## 4 Method Validation
### Intra-Day and Inter-Day Precision and Accuracy  
The IP-RP-HPLC/ELSD method was validated using two Leu external control solutions as fully described in Reference [21]. In the following paragraphs, a brief summary of the results is reported.  
The developed chromatographic method was validated in terms of precision and accuracy, in both the short- (intra-day) and the long-term (inter-day) period.  
The intra-day precision was assessed by using the regression Equation 2 reported in Table 4. Two control solutions used as external set were run in triplicate each day, for three consecutive days. Equation 2 was then used to calculate the concentrations of the control solutions. The intra-day precision was determined as the relative standard deviation (RSD%) among the concentration values achieved from consecutive injections. An appreciable precision of the method was obtained in the short-period, with RSD% values ranging from 1.05 to 9.5%, thus producing evidence on the good stability of the employed analytical method. The resulted rather high RSD% value (that is, 9.5%) can be ascribed to the so-called \"detector fatigue\" [21; 33]. Indeed, a progressive reduction in the output stability after a prolonged use is a rather common issue with ELSDs.  
For each control solution, the variation within replicate injections performed during the three-consecutive days of analysis (which means a total of nine injections) was used to calculate  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Amino Acid** & **Regression Equation** & **R\({}^{2}\)** & **Linearity Range (\(\mu\)g/mL)** & **Eq.** \\ \hline Ile & y = 2.0528x + 0.8474 & 0.987 & 62.5–500 & 1 \\ Leu & y = 1.5153x + 2.3829 & 0.995 & 15.6–250 & 2 \\ Val & y = 1.2477x + 2.7443 & 0.993 & 50–1000 & 3 \\ Tyr & y = 1.4012x + 2.2447 & 0.993 & 62.5–500 & 4 \\ \hline \hline \end{tabular}
\end{table}
Table 4: Calibration data for Ile, Leu, Val and Tyr, with the developed IP-RP-HPLC/ELSD method.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Amino Acid** & \multicolumn{3}{c}{**Selected Chromatographic Parameters**} \\ \cline{2-4}  & **k** & **\(\alpha\)** & **R\({}_{\text{S}}\)** \\ \hline Val & 7.78 & & \\ Tyr & 9.74 & 1.25 & 25.25 \\ Ile & 10.02 & 1.03 & 4.37 \\ Leu & 10.35 & 1.03 & 4.48 \\ \hline \hline \end{tabular}
\end{table}
Table 3: Selected chromatographic parameters (retention factor k, separation factor \(\alpha\) and resolution factor R\({}_{\text{S}}\) values) obtained in the analysis of the investigated amino acids according to their elution order.  
the inter-day precision. In this case, more than acceptable RSD% values, equal to 6.72 and 7.13%, were respectively found in the long-term (inter-day) of analysis for the higher and lower concentrated control solutions.  
The Recovery% [34] was employed as test to estimate the accuracy of the applied IP-RP HPLC-ELSD method. Intra-day and inter-day accuracy were determined with the same external solutions previously used to apprise the precision. The short-term accuracy was determined by considering the three replicated runs for each control solution within a single day. In order to evaluate the long-term accuracy, the average value from nine determinations, along three consecutive days of analysis, was considered. Acceptable percentages of recovery were obtained, ranging from 89.38 up to 107.28% in the case of the intra-day analyses. Recovery% values, equal to 93.61 and 102.46%, were obtained for the lower and higher concentrated control solutions, respectively.