. 2018 Aug 14;6:334. doi: 10.3389/fchem.2018.00334

Table 1.

Proposed identifications of components producing CAD peaks in the UHPLC-UV-CAD-HRMS analysis of GSE-1.

CAD Peak	RT (min)	Proposed Identification	Molecular Formula	Amt. μg per 210 mg dose	Confidence (A-D)	Accurate mass matches formula	MS/MS matches structure	Literature/database match	Reference match
1a	1.04	Magnesium salts	Mg_xFm_y	700	Partial	✓ (E)
1b		Calcium salts	Ca_xFm_y		Partial	✓ (E)
2a	1.18	Sodium salts	Na_xFm_y	4,600	Partial	✓ (E)
2b		Potassium salts	K_xFm_y		Partial	✓ (E)
3a	1.37	Monosaccharide	C₆H₁₂O₆	16,000	Matched	✓	✓ (F)	✓ Eyduran et al., 2015; Musingarabwi et al., 2016
3b		Gluconic Acid	C₆H₁₂O₇		Matched	✓	✓	✓ Larcher et al., 2009; 2017c
3c		Glutamic Acid	C₅H₉NO₄		Reference	✓	✓	✓ Bouloumpasi et al., 2002	✓
3d		Choline hexoside	C₁₁H₂₃NO₆		Tentative	✓	✓(G)	✓ Eyduran et al., 2015
3e		Arginyl fructose	C₁₂H₂₄N₄O₇		Tentative	✓	✓	✓ Ryu et al., 2001; Joo et al., 2008
3f		Cellobiosan	C₁₂H₂₀O₁₀		Partial	✓	✓(G)	Hurt et al., 2013
3g		Isovaline	C₅H₁₁NO₂		Matched	✓	✓	✓2016
4a	1.53	Tartaric Acid	C₄H₆O₆	5,700	Matched	✓	✓	✓ Eyduran et al., 2015; Musingarabwi et al., 2016; 2017c
4b		Disaccharide	C₁₂H₂₂O₁₁		Matched	✓	✓	✓ Eyduran et al., 2015; Musingarabwi et al., 2016; 2017c
4c		Proline	C₅H₉NO₂		Reference	✓	✓	✓ Bouloumpasi et al., 2002
4d		N-methylnicotinate	C₇H₇NO₂		Matched	✓	✓	✓ Eyduran et al., 2015; Musingarabwi et al., 2016
5a	2.17	Malic Acid	C₄H₆O₅	1,700	Reference	✓	✓	✓ Musingarabwi et al., 2016	✓
5b		Unidentified	–		Unknown		(H)
6a	4.46	Citric Acid	C₆H₈O₇	51	Reference	✓	✓	✓ Eyduran et al., 2015	✓
6b		Pyroglutamic Acid	C₆H₈O₇		Matched	✓	✓	✓ 2017c
7a	5.56	Succinic Acid	C₄H₆O₄	540	Reference	✓	✓	✓
7b		Tyrosine	C₉H₁₁NO₃		Reference			Eyduran et al., 2015
7c		Uridine	C₉H₁₂N₂O₆		Matched	✓	✓	✓2017c
7d		5′-O-(β-D-Glucopyranosyl) pyridoxine	C₁₄H₂₁NO₈		Tentative	✓	✓ (G)	Nikolić et al., 2012
7e		Adenosine	C₁₀H₁₄N₅O₄		Matched	✓	✓	✓ 2017c
7f		Leucine-Fructose	C₁₂H₂₃O₇		Tentative	✓	✓
7g		Unidentified	C₉H₁₆O₈		Partial	✓
7h		Unidentified	C₉H₁₈O₈		Partial	✓
7i		Unidentified	C₁₀H₁₄O₈N₂		Partial	✓
7j		Unidentified	C₁₁H₂₁N₂O₃		Partial	✓
7k		Unidentified	C₁₂H₂₁N₂O₃		Partial	✓
7l		Unidentified	C₁₉H₁₂N₃O₂		Partial	✓
8	9.46	Gallic acid	C₇H₆O₅	210	Reference	✓	✓	✓ Lin et al., 2014	✓
9a	13.39	Glucogallin	C₁₃H₁₆O₁₀	180	Matched	✓	✓	✓ Li et al., 2016
9b		Unidentified			Unknown	(I)
10	16.39	Tryptophan	C₁₁H₁₂N₂O₂	92	Reference	✓	✓	✓ Bouloumpasi et al., 2002	✓
11	18.62	Proanthocyanidin B1	C₃₀H₂₆O₁₂	1,400	Reference	✓	✓	✓ Lin et al., 2014	✓
12	19.02	Proanthocyanidin B	C₃₀H₂₆O₁₂	110	Matched	✓	✓	✓ Lin et al., 2014
13	19.57	Proanthocyanidin B	C₃₀H₂₆O₁₂	510	Matched	✓	✓	✓ Lin et al., 2014
14	20.25	Catechin	C₁₅H₁₄O₆	4,100	Reference	✓	✓	✓ Lin et al., 2014	✓
15a	20.57	Proanthocyanidin B	C₃₀H₂₆O₁₂	83	Matched	✓	✓	✓ Lin et al., 2014
15b		Gallocatechin Gallate isomer	C₂₂H₁₈O₁₁		Tentative	✓	✓(J)	✓ Lin et al., 2014
15c		Unidentified			Unknown	(K)
16	20.69	Proanthocyanidin C	C₄₅H₃₈O₁₈	180	Matched	✓	✓	✓ Lin et al., 2014
17	21.01	Benzyl alcohol	C₁₈H₂₆O₁₀	97	Tentative	✓	✓(G)	Amessis-Ouchemoukh et al., 2014
