SupplementaryInformation

Identification number (id), m/z (in negative or positive ionization mode), retention time (rt, in minutes), tentative identification and fragmentation (ms/ms of the molecular and ms3 of the fragment highlighted in red).*= molecule identified through matching of retention time, m/z and fragmentation tree with those of authentic standards; **= molecule tentatively identified through fragmentation tree analysis: sugars were determined by neutral loss, while the aglycones were determined by comparison with m/z and fragmentation of authentic standards; ***= molecule tentatively identified through comparison of m/z and fragmentation trees with those reported in public databases or in the literature. The id are the same of figure 3 and table 3.

id / m/z (-) / m/z (+) / rt / tentative identification / details / ms/ms / I % / ms3 / I%
1 / 190,6 / 3,2 / citric acid / * / 110,6 / 100 / 108,6 / 100
172,5 / 30 / 107,8 / 14
2 / 131,9 / 3,5 / leucine/isoleucine / * / 86,1 / 100
3 / 165,9 / 4,7 / phenylalanine / * / 119,9 / 100
4 / 204,9 / 10,8 / tryptophan / * / 187,8 / 100
5 / 352,7 / 12,9 / chlorogenic acid / * / 190,5 / 100 / 126,6 / 100
191,6 / 6 / 170,6 / 55
178,5 / 5 / 172,5 / 47
84,7 / 44
190,5 / 44
108,6 / 31
6 / 324,7 / 12,9 / coumaric acid-O-hexose / ** / 162,6 / 100 / 118,6 / 100
118,6 / 20 / 119,6 / 6
7 / 386,8 / 411,0 / 14,9 / dihydrosinapic acid-O-hexose / **, *** / 206,6 / 100 / 162,6 / 100
162,6 / 73 / 163,6 / 8
163,7 / 11 / 188,5 / 3
368,7 / 8
224,6 / 7
8 / 336,7 / 15,5 / coumaroylquinic acid-1 / ** / 190,5 / 100 / 172,6 / 100
162,6 / 7 / 126,6 / 69
191,6 / 5 / 110,6 / 36
118,5 / 0 / 108,6 / 27
9 / 550,7 / 15,6 / ferulic acid derivative / ** / 192,6 / 100 / 192,6 / 100
388,6 / 88 / 148,6 / 47
194,5 / 22 / 164,6 / 16
177,6 / 16 / 177,5 / 9
133,6 / 8
10 / 336,7 / 17,4 / coumaroylquinic acid-2 / ** / 190,5 / 100 / 126,6 / 100
162,5 / 6 / 172,5 / 69
191,6 / 3 / 190,5 / 43
118,6 / 1 / 110,6 / 36
11 / 446,7 / 448,9 / 17,8 / orientin (luteolin-6-C-glucoside) / * / 326,6 / 100 / 326,6 / 100
356,6 / 38 / 265,6 / 10
327,5 / 13 / 298,7 / 10
357,6 / 12
12 / 430,7 / 432,9 / 19,1 / vitexin (apigenin-8-C-glucoside) / * / 310,7 / 100 / 310,7 / 100
311,6 / 17 / 282,6 / 18
340,7 / 11 / 283,7 / 4
282,7 / 6
13 / 608,7 / 19,3 / quercetin-O-hexosedesoxyhexose / ** / 300,6 / 100 / 300,6 / 100
299,8 / 22 / 299,6 / 49
178,5 / 10 / 150,5 / 41
301,6 / 10 / 178,6 / 29
14 / 488,6 / - / 19,7 / acetylorientin / **,*** / 326,6 / 100 / 326,6 / 100
327,7 / 15 / 298,7 / 7
356,8 / 14 / 271,7 / 3
428,6 / 8
369,6 / 7
446,4 / 1
15 / 532,6 / 534,9 / 19,8 / acetyl orientin derivative / ** / 488,7 / 100 / 326,6 / 100
