Development and validation of a precise and accurate method to determine polyamine levels in cultured cells.
Cabrera, D.; Pujana Vaquerizo, M.; Martinez La Osa, B.; Zabala Letona, A.; Manuel Falcon, J.; Carracedo, A.; van Liempd, S.
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We describe a robust, fast and accurate method for the quantification of intra-cellular concentrations of the polyamines, putrescine, spermidine and spermine in cultured cells. Hydrophilic interaction liquid chromatography in combination with Time-of-Flight mass spectrometry was used to obtain high resolution data for the analytes. Assay performance was determined with respect to chromatographic resolution, quantification, analyte recovery and matrix effects. Furthermore, assay variability was determined in a biological context. Based on these variability measurements, minimal detectable effects (MDEs) which would lead to significant differences in a null-hypothesis significance test, were calculated. As such, changes in spermine could be determined with the highest sensitivity with point estimates for the MDEs of 32% between-days and 10% within-days. For spermidine, these values were 38% between-days and 16% within-days. Finally, effects for putrescine were measured least sensitive with 43% between-days and 36% within-days. Finally, we employed the method to analyze the impact of polyamine synthesis pathway inhibition and cell culture conditions which are relevant aspects for the interpretation of the biological role of polyamines.
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