18	21.14	Galloylated Proanthocyanidin (tetramer)	C₆₇H₅₄O₂₈	120	Matched	✓	✓	✓ Lin et al., 2014
19	21.82	Proanthocyanidin B2	C₃₀H₂₆O₁₂	1,700	Reference	✓	✓	✓ Lin et al., 2014	✓
20a	22.39	Proanthocyanidin B	C₃₀H₂₆O₁₂	180	Matched	✓	✓	✓ Lin et al., 2014
20b		Lariciresinol Glucoside analog	C₂₆H₃₄O₁₁		Tentative	✓	✓(L)	Baderschneider and Winterhalter, 2001
20c		Unidentified	C₁₃H₁₄O₃		Partial	✓	(M)
21	22.73	Proanthocyanidin B	C₃₀H₂₆O₁₂	87	Matched	✓	✓	✓ Lin et al., 2014
22	23.02	Epicatechin	C₁₅H₁₄O₆	3,500	Reference	✓	✓	✓ Lin et al., 2014	✓
23a	23.36	1-(3′,4′-dihydroxy-phenyl)-3-(2″,4″,6″-trihydroxyophenyl) propan-2-ol	C₁₅H₁₆O₆	180	Matched	✓	✓	✓ Appeldoorn et al., 2009; Sánchez-Patán et al., 2012
23b		Dihydrokaempferol 3-O-ß-D-glucoside	C₂₁H₂₂O₁₁		Matched	✓	✓	✓ Pati et al., 2014
24a	23.54	Epigallocatechin Gallate	C₂₂H₁₈O₁₁	270	Reference	✓	✓	✓ Lin et al., 2014	✓
24b		Galloylated Proanthocyanidin (dimer)	C₃₇H₃₀O₁₆		Matched	✓	✓	✓ Lin et al., 2014
24c		Proanthocyanidin (tetramer)	C₆₀H₅₀O₂₄		Matched	✓	✓	✓ Lin et al., 2014
25	23.99	Proanthocyanidin C	C₄₅H₃₈O₁₈	240	Matched	✓	✓	✓ Lin et al., 2014
26a	24.24	Proanthocyanidin C1	C₄₅H₃₈O₁₈	890	Reference	✓	✓	✓ Lin et al., 2014	✓
26b		Leptolepisol D	C₂₇H₃₂O₁₀		Tentative	✓	✓	Liu et al., 2016
27a	24.94	Proanthocyanidin B	C₃₀H₂₆O₁₂	230	Matched	✓	✓	✓ Lin et al., 2014	✓
27b		Galloylated Proanthocyanidin (trimer)	C₅₂H₄₂O₂₂		Matched	✓	✓	✓ Lin et al., 2014
28a	25.07	Galloylated Proanthocyanidin (dimer)	C₃₇H₃₀O₁₆	950	Matched	✓	✓	✓ Lin et al., 2014
28b		Proanthocyanidin (tetramer)	C₆₀H₅₀O₂₄		Matched	✓	✓	✓ Lin et al., 2014
29a	25.30	Galloylated Proanthocyanidin (trimer)	C₅₂H₄₂O₂₂	85	Matched	✓	✓	✓ Lin et al., 2014
29b		Lariciresinol Glucosides analog	C₂₆H₃₄O₁₁		Tentative	✓	✓(L)	Baderschneider and Winterhalter, 2001
30a	25.91	Galloylated Proanthocyanidin (trimer)	C₅₂H₄₂O₂₂	240	Matched	✓	✓	✓ Lin et al., 2014
30b		Proanthocyanidin (pentamer)	C₇₅H₆₂O₃₀		Matched	✓	✓	✓ Lin et al., 2014
31	26.32	Proanthocyanidin B	C₃₀H₂₆O₁₂	93	Matched	✓	✓	✓ Lin et al., 2014
32a	27.37	Galloylated Proanthocyanidin (trimer)	C₅₂H₄₂O₂₂	110	Matched	✓	✓	✓ Lin et al., 2014
32b		Rutin	C₂₇H₃₀O₁₆		Reference	✓	✓	✓ Iacopini et al., 2008
32c		Galloylated Proanthocyanidin (dimer)	C₄₄H₃₄O₂₀		Matched	✓	✓	✓ Lin et al., 2014
32d		2-(3,4-dihydroxyphenyl)-4-(3-(3-(3,4-dihydroxyphenyl)-2-hydroxypropyl)-2,4,6-trihydroxylphenyl) chromane-3,5,7-triol	C₃₀H₂₈O₁₂		Tentative	✓	✓(N)	Köhler et al., 2008
32e		Methylated Proanthocyanidin B-type analog	C₃₁H₂₈O₁₂		Tentative	✓	✓	Lin et al., 2014
33a	28.00	Epicatechin gallate	C₂₂H₁₈O₁₀	320	Reference	✓	✓	✓ Lin et al., 2014	✓
33b		Proanthocyanidin B	C₃₀H₂₆O₁₂		Matched	✓	✓	✓ Lin et al., 2014
34a	28.41	Catechin gallate	C₂₂H₁₈O₁₀	100	Reference	✓	✓	✓ Lin et al., 2014	✓
34b		Galloylated Proanthocyanidin (trimer)	C₅₂H₄₂O₂₂		Matched	✓	✓	✓ Lin et al., 2014
34c		Embigenin	C₂₃H₂₄O₁₀		Tentative	✓	✓ (G)	Bakhtiar et al., 1994
35	66.70	Ursolic Acid	C₃₀H₄₈O₃	690	Tentative	✓	✓ (O)	✓ 2017c
36	67.04	Linoleic Acid	C₁₈H₃₂O₂	200	Matched	✓	✓	✓ 2017c
37	68.37	Palmitic Acid	C₁₆H₃₂O₂	170	Matched	✓	✓	✓ 2016
38	68.66	Oleic Acid	C₁₈H₃₄O₂	110	Matched	✓	✓	✓2016; 2017c
39	15-45	Tannins		~160,000	Matched	✓	✓	✓ Peng et al., 2001

