Analysis of adenosine by RP-HPLC method and its application to the study of adenosine kinase kinetics.
An RP-HPLC method for the analysis of adenosine (ADO) has been developed and validated. In the present study, we report an RP-HPLC-based method with modifications of mobile phase and shorter retention time that substantially improved the efficiency of ADO analysis. The HPLC separation of the ADO was achieved on a C18 column, using a mobile phase consisting of water, containing 7% v/v ACN, at a flow rate of 0.8 mL/min. The column effluent was monitored by UV detection at 260 nm. A linear response was achieved over the concentration range of 0.25-100.00 micromol/L. The analytical method inter- and intra-run accuracy and precision were better than +/- 15%. The LOQ was 0.25 micromol/L, with ADO detection in the range of 6.25 pmol per sample. The method has been applied to the study of adenosine kinase (AK) kinetics.
Marin
Rodrigo Miguel
RM
Department of Internal Medicine, School of Medicine, UNICAMP, Campinas, SP, Brazil.
Franchini
Kleber Gomes
KG
Rocco
Silvana Aparecida
SA
D000241:Adenosine / Q000032:analysis*; D000248:Adenosine Kinase / Q000378:metabolism*; D000818:Animals; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007700:Kinetics; D051379:Mice; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D014867:Water / Q000737:chemistry*
D014867:Water; D000248:Adenosine Kinase; D000241:Adenosine