Abstract
A signalling molecularly imprinted polymer was synthesised for easy detection of tamoxifen and its metabolites. 6-Vinylcoumarin-4-carboxylic acid (VCC) was synthesised from 4-bromophenol to give a fluorescent monomer, designed to switch off upon binding of tamoxifen. Clomiphene, a chlorinated analogue, was used as the template for the imprinting, and its ability to quench the coumarin fluorescence when used in a 1:1 ratio was demonstrated. Tamoxifen and 4-hydroxytamoxifen were also shown to quench coumarin fluorescence. Imprinted and non-imprinted polymers were synthesised using VCC, methacrylic acid as a backbone monomer and ethylene glycol dimethacrylate as cross-linker, and were ground and sieved to particle sizes ranging between 45 and 25 μm. Rebinding experiments demonstrate that the imprinted polymer shows very strong affinity for both clomiphene and tamoxifen, while the non-imprinted polymer shows negligible rebinding. The fluorescence of the imprinted polymer is quenched by clomiphene, tamoxifen and 4-hydroxytamoxifen. The switch off in fluorescence of the imprinted polymer under these conditions could also be detected under a UV lamp with the naked eye, making this matrix suitable for applications when coupled with a sample preparation system.
Highlights
Tamoxifen belongs to a family of drugs known as selective oestrogen receptor modulators (SERM), which are characterised by agonist or antagonist activity depending on the different tissues
For the past 15 years, tamoxifen has been classified as a prohibited substance by the World Anti-doping Authority (WADA) and the International Olympic Committee (IOC); as a result of its antagonist activity in breast tissues, this drug has been inappropriately used by athletes to mask the side effects of using growth hormones and anabolic androgenic steroids (AAS), well known for causing gynecomastia, i.e. enlargement of breast in man [2]
Clomiphene (2) is structurally very similar to the target analyte tamoxifen (1), with three phenyl groups providing multiple opportunities for hydrophobic interactions, and the replacement of the ethyl group with the chlorine atom being the only structural difference. This is not expected to have a significant impact on molecular recognition, as the main interactions between template and monomer will derive from the ionic bond of the tertiary amine in tamoxifen and the carboxylic acid in the polymer, together with the hydrophobic interactions derived from π-π stacking
Summary
Tamoxifen belongs to a family of drugs known as selective oestrogen receptor modulators (SERM), which are characterised by agonist or antagonist activity depending on the different tissues. MIPs have been developed in sample preparation prior to HPLC-UV analysis to ensure efficient extraction and enrichment of tamoxifen and its polar metabolites following the hydrolysis step [7] and the stability of the matrix to harsh environments offers an interesting alternative to proteins. The retention time of the clomiphene in the presence of MIP/NIP was obtained with a mobile phase of 5:95 acetic acid/acetonitrile solution. 6-Vinylcoumarin-4-carboxylic acid was selected as functional monomer for this work, as it satisfied two important requirements: (i) the presence of the COOH group, which allows strong ionic interactions to take place with the tertiary amine unit of tamoxifen, 4-hydroxytamoxifen and clomiphene; (ii) the strong fluorescence of the coumarin unit, which was previously shown to be quenched when in contact with ephedrine [14]
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