# Testing Clean-Up Methods for the Quantification of Monosaccharides and Uronic Acids
## 4 Discussion  
In this paper, two distinct methods for suppressing inorganic ion interference in the quantification of monosaccharides and uronic acids by GC-MS were investigated, employing two ion exchange resins and a solid-phase extraction. The use of the two different ion exchange resins, Amberlite MB-6113 and IRN-150, did not give satisfactory results, even though the Amberlite IRN-150 resin performed better under acid elution conditions using 3 mL of TFA 2 M. The very low recoveries and the lack of reproducibility can be also due to the non-efficient packing of the Pasteur column (Eddy-diffusion parameter (A) of the Van Deemeter equation) and to the variable amount of resin introduced. \begin{table}
\begin{tabular}{c c} \hline
**Analyte** & **Concentration (ppm)** \\ \hline Xylose & 240 \\ Arabinose & 895 \\ Rhamnose & 0 \\ Fucose & 0 \\ Galacturonic acid & 957 \\ Glucuronic acid & 1145 \\ Glucose & 124 \\ Mannose & 427 \\ Galactose & 222 \\ \hline \end{tabular}
\end{table} 
Table 4: Concentrations (ppm) of monosaccharides and uronic acids determined in a sample collected from the preparation layer of an ancient mural painting (3.7 mg). Figure 6: GC-MS chromatogram in SIM mode of the preparation layer of the archaeological sample subjected to the SPE column clean up under optimum elution conditions (experiment C). The peaks in the chromatogram with no assignment were not identified. I.S. : internal standard. The aim of the paper was addressed by the determination of the best clean-up method. Thus, the best results were obtained with an SPE using a Macherey-Nagel(tm) Chromabond(tm) NH2 Polypropylene SPE column eluting with 1 mL of trifluoroacetic acid 400 mM in a 40% acetonitrile solution, allowing the simultaneous elution of both monosaccharides and uronic acids. Indeed, the acidic elution enables good recoveries of uronic acids, which, in acidic pH, are present in their undissociated form. These uronic acids are thus efficiently eluted (recoveries higher than 60%) from the stationary phase of the SPE column. Under these conditions, recoveries of more than 70% were obtained for xylose, arabinose, glucuronic acid, and glucose. For the other analytes, recoveries are typically greater than 50%. Finally, the preparation layer of a wall paint sample with a high content of inorganic fraction, assessed by preliminary ATR-FT-IR analysis, was successfully purified and analyzed by GC-MS allowing confirmation of the effectiveness of the method in the removal of inorganic ions and to ascribe the saccharide profile of the hydrolyzed sample to that of a fruit tree gum. In conclusion, this paper provides a first insight and comparison of clean-up procedures for quantifying monosaccharides and uronic acids in mural paintings. These results fill a gap in the literature related to the quantitative assessment of the removal of inorganic interferences from complex matrices containing saccharides such as paintings, where the presence of inorganic pigments is well known. Nevertheless, future studies need to be carried out to test the efficiency of the optimized method on other matrices not only concerning artistic fields but also foodstuff and writing nks. Moreover, our next steps will be focused on developing and optimizing protocols for the removal of other types of interferences, along with testing SPE columns with different stationary phases and exchanger properties, assessing the most detrimental for the profiling of polysaccharides. Conceptualization, J.J.L., F.S., F.M. and I.B. ; methodology, I.B., F.S. and J.J.L. ; investigation: I.B. ; writing--original draft preparation: I.B. ; writing--review and editing, F.S., J.J.L. and F.M. ; supervision, J.J.L. All authors have read and agreed to the published version of the manuscript. This research received no external funding. Not applicable. Data will be made available on request. [PERSON] (Department of Chemistry and Industrial Chemistry, University of Pisa) is acknowledged for her technical assistance with the ATR-FT-IR measurement.