Absolute quantitative imaging of sphingolipids in brain tissue by exhaustive liquid microjunction surface sampling-liquid chromatography-mass spectrometry.

It is challenging to achieve absolute quantitation and accurate identification with mass spectrometry imaging (MSI) techniques. The issues facing these techniques include the uncertainty of sampling and ionization efficiencies in a localized environment, the difficulty in defining the concentration of spiked standard on tissue, and the low identification capability of MS in distinguishing isobaric compounds. In this study, we coupled continuous flow liquid microjunction surface sampling (LMJSS) with ultrahigh-performance liquid chromatography (UPLC)-MS for quantitative MSI of brain tissue. LC separation could increase the dimensions of identification and reduce the matrix effects in the tissue. A new LMJSS extraction solvent was developed to achieve exhaustive surface sampling; therefore, direct internal standard addition in the extraction solvent could be used for spot-to-spot absolute quantitation. The results showed that sphingolipids were successfully separated from their isobaric counterparts with LC-MS, and 10 sphingolipids were identified and imaged in brain tissue. The matrix effects in different locations of brain tissue with the new solvent were all in the range of 80%-150%. Compared with the traditional LMJSS solvent (90% methanol-water), the new solvent (10% 1,1,1,3,3,3-Hexafluoro-2-propanol-10% isopropanol-80% methanol) led to higher detection coverage (more lipid features) and 6-10-fold higher sensitivity for 6 identified metabolites in brain tissue. Moreover, extraction efficiencies of 80-98% for targeted sphingolipids in brain tissue were obtained with the new solvent, which allowed direct standard addition in extracts for absolute quantitation. Finally, the absolute quantitation results with LMJSS-LC-MSI were compared with those using traditional bulk tissue extraction-LC-MS, and similar quantitation results with these two methods (relative recoveries=64-119%) were obtained for sphingolipids. The absolute quantitative spatial distributions of targeted metabolites largely matched previously reported results. The method was applied to a study on the quantitative spatial changes of sphingolipids and creatine in brain after traumatic brain injury (TBI). The biological replicate results showed that 2 metabolites had significant changes after TBI in several specific regions of brain tissue.