## 3 Results and Discussion
### Method Validation
#### 3.2.7 Evaluation Method Applicability to Real Serum Samples  
The current method was applied to real serum samples of 50 healthy adult volunteers, with a mean age of 52.2 \(\pm\) 6.2. For the purpose of quantitative analysis of the samples, a calibration graph was constructed, and the concentrations of the samples were back-calculated from the standard curve. Sample blank and reagent blank were analyzed with each run. The serum concentrations of arachidonic acid in the tested samples ranged between 0.34 and 4.32 \(\upmu\)g/mL, with a mean value of 2.05 \(\pm\) 0.90 \(\upmu\)g/mL. This range is consistent with expected values for healthy individuals reported by other studies [40; 41], and indicates that the current method can effectively detect and quantify AA in real serum samples. Further, method robustness and reproducibility were assessed by overlaying the chromatograms of 50 serum samples. A clear arachidonic acid peak consistency across the samples was seen with RT variations between 4.75-4.76 min (Figure 6). The variability among samples may be attributed to individual physiological differences, dietary factors, or other biological variables. In addition, a few samples of ischemic stroke patients were analyzed with the presented method. The results were in the linear range of the method, showing AA concentration 1.32 \(\pm\) 0.29 \(\upmu\)g/mL. Currently, there is no established and standardized analytical method for analyzing arachidonic acid in blood serum/plasma. There are also no available commercial calibrators and controls. The methods described in the literature have been developed in-house for the needs of specific studies [33; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50]. Since most of them use GC-MS for quantitative analysis, this requires the derivatization of free AA to more volatile esters [45; 48; 49]. Sample preparation usually involves the extraction of the analyte from the matrix by liquid-liquid or solid-phase extraction and subsequent derivatization. This is a relatively time-consuming procedure, burdened by potential analyte loss, which can compromise the reproducibility and accuracy of the method, making it difficult to apply in routine clinical testing. In recentyears, LC-MS platforms have become increasingly widespread in clinical laboratories due to their potential for automation, high analytical specificity, often superior to immunoassayassays, and higher throughput than GC-MS. Some of the main requirements for these analytical techniques are easy and fast sample preparation, high analytical reliability, and short analysis time. We have developed and validated a simple, robust, and selective LC-MS method for the quantification of free arachidonic acid (AA) in human serum. The method requires only a small sample volume and involves a straightforward liquid-liquid extraction (LLE) procedure, eliminating the need for derivatization steps commonly required in GC-MS and even some LC-MS/MS protocols [32; 33]. A key advantage of this method is the use of a single-quadrupole (QDa) mass detector, which is more cost-effective and accessible than advanced tandem or high-resolution instruments. Despite its simplicity, the method demonstrates excellent analytical performance, with accuracy and precision comparable to those of more complex triple-quadrupole systems reported by [PERSON] et al. [43] (77.7-109.7%), [PERSON] et al. [40] (97.7-113.6%), [PERSON] et al. [48] (97.9-101.7%), and [PERSON] et al. [50] (94.8-106.8%). Furthermore, the validated lower limit of quantification (LLOQ) is well below the physiological and pathological concentrations of AA reported in the literature [42]. The method was successfully applied to serum samples from healthy individuals, producing AA levels consistent with previously published data [40; 41]. Additional analysis of samples from ischemic stroke patients confirmed that their AA concentrations fall within the method's validated linear range.