Validation of a method for the determination of aminoglycosides in different matrices and species based on an in-house concept.

A simple, rapid, and sensitive method for the determination and confirmation of the aminoglycosides streptomycin, dihydrostreptomycin, spectinomycin, apramycin, kanamycin, paromomycin, gentamicin and neomycin in cow's milk as well as in bovine and porcine muscle and kidney was developed. Validation was performed on the basis of an in-house concept with different factor-level combinations in accordance with Commission Decision 2002/657/EC. After extraction with trichloroacetic acid solution, clean-up was performed by way of SPE. LC-MS/MS analysis was carried out by means of an HILIC column for the separation of the analytes, and by using MS/MS in positive ESI mode to measure the transitions of the substances in MRM mode. For quantification, matrix calibration curves in the linear range around the MRLs as well as the internal standard tobramycin were used. The calculated validation parameters like CCα, CCß, recovery (94-103%), relative repeatability RSDr (3.6-9.7%), and relative within-laboratory reproducibility RSDwR (4.6-10.0%) fulfilled the requirements of Commission Decision 2002/657/EC.

Endogenous and synthetic steroids in bovine urine--preparation of in-house reference material, stability studies and results of a proficiency test.

Within the framework of the German National Residue Control Plan a specific number of samples of animal origin have to be analysed for natural and synthetic steroids each year. As a measure of external quality control of the methods applied in routine analysis a proficiency test was carried out. To this end, in-house reference material containing incurred residues of 17α- and 17ß-nortestosterone and 17α- and 17ß-estradiol as well as fortified residues of 17α-methyltestosterone and 17α-trenbolone in bovine urine were produced. Before sending the proficiency test material to the participants, the homogeneity of all samples was tested and confirmed. Furthermore extensive short- and long-term stability studies were carried out. The statistical evaluation of the proficiency test was performed by applying robust statistics as described in standard DIN 38402. Based on the target value and standard deviation z-scores were calculated as standardised measure of the laboratory performance. The evaluation of the proficiency test showed that nine laboratories submitted quantitative results within the tolerance limits for all analytes. Taking into account the individual decision limits, there were no false negative results. In overall evaluation, 11 of 12 laboratories participated successfully.

Confirmatory method for the determination of streptomycin and dihydrostreptomycin in honey by LC-MS/MS.

A method was specifically developed for the determination and confirmation of streptomycin and dihydrostreptomycin in different types of honey. The method is simple, rapid, sensitive and was validated for streptomycin and dihydrostreptomycin in accordance with Commission Decision 2002/657/EC. After extraction with phosphate buffer and a pH change, clean-up was performed via SPE with polymeric phase. LC-MS/MS analysis was carried out using two different HILIC columns for the separation of the analytes and using a triple quadrupole mass spectrometer in positive ESI mode to measure the transitions of the substances in MRM mode. For the quantification of both substances, matrix calibration curves in a linear range of 5-80 g kg(-1) were used. The validation parameters established for streptomycin and dihydrostreptomycin, CCα (11.8 and 11.5 µg kg(-1), respectively), CCß (18.9 and 19.9 µg kg(-1), respectively), recovery (97 and 101%, respectively) and the relative within-laboratory reproducibility RSD(wR) (16.4 and 20.8%, respectively) at the recommended concentration of 40 µg kg(-1), fulfil the requirements of Commission Decision 2002/657/EC.

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).

Multi-method for the determination of antibiotics of different substance groups in milk and validation in accordance with Commission Decision 2002/657/EC.

The presented method is able to analyse 47 substances of the antibiotic groups tetracyclines, quinolones, macrolides, sulfonamides, diamino-pyrimidine derivatives and lincosamides simultaneously in a single analytical run. Applying an in-house validation concept, the validation of the multi-method was successfully accomplished with a low number of experiments. Each substance was validated at least at the concentrations 0.5, 1.0 and 1.5MRL (maximum residue limit), or respectively, at concentrations as low as possible for substances without MRL. The calculated relevant validation parameters, e.g. the decision limit CCalpha, the detection capability CCbeta, the repeatability, the within-laboratory reproducibility and the recovery, are in an acceptable range and in compliance with the requirements of Commission Decision 2002/657/EC. A proficiency test and the implementation of the method by other laboratories were performed successfully.

Validation of a confirmatory method for the determination of residues of four nitrofurans in egg by liquid chromatography-tandem mass spectrometry with the software InterVal.

