# Testing Clean-Up Methods for the Quantification of Monosaccharides and Uronic Acids
## 3 Results  
In order to find the most selective and sensitive stationary phase for the seven monosaccharide and two uronic acids in terms of recovery, the results collected using the two commercial ionic exchange resins were compared and discussed, drawing consideration on the advantages of the application of SPE to the profiling of saccharides in complex matrices containing polysaccharides. The results are expressed as percentage recoveries, calculated as a percentage ratio between the concentration determined by GC-MS in standard solution subjected to a clean-up procedure with the ion exchange resins or the SPE, and the concentration in the standard solution without a clean-up step. The concentration of the analytes eluted from the ion exchange resins or the SPE cartridge was obtained, integrating the areas in the SIM chromatogram for each analyte and using calibration curves. Each experiment was conducted in triplicate and average values are provided. Figure 1 shows the GC-MS chromatogram in SIM mode collected for a standard sugar solution of seven monosaccharides and two uronic acids. Different conditions were tested, and the working plan was adapted step by step on the basis of the recoveries determined. The results of the tests carried out with the two types of Amberlite ionic exchange resins are summarized in Table 2 and carefully explained following. Elution with 1 mL and 3 mL of water from Amberlite MB-6113 resin provided low recoveries and poor reproducibility for each analyte. The first experiment with Amberlite IRN-150 involved the elution of the analytes with 3 mL of bi-distilled water to compare the recoveries to those obtained by Amberlite MB-6113. The recoveries collected by IRN-150 are higher than those achieved by MB-6113, but not yet satisfactory (Table 2). The elution in strongly acidic conditions, using a TFA 2 M solution (pH < 0), shows a significant improvement in monosaccharides recoveries. To maximize the recoveries, further tests were performed by eluting the analytes at pH 4 and 9, since Amberlite IRN-150 resin provides optimal performance in this pH range [31]. In particular, the elution with 3 mL of 10\({}^{-4}\) M TFA solution (pH = 4) and with 3 mL NH\({}_{3}\) 6\(\cdot\)10\({}^{-6}\) M (pH = 9) was tested. In both cases, there was no improvement in monosaccharide recoveries compared to those obtained by the elution with a TFA 2 M solution. Given the results obtained with two Amberlite resins, the purification with ion exchange resin with solid-phase extraction was replaced with an aminopropyl-modified silica phase. The potentiality of this type of column to also remove phenolics compounds that could affect derivatization can be a useful advantage when we deal with specific matrices [5]. In the literature, the elution of monosaccharides from this type of column is carried  
Figure 1: GC-MS chromatogram in SIM mode of a standard sugar solution of 7 monosaccharides and 2 uronic acids (each at 10 ppm), where IS internal standard—Mannitol (10 ppm). out with 1 mL of 40% ACN solution [5]; thus, these conditions were initially adopted. The recoveries in the first, second, and third milliliter of the elution with a 40% ACN solution, collecting and analyzing the eluates separately, are summarized in Table 3 (Experiment A). The results, based on three replicates, are reported as average recovery, and confidence intervals are defined by the product between the t student value (95% confidence) and the standard deviation divided by the root of the number of measurements. The histograms relative to monosaccharides recoveries in each fraction, depicted in Figure 2, show that 1 mL of acetonitrile solution recovers almost the total amount of saccharides, leading to a nearly better performance than those of both ion exchange resins. Nevertheless, uronic acids are poorly recovered by the aminopropyl-modified phase and, in particular, for galacturonic acid, the recovery tends to zero.