality in many other types of chemical analysis. The analyst must also have a sufficient understanding of the nature of the targeted veterinary drug residues to ensure that the method used is fit for the purpose. That is, the method used should be developed and validated for an appropriate concentration range for the right analyte and should be directed at a matrix where residues are likely to be found. In addition, the analyst might reasonably be expected to provide advice on the significance of the results generated with respect to regulatory limits to clients with limited scientific knowledge.

In this chapter, we discuss some of the terminology that is commonly applied in veterinary drug residue analysis, as well as some of the basic information on pharmacokinetics, metabolism, and distribution that help with direct choices of analyte and matrix and that also inform the interpretation of analytical results. We also briefly review the common national and international approaches to the regulation of veterinary drug residues in foods and the establishment of maximum residue limits (MRLs).

1.2 Pharmacokinetics

The term pharmacokinetics is used to describe studies related to quantitative changes in the concentrations of an administered drug in a body over time. Basic parameters associated with a dose are Cmax, the maximum concentration attained following the receipt of a dose of a drug, and t½, the half-life of the drug in the body. These may be determined in the blood or in specific tissues. For the residue analyst, some knowledge of these factors is required to help target analysis at a matrix where residues are likely to be found and to interpret the significance of a residue finding. If the half-life (t½) of a drug in a body fluid or a tissue is measured in minutes or a few hours, there is very probably little to be gained by testing that matrix for residues in an animal slaughtered days or weeks after the drug administration.

The means by which a drug is administered may influence the pharmacokinetics. Veterinary drugs may be available in a variety of formulations, which include injectables, feed additives, sprays, pour-ons, and dips. Injections may be via routes which included intravenous, intramuscular (i.m.), subcutaneous (s.c.), and intramammary. In some cases, the injection may lead to the occurrence of a depot at the injection site, with a low rate of absorption, leading to the presence of significant residues at the injection site for an extended period. The residues at the injection site will not be representative of residues found in muscle tissue away from the site of injection. Thus, a finding of high residue concentrations in muscle tissue, for example, should lead the analyst to suspect that the tissue analyzed may be from an injection site, and therefore additional analyses should be conducted on muscle samples from other parts of the carcass or lot before concluding that the initial results are truly representative.

For example, the 47th Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) recommended MRLs of 10 µg/kg for doramectin residues in beef muscle.1 It also noted that residues were slightly higher in the muscle from cattle given an s.c. dose when compared to cattle which received treatment by i.m. injection. In addition, after 35 days withdrawal, residues in muscle were < 3 and < 2 µg/kg from the s.c. and i.m. treatment groups, respectively. However, injection site muscle from these animals contained 930 µg/kg (s.c. group) and 177 µg/kg (i.m. group) at 35 days post-treatment. The committee in recommending MRLs for doramectin in cattle noted that high concentrations of residues may remain at the injection site after treatment according to approved uses. In adopting the MRL recommendations, the Codex Alimentarius Commission (CAC) included a note with the MRLs for beef muscle and fat that there was a potential that residues of doramectin in excess of the MRLs could persist at injection sites following recommended treatment.2

Subsequently, in reviewing data for the use of doramectin in the treatment of pigs, the 52nd Meeting of the JECFA recommended an MRL of 5 µg/kg in pork muscle, based on twice the limit of quantification (LOQ) of a method judged to be suitable for routine regulatory use.3 In a depletion study reviewed by the 52nd Meeting of the JECFA, pigs were treated by i.m. injection at 1.25 times the recommended dose and subjected to a 28-day withdrawal period, as per the approved use from a Codex Alimentarius member state.3 No quantifiable residues were detected in "normal" muscle tissue, meaning that residues in the muscle tissue should be below this limit if the drug is used according to the established Good Veterinary Practices (GVP). The committee again noted that higher concentrations could be found in the injection site tissue from pigs. A finding of residues in excess of the MRL for doramectin in muscle or fat may therefore mean that the tissue sample is from a site of injection and does not represent the residues present in "normal" muscle or fat. Such a finding indicates that additional sample material should be obtained to determine if the initial sample analyzed was truly representative of tissues from the animal or lot. Thus, knowledge of the pharmacokinetics and depletion of a drug is required when interpreting the results of analysis.

1.3 Metabolism and Distribution

The term metabolism refers to the chemical processes which occur in a living organism and which can transform an administered drug into other chemical compounds, while the term distribution refers to the manner in which residues are distributed to different tissues and body fluids. Knowledge of these elements is critical to determining the nature of the residues which should be determined by a method and the matrix or matrices in which these residues are most likely to be found.

This brings us to two fundamental terms frequently used in the analysis of veterinary drug residues: the marker residue and the target tissue. The CAC has defined the marker residue as the "residue whose concentration decreases in a known relationship to the level of total residues in tissues, eggs, milk or other animal tissues."4 CAC guidelines for the design and implementation of a program for the control of veterinary drug residues in foods note that the marker residue "may be the parent drug, a major metabolite, a sum of parent drug and/or metabolites or a reaction product formed from the drug residues during analysis" and that "the parent drug or the metabolite may be present in the form of a bound residue which requires chemical or enzymatic treatment or incubation to be released for analysis."5 The target tissue is usually "the edible tissue in which residues of the marker residue occur at the highest concentrations and are most persistent." Knowledge of the appropriate marker residue and target tissue is usually obtained from controlled studies to investigate the metabolism and distribution of residues of a drug following administration to an animal species. For veterinary drugs which have been reviewed by the JECFA as part of the process of the development of international standards (MRLs) through the CAC, monographs detailing the pharmacokinetics, metabolism, distribution, and depletion studies may be found on the Food and Agriculture Organization (FAO) JECFA website.6

It was common practice in most countries until about 2000 to monitor nitrofuran use by testing for parent compounds, although it had been shown in the 1980s that these compounds were rapidly metabolized, as noted in a JECFA review of residues of furazolidone,7 and that monitoring for parent compounds was therefore highly unlikely to produce positive results. However, when methods became available to monitor for bound residues of the metabolites of these compounds, the use of which had been banned in food-producing animals in most countries, detection of use became practical and positive results were reported.8 This provides an example of the importance of identifying the appropriate marker residue. Some drugs, such as lasalocid sodium9 and ractopamine hydrochloride,10 are administered as salts but are rapidly transformed to the free parent drug (lasalocid or ractopamine) on injection, and it is the free parent drug, not the salt, which is the appropriate marker residue. Other drugs are rapidly transformed into new active substances immediately following injection. The organophosphate trichlorfon is used orally or topically to treat parasites in various animal species. Following administration, it is rapidly transformed to the insecticide dichlorvos, and it was noted in the JECFA evaluation that trichlorfon is "metabolized so extensively and rapidly that the ratio of marker residue to total residues cannot be defined."11 However, despite the extensive metabolism, it was determined by JECFA that trichlorfon parent drug was the most appropriate marker residue.

Metabolism can also convert parent drugs into...