Peter Batjoens

Determination of Dexamethasone in Urine and Faeces of Treated Cattle with Negative Chemical Ionization - Mass Spectrometry.

For several years, the misuse of dexamethasone and its esters in livestock production has been clearly demonstrated. The first part of the present study deals with the elaboration of a sensitive and specific method for the determination of residues of dexamethasone in excreta at the ppb level. Sample preparation for urine and faeces, including high-performance liquid chromatography (HPLC) fractionation, was carried out. The detection was based on established methodology employing negative chemical ionization-mass spectrometry (NCI-MS) after oxidation of the dexamethasone. In comparison with previous literature, the yield of oxidized dexamethasone was substantially improved and the oxidation procedure was made more simple and robust. In the second part of the study, the relationship between the dose of dexamethasone administered and the levels of the drug in excreta was investigated using this method, as was the ratio between drug levels in urine and faeces. Treatment was carried out for 7 d with an oral dose of 50 mg d-1, the maximum levels found in urine and faeces were 980 and 744 ppb, respectively. While the elimination via faeces responded much slower at the start and the end of treatment, the final part of both excretion profiles were very similar and a level of 1 ppb was reached in both matrices 9 d after the end of treatment. Gas chromatography-mass spectrometry (GC-MS) results obtained for the urine samples were compared with those obtained with direct enzyme immunoassay.

Rapid and high-performance analysis of thyreostatic drug residues in urine using gas chromatography-mass spectrometry

A more sensitive method was developed using the hyphenated technique of gas chromatography-mass spectrometry (GC-MS) supplementary to the official high-performance thin-layer chromatography (HPTLC) method. Even combined with less efficient extraction and clean-up methods, GC-MS is able to lower the detection limit to less than 50 ppb. The powerful technique of GC-MS-MS is tried out to reduce the detection limit even more, in combination with simplified extraction methods. This time-saving approach combined with the increase in sensitivity is of great importance for a routine technique.

Ion chromatographic determination of perchlorate in cattle urine

Abstract Much research has been done over the years on the subject of thyreostatic drugs of the thiouracil type in cattle fattening. These substances increase body weight by enlarged filling of the gastro-intestinal tract and by augmented water retention. On the other hand, little work has been done on the elimination and the presence of residues of inorganic thyreostatics such as perchlorate. The aim of this study was to evaluate the elimination of ammonium perchlorate in cattle urine according to the concentration given and the duration of administration. Perchlorate concentrations were measured by mobile phase ion chromatography. The detection limit was 0.1 mg kg −1 for tenfold diluted urine.

Endogenic nortestosterone in cattle

When residues of nortestosterone (NT) were found in the urine of cattle, racehorses or bodybuilders, exogenic administration was thought to be proven. In previous literature, no records were found of the endogenic presence of this molecule. In the horse-racing world, Houghton and Courthot found that NT is normally present in the urine of the stallion. Belgian and Dutch researchers found that NT is also present in the urine and edible parts of the intact boar. Vandenbroeck et al. (1991) suggested the endogenous presence of NT (in the beta form) in the pregnant cow. Meyer (1992) reported the presence of NT (in the alpha form) in relatively high amounts in the urine of the cow peri-partum and the neo-natal calf. These observations may have important consequences for veterinary meat inspection in the EU. Therefore, in Belgium a large scale experiment was set up in co-operation with the EU Community Reference Laboratory (RIVM). In this paper the present state of the results in this area is presented. A large number of urine samples (> 50) of pregnant non-treated cows were collected and analysed by gas chromatography-mass spectrometry (GC-MS) in 4 different laboratories. Further samples (> 100) were taken, but only analysed in one laboratory. The results proved clearly that NT may indeed be detectable in the alpha form in the urine of pregnant cows, from at least 2 months, but most probably from 4-5 months before partus.

Comparison of the possibilities of gas chromatography-mass spectrometry and tandem mass spectrometry systems for the analysis of anabolics in biological material

Chromatographic techniques such as GC-MS play a most important role in modern multi-residue analysis of anabolic steroids. The major difference between GC-MS apparatus from different manufacturers is the way of detection and recording. Most apparatus use selected-ion monitoring (SIM) for the determination of low concentrations. Systems based on ion trap technology record in full-scan to even picogram concentrations using a computer algorithm to compare the most important peaks of the mass spectrum of the unknown to those of the standard. In this investigation the possibilities of ion trap GC-MS and the recently released GCQ MS and MS2 for the analysis of anabolics in biological material are compared.