489,3 / 14 / 356,6 / 19
356,9 / 3 / 327,6 / 9
326,6 / 2
16 / 446,7 / 20,0 / luteolin-7-O-glucoside / * / 284,6 / 100 / 284,8 / 100
285,6 / 20
356,5 / 4
326,6 / 2
174,4 / 1
17 / 460,6 / 462,8 / 20,4 / luteolin-O-glucuronide / **,*** / 284,6 / 100 / 284,6 / 100
285,4 / 14 / 285,5 / 6
380,5 / 3 / 175,7 / 2
326,6 / 3
18 / 592,7 / 20,9 / kaempferol-O-hexosedesoxyhexose / ** / 284,6 / 100 / 284,6 / 100
285,4 / 8 / 256,5 / 67
283,6 / 8
256,7 / 5
430,8 / 1
19 / 472,7 / - / 21,3 / acetylvitexin / ** / 310,7 / 100 / 310,6 / 100
311,6 / 25 / 282,6 / 29
412,7 / 22 / 283,8 / 7
282,7 / 12 / 311,3 / 7
340,7 / 9 / 147,8 / 3
20 / 516,7 / 518,9 / 21,3 / acetylvitexin derivative / ** / 472,7 / 100 / 310,6 / 100
473,5 / 24 / 412,6 / 22
282,8 / 12
311,6 / 10
340,6 / 9
413,7 / 7
190,6 / 3
21 / 574,6 / 21,4 / vitexin derivative / ** / 430,6 / 100 / 310,6 / 100
310,6 / 89 / 311,6 / 56
340,6 / 81
311,5 / 38
22 / 400,7 / 21,5 / apigenin-C-pentose / ** / 310,6 / 100 / 310,6 / 100
311,5 / 21 / 283,6 / 14
340,7 / 15 / 282,6 / 10
282,7 / 3 / 311,7 / 4
23 / 418,7 / 21,6 / ellagic acid-O-hexose / ** / 256,6 / 100 / 256,6 / 100
257,6 / 20 / 254,7 / 33
212,6 / 6 / 106,7 / 23
24 / 430,7 / 22,0 / apigenin-7-O-glucoside / * / 268,6 / 100 / 268,6 / 100
269,5 / 20 / 267,7 / 12
267,8 / 11 / 224,5 / 3
310,6 / 3
25 / 446,7 / 22,2 / luteolin O-hexose / ** / 284,6 / 100 / 284,6 / 100
326,5 / 29 / 224,7 / 12
268,6 / 29
174,5 / 25
26 / 444,7 / 22,5 / apigenin-O-glucuronide / ** / 268,5 / 100 / 268,6 / 100
174,6 / 13 / 269,5 / 7
27 / 358,7 / 23,0 / rosmarinic acid / * / 160,6 / 100 / 160,4 / 100
178,5 / 25
196,5 / 19
222,5 / 8
132,6 / 4
28 / 314,7 / 23,7 / quercetin methyl ether / ** / 299,5 / 100 / 299,6 / 100
300,6 / 21 / 255,4 / 16
314,6 / 20
160,6 / 1
270,7 / 0
29 / 372,7 / 24,8 / caffeic acid derivative / ** / 178,6 / 100 / 134,7 / 100
134,6 / 44
160,4 / 29
272,5 / 22
196,6 / 22
30 / 758,6 / 25,8 / orientin derivative / ** / 446,7 / 100 / 326,6 / 100
326,6 / 82 / 356,6 / 33
578,6 / 77
356,7 / 33
447,6 / 17
458,5 / 16
579,4 / 13
638,8 / 13
310,8 / 9
31 / 270,6 / 26,4 / naringenin / * / 150,5 / 100 / 150,5 / 100
176,5 / 17 / 106,6 / 50
151,6 / 7
106,6 / 5
268,5 / 4
32 / 312,7 / 26,6 / Salvianolic acid F isomer / *** / 160,6 / 100 / 160,4 / 100
268,6 / 17
33 / 328,7 / 26,9 / dimethylquercetin (quercetindimethyl ether) / *** / 313,6 / 100 / 312,6 / 100
314,5 / 20 / 313,5 / 93
298,6 / 12 / 298,4 / 88
313 / 5 / 310,8 / 41
312,8 / 4 / 299,6 / 30
328,5 / 3 / 314 / 27
284,7 / 3 / 161,5 / 26
284,5 / 25
34 / 670,7 / 27,2 / rosmarinic acid derivative / ** / 358,7 / 100 / 160,6 / 100
310,7 / 59 / 178,6 / 27
508,6 / 50 / 196,5 / 12
490,7 / 23 / 132,6 / 12
161,6 / 11
222,5 / 11

