Gijsbert Zomer

Multi-immunoaffinity chromatography: a simple and highly selective clean-up method for multi-anabolic residue analysis of meat.

A method for the detection of nortestosterone (NT) in bovine muscle at levels below 1 microgram/kg is described, based on enzymatic digestion of the sample, clean-up by immunoaffinity chromatography after defatting and detection by gas chromatography-mass spectrometry (selected-ion monitoring). The immunoaffinity matrix was prepared after combining the isolated immunoglobulin G fractions from a rabbit antiserum raised against NT and methyltestosterone (MT). Its capacity per millilitre of gel was approximately 10 ng for each of the two steroids. Results for samples containing 0.1 microgram/kg NT and above are described. It is concluded that for multi-residue analysis of samples of muscle at levels as low as 0.1 microgram/kg, multi-immunoaffinity chromatography is a very suitable method of sample clean-up. For purposes of quantification the trideuterated internal standard [16,16,17 alpha-2H3] nortestosterone was synthesized.

Development and validation of a gas chromatographic—mass spectrometric procedure for the identification and quantification of residues of chloramphenicol

Abstract A method for the identification and quantification of residues of the antibiotic chloramphenicol was developed and validated. The method is based on combined gas chromatography-mass spectrometry with negative-ion chemical ionization and the use of [ 37 Cl 2 ]chloramphenicol as an internal standard. A set of identification criteria, in accordance with guidelines of the European Community, is described. For urine, muscle and eggs limits of detection and quantification of 0.1 μg kg −1 are obtained. The method shows good repeatability and reproducibility. Results for urine were compared with those obtained with a radioimmunochemical procedure and an enzyme immunoassay (Quik-Card). Screening with an immunochemical procedure followed by confirmation with gas chromatography-mass spectrometry was found to be an effective strategy for monitoring residues of chloramphenicol in biological matrices.

The synthesis of [1,2,3,4-13C] cortisol

The preparation of [1,2,3,4-13C] cortisol 21 acetate by total synthesis is described. The C labels are introduced in a way analogous to the one described by us previously for the synthesis of testosterone and estradiol. The cortisol dihydroxyacetone side chain was elaborated using known methods. The 11 beta-hydroxyl function was introduced by addition of hypobromous acid to a 9-double bond followed by reductive debromination. 13C-labeled cortisol can be used as a tracer for the determination of cortisol production rates.

[1,2,3,4-13C] testosterone and [1,2,3,4-13C] estradiol

The preparation of [1,2,3,4-13C] testosterone and of [1,2,3,4-13C] estradiol by total synthesis is described. The 13C labels are introduced by alkylating intermediate 1 with [1,2,3,4-13C]l-iodo-3,3-ethylenedioxybutane (2) to obtain intermediate 10. Hydrolysis of the ketal function, cyclization, aromatization and removal of protective groups gave [1,2,3,4-13C] estradiol. Labeled testosterone was prepared by methylating intermediate 10 and by subsequent treatment with acid. The labeled steroids can be used as tracers for in vivo metabolic studies and as internal standards for the development of definitive gc-ms quantitative methods.

Synthesis of 13C-labeled steroid hormones

The synthesis of 13C-labeled steroid hormones is reviewed. Two general approaches are highlighted: partial synthesis in which part of the steroid nucleus is replaced with 13C-labeled synthons, and total synthesis. Examples from both approaches, leading to (3-(3)C)-, (4-(13)C)-, (3,4-(13)C2)-, and (1,2,3,4-(13)C4)- labeled steroid hormones (e.g., testosterone, estradiol, progesterone, and cortisol), are presented.

Synthesis of deuterium-labelled medroxyprogesterone, megestrol and melengestrol derivatives

The deuterium-labelled medroxyprogesterone derivatives 1 and 2, were prepared by opening of the epoxide in 5β, 6β-epoxy-17α-hydroxypregnane-3,20-dione 3,20-bis(ethyleneketal)(9β) with [2H3]methyl magnesium iodide and further processing of the thus obtained 6-C-pregnane derivative, 10. [2H3]megestrol acetate (4) was prepared by p-chloranil oxidation of [2H3]medroxyprogesterone acetate (2). Subsequent deacetylation of 4 gave [2H3]megestrol (3). In a similar fashion, 16-dehydro-16-methylprogesterone (11) was converted into [2H3]melengestrol (5) and its acetate, 6. In addition, the application of the labelled steroids in the detection of gestagens in samples of kidney fat was shown.