In-house validation and factorial effect analysis of a liquid chromatography-tandem mass spectrometry method for the determination of corticosteroids in bovine and porcine muscle tissue.

A sensitive and robust liquid chromatography-tandem mass spectrometry method allowing the rapid screening and confirmation of ten synthetic corticosteroids in bovine and porcine muscle tissue was developed and validated. The validation was conducted according to Commission Decision 2002/657/EC, Sect. 3.1.3 ("Validation according to alternative models"), by applying a matrix-comprehensive in-house validation concept. The decision limit, detection capability, recovery, repeatability, within-laboratory-reproducibility and measurement uncertainty were calculated. Furthermore, a factorial effect analysis was conducted to identify factors that have a significant influence on the method. To this end, factors considered to be relevant for the method in routine analysis (e.g. operator, duration of storage of the extracts before measurement, different lots of the cartridges and different species) were systematically varied on two levels during the validation study. Subsequently, the extent to which these factors influence the measurement results of the individual analytes was examined.

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.

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.

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.

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).

In-house validation and factorial effect analysis of a liquid chromatography-tandem mass spectrometry method for the determination of steroids in bovine muscle.

Anabolic steroids are banned from use in food-producing animals in the EU (Council Directive 96/22/EC). To control the zero-tolerance concept a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the screening and confirmation of most of the relevant natural and synthetic estrogenic and androgenic steroids in bovine muscle was developed and validated. The method permits to confirm and quantify almost all steroids below 1 microgkg(-1). The validation was carried out according to Commission Decision 2002/657/EC, chapter 3.1.3 "alternative validation", by applying a matrix-comprehensive in-house validation concept. Decision limit CCalpha, detection capability CCbeta, recovery, repeatability, within-laboratory reproducibility and the uncertainty of measurement were calculated. Furthermore, a factorial effect analysis was carried out to identify factors that have a significant influence on the method. Factors considered to be relevant for the method in routine analysis (e.g., operator, storage duration of the extracts before measurement, different cartridge lots) were systematically varied on two levels. The factorial analysis showed that different cartridge lots and different storage durations of the extracts before measurement can exert a relevant influence on the method.

Development and in-house validation of an LC-MS/MS method for the determination of stilbenes and resorcylic acid lactones in bovine urine.

Stilbenes and zeranol are nonsteroidal estrogenic growth promoters which are banned in the European Union (EU) for use in food-producing animals by Council Directive 96/22/EC. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the screening and confirmation of stilbenes (diethylstilbestrol, dienestrol, hexestrol) and resorcylic acid lactones (zeranol and its metabolites taleranol and zearalanone as well as the mycotoxins alpha-zearalenol, beta-zearalenol and zearalenone) in bovine urine. The method permits the confirmation and quantification of stilbenes and resorcylic acid lactones at levels below 1 microg L(-1) and 1.5 microg L(-1), respectively. The validation was carried out according to Commission Decision 2002/657/EC, Chap. 3.1.3 "alternative validation" by a matrix-comprehensive in-house validation concept. Decision limit CCalpha, detection capability CCbeta, recovery, repeatability, within-laboratory reproducibility and the uncertainty of measurement were calculated. Furthermore, a factorial effect analysis was carried out to identify factors that have a significant influence on the method. Factors considered to be relevant for the method in routine analysis (e.g. operator, matrix condition, storage duration of the extracts before measurement, different cartridge lots, hydrolysis conditions) were systematically varied on two levels. The factorial analysis showed that different cartridge lots, storage durations and matrix conditions can exert a relevant influence on the method.

Matrix-comprehensive in-house validation and robustness check of a confirmatory method for the determination of four nitrofuran metabolites in poultry muscle and shrimp by LC-MS/MS.

An already well-described method for the determination of nitrofuran metabolites 3-amino-5-methyl-morpholino-2-oxazolidinone (AMOZ), 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM) and 1-aminohydantoin (AHD) was adapted to the needs of our laboratory and checked for its robustness regarding sample conditions and the processing step. Using the same data, the method was validated and the measurement uncertainty was estimated. All criteria and requirements of Commission Decision 2002/657/EC were fulfilled. The CC(alpha) determined lies between 0.1 and 0.7 microg/kg, the CC(beta) lies between 0.1 and 0.9 microg/kg, the measurement uncertainty was estimated as being between 7 and 17% taking into account matrix, time and sample preparation influences.