An HPLC method for the determination of adenosine diphosphate: an important marker of hexokinase activity in metabolic diseases.

Hexokinases play a critical role in the cellular uptake and utilization of glucose. As such, they are of fundamental importance to all cells. By catalyzing of glucose to produce glucose-6-phosphate, hexokinases control the first irreversible step of glucose metabolism and initiating all major pathways of glucose consumption. Our objective was to develop and validate highly sensitive and selective high performance liquid chromatography with photodiode array detector (HPLC-PDA) assays allowing the determination of adenosine diphosphate (ADP), which was used for determination of the hexokinase activity. Samples were analyzed by HPLC-PDA using a C18 analytical column (250 x 4.6 mm) for chromatographic separation. Optimal detection was achieved based on isocratic elution with a mobile phase consisting of mixture sodium phosphate monobasic buffer-methanol. This method met all requirements of specificity, sensitivity, linearity, precision, accuracy, and stability generally accepted in bioanalytical chemistry and was successfully applied to study hexokinase activity in alloxan-induced diabetic rat model. Determination of hexokinase activity will permit characterization of cellular metabolic state in many diseases, such as cancer and diabetes.