Analysis of synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and laser-induced fluorescence detection.
Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were used for the analysis of new synthetic derivatives of hypophysis neurohormones--vasopressin and oxytocin, and pancreatic hormone--human insulin (HI) and its octapeptide fragment, derivatized by fluorescent probe, 4-chloro-7-nitrobenzo[1,2,5]oxadiazol (NBD). The suitable composition of background electrolytes (BGEs) was selected on the basis of calculated pH dependence of effective charge of analyzed peptides. Basic ionogenic peptides were analyzed by CZE in the acidic BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The ionogenic peptides with fluorescent label, NBD, were analyzed in 0.5 M acetic acid, pH 2.5. The best MEKC separation of non-ionogenic peptides was achieved in alkaline BGE, 20 mM Tris, 5 mM H3PO4, with micellar pseudophase formed by 50 mM sodium dodecylsulfate (SDS), pH 8.8. Selected characteristics (noise, detectability of substance, sensitivity of detector) of the UV-absorption detectors (single wavelength detector, multiple-wavelength photodiode array detector (PDA), both of them operating at constant wavelength 206 nm) and laser-induced fluorescence (LIF) detector (excitation/emission wavelength 488/520 nm) were determined. The detectability of peptides in the single wavelength detector was 1.3-6.0 micromol dm(-3) and in the PDA detector 1.6-3.1 micromol dm(-3). The LIF detection was more sensitive, the applied concentration of NBD derivative of insulin fragment in CZE analysis with LIF detection was three orders lower than in CZE with UV-absorption detector, and the detectability of this peptide was improved to 15.8 nmol dm(-3).
Solínová
Veronika
V
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo 2, 166 10 Prague 6, Czech Republic.
Kasicka
Václav
V
Koval
Dusan
D
Barth
Tomislav
T
Ciencialová
Alice
A
Záková
Lenka
L
D000595:Amino Acid Sequence; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006728:Hormones / Q000032:analysis*; D007834:Lasers; D010455:Peptides / Q000032:analysis*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D006728:Hormones; D010455:Peptides