HILIC-MS Rat Brain Analysis, A New Approach for the Study of Ischemic Attack

Abstract Clinicians often rely on selected small molecular compounds from body fluids for the detection, screening or monitoring of numerous life-threatening diseases. Among others, important monoamines – biogenic amines (BAs) – and their metabolites serve as sensitive biomarkers to study the progression or even early detection of on-going brain pathologies or tumors of neuroendocrine origins. Undertaking the task to optimize a reliable method for the simultaneous analysis of the most relevant BAs in biological matrices is of utmost importance for scientists. Hydrophilic interaction liquid chromatography (HILIC) with mass spectrometry (MS) detection provides a specific and sensitive technique for the separation and assessment of several neurotransmitter concentrations in body fluids (blood, urine, tissues). The present study was focused on the optimization of a straightforward, sensitive and reliable method for the simultaneous analysis of the ten most important BAs and their acidic metabolites from homogenates of rat brain tissues by use of HILIC-MS. Here, we present the optimized experimental workflow in terms of sample preparation, buffer compositions, HILIC and MS settings and data analysis. The presented method is reliable, straightforward and sensitive. Our method permits the unbiased, qualitative and quantitative determination of several BAs and their metabolites simultaneously. The optimized method was applied to the analysis of rat brain tissue samples from healthy hemispheres or those with induced transient ischemic attack (TIA). The undertaken pilot study demonstrated that the proposed approach could be applied to reveal the perturbation in neurotransmitters concentration after TIA in rat brains.


Introduction
Biogenic amines (BAs) are known to be associated with numerous life-threatening diseases such as neuroendocrine tumors (NET), neurodegenerative disorders like the above-mentioned biomarkers are sufficient alone to describe the nature and predict the development of listed pathophysiological stages of the disease. Moreover, for some of the pathologies where an unbalanced secretion of neurotransmitters could be observed, the usefulness of BAs assessment has not been evaluated yet.
Among those diseases where the importance of BA assessment from biological samples has not yet been fully investigated, stroke could be highlighted. The incidence of stroke dangerously affects the lives of people all around the world and could cause long-term disability [4]. One event that is highly connected to stroke is transient ischemic attack (TIA). TIA is caused by a temporary clot and is often called a "mini stroke" by the American Heart Association. It should be taken very seriously. The high disagreement in the diagnosis of TIA, by history, varies between 42% and 86% among neurologists.
A description of potential new biomarkers for TIA and/or ischemic stroke could be useful in everyday clinical practice. Recent studies revealed the potential ability of some molecules (cytokines, peptides) in easily accessible body fluids, to be applied as predictive biomarkers of poor outcomes (disability or death) after stroke [5,6]. Despite [3,9,10]. HILIC allows the separation of compounds that are poorly retained on the RP-LC column. Moreover, the use of ion-pairing reagents that are often necessary for PR-LC is omitted, which simplifies the entire analytical platform.
Most commonly, LC is coupled to mass spectrometry (MS) [8], a diode array detector (DAD) [11] and fluorymetric or electrochemical detectors [12,13] for BA analysis. With these methods, even nanomolar concentrations of each BA could be detected in biological samples [12,14,15]. BAs possess weak chromophores in their structure and often derivatization is necessary before analysis (for example when   ChemStation software (Agilent Technologies, Santa Clara, CA, USA) was used to collect HPLC data.

Sample preparation for the HILIC-MS analysis
The LC-MS technique is very sensitive to all impurities, salts and detergents that are present in biological matrices. Signals from the background could cause a "matrix effect" and this tends to be the main analytical problem when using the LC-MS technique. This affects the detection capability and accuracy, and suppresses the ions, which are essential for conclusive small, polar molecule detection [19]. Therefore, the purification and extraction procedure were optimized to provide high recovery of the studied compounds and allow their simultaneous analysis by HILIC-ESI-MS.
In the present study, the extraction of brain The presence of salt in the MP is important for ionic analyte elution and good peak shape.
Here, ammonium acetate and ammonium  [1,16]. In the presented study, to achieve an acceptable peak shape and resolution, different information to be obtained about the optimal fragmentor voltage for each of the analytes, which will enable its effective and most favorable fragmentation.
To conduct FIA, at first the most intense ion for each analyte from the scan ion chromatogram was chosen. Subsequently, optimization of the fragmentor voltage was carried out for each of the chosen ions (see Table 2 were differentially secreted while the animal experienced the TIA incidence ( Table 3). The

Conclusions
The HILIC-ESI-MS method for the simultaneous separation and detection of several BAs and their acidic metabolites from biological matrices was proposed. The method allowed the analysis of nanomolar concentrations of BAs in rat brain