New approach to derivatisation for oestradiol esters detection in animal blood plasma using negative chemical ionisation GC-MS.

In 1996, the EU prohibited the use of substances with anabolic action for food-producing animals (EU Directive 96/22/EC). In cases of illegal use of steroid hormones, these substances are usually applied to the animals in the form of esters. The reliable determination of intact steroid esters in animal tissues or body fluids is an unequivocal proof of illegal treatment of animals with EU prohibited anabolic substances. Previously our laboratory developed a sensitive method for determination of oestradiol benzoate and other steroid esters in blood plasma using LC-MS/MS, validated according to Commission Decision 2002/657/EC. This study describes a GC-MS method which has been developed for five oestradiol esters in blood plasma. The sample preparation procedure consisted of protein precipitation, phospholipids removal and cleaning on an alumina column. Oestradiol esters were derivatised with 2, 3, 4, 5, 6-pentafluorobenzoyl chloride (PFBCl) and pyridine in dichloromethane. The measurement of oestradiol esters was carried out by GC-MS/NCI with Cool On-Column injection. Methane was used as a negative chemical ionisation reagent gas. The method for determination of oestradiol esters in blood plasma has been validated according to Commission Decision 2002/657/EC. Decision limits for all analytes were observed below 0.05 ng mL-1. The method is robust for bovine and porcine plasma analyses and can be applied both for screening and confirmatory determination in routine residue monitoring.

Residue study of nitroimidazoles depletion in chicken feathers in comparison with some other selected matrixes.

This paper presents the results from a residue study conducted on a statistically representative number of chicken broilers that were individually orally treated with the selected nitroimidazoles (metronidazole, ornidazole and ipronidazole) in an appropriate amount close to the theoretical therapeutic dose. A mutual persistence comparison of the monitored analytes in feathers, serum, muscle and shanks was performed and attention was also paid to selected metabolites (hydroxymetronidazole and hydroxyipronidazole). An analytical LC/MS/MS method using SupelMIP SPE nitroimidazoles cartridges was developed for the determination of nitroimidazoles residues in poultry feathers, serum, muscle and shanks and the method was validated according to Commission Decision 2002/657/EC. High concentrations of nitroimidazoles residues in feathers were observed 19 days after the broilers' treatment unlike the muscle and serum samples, where nitroimidazoles depletion was significantly faster (residue concentrations were below detection limits in 5 days in muscle and in 12 days in serum). Shanks (chicken claws) also proved to be a very useful matrix for the detection of nitroimidazoles drugs misuse due to the longer persistence of these drugs residues and their metabolites in this matrix (determinable concentrations were observed 19 days after the broilers' last treatment). Feathers and shanks appear to be suitable matrixes for the screening of various nitroimidazoles in poultry because long-term persistence of residues enables reliable detection of the illegal use of nitroimidazoles compounds in official checks.

Determination of testosterone esters and nortestosterone esters in animal blood serum by LC-MS/MS.

A sensitive and robust confirmatory method for determination of steroid esters in blood serum is essential for reliable monitoring of possible illegal use of steroid hormones as growth promoters in meat production. A previously used sample preparation methodology was improved. The procedure consists of protein precipitation and removal of phospholipids by dispersive SPE Supel™ QuE Z-Sep (Sigma-Aldrich) followed by clean-up on alumina column and LC-MS/MS measurement. The modified method has been validated according to Commission Decision 2002/657/EC. Validation parameters for determination of six testosterone esters and five nortestosterone esters in bovine and porcine blood serum are presented in this article. Decision limits for all analytes were observed in the range 10-20 pg mL-1. The method described is considerably robust for bovine and porcine serum analyses and can be applied both for screening and confirmatory determination in routine residue monitoring.

Determination of testosterone esters and estradiol esters in bovine and porcine blood serum.

Monitoring of steroid esters in blood serum is desirable in order to detect the possible illegal use of natural hormones as growth promoters. A method for the determination of testosterone propionate, testosterone benzoate, testosterone isocaproate, testosterone decanoate and estradiol benzoate in bovine and porcine blood serum was developed. The procedure consists of protein precipitation and removal of phospholipids using a HybridSPE®-Phospholipid column followed by clean-up on a hydrophilic modified styrene polymer SupelTM-Select HLB column and LC-MS/MS measurement. The method was validated according to Commission Decision 2002/657/EC. Decision limits for all analytes were observed in the range 5-30 pg ml-1. The method was shown to be robust for bovine and porcine serum analyses and can be applied for both screening and confirmatory determination in routine residue monitoring.