Gas chromatography–mass spectrometric confirmation of anabolic compounds in injection sites

Intramuscularly administered, many anabolic residues are present in injection sites in an esterified form. Routine high-performance thin-layer chromatography (HPTLC) results show different spots recognizable as the esters of a certain anabolic compound, e.g., testosterone or estradiol, without giving the exact identity of the ester. Hydrolysis of the extract and respotting on HPTLC plates can confirm these spots to be esters owing to the absence of such spots on the plate after hydrolysis. The residues are then seen in their unesterified form. Identification is possible after respotting on reversed phase plates. As this method is complex and time consuming, an alternative method was tried out using gas chromatography–mass spectrometry (GC–MS). GC–MS analysis of injection site extracts is not only an independent method to confirm HPTLC results, but also provides the opportunity to identify the specific ester of the anabolic steroid. Identification is only possible when a standard of the ester is available for comparing the respective mass spectra. This report presents the mass spectra of some esters of testosterone, estradiol and nortestosterone found during the confirmation analysis of 35 injection sites by using GC–MS.

Gas chromatographic-tandem mass spectrometric analysis of clenbuterol residues in faeces

In all EU member states, the use in livestock farming of certain substances having a hormonal action is prohibited. Clenbuterol, the beta-adrenergic agonist, has some growth promoting characteristics. Screening for clenbuterol can be carried out by an immunoassay. Gas chromatography-mass spectrometry (GC-MS) is very valuable for confirmatory purposes. In full scan MS it is impossible to fulfil the EU criteria of four diagnostic ions with one single ionisation mode. Some alternative possibilities are: (1) the use of two different ionisation modes, (2) the use of different derivatization methods or (3) the use of tandem MS. Each derivatisation or ionisation mode on its own did not give a sufficient number of ions. By combining these different possibilities we were able to obtain four ions, fulfilling the EU criteria.

Multi-residue analysis of tranquillizers in meat: confirmatory assays using mass spectrometry†

A rapid and sensitive multi-residue method was developed to attempt to confirm the presence of the beta-blocker carazolol and the tranquillizers acepromazine, azaperone, chlorpromazine, propionylpromazine and xylazine in pig muscle tissues. The procedure involves determination by liquid chromatography coupled with tandem mass spectrometry. The liquid chromatographic separation was performed on a Symmetry C18 column with gradient elution. A mixture of aqueous buffer, containing 0.01% m/v trifluoroacetic acid (pH 3.5), and acetonitrile at a flow rate of 0.4 ml min-1 was used as the mobile phase. The abundant parent ions [M+ H+] produced by positive electrospray ionisation were selected for collisional dissociation with argon. Fragment ions were recorded with daughter ion scan and multiple reaction monitoring. The analytes were identified unambiguously by assessing retention times and diagnostic ions in meat samples spiked from 50 micrograms kg-1 [maximum residue limit (MRL) for azaperone and azaperol] to 5 micrograms kg-1 (MRL for carazolol).

Pitfalls in Selected Ion Monitoring in Gas-chromatography mass-spectrometry, a theoretical example.

For the routine determination of residues of growth promoters two important types of low-resolution gas chromatograph—mass spectrometer may be distinguished: the ultra-trace full-scan instrument [e.g., the ion trap mass spectrometer (ITS40)] and most other quadrupole apparatus using the selected ion monitoring (SIM) mode for detecting very small amounts (< 1–10 ng). In analysing biological extracts interference between matrix components, present at high concentrations, and analytes, present at low concentrations, should be avoided. In this investigation theoretical examples of pitfalls in SIM due to isotope interference (13C) from matrix components with the analyte were considered. These interferences may lead to false-positive and -negative results and false quantification.

Qualitative or quantitative methods for residue analysis

Abstract Qualitative methods are used for illegal drugs (e.g. hormones) which have a so-called zero tolerance (i.e. the presence of even a very small amount is not tolerated). In practice a limit of determination is used to prove the presence or absence of an analyte. Quantitative analysis is necessary especially for residues with a maximum residue limit (MRL). The method used in this case must have a limit of quantification (much) lower than the MRL. In any case, quality criteria must be fulfilled before quantification. In this investigation the pros and cons of different strategies towards the use of qualitative and quantitative methods are discussed from the point of view and the daily experience of routine control in two field laboratories and one national reference laboratory.