A method for the detection and determination of nitrofuran derivatives in egg by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was validated with the software InterVal and can be applied for the confirmation of nitrofuran metabolites in fresh or lyophilised eggs. The validation study comprises variations in operator, storage condition, breeding, equipment and duration of sample preparation. A comprehensive overview of the robustness of the method is obtained by analysing eight samples at six concentration levels. First results of short- and medium-term investigations for stability of analytes in solution show that standard solutions of nitrofuran metabolites are stable for at least 1 year when stored at +4 degrees C in the dark. The decision limit CCalpha expressed for the underivatised metabolite is 0.05 microg kg(-1) for 3-amino-5-methyl-morpholino-2-oxazolidinone, 0.03 microg kg(-1) for 3-amino-2-oxazolidinone, 0.20 microg kg(-1) for semicarbazide and 0.22 microg kg(-1) for 1-amino-hydantoin.

Determination of chloramphenicol residues in crustaceans: preparation and evaluation of a proficiency test in Germany.

Chloramphenicol (CAP) is banned for use in food-producing animals and is, thus, controlled on the basis of the National Residue Control Plans in the European Union. Due to current problems with residues of CAP in shrimp, crayfish and prawns, a sensitive GC/NCI/MS method was optimised and in-house validated. The validation study resulted in a decision limit (CCalpha) of 0.07 microg kg-1, a recovery of 95% and a within-laboratory reproducibility of 9%. The method was used for preparing a proficiency test to assess the quality of residue control in Germany. The proficiency test involved analysis of five samples and the results were very satisfactory. The reproducibility standard deviation for five samples ranged from 17 to 24%, and the median concentrations lay between 0.43 and 0.51 microg kg-1 CAP. These values are clearly below the corresponding Horwitz standard deviation of about 50%. From the study, it can be concluded that there are, irrespective of the method applied, well-established and proper working analytical procedures for the control of CAP around the minimum required performance limit (MRPL) of 0.3 microg kg-1.

Validation of a method for the detection and confirmation of nitroimidazoles and corresponding hydroxy metabolites in turkey and swine muscle by means of gas chromatography-negative ion chemical ionization mass spectrometry.

The results of a validation study of a GC-NCI-MS method for the quantitative determination of 5-nitroimidazoles [1,2-Dimethyl-5-nitroimidazole (dimetridazole, DMZ), 1-methyl-2-[(carbamoyloxy)methyl]-5-nitroimidazole (ronidazole, RNZ), 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole, MNZ) and 2-isopropyl-1-methyl-5-nitroimidazole (ipronidazole, IPZ)] including the hydroxy metabolites of these agents [2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI), 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole (MNZOH), and 1-methyl-2-(2'-hydroxyisopropyl)-5-nitroimidazole (IPZOH)] in turkey and swine muscle are presented. The validation was carried out according to the requirements of the draft for the revision of Commission Decision 93/256/EC, which is expected to be adopted by the European Commission in due course. The determination of the method's performance parameters revealed decision limits (CCalpha) between 0.65 and 2.8 microg/kg for DMZ, RNZ/HMMNI, MNZ and MNZOH. Confirmatory analyses according to the requirements of the forthcoming EC decision are possible for all analytes except for IPZ and IPZOH where already the decision limits (CCalpha) were higher (5.2 microg/kg) than for the above-mentioned nitroimidazoles. The within-laboratory reproducibility and the mean recovery were in an acceptable range for all analytes.

Measurement of beta-agonist residues in retinal tissue of food producing animals.

This report presents the results of an investigation on the accumulation of beta-agonist residues in the retinal tissue of food producing animals. Three different species, calf, pig and turkey, were treated with six different beta-agonists and analysed for beta-agonist residues in retinal tissue applying a newly developed retina preparation procedure which provides sufficient sample material for multiple analyses. The results show that all selected beta-agonists accumulate in the retina, though in varying concentrations. The results are discussed on the basis of existing binding theories and with regard to their impact on the existing residue control strategy for beta-agonists.

Multi-residue method for non-steroidal anti-inflammatory drugs in plasma using high-performance liquid chromatography-photodiode-array detection. Method description and comprehensive in-house validation.

A multi-residue high-performance liquid chromatography (HPLC) method with photodiode-array detection is presented for the determination of 12 non-steroidal anti-inflammatory drugs (NSAIDs) in plasma. This method has been validated under the consideration of actual alpha- and beta-errors according to an in-house validation concept based on a fractional factorial experiment. A wide range of matrices and other influencing factors have been included in the validation experiment. In order to assess the method's performance the power curve, which demonstrates the detection power of the analytical method, was computed and CCalpha and CCbeta values were calculated.