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Chapter 1
Antibiotics: Groups and Properties
Philip Thomas Reeves
1.1 Introduction
The introduction of the sulfonamides in the 1930s and benzylpenicillin in the 1940s completely revolutionized medicine by reducing the morbidity and mortality of many infectious diseases. Today, antimicrobial drugs are used in food-producing animals to treat and prevent diseases and to enhance growth rate and feed efficiency. Such use is fundamental to animal health and well-being and to the economics of the livestock industry, and has seen the development of antimicrobials such as ceftiofur, florfenicol, tiamulin, tilmicosin, tulathromycin, and tylosin specifically for use in food-producing animals.[1, 2] However, these uses may result in residues in foods and have been linked to the emergence of antibiotic-resistant strains of disease-causing bacteria with potential human health ramifications.[3] Antimicrobial drug resistance is not addressed in detail in this text, and the interested reader is referred to an excellent overview by Martinez and Silley.[4]
Many factors influence the residue profiles of antibiotics in animal-derived edible tissues (meat and offal) and products (milk and eggs), and in fish and honey. Among these factors are the approved uses, which vary markedly between antibiotic classes and to a lesser degree within classes. For instance, in some countries, residues of quinolones in animal tissues, milk, honey, shrimp, and fish are legally permitted (maximum residue limits [MRLs] have been established). By comparison, the approved uses of the macrolides are confined to the treatment of respiratory disease and for growth promotion (in some countries) in meat-producing animals (excluding fish), and to the treatment of American foulbrood disease in honeybees. As a consequence, residues of macrolides are legally permitted only in edible tissues derived from these food-producing species, and in honey in some countries. Although a MRL for tylosin in honey has not been established, some countries apply a safe working residue level, thereby permitting the presence of trace concentrations of tylosin to allow for its use. Substantial differences in the approved uses of antimicrobial agents also occur between countries. A second factor that influences residue profiles of antimicrobial drugs is their chemical nature and physicochemical properties, which impact pharmacokinetic behavior. Pharmacokinetics (PK), which describes the timecourse of drug concentration in the body, is introduced in this chapter and discussed further in Chapter 2.
Analytical chemists take numerous parameters into account when determining antibiotic residues in food of animal origin, some of which are discussed here.
1.1.1 Identification
A substance needs to be identified by a combination of the appropriate identification parameters including the name or other identifier of the substance, information related to molecular and structural formula, and composition of the substance.
International nonproprietary names (INNs) are used to identify pharmaceutical substances or active pharmaceutical ingredients. Each INN is a unique name that is internationally consistent and is recognized globally. As of October 2009, approximately 8100 INNs had been designated, and this number is growing every year by some 120–150 new INNs.[5] An example of an INN is tylosin, a macrolide antibiotic.
International Union of Pure and Applied Chemistry (IUPAC) names are based on a method that involves selecting the longest continuous chain of carbon atoms, and then identifying the groups attached to that chain and systematically indicating where they are attached. Continuing with tylosin as an example, the IUPAC name is [(2R,3R,4E,6E,9R,11R,12S,13S,14R)-12-{[3,6-dideoxy-4-O-(2,6-dideoxy-3- C-methyl- α-L-ribohexopyranosyl)-3- (dimethylamino)-β-D-glucopyranosyl]oxy}-2-ethyl-14-hydroxy-5, 9,13-trimethyl- 8, 16-dioxo-11- (2-oxoethyl)oxacyclohexadeca-4,6-dien-3-yl]methyl 6-deoxy-2,3-di-O-methyl-β-D-allopyranoside.
The Chemical Abstract Service (CAS) Registry Number is the universally recognized unique identifier of chemical substances. The CAS Registry Number for tylosin is 1401-69-0.
Synonyms are used for establishing a molecule's unique identity. For the tylosin example, there are numerous synonyms, one of which is Tylan.
1.1.2 Chemical Structure
For the great majority of drugs, action on the body is dependent on chemical structure, so that a very small change can markedly alter the potency of the drug, even to the point of loss of activity.[6] In the case of antimicrobial drugs, it was the work of Ehrlich in the early 1900s that led to the introduction of molecules selectively toxic for microbes and relatively safe for the animal host. In addition, the presence of different sidechains confers different pharmacokinetic behavior on a molecule. Chemical structures also provide the context to some of the extraction, separation, and detection strategies used in the development of analytical methods. Certain antibiotics consist of several components with distinct chemical structures. Tylosin, for example, is a mixture of four derivatives produced by a strain of Streptomyces fradiae. The chemical structures of the antimicrobial agents described in this chapter are presented in Tables 1.2–1.15.