Observation of residues in tissues of chickens exposed to low dietary concentrations of chloramphenicol.

To investigate potential residues in tissues arising from naturally occurring low levels of chloramphenicol in plant material, feeding studies were conducted with chickens. A common chicken feed was prepared containing 0, 10, 50 and 200 µg kg-1 chloramphenicol and levels were confirmed by LC-MS/MS. Four separate groups of broiler chickens, eight animals in each group, were fed all their 35-day life with this contaminated feed. They were allowed ad libitum access to this feed and fresh water. After slaughtering the chickens, the residues in muscle and liver tissues were determined using GC/MS-NCI method. No residues were detected in tissues of animals from groups fed with feed containing 0, 10 or 50 µg kg-1. Low chloramphenicol residual concentrations were observed in a few of the muscle samples obtained from the group of chickens fed with feed containing chloramphenicol in added concentration 200 µg kg-1. No residues were detected in the remaining samples of this group. These results indicate that when residues of chloramphenicol are detected it is in all probability through illegal use.

Persistence of chloramphenicol residues in chicken muscle tissue after a therapeutic dose administration.

One-day-old chickens were individually orally treated with chloramphenicol at a dose of 100 mg per kg of body weight per day for three consecutive days. After the final treatment, the groups of six birds were sacrificed in seven-day intervals up to 42 days. The muscle tissue collected from the breasts and legs of each bird was individually examined for the presence of chloramphenicol residues using a GC/MS-NCI analytical method, which was validated according to Commission Decision 2002/657/EC. The decision limit (CCα) obtained for the method was 0.05 ng g-1. The results showed a rapid decrease of chloramphenicol concentration in the muscle tissue after termination of the treatment, but also showed a relatively long persistence of low residue concentrations. Levels of chloramphenicol in muscle tissue averaged 64 ng g-1 seven days after the final treatment and fell to 0.21 ng g-1 after 35 days. All animals tested on the 35th day after the final treatment showed detectable chloramphenicol concentrations above the decision limit of the method used. No residues were detected in the animal tissues 42 days after the end of the treatment.

Development and validation of an LC-MS/MS method for the determination of six gestagens in kidney fats.

A method for the determination of residues of six steroids with gestagenic action (altrenogest, medroxyprogesterone acetate, megestrol acetate, melengestrol acetate, acetoxyprogesterone and chlormadinone acetate) in animal fat tissue was developed. The procedure consists of methanol extraction, clean-up on an alumina column and LC-MS/MS measurement. The method has been validated according to Decision 2002/657/EC. Decision limits for all analytes were observed within the range from 0.3 to 1.7 ng g(-1), and recoveries were between 80% and 105%. The method is robust and can be applied for both screening and confirmatory analyses in routine residue monitoring.

Determination of 5-nitroimidazoles in various types of matrices using molecular imprinted polymer purification.

Rapid and sensitive methods based on LC-MS/MS using positive electrospray ionisation have been developed for the determination of nine 5-nitroimidazoles and their three hydroxylated metabolites in blood serum, egg and muscle samples. The methods use a new type of column based on molecularly imprinted polymer for the cleanup of primary extracts of samples. A validation study was carried out according to criteria (accuracy, linearity, repeatability, reproducibility, ruggedness and specificity) and the requirements of Commission Decision 2002/657/EC. The methods with molecularly imprinted polymers are simple, fast and selective for the extraction of 5-nitroimidazoles from different matrices. They provide high recovery, good reproducibility, clean extracts and low background signals. The decision limits and detection capabilities were lower than the recommended minimum required performance limits in every matrix. These procedures could also be used as screening and confirmatory methods for the monitoring of veterinary drug residues.

Determination of chloramphenicol in urine, feed water, milk and honey samples using molecular imprinted polymer clean-up.

A method for determination of low concentrations of chloramphenicol in urine, feed water, milk and honey was developed. A comparison was carried out between a routinely used analytical method based on solid phase extraction (SPE-C18) for cleaning the extract and the new procedure for the sample preparation using columns based on the molecular imprinted polymers (MIP) principle. The extracts obtained from the MIP clean-up procedure were clean enough for chromatografic analyses. Confirmatory analyses were conducted using GC/MS-NCI after derivatisation (silylation). The described method was fully validated according to CD 2002/657/EC. This method is considerably robust and allows very dirty samples to be processed. The described MIP procedure is very simple and low-time-consuming, and provides high throughput of the samples examined. This could be used for routine screening and confirmatory analyses as well.