The details of the tentative identification of those molecules that were not identified through a comparison with the authentic standards are reported below.

Id 6, m/z 325: the neutral loss of 162 is typical of an O-hexose loss, while the fragment of m/z 163, which generates a further fragment of m/z 119 correspond to coumaric acid. The metabolite was tentatively identified as coumaric acid O-hexose.

ID 7, m/z 387:the neutral loss of 162 is typical of an O-hexose loss, while the fragment of m/z 225 correspond to dihydrosinapic acid; in turn, the fragment with m/z 207 is typically generated by dihydrosinapic acid, as reported by Narváez-Cuenca et al. (2012). The metabolite was tentatively identified as dihydrosinapic acid-O-hexose.

Id 8 and 10, m/z 337: The two metabolites generates fragments with m/z 191 (corresponding to quinic acid), 163 (corresponding to coumaric acid), 119 (corresponding to the typical fragment of coumaric acid). The m/z 191 fragment was further fragmented (ms3) showing the typical fragmentation of quinic acid. The two metabolites were tentatively identified as coumaroylquinic acid.

Id 9, m/z 551: the ms/ms generates a m/z 193 fragment which was further fragmented (ms3) showing the typical fragmentation of ferulic acid. The metabolite was tentatively identified as ferulic acid derivative.

Id 13, m/z 609: the neutral loss of 308 corresponds to an O-hexose-desoxyhexose, while the fragment of m/z 301 correspond to quercetin. The further fragmentation (ms3) of 301 showed a pattern identical to that of quercetin. The metabolite was tentatively identified as quercetin-O-hexosedesoxyhexose

Id 14, m/z 489: fragmentation pattern similar to that obtained in this research has been described by Wu et al., 2013, and correspond to acetyl orientin. The fragment 327 (ms/ms) which generate 299 (ms3) is typical of orientin, and is generated from orientin by the loss of a fragment of 120 AMU typical of the C-linked glucose.; here, the neutral loss of 162 probably correspond to the loss of an acetylated fragment of C-linked glucose (120+42).

Id 15, m/z 533: the fragments 489 (corresponding to acetyl orientin, and to the neutral loss of an acetyl group) and 327 (the main fragment generated buy orientin) suggests that this is a derivative of acetyl orientin.

Id 17, m/z 461: the neutral loss of 176 corresponds to an O-glucuronic acid, while the fragment of m/z 285, and its fragmentation pattern (ms3), correspond to luteolin. The metabolite was tentatively identified as luteolin-O-glucuronide. This molecules has been reported in salvia officinalis by Zimmermann et al., 2011.

Id 18, m/z 593: the neutral loss of 308 correspondsto an O-hexosedesoxyhexose,, while the fragment of m/z 285, and its fragmentation pattern (ms3) correspond to kaempferol. The metabolite was tentatively identified as kaempferol-O-hexosedesoxyhexose.

Id 19, m/z 473: The neutral loss of 60, with the generation of a fragment of m/z 413, correspond to the loss of a piece of a C-linked hexose; the fragment of m/z 311 (ms/ms) which generate a 283 fragment(ms3) is typical of vitexin (apigenin-8-C-glucoside); here, the neutral loss of 162 that generates the m/z 311 fragment, probably correspond to the loss of an acetylated fragment of C-linked glucose (120+42). These fragments have also been described by Simirgiotis et al., 2013, and correspond to acetyl vitexin.

Id 20, m/z 517: the fragments 473 (corresponding to acetylvitexin, and to the neutral loss of an acetyl group) and 311 (the main fragment generated by vitexin) suggests that this this is a derivative of acetylvitexin.

Id 21, 575: the fragment of m/z 431 (ms/ms), and its fragmentation pattern (ms3), correspond to vitexin. The metabolite was tentatively identified as vitexin derivative.

Id 22, m/z 401: the neural loss of 90, generating a m/z 401 fragment, is typical of C-llinked sugars;: the metabolite was tentatively identified as apigenin-C-pentose.

Id 23, m/z 419: the neutral loss of 162 is typical of an O-hexose, while the fragment of m/z 256, and its fragmentation pattern (ms3), correspond to ellagic acid. The metabolite was tentatively identified as ellagicacid-O-hexose.

Id 25, m/z 447: the neutral loss of 162 is typical of an O-hexose, while the fragment of m/z 285, and its fragmentation pattern (ms3), correspond to luteolin. The metabolite was tentatively identified as luteolin -O-hexose.

Id 26, m/z 445: the neutral loss of 176 is typical of an O-glucuronic acid, while the fragment of m/z 269, and its fragmentation pattern (ms3), correspond to apigenin. The metabolite was tentatively identified as apigenin-O-glucuronide.

Id 28, m/z 315: the fragment of m/z 300 correspond to the loss of a methyl group; the fragmentation pattern, but not the retention time, corresponds to that of isorhamnetin (quercetin-3’-methy ether) and quercetin-3-methyl ether; the metabolite is probably an isomer of isorhamnetin and quercetin-3-O-methyl ether, and it was tentatively identified as quercetin methyl ether.

Id 29, m/z 373: the fragment of m/z 179 and its fragmentation pattern (ms3) corresponds to caffeic acid. The molecule was tentatively identified as caffeic acid derivative.

Id 30, m/z 759: the fragment of m/z 447 (ms/ms) and its fragmentation pattern (ms3) corresponds to orientin. The molecule was tentatively identified as orientin derivative.

Id 32, m/z 313: a metabolite with the same m/z value and very similar fragmentation pattern have been observed also by Barros et al. (2013) in Melissa officinalis, and was tentatively identified as an isomer of Salvianolic acid F.

Id 33, m/z 329: a metabolite with the same m/z value and very similar fragmentation pattern have been observed also by Bertransw et al. (2013) in Propolis samples, and was tentatively identified as quercetindimethyl ether.

Id 34, m/z 671: the fragment of m/z 359 (ms/ms) and its fragmentation pattern (ms3) are identical to rosmarinic acid. The molecule was tentatively identified as rosmarinicacid derivative.

References

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