Untitled - MendelNet 2013 - Mendelova zemědělská a lesnická ...

MENDELNET 2013Optimization of FIA-ED conditionsFlow injection analysis with electrochemical detection (FIA-ED) was used for analysis ofdoxorubicin and subsequently for analysis of its release from liposomes. The effect of differentbuffers on detection of doxorubicin itself was evaluated before the carrying out this kind ofanalysis. Standard solution of doxorubicin was always diluted to 50 µg.ml -1 concentration using abuffer which was also used as mobile phase in FIA-ED. Each buffer was used in its naturalbuffering range: Britton-Robinson buffer (pH 2, 3, 4, 5, 6, 7, 8, 9, 10), acetate buffer (pH 3.5, 4.5,5.5), phosphate buffer (pH 5.5, 6.5, 7.5) and borate buffer (pH 7.5, 8.5, 9.5). The largest peak areawas achieved using the highest pH – a Britton-Robinson buffer at pH 10 (Fig. 2D). With decreasingpH the peak area of doxorubicin was also decreasing. This effect of pH was surprising because withother types of electrochemical detection the lower pH is usually used [9]. For similar types ofdetection (in HPLC), the lower pH is also used. Often a phosphate buffer with addition oftriethylamine is used at pH lower than 5 [10].Fig. 2: FIA-ED analysis of doxorubicin (50 mg.ml -1 ) in different buffers. The buffer used fordilution of doxorubicin’s aliquot was also used as a mobile phase. The potential range was 100-1200 mV with 100-mV step. Blue marks in graphs represent maximal measured values. (A)Phosphate buffer (PB) with pH 5.5, 6.5 and 7.5. (B) Acetate buffer (AB) with pH 3.5, 4.5 and 5.5.(C) Borate buffer (BB) with pH 7.5, 8.5 and 9.5. (D) Britton-Robinson buffer (BR) with pH 2.0, 3.0,4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0.Electrochemical monitoring of doxorubicin releasingThe electrochemical characterization of liposomes was performed under optimized conditionsaccording to the results obtained above. Differential HDVs of all samples are shown in Fig. 3.Differential HDV’s curves were obtained by subtracting the peak areas of blank samples(liposomes without doxorubicin) from the peak areas of doxorubicin encapsulated in liposomes. InFig. 3D, 3H are shown the maximal differences of peak areas at 900 mV potential, which providedthe highest response of detector. In Fig. 3A, 3B, 3C and 3D the biggest differences showedliposome 9 with all concentrations of doxorubicin. It’s interesting, that liposome 8 with higherconcentration of cholesterol showed smaller difference of peak areas than liposome 9. Cholesterolhas probably a role in the improvement of electrochemical detection of encapsulated doxorubicin,but this improvement has a limitation factor in the concentration of cholesterol (criticalconcentration). It’s possible, that cholesterol enhances the electron transfer at the appliedconditions, but further experiments are necessary to prove it. In Fig. 3E, 3F, 3G and 3H theelectrochemical detection was influenced by the addition of SDS in the way that increaseddifferences were obtained in the case of the highest applied concentration of doxorubicin.A significant increase from 13.2 to 29.4 µC in maximal difference of peak areas occurred atliposome 10. In contrast, liposomes with cholesterol provided decreased differences of peak areasand thus the detection was deteriorated. Decreasing trend is in correlation with a concentration ofcholesterol in phospholipid bilayer of liposomes.916 | P age