1.1.3 Molecular Formula
By identifying the functional groups present in a molecule, a molecular formula provides insight into numerous properties. These include the molecule's water and lipid solubility, the presence of fracture points for gas chromatography (GC) determinations, sources of potential markers such as chromophores, an indication as to the molecule's UV absorbance, whether derivatization is likely to be required when quantifying residues of the compound, and the form of ionization such as protonated ions or adduct ions when using electrospray ionization. The molecular formulas of the antimicrobial agents described in this chapter are shown in Tables 1.2–1.15.
1.1.4 Composition of the Substance
Regulatory authorities conduct risk assessments on the chemistry and manufacture of new and generic antimicrobial medicines (formulated products) prior to granting marketing approvals. Typically, a compositional standard is developed for a new chemical entity or will already exist for a generic drug. A compositional standard specifies the minimum purity of the active ingredient, the ratio of isomers to diastereoisomers (if relevant), and the maximum permitted concentration of impurities, including those of toxicological concern. The risk assessment considers the manufacturing process (the toxicological profiles of impurities resulting from the synthesis are of particular interest), purity, and composition to ensure compliance with the relevant standard. The relevant test procedures described in pharmacopoeia and similar texts apply to the active ingredient and excipients present in the formulation. The overall risk assessment conducted by regulatory authorities ensures that antimicrobial drugs originating from different manufacturing sources, and for different batches from the same manufacturing source, have profiles that are consistently acceptable in terms of efficacy and safety to target animals, public health, and environmental health.
1.1.5 pKa
The symbol pKa is used to represent the negative logarithm of the acid dissociation constant Ka, which is defined as [H+][B]/[HB], where B is the conjugate base of the acid HB. By convention, the acid dissociation constant (pKa) is used for weak bases (rather than the pKb) as well as weak organic acids. Therefore, a weak acid with a high pKa will be poorly ionized, and a weak base with a high pKa will be highly ionized at blood pH. The pKa value is the principal property of an electrolyte that defines its biological and chemical behavior. Because the majority of drugs are weak acids or bases, they exist in both ionized and un-ionized forms, depending on pH. The proportion of ionized and un-ionized species at a particular pH is calculated using the Henderson–Hasselbalch equation. In biological terms, pKa is important in determining whether a molecule will be taken up by aqueous tissue components or lipid membranes and is related to the partition coefficient log P. The pKa of an antimicrobial drug has implications for both the fate of the drug in the body and the action of the drug on microorganisms. From a chemical perspective, ionization will increase the likelihood of a species being taken up into aqueous solution (because water is a very polar solvent). By contrast, an organic molecule that does not readily ionize will often tend to stay in a non-polar solvent. This partitioning behavior affects the efficiency of extraction and clean-up of analytes and is an important consideration when developing enrichment methods. The pKa values for many of the antimicrobial agents described in this chapter are presented in Tables 1.2–1.15. The consequences of pKa for the biological and chemical properties of antimicrobial agents are discussed later in this text.
1.1.6 UV Absorbance
The electrons of unsaturated bonds in many organic drug molecules undergo energy transitions when UV light is absorbed. The intensity of absorption may be quantitatively expressed as an extinction coefficient ε, which has significance in analytical application of spectrophotometric methods.
1.1.7 Solubility
From an in vitro perspective, solubility in water and in organic solvents determines the choice of solvent, which, in turn, influences the choice of extraction procedure and analytical method. Solubility can also indirectly impact the timeframe of an assay for compounds that are unstable in solution. From an in vivo perspective, the solubility of a compound influences its absorption, distribution, metabolism, and excretion. Both water solubility and lipid solubility are necessary for the absorption of orally administered antimicrobial drugs from the gastrointestinal tract. This is an important consideration when selecting a pharmaceutical salt during formulation development. Lipid solubility is necessary for passive diffusion of drugs in the distributive phase, whereas water solubility is critical for the excretion of antimicrobial drugs and/or their metabolites by the kidneys.
1.1.8 Stability
In terms of residues in food, stability is an important parameter as it relates to (1) residues in biological matrices during storage, (2) analytical reference standards, (3) analytes in specified solvents, (4) samples prepared for residue analysis in an interrupted assay run such as might occur with the breakdown of an analytical instrument, and (5) residues being degraded during chromatography as a result of an incompatible stationary phase.
Stability is also an important property of formulated drug products since all formulations decompose with time.[7] Because instabilities are often detectable only after considerable storage periods under normal conditions, stability testing utilizes high-stress conditions (conditions of temperature, humidity, and light intensity, which are known to be likely causes of breakdown). Adoption of this approach reduces the amount of time required when determining shelf life. Accelerated stability studies involving the storage of products at elevated temperatures are commonly conducted to allow unsatisfactory formulations to be eliminated early in development and for a successful product to reach market sooner. The concept of accelerated stability is based on the Arrhenius equation:
where k is the rate constant of the chemical reaction; A, a pre-exponential factor; Ea, activation energy; R, gas constant; and T,...
