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The penicillin-binding protein PBP 2x* from Streptococcus pneumoniae has been utilised to develop a novel microplate assay for the detection and determination of penicillins and cephalosporins with intact beta-lactam structure in milk, bovine and porcine muscle juice, honey and egg. In the assay, the receptor protein is immobilised to a microplate in the first step. To each sample a bifunctional reagent is added, with ampicillin and digoxigenin as functional groups (DIG-AMPI). The amount of bifunctional reagent, which is bound via its ampicillin part to the receptor protein, decreases with increasing beta-lactam concentration in the sample. The detection step uses anti-digoxigenin F(ab) fragments marked with horseradish peroxidase. The more bifunctional reagent is bound to the receptor protein, the more antibody fragments are bound via the digoxigenin part of the reagent. A maximum colour development with tetramethylbenzidine as chromogen for the peroxidase reaction is achieved, when no beta-lactam residues are present. A fractional factorial design was applied to detect chemometrically effects and interactions of the assay parameters. For optimisation of the significant parameters a Box-Behnken design was used. The assay has been developed for various food matrices as screening test with the option for a quantitative assay, when the identity of the residual beta-lactam is known (e.g. elimination studies). Cefoperazon, cefquinome, cefazolin, cloxacillin, ampicillin and benzylpenicillin could be detected at levels corresponding to 1/2 EU maximum residue limit (MRL) in milk, meat juice from muscle tissue of different species, egg and honey (where applicable) without needing lengthy and elaborate sample pre-treatment. Matrix calibration curves are presented, which show that quantitative analyses are possible.

Veterinary drugs and feed additives (especially some coccidiostats) can be absorbed by the digestive tract of laying hens and transferred to the egg. Physicochemical characteristics of these compounds determine their pharmacokinetic behavior and distribution to and within the egg. Traditionally the quite lipid soluble drugs and additives are expected to yield residues only in the fat-rich yolk. However, the quite lipid soluble drug doxycycline--as well as many other drugs--showed during long-term administration higher residues in white than in yolk. In a model study with 11 sulfonamides differing in pK(a) value and lipid solubility, their distribution in vivo between yolk and white was determined. Neither differences in pK(a) values nor those in lipid solubility could explain the distributions found. Binding to egg white macromolecules in vivo as an explanatory factor was tested with five sulfonamides, and no correlation between binding and the distribution of sulfonamides between white and yolk was found. Literature data on the distribution of drugs between egg white and yolk showed a reasonable consistency within drugs and a large variability among drugs (as could be expected). This larger database also did not provide a clue as to what factor determines the distribution of a drug between egg white and yolk when given to laying hens.

The German provisional prevalidated official method for the determination of residues of benzylpenicillin in muscle (beef) which uses gas chromatography with nitrogen-specific detection (GC-NPD) was successfully adapted to gas chromatography-mass spectrometry (GC-MS) with selected-ion monitoring. The programmed temperature vaporisation injection technique used in the original method was substituted by on-column injection. An isotope dilution procedure for benzylpenicillin using 13C2-labelled benzylpenicillin as internal standard was developed to improve the reliability of the GC-MS results. The spectral overlap of this labelled standard and the analyte caused a non-linear relationship in calibration for which linear bracketing and single-point calibration were compared. Single-point calibration proved to be the superior method, showing less deviation from the 'true' value, while at the same time requiring fewer calibration measurements. A comparison of the results obtained by using (i) external standardisation only, (ii) a close analogue of the analyte (phenoxymethylpenicillin) and (iii) 13C2-labelled Pen G as internal standard showed, as was to be expected, that varying recoveries are best corrected by using the isotope-labelled standard.

Milk samples from 28 cows were analysed for residues of oxacillin after drying off with Stapenor Retard TS (oxacillin). Analysis was performed with an automated HPLC system consisting of an on-line solid-phase extraction and photochemical post-column derivatization with UV-detection at 300 nm. Although the time interval between treatment and parturition was less than the demanded 55 days, the maximum residue limit of 30 micrograms kg-1 was only exceeded in one case, in which the withdrawal time was 28 days.

A high-performance liquid chromatographic (HPLC) method for the determination of the macrolide antibiotic erythromycin in eggs, milk, swine muscle, kidney and liver was developed. The drug was extracted from the matrix with acetonitrile. The raw extract was purified by liquid-liquid partitioning and fractionation by reversed-phase HPLC for additional cleanup. Erythromycin was reacted in a pre-column procedure with 9-fluorenylmethylchloroformate (FMOC) to enable fluorimetric detection (excitation 255 nm, emission 315 nm) after isocratic separation on an analytical RP-18 HPLC column. Mean recoveries ranged from 99% at fortification levels of 0.03 mg/kg in egg to 38% at 0.06 mg/kg in liver. With the exception of liver all detection limits were below 0.01 mg/kg and precision for all other matrices and tested concentrations (0.015-0.09 mg/kg) better than 20% (coefficient of variation).

A capillary gas chromatographic method is described for the determination of residues of benzylpenicillin, phenoxymethylpanicillin, methicillin, oxacillin, cloxacillin, dicloxacillin and nafcillin in bovine muscle, liver, kidney, adipose tissue and milk. The samples are extracted with acetonitrile under slightly acidic conditions, the co-extracted water is separated with the addition of sodium chloride and dichloromethane and discarded. Clean-up is performed by liquid/liquid partitioning steps and anion exchange chromatography. The penicillin residues are methylated with diazomethane. After derivatization, only the extracts from liver and kidney needed further clean-up using cartridges with a polar diol sorbent. The gas chromatographic procedure is based on split/splitless injection, programmed temperature vaporization, separation on a methyl silicone fused silica column and nitrogen-specific thermionic detection. Internal standardization is used for quantification. The limits of detection for all penicillins are well below 3 microgram/kg in milk and all tissues. Recoveries of spiked samples at 3 and 10 micrograms/kg are in the range of 65-80% for milk and 50-70% for bovine tissues.