Molecularly Imprinted Solid Phase Extraction Before Capillary Electrophoresis for the Analysis of Estrogens in Serum Samples

CONTIN M.; FLOR S.; MARTINEFSKI M.,; LUCANGIOLI S.; TRIPODI V.

Buenos Aires

Simposio; 18 th Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical and Industrial Applications of Capillary Electrophoresis and Microchip Technology; 2012

LACE

An estrogen selected molecular imprinted polymer (MIP) has been developed to be used as an alternative sorbent in solid phase extraction of serum samples before capillary electrophoresis analysis. An initial series of polymers were prepared to determine the best condition to obtain an appropriate MIP for the estrogens extraction. The study of UV spectra was used for evaluating and optimizing the template: monomer ratio. On the other hand,  fixing the template amount, different monomer - cross-linker molar ratios and the appropriate porogen, were tested. Following a heat polymerization, MIP has been synthesized using methacrylic acid (MAA) as functional monomer, ethyleneglycol dimethacrylate (EGDMA) as crosslinker, benzoyl peroxide as radical initiator, 17β- estradiol as template, and toluene as porogen. The optimized molecular imprinted solid phase extraction (MISPE), using the MIP developed, was compared to a non imprinted polymer (NIP) extraction and to the traditional C18 solid phase extraction to purify estrogens from complex matrices. Serum samples were spiked with estrona (E1), estradiol (E2) and estriol (E3). Capillary electrophoresis (CE) analysis was carried out with a P/ACE Ô MDQ capillary electrophoresis. Uncoated fused silica capillaries of 50 cm (40 cm length to the detector) x 75 µm i.d., were used. The separation was performed by a MEKC system consisting of 44 mM cholic acid, 10 mM SDS, 0.05% w/v tetronic Ò 1107, 2.5% v/v methanol, 2.5% v/v tetrahydrofuran and 5 mM borate- 5 mM phosphate buffer (pH=8.0) as background electrolyte. The capillary temperature was 25°C, and UV detection was set at 210 nm. Samples were injected under 0.5 psi pressure for 3s and electrophoretic system was operated under positive polarity and a constant voltage of 18 kV. It was showed that using MISPE for a quantitative extraction, an excellent selectivity was obtained with E1, E2 and E3. The recovery using MIP cartridge was 93 % for E3, 42 % for E1 and 100 % for E2, higher than that of NIP cartridge (12 %, 30 %, and 65 %, for E3, E1 and E2 respectively) and C18 cartridge (75%, 0% and 27%). Moreover, if C18 cartridges are employed, additional peaks together with a co-elution of serum interferences appear in the electropherogram. It was concluded that the MISPE system developed is useful for the selective extraction of 17β- estradiol and its major natural related estrogens, estriol and estrone, from serum samples with minimal interferences and high recoveries.