Corticosteroids cleanup

A generic enzyme immunoassay for the determination of several synthetic corticosteroids including dexamethasone, betamethasone, flumethasone, triamcinolone, prednisolone, and methylprednisolone in milk, liver, kidney, and muscle samples was recently developed (156). Antibodies raised against dexamethasone-21-hemisuccinate-bovine serum albumin were used in this assay, whereas dexa-methasone-horseradish peroxidase was the label conjugate. Skimmed milk could be directly screened for the presence of corticosteroids at limits of detection of 0.1 ppb for dexamethasone, betamethasone, and flumethasone, 0.3 ppb for triamcinolone and 0.5 ppb for prednisolone. Tissue samples were submitted, prior to the immunoassay, to an extraction/cleanup procedure involving liquid-liquid partitions with acetonitrile-water followed by hexane-chloroform. Background values for bovine liver, swine kidney, and calf muscle were determined to be 0.26, 0.26, and 0.07 ppb, respectively, of dexamethasone equivalents. 

Very few methods have appeared in the literature with respect to the extraction, cleanup, and subsequent determination of corticosteroids in food samples (Table 29.17). Milk analysis usually requires a pretreatment step for fat elimination (527). Centrifugation for 20 min at about 7000 g at 4 C is the usually applied procedure for making the fat floating on the top of the sample. Tissue analysis also requires a pretreatment step for matrix break-up that can be accomplished by means of a mincing and/or a homogenizing apparatus. 

Cleanup and concentration of corticosteroids from coextracted matrix constituents can also be accomplished using solid-phase extraction cartridges that contain nonpolar Cig (527, 535), or Cg sorbents (452), as shown in Table 29.17. In addition, Cig cartridges have been used to extract endogenous and synthetic corticosteroids from swine plasma (536), and to isolate cortisol from plasma or serum samples (537). Other workers employed conventional columns containing deactivated Florisil to isolate cortisol in milk (529) or fluoroprednisolone in bovine tissues (533). 

Following extraction and cleanup, corticosteroid residues in sample extracts can be determined by thin-layer, liquid, or gas chromatographic methods. Spectrophotometric, fluorometric, electron capture, or mass spectrometric detection systems have all been successfully used in corticosteroids analysis. 

Solid-phase extraction for milk, urine, and feces samples is carried out by washing the loaded Cig cartridge successively with 5 ml water, 5 ml acetone/ water (20 80), 5 ml methanol/water (20 80), 5 ml dichloromethane/hexane (20 80), and 5 ml ethyl acetate/hexane (10 90). The corticosteroids are eluted with 3 ml ethyl acetate. The eluate is evaporated, and the residual is reconstituted in 0.5 ml ethanol and 5 ml phosphate-buffered saline, pending subsequent immunoaffinity column cleanup. The solid-phase extraction procedure differs for liver samples. In that case, washing of the cartridge is performed with 5 ml water, 5... 

Following solid-phase extraction, all extracts are adjusted in the pH range 7-7.5, and submitted to additional cleanup on an immunoaffinity column containing a mixture of dexamethasone- and prednisolone-specific gels. Column washing is performed with water, while elution of the analytes with 3 ml methanol/water (80 20). Aliquots of the eluates are submitted to oxidative reaction with pyridin-ium chlorochromate, and the oxidized corticosteroid derivatives are then analyzed by gas chromatography-mass spectrometry under the conditions shown in Table... 

East sample preparation without cleanup. Separation of the two isomers betamethasone and dexamethasone Performance fit-for-purpose for low concentrations of corticosteroids. 

The sample cleanup for corticosteroids is rather similar to that used for anabolic steroids. The polarity is somewhat higher, allowing the possibility to separate the two groups. Also lAC has been described [50]. 

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