Separation and quantitation of fructose-6-phosphate and fructose-1 ,6-diphosphate by LC-ESI-MS for the evaluation of fructose-1,6-biphosphatase activity.
An LC-ESI-MS method was developed for the identification and quantification of fructose-1,6-biphosphate (F1,6BP) and fructose-6-phosphate (F6P), respectively the substrate and the product of the enzymatic reaction catalysed by fructose-1,6-bisphosphatase (F1,6BPase). F1,6BPase, expressed predominantly in liver and kidney, is one of the rate-limiting enzymes of hepatic gluconeogenesis and has become a target for the development of new drugs for type 2 diabetes. The two sugar phosphates were separated on a Phenomenex Luna NH2 column (150 mm x 2.0 mm id) using the following mobile phase: 5 mM triethylamine acetate buffer/ACN (80:20) v/v in a linear pH gradient (from pH = 9 to 10 in 15 min) at the flow rate of 0.3 mL/min. The detection was performed with an IT mass spectrometer in negative polarity (full scan 100-450 m/z) and in SIM mode on the generated anions at m/z = 339 (F1,6BP) and m/z = 259 (F6P). Under the optimised final conditions, the method was validated for accuracy, specificity, precision (inter- and intradays RSD comprised between 1.0 and 6.3% over the range of concentrations used), linearity (50-400 microM), LODs (0.44 microM) and LOQs (1.47 microM), and the method was applied to F6P determination in the F1,6BPase catalysed hydrolysis of F1,6BP.
Mancini
Francesca
F
Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy.
Fiori
Jessica
J
Cavrini
Vanni
V
Andrisano
Vincenza
V
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D006597:Fructose-Bisphosphatase / Q000032:analysis* / Q000378:metabolism; D005635:Fructosediphosphates / Q000032:analysis / Q000302:isolation & purification*; D005636:Fructosephosphates / Q000032:analysis / Q000302:isolation & purification*; D066298:In Vitro Techniques; D007700:Kinetics; D011817:Rabbits; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D005635:Fructosediphosphates; D005636:Fructosephosphates; C027618:fructose-6-phosphate; D006597:Fructose-Bisphosphatase; C029063:fructose-1,6-diphosphate