3.2.3 Mass spectrometry
The high-resolution MS/MS spectrum of commercial ethyl lauroyl arginate, obtained by infusion into a Orbitrap mass spectrometer instrument, exhibit the fragmentation pattern shown in Figure 7 . The product ions, labeled on the spectrum as A, B, and C, are consistent with the calculated masses for the compound fragments shown on the structure of Figure 7 , providing evidence for the identification of the arginate derivative. The A ion resulted from the elimination of ammonia, and the B ion from the elimination of iminourea. The C ion is the ethyl arginate fragment after cleavage at the amide side producing the fragment at m/z 186, with the subsequent fragmentations leading to the lower mass ions at m/z 169, 158, 112, and 70 as shown in the proposed fragmentation pathway inFigure 7 .
Fatty acid derivatives are usually analyzed by gas chromatography-mass spectrometry, however, the polarity and high mass of the ethyl arginate compounds synthesized in this study limited the utilization of this approach. Consequently, the products were analyzed using an LC-MS-Q-Tof method. All the ethyl arginate derived product showed the characteristic MS/MS spectrum with the fragmentation pattern exhibit by the ethyl lauroyl arginate, exhibiting the ions A, B, and C, with the subsequent fragmentation of the ion C. STable 1 (Supplementary Information) shows the structures of the synthesized ethyl arginate derivatives for lauroyl, iso -oleoyl, n -oleoyl,iso -stearoyl, and n -stearoyl products with the corresponding calculated and experimental masses for the A, B, and C ions, confirming the structures for these derivatives. It should be noticed that the iso -oleoyl and iso -stearoyl products derivatives were synthesized from a mixture of different isomers, as was reported before (Ngo, Hoh, Foglia, 2012), and the structures presented in STable 1 (Supplementary Information) are, for simplicity, one of the possible formed. The iso -product cannot be distinguished by mass spectrometry from the linear form because the spectra are almost identical. However, the LC analysis for theiso -products showed that the peak corresponding to these iso-structures are broader than the peak for linear isomer, due to the presence of different isomeric forms of the iso -product. This effect can be seen in the chromatograms for the iso -oleoyl, eluting with a broad peak in comparison with the linearn -stearoyl with a narrow peak SFigure 3 (Supplementary Information) .
Although these analytical results have clearly demonstrated the formation of the desired products with high purities, it can’t be determined if the hydrochloride is protonated at the arginine side chain. Thus, an ion chromatography technique using Dionex ICs 6000 was utilized to determine the chloride content (samples ran by ALFA company). Two samples (ethyl lauroyl arginate hydrochloride and ethyl iso-oleoyl arginate hydrochloride) were evaluated, and the results confirmed that the products did contain chloride (0.58% in lauroyl arginate and 0.41% in iso-oleoyl arginate). Since these are protonated compounds, this explains why the products are soluble in water (about 4.4%) for compounds 1, 3, 4, 5 in Figure 2 and 0.46% for ethyl iso -stearoyl arginate (Fig. 2, structure 2) .