title: Molecularly imprinted polymer applied to the selective isolation of urinary steroid hormones: an efficient tool in the control of natural steroid hormones abuse in cattle.
abstract: The use of anabolic substances to promote growth in livestock is prohibited within the European Union as laid down in Directive 96/22/EC. Nowadays, efficient methods such as steroid profiling or isotopic deviation measurements allow to control natural steroid hormones abuse. In both cases, urine is often selected as the most relevant matrix and, due to its relatively high content of potential interferents, its preparation before analysis is considered as a key step. In this context, the use of a selective sorbent such as molecularly imprinted polymer (MIP) was investigated. A MIP was synthesized based on 17β-estradiol, methacrylic acid and acetonitrile as template, monomer and porogen, respectively. Two approaches were then tested for non-conjugated (aglycons and glucuronides deconjugated) steroid purification: (i) molecularly imprinted solid phase extraction (MISPE) and (ii) semi-preparative supercritical fluid chromatography with a commercial MIP as stationary phase (SFC-MIP). Parameters for both approaches were optimized based on the main bovine metabolites of testosterone, estradiol, nandrolone and boldenone. The MISPE protocol developed for screening purposes allowed satisfactory recoveries (upper 65% for the 12 target steroids) with sufficient purification for gas chromatography-mass spectrometry (GC-MS) analysis. For confirmatory purposes, the use of isotopic ratio mass spectrometry (IRMS) requires a higher degree of purity of the target compounds, which can be reached by the SFC-MIP protocol with three steps less compared to the official and current method. Purity, concentration and absence of isotopic fractionation of target steroids extracted from urine of treated cattle (treated with testosterone, estradiol, androstenedione, and boldenone) allowed the measurement of (13)C/(12)C isotopic ratios of corresponding metabolites and endogenous reference compounds (ERC) and proved the relevance of the strategy.
authors.affiliation: LUNAM Université, Oniris, Laboratoire d'Etude des Résidus et Contaminants dans les Aliments (LABERCA), Nantes, F-44307, France. laberca@oniris-nantes.fr
authors.forename: Mickael
authors.initials: M
authors.lastname: Doué
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Bichon
authors.forename: Gaud
authors.initials: G
authors.lastname: Dervilly-Pinel
authors.forename: Valérie
authors.initials: V
authors.lastname: Pichon
authors.forename: Florence
authors.initials: F
authors.lastname: Chapuis-Hugon
authors.forename: Eric
authors.initials: E
authors.lastname: Lesellier
authors.forename: Caroline
authors.initials: C
authors.lastname: West
authors.forename: Fabrice
authors.initials: F
authors.lastname: Monteau
authors.forename: Bruno
authors.initials: B
authors.lastname: Le Bizec
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D000822:Animal Husbandry / Q000592:standards*; D000818:Animals; D002417:Cattle; D025924:Chromatography, Supercritical Fluid / Q000379:methods; D059022:Food Safety; D008401:Gas Chromatography-Mass Spectrometry; D000432:Methanol / Q000737:chemistry; D054802:Molecular Imprinting / Q000379:methods*; D011108:Polymers / Q000737:chemistry; D052616:Solid Phase Extraction / Q000379:methods; D013256:Steroids / Q000302:isolation & purification / Q000652:urine*; D019155:Veterinary Drugs / Q000302:isolation & purification / Q000652:urine*
chemical_list: D000097:Acetonitriles; D011108:Polymers; D013256:Steroids; D019155:Veterinary Drugs; D000432:Methanol; C032159:acetonitrile