# UHPLC-MS Method for the Analysis of the Molecular Adjuvant Sulfavant [PERSON]
## 3 Results and Discussion
### Linearity, Accuracy, Precision and Matrix Effect  
The liquid chromatography-mass spectrometry (LC-MS) method was validated for linearity and by an evaluation of intra-and inter-day precision and accuracy in accordance with the currently-approved EMEA and FDA guidelines for the validation of bioanalytical methods [22; 23]. Moreover, with the aim to demonstrate the consistency and robustness for the analysis of biological samples, we also tested the above-mentioned parameters in five different replicates of lipid extracts derived from mice lung tissues. Samples of SULF A and d\({}_{70}\)-SULF A were tested in triplicates between 1 and 2000 ngmL\({}^{-1}\). The resulting calibration curve was found to be linear in the concentration range between 2.5 and 2000 ngmL\({}^{-1}\), as shown in Figure 4. Analogously, SULF A and d\({}_{70}\)-SULF A were spiked at the maximum concentration into MeOH and in a lipid extract of mice lung in MeOH in order to evaluate matrix effect (one of the five extracts available). Samples of the two calibration curves (in pure MeOH and in matrix) were carried out by dilution from the more concentrated solution. Table 2 outlines the slope values and R\({}^{2}\) parameters of the calibration curves acquired at different days (T0 and T48). The R\({}^{2}\) was found to be 0.999 and stable during the days (slight differences in the slope could be appointed to the variability linked to the instrument response). The difference in the mean ordinate between SULF A and d\({}_{70}\)-SULF A is in agreement with the isotope effect that can cause a different degree of ion suppression. However, although the ionization and chromatographic response was not identical, the peak area ratio of the analyte versus the IS was constant over the range of tested concentrations, with a correlation coefficient (r\({}^{2}\)) that is always higher than 0.9991. The matrix effect (ME) is one of the most important parameters to investigate during the development of an analytical method. In order to evaluate the ME, five mice lung lipid extracts were aliquoted and spiked with three different quantities (30, 300, and 1500 ng) of SULF A (Figure 5), while the same known amount of d\({}_{70}\)-SULF A (50 ng per sample) as IS was added to compare the area of the analyte in matrix with the area measured in pure MeOH (normalized matrix effect-nME). A higher CV was observed for the high concentrations in comparisons with the low ones, as can be seen in Table 3. However, all of the datasets (intra- and inter-day) passed the 15% acceptance criterion, with an overall nME average of 10%. This finding was the first evidence of the robustness of the proposed analytical procedure. \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{2}{c}{**T0**} & \multicolumn{2}{c}{**T48**} \\ \hline  & **MeOH** & **Matrix** & **MeOH** & **Matrix** \\ \hline \multirow{2}{*}{**SULF A**} & y = 40229x & y = 47981x & y = 50537x & y = 46508x \\  & R\({}^{2}\) = 0.9996 & R\({}^{2}\) = 0.9997 & R\({}^{2}\) = 0.9991 & R\({}^{2}\) = 0.9993 \\ \hline \multirow{2}{*}{**d\({}_{70}\)-SULF A**} & y = 23689x & y = 24546x & y = 28250x & y = 24185x \\  & R\({}^{2}\) = 0.9998 & R\({}^{2}\) = 0.9991 & R\({}^{2}\) = 1 & R\({}^{2}\) = 0.9996 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Calibration curve of d\({}_{70}\)-Sulfavant A and Sulfavant A at T0 and T48, both in MeOH and in matrix. Figure 4: Calibration curve of d\({}_{70}\)-Sulfavant A (**a**) and Sulfavant A (**b**). This analytical method allowed for the detection of SULF A at nanomolar concentration (LOD was below 2.5 ngmL\({}^{-1}\)) with a LOQ of 6.5 ngmL\({}^{-1}\), in both only MeOH and in the presence of matrix. Indeed, at the LOD the analyte in the samples can be identified by only HR-MS, but it cannot be quantitatively determined with appropriate precision and accuracy, whereas, at starting from the calibration point at 5 ngmL\({}^{-1}\), the peak of SULF  
Figure 5: Chromatograms and mass spectrum acquired at 3 min.