## 1 Introduction  
The brain is highly susceptible to ischemic injury due to its high metabolic activity, energy demands, and reliance on aerobic metabolism. Brain ischemia can lead to irreversible neuronal death within minutes. Therefore, timely diagnosis and treatment are critical in mitigating brain injury [1]. After adipose tissue, the brain is the second-most lipid-rich tissue in the human body. Brain lipids are involved in several key functions, such as maintaining membrane fluidity and permeability, energy storage, and signal transduction [2]. Altered metabolism of brain lipids could be associated with pathogenesis of brain disorders, and thus serve as a diagnostic biomarker [3, 4]. Among the different classes of lipid biomarkers for assessing brain function, free fatty acids separated from complex lipids could be a valuable marker for monitoring the progression of neurodegenerative diseases and brain injuries, including cerebral ischemia [5]. Arachidonic acid (AA) is a key component of brain phospholipids, accounting about 20% of brain fatty acid composition [6]. AA is extremely important for brain development,membrane fluidity, selective permeability, and cell-cell signaling throughout life [7]. It has been estimated that the products of AA metabolism are involved in modulating synaptic plasticity and neurotransmission [8]. Various brain pathologic conditions, including stroke, are associated with an uncontrolled release of free AA from brain phospholipids following its extensive oxidative metabolism [9]. The impaired equilibrium between AA release and incorporation in membrane phospholipids is crucial for neuroinflammation and neuronal dysfunction. Overexpression of the enzymes metabolizing AA to its bioactive products, such as COX-2 and 5-LOX, is found to play a significant role in the pathogenesis of neurodegenerative diseases and in the progression of cerebral ischemia [7]. There are inconsistent findings about the role of individual free fatty acids (FFAs), including AA, in stroke. Some reports indicate an elevation of circulating FFAs in stroke patients, while others did not find such relations [10]. These discrepancies may be due to differences in study design. It has been shown that the concentration of AA as a separate marker and in correlation with other serum lipids, including classical lipid biomarkers and omega-3 polyunsaturated fatty acids (n-3 PUFA), can vary depending on age, gender, and stroke subtype [11; 12]. It has been demonstrated that a low serum n-3 PUFA/AA ratio may predict neurological deterioration in acute ischemic stroke if measured within 24 h of incidence onset [11]. In addition, serum AA levels may be a valuable marker for interpreting the effectiveness of therapies during recovery after ischemic stroke [13; 14]. Despite the potential importance of AA as a biomarker, no commercial chromatography kits have been developed for its determination, and reference ranges have not been established. Therefore, reliable estimation of plasma AA levels could be a valuable prognostic biomarker in stroke patients. The conventional analytical method for the determination of free fatty acids, including AA, in biological samples is by gas chromatography with flame-ionization (FID) or mass-spectrometry (MS) detector [15; 16; 17]. In both cases, a preliminary derivatization step of fatty acids to corresponding methyl esters is required. Derivatization is a time-consuming procedure and carries the risk of errors and variations in the result. Direct methods for AA analysis in biological samples, such as liquid chromatography with mass-selective detection, have a number of advantages, the most important of which is that the derivatization step is avoided. Most studies use UPLC coupled with a triple quadrupole MS detector or more sophisticated platforms such as an Orbitrap mass spectrometer [14; 18] or high-resolution mass spectrometry (HRMS) with a Triple-TOF mass spectrometer [18].