Intracellular metabolite profiling of platelets: Evaluation of extraction processes and chromatographic strategies

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2012-06, Vol.898, p.111-120
• Main Authors: Paglia, Giuseppe, Magnúsdóttir, Manuela, Thorlacius, Steinunn, Sigurjónsson, Ólafur E., Guðmundsson, Sveinn, Palsson, Bernhard Ø., Thiele, Ines
• Format: Article
• Language: English
• Subjects: Blood Platelets - chemistry; Blood Platelets - metabolism; Chemical Fractionation - methods; Chromatography, Liquid - methods; Extraction; HILIC; Humans; hydrophilic interaction chromatography; Mass Spectrometry; metabolites; Metabolome; Metabolomics; Metabolomics - methods; Methanol; physicochemical properties; Platelets; Principal Component Analysis; Reproducibility of Results; Reversed phase liquid chromatography; ultra-performance liquid chromatography; UPLC–MS; Water
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2012.04.026

► This is the first metabolomic study reporting analytical protocols for platelets. ► Seven different extraction methods and two different UPLC–MS methods were tested. ► MeOH–H2O extraction coupled with HILIC–MS method identified 107 metabolites. An extraction method for intracellular metabolite profiling should ideally be able to recover the broadest possible range of metabolites present in a sample. However, the development of such methods is hampered by the diversity of the physico-chemical properties of metabolites as well as by the specific characteristics of samples and cells. In this study, we report the optimization of an UPLC–MS method for the metabolite analysis of platelet samples. The optimal analytical protocol was determined by testing seven different extraction methods as well as by employing two different LC–MS methods, in which the metabolites were separated by using hydrophilic interaction liquid chromatography (HILIC) and reversed phase liquid chromatography (RPLC). The optimal conditions were selected using the coverage of the platelets’ metabolome, the response of the identified metabolites, the reproducibility of the analytical method, and the time of the analysis as main evaluation criteria. Our results show that methanol–water (7:3) extraction coupled with HILIC–MS method provides the best compromise, allowing identification of 107 metabolites in a platelet cell extract sample, 91% of them with a RSD% lower than 20. A higher number of metabolites could be detected when analyzing the platelet samples with two different LC–MS methods or when using complementary extraction methods in parallel.