# UHPLC-MS Method for the Analysis of the Molecular Adjuvant Sulfavant [PERSON]
## 3 Results and Discussion
### Linearity, Accuracy, Precision and Matrix Effect
of pure matrix extract (**A**), matrix spiked with 300 ng (**B**) and 1500 ng (**C**) of Sulfavant A. Chromatograms were obtained as \(m/z\) mass range between 400 and 1200. \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{4}{c}{**SULF A in MeOH**} \\ \hline  & **Nominal Concentration (ngmL\({}^{-1}\))** & **Mean \(\pm\) SD a** & **Accuracy (\%) b** & **Precision (CV \%) c** \\ \hline  & 30 & 27.8 \(\pm\) 2.2 & 92.6 & 8.0 \\ \cline{2-5} T0 & 300 & 315.0 \(\pm\) 19.4 & 105 & 6.1 \\ \cline{2-5}  & 1500 & 1247.8 \(\pm\) 69.2 & 84.2 & 5.5 \\ \hline  & 30 & 27.7 \(\pm\) 1.14 & 92.4 & 4.1 \\ \cline{2-5} **(Intra-day)** & 300 & 313.9 \(\pm\) 17.6 & 104.6 & 5.6 \\ \cline{2-5}  & 1500 & 1265.2 \(\pm\) 27.6 & 84.3 & 2.2 \\ \hline  & 30 & 30.7 \(\pm\) 1.5 & 102.2 & 4.9 \\ \cline{2-5} **(Inter-day)** & 300 & 327.7 \(\pm\) 16.3 & 109.2 & 5.0 \\ \cline{2-5}  & 1500 & 1353.6 \(\pm\) 15.0 & 90.2 & 1.1 \\ \hline  & \multicolumn{4}{c}{**SULF A in Matrix**} \\ \hline  & **Nominal Concentration (ngmL\({}^{-1}\))** & **Mean \(\pm\) SD a** & **Accuracy (\%) b** & **Precision (CV \%) c** \\ \hline  & 30 & 26.0 \(\pm\) 2.2 & 86.6 & 8.6 \\ \cline{2-5} T0 & 300 & 283.1 \(\pm\) 6.5 & 94.4 & 2.3 \\ \cline{2-5}  & 1500 & 1459.9 \(\pm\) 79.7 & 97.3 & 5.4 \\ \hline  & 30 & 28.6 \(\pm\) 2.0 & 95.2 & 7.1 \\ \cline{2-5}
**(Intra-day)** & 300 & 274.4 \(\pm\) 16.6 & 91.5 & 6.1 \\ \cline{2-5}  & 1500 & 1457.3 \(\pm\) 84.1 & 97.1 & 5.8 \\ \hline  & 30 & 27.0 \(\pm\) 1.2 & 90.0 & 4.3 \\ \cline{2-5}
**(Inter-day)** & 300 & 279.8 \(\pm\) 22.0 & 93.3 & 7.9 \\ \cline{2-5}  & 1500 & 1432.5 \(\pm\) 46.2 & 95.5 & 10.2 \\ \hline \hline \end{tabular}  
* SD = Standard Deviation b [Measured mean concentration/ nominal concentration] \(\times\) 100. c [Standard deviation/ measured mean concentration] \(\times\) 100.  
\end{table} 
Table 3: Accuracy and precision of quality control (QC) samples of Sulfavant A during intra-day and inter-day analysis. A is also identifiable with MSMS and the signal appears to be defined and reproducible. Moreover, no carryover effect was detected during analysis of a blank sample after the injection of a standard sample at the highest calibration point (2000 ngmL\({}^{-1}\)). The reliability of this UHPLC-MS method was evaluated by measuring intra-day and inter-day accuracy and precision (n = 3 in MeOH; n = 5 in matrix) of SULF A in quality control (QC) samples at three different concentrations (30, 300, and 1500 ngmL\({}^{-1}\)). Table 3 reports the results in intra-day and inter-day accuracy and precision. The accuracy of QC samples ranged from 84.2 to 109.2 %, and precision ranged from 1.1 to 10.2% (Table 3). The overall assay accuracy (mean of accuracy estimates of all quality control samples) was 96% in MeOH and 93.4% in matrix, and overall assay precision (mean of precision estimates of all quality control samples) was 4.7% in MeOH and 6.4% in matrix. Revalidation using two different batches of synthetic SULF A (namely, BS90A and BS90A1) was carried out at the same concentrations in the range between 2.5 and 2000 ngmL\({}^{-1}\) to further support the reliability of the method. The results showed an absolute reproducibility of the analysis with two superimposable calibration curves (BS90A: y = 31579x, R\({}^{2}\) = 0.9999; BS90A1: y = 31331x, R\({}^{2}\) = 0.9999).