## 2 Results and Discussion  
Sample manipulation can significantly affect the reliability of a bioassay guided fractionation. The process, which has to cover the range of polarity of the metabolites in the sample, allows recovery of minor components in enriched fractions, the activity of which may be masked in the raw extract. In marine samples, this is the most critical issue, since the overwhelming presence of salts can significantly lead to overestimation of the extract weight and misinterpretation of the hypothetical level of the bioactive components. To address this aim, extracts of marine samples have been traditionally partitioned with organic solvents of increasing polarity in order to enrich the amount of secondary metabolites recovered and reduce the masking effect of salt. Independently of the efficiency, this procedure can be highly time consuming, solvent demanding, poorly reproducible and expensive. Since the pioneering applications in early 80s, materials based on poly(styrene-divinylbenzene) (PS-DVB) have been largely used as chromatographic support for fractionation and purification of organic components from complex matrices [7; 8; 9]. In particular, PS-DVB resin is an attractive adsorbent for extraction of various types of organic compounds due to pH stability and the hydrophobic surfaces that highly retain non-polar molecules. For these properties, different commercial types of PS-DVB absorbent (e.g., Amberlite(r) XAD(r)-2, Diaion(r) HP-20, Sigma-Aldrich, Milan, Italy) have been long used by independent authors to desalt and fractionate marine extracts as non-exhaustive examples, see [10, 11, 12, 13, 14]. More recently, the potential of PS-DVB (Diaion(r) HP-20 SS, Sigma-Aldrich, Milan, Italy) as an alternative to prepare purified natural product libraries has been carefully evaluated by [PERSON] and co-workers [15]. By using selected plant extracts as test panel, these authors concluded that cross-linked polystyrene matrix retained considerable bioactivities and reduced loss of bioactive natural products in comparison with normal-phase sorbents. Starting from this consideration, we explored the use of a spherical PS-DVB resin for SPE (Chromabond(r) HR-X, Duren, Germany) together with an alternative stepwise elution in order to desalt the samples and to obtain a reproducible resolution of the main chemical classes of common marine extracts. In comparison with other PS-DVB resins (e.g., Diaion(r) HP-20, Sigma-Aldrich, Milan, Italy) that have been used with the same aim [11, 13], Chromabond(r) HR-X offers the advantage to have narrow pores (260 A) and regular, little particles (around 65 \(\upmu\)m) that increase the surface area. After a careful analysis of the material that was available in our laboratory, three organisms containing chemical known products that both represent common marine metabolites and cover a wide range of polarity were selected to test the procedure (Table 1). \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Phylum** & **Sample Name** & **Main Secondary Component** & **Ref. ** \\ \hline Porifera & _Dendrillamem_ & 9,11-dihydroracilin A & [16] \\ Porifera & _Renierasarai_ & & [17, 18] \\ Porifera & _Renierasarai_ & & [17, 18] \\  & & & \\  &  
The methanol extracts of these organisms were dried at reduced pressure and re-suspended in a minimal volume (1 mL) of distilled water. The aqueous suspensions were then loaded onto the pre-packed CHROMABOND(r) HR-X cartridges (6 mL/500 mg) and submitted to a five-step elution protocol. After a preliminary desalting step with 2 mL of distilled water, fractionation of the organic components was achieved by elution with 100% H\({}_{2}\)O (fraction A, 18 mL) followed by solvent mixtures of increasing chromatographic strength from CH\({}_{3}\)OH/H\({}_{2}\)O (fraction B, 50:50, 24 mL) to CH\({}_{3}\)CN/H\({}_{2}\)O (fraction C, 70:30, 18 mL), 100% CH\({}_{3}\)CN (fraction D, 18 mL) and, finally, CH\({}_{2}\)Cl\({}_{2}\)/CH\({}_{3}\)OH (fraction E, 90:10, 18 mL).