EnhaNcement of sensitivity in separation techniques with fluorescence detection using deuterated
                                               water


                  Josef Kučera^1, Ondřej Peš^2, Ivona Voráčová^3, Petr Táborský^1

                                                 ^

^1Department of Chemistry, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech
Republic

^2Department of Biochemistry, Faculty of Medicine, Masaryk University, Kamenice 5, 62500 Brno,
Czech Republic

^3Department of Bioanalytical Instrumentation, Institute of Analytical Chemistry, Czech Academy of
Sciences, Veveří 967/97, 60200 Brno, Czech Republic


Email of presenting author: 408285mail.muni.cz



Fluorescent molecules with emission maxima above 500 nm are often dynamically quenched by OH
oscillators present in water molecules. We have changed H[2]O for D[2]O to obtain higher
fluorescence intensity signal. Our experiments carried out on spectrofluorometer have proved that
anthracyclines can achieve about 300 % higher response in D[2]O as a solvent. The affect can be
employed in various analytical methods including HPLC and capillary electrophoresis coupled to
fluorescence detection. Fluorescence intensity in D[2]O based mobile phase of doxorubicin had
increased about 2.87 times. An advantage of capillary electrophoresis compared to HPLC is the lower
consumption of background electrolyte due to small volume of capillary. We achieved the separation
of daunorubicin and epirubicin using capillary electrophoresis coupled with laser induced
fluorescence detection (Ar^+ laser 488 nm). The separation was carried out using borate buffer (105
mM, pH 9.0) prepared in H[2]O and D[2]O.  We obtained 2.3 times larger peak area in buffer prepared
in D[2]O. Changing normal water for deuterium water is a simple way to increase LODs in separation
of anthracyclines, drugs used in cancer chemotherapy, by HPLC or CE coupled with fluorescence
detection.