Analysis of diethylstilbestrol, dienestrol and hexestrol in biological samples by immunoaffinity extraction and gas chromatography-negative-ion chemical ionization mass spectrometry

Renzo Bagnati, Maria Grazia Castelli, Luisa Airoldi, Maurizio Paleologo Oriundi, Antonio Ubaldi, Roberto Fanelli

Research output: Contribution to journalArticle

Abstract

A method has been developed for the detection of diethylstilbestrol, together with dienestrol and hexestrol, using extraction with a single immunoaffinity column containing antibodies raised against diethylstilbestrol, followed by gas chromatography-negative-ion chemical ionization mass spectrometry. Immunoaffinity columns were prepared by coupling immunoglobulin G fractions obtained from rabbit antisera with a Sepharose matrix. The immunizing agent was synthesized by introducing a carboxyl group into the diethylstilbestrol molecule and coupling this product to bovine serum albumin. The columns were used for immunoadsorption of diethylstilbestrol and other estrogens, after dilution of samples with phosphate buffer, and were eluted with acetone-water (95:5 v/v). A derivatization method suitable for gas chromatographic-mass spectrometric analysis of diethylstilbestrol and other estrogens was developed using pentafluorobenzyl bromide and ethanolic potassium hydroxide as reagents. The derivatives obtained were detectable at the sub-picogram level using gas chromatography with negative-ion chemical ionization mass spectrometry. Recoveries of cis- and trans-diethylstilbestrol, dienestrol and hexestrol from the immunoaffinity columns, determined after extraction from urine, plasma and buffer, ranged from 28 to 96%. The sensitivity for diethylstilbestrol in urine samples was ca. 10 ppt. The method was applied to the analysis of urine from calves given a single dose of 10 mg of diethylstilbestrol. Free and glucuronic acid conjugated diethylstilbestrol decreased with time, but their ratio was variable.

Original languageEnglish
Pages (from-to)267-278
Number of pages12
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume527
Issue numberC
DOIs
Publication statusPublished - 1990

ASJC Scopus subject areas

  • Chemistry(all)
  • Clinical Biochemistry
  • Molecular Medicine

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