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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
Author(s) -
Mancini Francesca,
Fiori Jessica,
Cavrini Vanni,
Andrisano Vincenza
Publication year - 2006
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200600077
Subject(s) - chemistry , chromatography , fructose , hydrolysis , biochemistry
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 NH 2 column (150 mm×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 μM), LODs (0.44 μM) and LOQs (1.47 μM), and the method was applied to F6P determination in the F1,6BPase catalysed hydrolysis of F1,6BP.