^APartial: The proposed identification has a molecular formula that matches in terms of accurate mass.

^BTentative: The proposed identification matches in terms of accurate mass and interpretation of MS/MS.

^CMatched: The proposed identification matches a literature reference and/or online database [ (2017c) or (2016)] in terms of accurate mass and MS/MS.

^DReference: The proposed identification matches a reference standard in terms of retention time, accurate mass, and tandem mass spectrometry (MS/MS).

^EObserved peaks for metal (M) clusters with formate (Fm) and acetonitrile (ACN) from the mobile phase. For example, [MFm + ACN]+ in positive-mode and [MFm3]– in negative-mode. It is believed that the anions are exchanging in solution (e.g., Cl- Fm-). The original anion is unknown. Common observance in other botanicals.

^FIdentified as a monosaccharide but could not be further narrowed down by UHPLC-MS/MS.

^GThe exact position and/or identity of the aglycone cannot be determined by UHPLC-MS/MS.

^HHRMS data was unable to assign formula, but positive-ion MS/MS data displays a loss of malic acid and the chromatographic trace follows exactly with malic acid standard.

^IHRMS data unable to assign formula, but chromatographic data follows exactly with glucogallin, even under different chromatographic conditions.

^JHRMS data consistent with formula. MS/MS data does not match with the reference standard with the same formula, but does contain fragments indicating it is related to the class of compounds. The gallate is believed to be on one of the remaining hydroxy groups of the A-ring based on interpretation of MS/MS.

^KHRMS data unable to assign formula.

^LLiterature reports another compound with same formula and matching MS/MS spectra. Could not differentiate between the two without a standard but the alternative structure was assigned to peak 29b/20b.

^MThere are several peaks (>10) matching this accurate mass throughout the run with identical MS/MS. Peaks are sharp, indicating that they aren't just background noise, but this is the only retention time where it is under a CAD peak of significance. Not believed to be anything of significant contribution to the CAD signal.

^NHRMS data consistent with formula. Negative-ion MS/MS similar to known compound but does not match [34]. Proposed structure is based on MS/MS interpretation.

^OThe exact position of some functional groups cannot be determined by UHPLC-MS/MS.