Gas Chromatographic Determination of Diethylstilbestrol and Its Glucuronide in Cattle Tissues

Abstract

A gas-liquid chromatographic (GLC) method is described for the determination of diethylstilbestrol (DES) and its glucuronide (DES-G) in cattle tissues. Muscle, liver, and kidney samples are extracted with methanol and centrifuged. An aliquot of extract equivalent to 50 g tissue is evaporated to remove methanol and hydrolyzed overnight, using β-glucuronidase. In the resulting sample, free DES is purified by alkaline liquid-liquid extraction and is reacted in base with dichloroacetyl chloride to form the DES diester. The diester is measured by GLC, using a 5% UC W-98 column and electron affinity detection. Fat samples are assayed for free DES by extracting with chloroform and processing as other tissues, beginning with the alkaline purification. Residues from tissues of steers fed DES, assayed by both GLC and bioassay procedures, showed excellent agreement. In these studies the GLC procedure had a sensitivity of about 0.001 ppm. Lean and fat contained no DES residue when animals were slaughtered at 0 time off medicated feed. Liver and kidney contained residues on the order of 0.02 ppm at 0 time. The DES declined to nondetectable levels in liver and kidney by 96 hr after withdrawal. The assay procedure has been used successfully on a routine basis for several months.

Radioimmunoassay for diethylstilbestrol and the monoglucuronide metabolite in bovine liver

“…The background for this method (0.05 ppb) was similar to that reported by Hoffmann and Laschutza (1980) and Vogt (1980). Compared with the GC method of Donoho et al (1973), upon which other GC methods for DES are based, all these RIA methods have superior specificity and lower background (0.01-0.05 vs. 0.4 ppb).…”

“…RIA tends to be more sensitive than most physical methods. Donoho et al (1973) encountered problems with quantitation below 2.0 ppb with GC, and Day et al (1975) were unable to document sensitivity below 1.0 ppb with GC-MS. We have employed our method down to 0.3 ppb. A detection limit of 0.09 ppb was calculated for our method by the formula of background plus 2 times the SD in order to make a comparison with the methods of Vogt (1980) and Hoffman and Laschutza (1980), who used this same formula.…”

“…Attempts to prepare a more specific antibody were unsuccessful and emphasis therefore was placed on finding satisfactory purification procedures to eliminate the interference. A purely liquid-liquid partition procedure similar to that used by Donoho et al (1973) was unsatisfactory, as was silica gel chromatography. A combination of the benzene extraction, which eliminated polar interference, and Sephadex LH-20 chromatography, which eliminated nonpolar interference, was found to be effective and reproducible.…”

“…Diethylstilbestrol (DES) is difficult to quantitate below 1.0 ppb in bovine liver, which is among the last tissues to contain DES after cattle are withdrawn from the drug (Donoho et al, 1973;Aschbacher, 1976). Methods to measure DES in bovine liver include gas chromatography (GC) (Donoho et al, 1973), GC with mass spectrometry (GC-MS) ( Day et al, 1975), and liquid chromatography (Kenyhercz and Kissinger, 1978), but quantitation below 1.0 ppb has not been documented for any of these methods.…”

Quantification of diethylstilbestrol residues in meat samples by gas chromatography-isotope-dilution mass spectrometry

“…They include bioassays, based on mouse uterus susceptibility to estrogens (Umberger et al, 1963a), colorimetry or fluorimetry (Umberger et al, 1963b), competitive protein-binding assays (Hoffmann, 1978;Hoffmann and Laschutza, 1980;Vogt, 1980;Gridley et al, 1983), and several gas chromatographic techniques with various detection systems. Among them, electron-capture (Coffin and Pilón, 1973;Donoho et al, 1973;Ryan and Pilón, 1976;Laitem et al, 1978;Tirpenou et al, 1983) and mass spectrometry (Day et al, 1975;Hollerer and Jahr, 1975;Stan and Abraham, 1980;Derks et al, 1983;Galli et al, 1983) are the most commonly employed techniques. The detection limit of the chromatographic methods is in the order of 1 ppb.…”