## 3 Results and Discussion
### Method Validation
#### 3.2.1 Accuracy and Precision  
One important challenge in the evaluation of accuracy and precision is that analyte is an endogenous molecule. According to the ICH guideline (2022), there are several approaches for calibrators and QCs preparation when a biological matrix free of endogenous analyte is not available [19]. One of them is the usage of a surrogate matrix to mimic the authentic one. For the preparation of the calibrators, we used bovine serum albumin in physiologic concentration as a surrogate matrix. We evaluated accuracy and precision by spiking the surrogate matrix with AA at three different concentrations, five replicates each. The accuracy, presented as percent recovery, ranged from 91.95% to 99.19%; % RSD from 5.94 to 14.44 for AA (Table 1).  
Precision was assessed by measuring both intra-day and inter-day variability at three spike levels using the same surrogate matrix-matched samples as for accuracy experiments. The relative standard deviation (%RSD) was below 15% for all tested spike levels, which supported the method's consistency and robustness in handling biological samples (Table 2).  
The current method fulfills the ICH criteria for accuracy and precision [19], which allows us to consider that it is reliable and reproducible. In addition, our results were consistent with the data of others reporting 95% and 105% recovery for AA at spike concentrations 0.1 \(\mu\)g/mL and 0.5 \(\mu\)g/mL, respectively, as well as 4.21% precision at spike concentration 0.5 \(\mu\)g/mL [39].  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Analyte**} & **Target** & **Mean Measured** & & **Mean Accuracy \(\pm\) SD** & **RSD** \\  & **Concentration** & **Concentration \(\pm\) SD** & **n** & **(\% Recovery)** & **(\%)** \\ \hline \multirow{3}{*}{AA spike} & 0.50 & 0.46 \(\pm\) 0.04 & 13 & 91.95 \(\pm\) 7.51 & 8.51 \\  & 1.00 & 0.91 \(\pm\) 0.13 & 13 & 92.75 \(\pm\) 12.11 & 14.44 \\ \cline{1-1}  & 5.00 & 4.97 \(\pm\) 0.3 & 15 & 99.19 \(\pm\) 6.07 & 5.94 \\ \hline \hline \end{tabular}  
* n = number of assays; accuracy was given as % recovery.  
\end{table}
Table 1: Accuracy data for AA in spiked serum samples.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multicolumn{3}{c}{**Intra-Day Precision**} & \multicolumn{3}{c}{**Inter-Day Precision**} \\ \hline \multirow{2}{*}{\begin{tabular}{c} **Target AA** \\ **Concentration** \\ **(\(\mu\)g/mL)** \\ \end{tabular} } & **Calculated AA** & & **Calculated AA** & & \\  & **Concentration** & **Precision** & **Concentration** & **Concentration** & **(\(\mu\)g/mL)** & **Precision** \\  & **Mean \(\pm\) SD** & & **(\(\mu\)g/mL)** & **Mean \(\pm\) SD** & **(\% RSD)** \\ \hline \multirow{2}{*}{\begin{tabular}{c} 0.5 AA (n = 5) \\ spike level \\ 1 \(\mu\)g/mL (n = 4) \\ \end{tabular} } & 0.51 \(\pm\) 0.07 & 13.29 & \begin{tabular}{c} endogenous AA \\ (n = 13) \\ \end{tabular} & 0.46 \(\pm\) 0.04 & 8.51 \\  & 1.03 \(\pm\) 0.09 & 9.17 & \
\begin{tabular}{c} spike level \\ 1 \(\mu\)g/mL (n = 13) \\ \end{tabular} & 0.88 \(\pm\) 0.12 & 13.19 \\ \cline{1-1}  & 6.37 \(\pm\) 0.52 & 8.20 &
\begin{tabular}{c} spike level \\ 5 \(\mu\)g/mL; \\ (n = 15) \\ \end{tabular} & 4.97 \(\pm\) 0.29 & 5.94 \\ \hline \hline \end{tabular}  
* \end{table}
Table 2: Precision data for AA in spiked samples.