Rapid extraction of clenbuterol from human and calf urine using empore C8 extraction disks.

In the present paper, disk extraction was evaluated for the rapid isolation of clenbuterol from human and calf urine, followed by high-performance liquid chromatography analysis with UV detection. A method was developed for the extraction with standard density C8 disks. The disks could be washed with 25% methanol in 0.01M sodium hydroxide without significant losses of clenbuterol. The recovery of denbuterol was about 85%, and the extracts were clean. The detection limit was about 10 ng/mL. The main advantages of these disks were the saving of time and the reduced amounts of organic solvents needed.

Multiresidue analysis of beta2-agonist in human and calf urine using multimodal solid-phase extraction and high-performance liquid chromatography with electrochemical detection.

Various beta2-agonists are used as illegal growth promoters in man and in animals. We developed a multiresidue procedure for the analysis of four beta-agonists in human and calf urine. The sample was pre-extracted with an Extrelut column at alkaline pH. The beta-agonists were eluted with a mixture of tert.-butylmethyl ether and hexane. Then the extract was further cleaned with a mixed mode SPE column, or with a combination of immunoaffinity chromatography (IAC) and the mixed mode SPE column. The IAC column contained antibodies against salbutamol, which were suitable for multiresidue extractions. The extract was then brought onto a mixed mode SPE column at an acidic pH. The column was washed with 70% methanol in water. Thereafter, the beta-agonists were eluted with ammoniated ethanol-hexane. The extract was analysed with an HPLC method with electrochemical detection. The beta-agonists were separated on a reversed-phase column using a mobile phase buffered at pH 5.5 and containing an ion-pair reagent. Recoveries were higher when the IAC procedure was not performed (90-105% vs. 65-75%), but the extracts were cleaner when the latter step was included. Detection limits in human and calf urine were in the low ng/ml range. The study indicated that beta2-agonists can be analysed in human and calf urine without the selectivity of a mass spectrometer, but that comprehensive clean-up is required to avoid the interference of urine matrix components.

Multi-residue analysis of anabolics in calf urine using high-performance liquid chromatography with diode-array detection.

We describe the development of an HPLC method with diode-array detection (DAD) for the analysis and identification of 20 substances with anabolic properties, that are considered as potential growth promoters, to be used for the analysis of extracts of calf urine samples. The substances are separated on an RP-Select B column using a mobile phase consisting of a mixture of acetonitrile and water. Gradient elution from 43-76% acetonitrile in water with a concave curve was used to achieve a good separation of the compounds with an acceptable analysis time. For the identification, a retention parameter and the UV spectrum were used. The retention parameter was the retention time corrected with a reference mixture. The latter reduced the standard deviations to about 25% of their original values. The limits of detection of the HPLC system ranged from 0.5-5 ng injected amount for the androgens, progestagens, stilbenes and resorcylic acid lactones and to 5-10 ng injected amount for the oestrogens. After extraction from urine the limits of detection were increased by the presence of matrix components, but they were between 5 and 10 ng injected amount for most of the substances.

Dramatic signal reduction in ion-mobility spectrometry by residues of solvents.

During our evaluation of ion-mobility spectrometry (IMS) screening for the abuse of the beta-agonist clenbuterol during fattening in cattle, we found that clenbuterol could not be detected with the instrument in extracts obtained by solid-phase extraction or ion-pair extraction, although the recovery for these procedures was at least 85%. Several experiments showed that the signal loss occurred mainly during the evaporation of the organic solvent. Again, it could be shown that no clenbuterol was lost during this step. On further investigation, we found that so-called solvent residues, which are left after the evaporation of the organic solvent, caused a significant reduction of the clenbuterol signal. Other reagents used in our sample pretreatment procedures were also found to reduce the signal. For example, the signal reductions caused by the different reagents used for the ion-pair extraction of clenbuterol from human urine were determined. We think that IMS is only useful for our purpose if a chromatographic preseparation is performed.