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) .