Separation and identification of enzymatically prepared dephosphorylated products of myo-inositolhexakisphosphate using LC-MS.

LC-MS technique described here is a new way for the separation and direct determination of UV-Vis insensitive inositol phosphates (InsP(2)-InsP(6)). This circumvents the need of radioisotopic labeling and post-column derivatization techniques. The method involves separation of various enzymatically dephosphorylated derivatives of InsP(6) on C(18)-column using MeOH/H(2)O (30:70 v/v) and their identification using electron spray ionization MS in positive ion mode (+pESI-MS). The LC-MS studies revealed that the purified phytase from Aspergillus niger van Teighem hydrolyzes InsP(6 )in a sequential manner leading to InsP(2 )(InsP(2) x 2Na, t(R) 4.4-4.54 min, base peak m/z 382.9) as the end product.