Analysis of dideoxyadenosine triphosphate by CE with fluorescence detection. I. Derivatization through the phosphate group.
A CZE method was developed, which separates 2',3'-dideoxyadenosine-5'-triphosphate (ddATP) from other metabolites and endogenous nucleotides at high concentrations (20-200 microg/mL) to allow UV detection. To enhance sensitivity, fluorescence detection which requires prior derivatization of compounds was examined. Precapillary derivatization of ddATP in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) with dansyl ethylenediamine (dansyl EDA) was faster and stable compared to that of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine (BODIPY FL EDA). Reaction conditions, reagent concentrations and detection parameters were optimized and highest derivatization efficiency was achieved in 0.1 M 1-methylimidazole buffer (pH 8.0) with 140 mM EDAC in 1-methylimidazole buffer and 30 mM dansyl EDA in DMF for 90 min at 60 degrees C. Dansyl EDA derivatives of ddATP, 2'-deoxyadenosine-5'-triphosphate (dATP) and ATP were comigrating with the CZE method; therefore, a MEKC method was developed and optimized for repeatable separations. Upon dansylation, sensitivity of ddATP with fluorescence detection (LOQ = 12 ng/mL) was 160 times higher than UV detection (LOQ = 1.9 microg/mL).
Mallampati
Swapna
S
Laboratory of Pharmaceutical Analysis, K.U. Leuven, Leuven, Belgium.
Van Aerschot
Arthur
A
Hoogmartens
Jos
J
Van Schepdael
Ann
A
D001896:Boron Compounds / Q000737:chemistry*; D002234:Carbodiimides / Q000737:chemistry*; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods; D003838:Deoxyadenine Nucleotides / Q000032:analysis* / Q000737:chemistry; D054306:Dideoxynucleotides / Q000032:analysis* / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D005029:Ethylenediamines / Q000737:chemistry*; D007202:Indicators and Reagents; D015394:Molecular Structure; D010710:Phosphates / Q000737:chemistry*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
C503730:4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine; D001896:Boron Compounds; D002234:Carbodiimides; D003838:Deoxyadenine Nucleotides; D054306:Dideoxynucleotides; D005029:Ethylenediamines; D007202:Indicators and Reagents; D010710:Phosphates; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; C037773:2',3'-dideoxyadenosine triphosphate