After acquiring the mass spectra, the PCA loadings indicated that the values of mass/charge (m/z) higher than 500 were not essential to differentiate between samples. So, the spectra were cut to be used from 50 to 510 (m/z), diminishing it to a 5845 variables dataset. A new PCA was applied to this new set, providing the separation projected in Fig. 1. As it was a non-targeted analysis, the PC1 provided a satisfactory pattern distinction between the samples regarding the process. To check which compounds (ions) were responsible for the differentiation, the loadings from PC1, presented in Fig. 2, were evaluated. These loadings were used to putatively identify the ions through manual comparison of their fragmentation spectra with mass spectra libraries and literature. Table 1 presents the most important ions identified in this study as responsible for differentiating samples.
Table 1
– Substances identified by mass spectrometry.
Compound | Drying Process | Aduct | Molecular Mass | tR (min) | [M + H]+ measured | [M + H]+ theoretical | Error (ppm) |
Arginine | Tradicional | [M + H]+ | C6H14N4O2 | 0.74 | 175.1186 | 175.1189 | -1.71 |
Tryptophan | Tradicional | [M + H]+ | C11H12N2O2 | 4.10 | 205.0965 | 205.0972 | -3.41 |
Proline | Tradicional | [M + H]+ | C5H9NO2 | 1.40 | 116.0704 | 116.0706 | -1.72 |
Phenylalanyl-alanine | Tradicional | [M + H]+ | C12H16N2O3 | 5.00 | 237.1236 | 237.1233 | 1.27 |
Glucotropaeolin | Tradicional | [M + H]+ | C14H19NO9S2 | 7.54 | 410.0568 | 410.0574 | -1.46 |
Coumaric acid | Tradicional | [M + H]+ | C9H8O3 | 1.53 | 165.0542 | 165.0546 | -2.42 |
Ferulic acid | Tradicional | [M + H-H20]+ | C10H10O4 | 9.26 | 177.0547 | 177.0546 | 0.56 |
12(13)-Epoxy-9Z-octadecenoic acid | Tradicional | [M + H]+ | C18H32O3 | 15.95 | 297.2416 | 297.2424 | -2.69 |
N-benzyl-(9Z,12Z)-octadecadienamide | Tradicional | [M + H]+ | C25H39NO | 17.10 | 370.3104 | 370.3104 | 0.00 |
Unknown | Oven | [M + H]+ | C18H26O4 | 13.21 | 307.1903 | 307.1903 | 0.00 |
Phenylalanine | Oven | [M + H]+ | C9H11NO2 | 2.96 | 166.0861 | 166.0862 | -0.60 |
5-oxo-6E, 8E-octadecadienoic acid (macaene) or isomer | Oven | [M + H]+ | C18H30O3 | 15.63 | 295.2263 | 295.2267 | -1.35 |
3-benzyl-1,2-dihydro-N-hydroxypyridine-4-carbaldehyde (macaridine) | Oven | [M + H]+ | C13H13O2N | 10.17 | 216.1017 | 216.1019 | -0.93 |
1,3-dibenzyl-2-pentyl-4,5-dimethylimidazilium | Oven | [M + H]+ | C24H30N2 | 10.57 | 347.2478 | 347.2481 | -0.86 |
N-benzyl-13-oxo-9E,11E-octadecadienamide | Oven | [M + H]+ | C25H37NO2 | 15.75 | 384.2891 | 384.2897 | -1.56 |
Lepidiline A | Oven | M+ | C19H21N2+ | 8.17 | 277.1701 | 277.1699 | 0.72 |
Lepidiline C | Oven | M+ | C20H23ON2+ | 8.49 | 307.1807 | 307.1811 | -1.30 |
Lepidiline D | Oven | M+ | C21H25ON2+ | 8.83 | 321.196 | 321.1967 | -2.18 |
3-benzyl-1,2-dihydro-N-hydroxypyridine-4-methoxy | Oven | [M + H]+ | C13H15O2N | 8.99 | 218.1174 | 218.1175 | -0.46 |
FIGURE 1
The loadings indicated that the drying process influences the content of the sample on the amino acids (arginine, tryptophan, proline, and phenylalanyl-alanine), glucosinolates (glucotropaeolin), hydroxycinnamic acids (coumaric and ferulic acids), and in the n-benzyl-(9Z,12Z)-octadecadienamide (which is one of the macamides), besides the 12(13)-Epoxy-9Z-octadecenoic acid, which is a polyunsaturated fatty acid. It can be said that it is somehow expected, once the high temperature used in the industrial ovens can degradate thermosensitive compounds such as these. On the other hand, one of the macamides, the n-benzyl-13-oxo-9E,11E-octadecadienamide, is suggested to resist oven temperatures. Moreover, most of the alkaloids are present in these samples, indicating that there are structures that are maintained after the heating.
FIGURE 2
Previous studies postulated that maca exhibits many biological activities being most of it due to the macamides, glucosinolates, saponins, alkaloids, and glucosinolates (Wang and Zhu 2019). The amino acids are also important nutrients and are susceptible to break depending on the heating temperature. According to (Wang and Zhu 2019), the most abundant amino acid found in yellow maca is arginine. In this study, the PCA indicated that arginine was the most important compound for the traditional dried maca, suggesting that it is influenced by maca processing.
Table 1
The main alkaloids are imidazole, which has shown biological activities related to protection against cytotoxicity and anti-microbial action. Two of the imidazole alkaloids were previously reported as being present in maca (Cui et al. 2003), being lepidiline A (1,3-dibenzyl-4,5-dimethylimidazolium chloride) and lepidiline B (1,3-dibenzyl-2,4,5- trimethylimidazolium chloride). As found by Jin et al (Jin et al. 2016), two of the rare imidazole alkaloids, lepidiline C and D, were also putatively identified. In this case, even though lepidiline B has not been identified, PCA indicates that the alkaloids lepidiline A, C and D are influencing the separation of the maca oven-dried. Nonetheless, according to a previous study (Wang and Zhu 2019), the total content of alkaloids is influenced by the area of cultivation and the maca variety (color). In this way, there was no specification to the commercial maca regarding its type.
Glucosinolates found in maca are anionic sulfur-rich secondary metabolites, and they are said to act in the plant defense system, imposing a pungent flavor on this food. In a maca compositional study, it was found that this compound is present in about 1% of fresh maca (Wang and Zhu 2019), and most of it is composed of glucotropaeolin (benzyl glucosinolate). In this study, the PCA results indicate that it is present in maca dried by traditional methods and is an important compound to differentiate between the drying process. Therefore, it is dependent on post-harvest processing.
The macamide identified as n-benzyl-(9Z,12Z)-octadecadienamide was found in maca traditionally dried. Nonetheless, the macamide n-benzyl-13-oxo-9E,11E-octadecadienamide was found in maca oven-dried. Therefore, it is suggested that oven processing does not eliminate macamides. Also, one substance identified by its m/z 307.1903 with molecular formula C18H26O4 as a polyunsaturated fatty acid (PUFA), besides the 5-oxo-6E,8E-octadecadienoic acid (macaene) or isomer, was found for the maca oven-dried. Nonetheless, the PUFA 12(13)-Epoxy-9Z-octadecenoic acid was found in the traditional dried maca. So, it suggests that oven processing does not consume all the fatty acids.