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The advent of mass spectrometric methods has facilitated the determination of multiple molecular species of cellular lipid classes including the polyphosphoinositides, though to date methods to analyse and quantify each of the individual three PtdInsP and three PtdInsP2 species are lacking. The use of imaging methods has allowed intracellular localization of the phosphoinositide classes but this methodology does not determine the acyl structures. The range of molecular species suggests a greater complexity in polyphosphoinositide signaling than yet defined but elucidating this will require further method development to be achieved. This article is part of a Special Issue entitled Tools to study lipid functions., (Copyright © 2014 Elsevier B.V. All rights reserved.)