Validation of a multi-residue method for the determination of several antibiotic groups in honey by LC-MS/MS.

The presented multi-method was developed for the confirmation of 37 antibiotic substances from the six antibiotic groups: macrolides, lincosamides, quinolones, tetracyclines, pleuromutilines and diamino-pyrimidine derivatives. All substances were analysed simultaneously in a single analytical run with the same procedure, including an extraction with buffer, a clean-up by solid-phase extraction, and the measurement by liquid chromatography tandem mass spectrometry in ESI+ mode. The method was validated on the basis of an in-house validation concept with factorial design by combination of seven factors to check the robustness in a concentration range of 5-50 µg kg(-1). The honeys used were of different types with regard to colour and origin. The values calculated for the validation parameters-decision limit CCα (range, 7.5-12.9 µg kg(-1)), detection capability CCß (range, 9.4-19.9 µg kg(-1)), within-laboratory reproducibility RSD(wR) (<20% except for tulathromycin with 23.5% and tylvalosin with 21.4 %), repeatability RSD(r) (<20% except for tylvalosin with 21.1%), and recovery (range, 92-106%)-were acceptable and in agreement with the criteria of Commission Decision 2002/657/EC. The validation results showed that the method was applicable for the residue analysis of antibiotics in honey to substances with and without recommended concentrations, although some changes had been tested during validation to determine the robustness of the method.

Validated determination of eight antibiotic substance groups in cattle and pig muscle by HPLC/MS/MS.

The described multimethod is suited for the determination of 53 substances of eight antibiotic groups-tetracyclines, quinolones, macrolides, sulfonamides, diphenylsulfones, diamino-pyrimidine derivatives, pleuromutilines, and lincosamides-in cattle and pig muscle. All substances were analyzed simultaneously with the same sample preparation and in one HPLC/MS/MS run. The validation of the multimethod was successfully accomplished with the help of an alternative in-house validation concept requiring only 48 experiments. The substances were validated at concentrations of 0.25, 0.5, 1.0, 1.5, and 2.0 x MRL (maximum residue limit) or 5, 10, 20, 30, and 40 microg/kg for substances without an MRL. The calculated relevant validation parameters were based on and comply with the requirements of Commission Decision 2002/657/EC, i.e., the decision limit, detection capability, repeatability, within-laboratory reproducibility, and recovery. The robustness of the method was demonstrated by varying seven factors of the analytical procedure. Several proficiency tests were carried out successfully to provide evidence for the applicability of the method.

Validation of a method for the determination of triphenylmethane dyes in trout and shrimp with superior extraction efficiency.

The described methods are able to analyse the triphenylmethane dyes malachite green (MG), crystal violet (CV) and brilliant green (BG) as well as their leuco metabolites leuco malachite green (LMG), leuco crystal violet (LCV) and leuco brilliant green (LBG) on the basis of a simple and fast extraction. The validation of the methods in two studies without and with a heated ultrasonic treatment during the extraction of fortified trout and shrimp samples was successfully performed applying an in-house validation concept. The evaluation of the relevant validation parameters, e.g. the decision limit CCα, the detection capability CCß, the repeatability, the within-laboratory reproducibility and the recovery for both extraction versions, showed results which fulfil the requirements of Commission Decision 2002/657/EC. The investigation of incurred material of trout containing the above compounds with an additional heated ultrasonic treatment during extraction leads to higher findings of MG and BG. This effect was also confirmed by other laboratories in the framework of a proficiency test. For CV and all three leuco metabolites no increase in the detected amounts could be observed after a heated ultrasonic treatment of the incurred trout material.

Preparation and characterisation of in-house reference material of tylosin in honey and results of a proficiency test.

The analysis of incurred material from animals treated with pharmacologically active substances is an efficient way to check the accuracy of a method. Tylosin A was chosen for the preparation of that material because it is highly effective in controlling active infections of American Foulbrood (AFB), a global threat to apiculture, but residues in honey are not allowed according to European legislation. For this reason an in-house reference material of honey containing the macrolide tylosin A and its degradation product desmycosin (tylosin B) was prepared. After the treatment of a beehive with the appropriate macrolide tylosin A, the honey samples were collected. The incurred honey material was diluted by mixing with blank honey. Concentrations of 25.81 µg kg(-1) for tylosin A and of 19.28 µg kg(-1) for its degradation product desmycosin (tylosin B) were reached. The homogeneity was checked by analysing 12 bottles in duplicate. The stability was tested at different defined temperatures and storage conditions. The reference material described above was homogeneous and stable. Samples of this in-house reference material were used for the realisation of a proficiency test with international participation. All participants accomplished satisfying results with the exception of one laboratory.

Confirmatory method for the determination of streptomycin in apples by LC-MS/MS.

The method was specifically developed for the determination and confirmation of streptomycin in apple samples using the whole mellow apple. The method is simple, rapid, sensitive and was validated for streptomycin in accordance with SANCO/3131/2007. After extraction with phosphate buffer and a pH change, the clean-up was performed by the way of SPE with polymeric phase. The LC-MS/MS analysis was carried out using a HILIC column for the separation of the analytes and a triple quadrupole mass spectrometer in positive ESI mode to measure the transitions of the substances in MRM mode. For the quantification of streptomycin a matrix calibration curve in the linear range of 1.0-20 microg kg(-1) and the internal standard dihydrostreptomycin (10 microg kg(-1)) were used. The calculated validation parameters like the recovery (101-105%) for 2, 5, 10 and 20 microg kg(-1) and the relative standard deviation (RSD, 4.1-11.4%) of the 6 replicates fulfil the requirements of SANCO/3131/2007. The LOQ was determined as 2 microg kg(-1).