# Testing Clean-Up Methods for the Quantification of Monosaccharides and Uronic Acids
## 3 Results
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & & \multicolumn{4}{c}{**Recoveries \%**} \\ \hline
**Experiment** & & **A** & & **B** & **C** \\ \hline
**Elution Condition** & **1\({}^{\circ}\) mL ACN 40\%** & **2\({}^{\circ}\) mL ACN 40\%** & **3\({}^{\circ}\) mL ACN 40\%** & **1 mL TFA 400 mM** & **1 mL ACN 40\%** \\ \hline
**Xylose** & 68.5 \(\pm\) 1.3 & 3.3 \(\pm\) 0.3 & 3.6 \(\pm\) 0.2 & 1.5 \(\pm\) 0.4 & 80.1 \(\pm\) 0.6 \\
**Arabinose** & 40.4 \(\pm\) 3.8 & 5.0 \(\pm\) 0.1 & 5.1 \(\pm\) 1.4 & 2.0 \(\pm\) 0.9 & 75.9 \(\pm\) 1.8 \\
**Rhamnose** & 55.6 \(\pm\) 1.4 & 1.6 \(\pm\) 1.7 & 4.4 \(\pm\) 0.6 & 0.8 \(\pm\) 0.2 & 55.0 \(\pm\) 3.5 \\
**Fucose** & 53.1 \(\pm\) 0.5 & 1.6 \(\pm\) 1.2 & 2.9 \(\pm\) 2.1 & 0.7 \(\pm\) 0.2 & 66.4 \(\pm\) 1.7 \\
**Galacturonic acid** & 0.4 \(\pm\) 0.1 & 0.4 \(\pm\) 0.1 & 1.4 \(\pm\) 0.2 & 41.6 \(\pm\) 2.7 & 62.6 \(\pm\) 4.2 \\
**Glucuronic acid** & 13.9 \(\pm\) 3.5 & 1.8 \(\pm\) 0.2 & 3.6 \(\pm\) 0.8 & 77.3 \(\pm\) 1.2 & 90.3 \(\pm\) 2.3 \\
**Glucose** & 72.9 \(\pm\) 1.9 & 1.5 \(\pm\) 0.2 & 10.0 \(\pm\) 0.6 & 1.2 \(\pm\) 0.03 & 74.8 \(\pm\)1.8 \\
**Mannose** & 53.4 \(\pm\) 1.9 & 5.0 \(\pm\) 0.7 & 6.0 \(\pm\) 1.7 & 1.9 \(\pm\) 0.1 & 57.8 \(\pm\) 6.1 \\
**Galactose** & 49.6 \(\pm\) 2.4 & 4.0 \(\pm\) 0.3 & 4.9 \(\pm\) 0.1 & 2.7 \(\pm\) 0.1 & 52.1 \(\pm\) 0.7 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Results obtained for a clean-up test using a Macherey-Nagel(tm) Chromabond(tm) NH2 Polypropylene SPE column applied to a standard solution of 7 monosaccharides and 2 uronic acids (each at 10 ppm). For the percentage recoveries, the relative confidence interval is reported. The measurements were conducted in triplicate. Experiment A and B refers to the optimization of the volume and the pH of elution respectively while experiment C represent the best results. Figure 2: Recoveries, expressed as a percentage with respect to the concentration of starting solution, reported for each fraction of eluate analyzed, relative to the clean-up test results (Experiment A) using an SPE column applied to a standard solution of 7 monosaccharides and 2 uronic acids (each at 10 ppm). The experiment was conducted in triplicate and the error bars represent the confidence interval. The poor recovery of uronic acids is due to the need of acidic conditions to shift the equilibrium towards their undissociated form to achieve their elution from the stationary phase. To improve their recoveries, after the elution with 1 mL of 40% ACN, a further elution with 1 mL of a slightly acidic aqueous solution of TFA 400 mM (pH 0.4) was tested. The recoveries (Experiment B, Table 3) and the histograms (Figure 3) highlight that the elution with TFA solution drastically increases the uronic acid recoveries (100 times more for galacturonic acid, 6 times for glucuronic acid). Finally, an elution with 1 mL of trifluoroacetic acid 400 mM in a 40% acetonitrile solution was tested in order to recover all the analytes in a single step. Eluting all analytes in a single step is considerably advantageous allowing us to shorten the analytical procedure (dry the aqueous solution under the nitrogen stream) and to collect the analytes in a lower volume, more suitable for analytical purposes. The results (Figure 4) show that all analytes were recovered with a single milliliter of elution. These latter conditions outperform all previous solid-phase extraction experiments conducted. Recoveries of more than 70% were obtained for xylose, arabinose, glucuronic acid, and glucose, while recoveries for the remaining analytes are typically greater than 50%. The recoveries achieved highlight the higher efficiency of the SPE methods for saccharide and uronic acid extraction with respect to results obtained for both the tested Amberlite resins. The solid-phase extraction using a Macherey-Nagel(tm) Chromabond(tm) NH2 Polypropylene SPE column under the optimized conditions was applied to the GC-MS analysis of a validation sample, rich in saccharides and inorganics and collected from the preparation layer of a mural painting.