3. Phenolic Profiling by UHPLC-ESI-QTOF-MS

In order to estimate the influence of the chemical composition on the antioxidant activity of rosemary infusions, a comprehensive phenolic profiling analysis was carried out based on the information provided by high-resolution mass spectrometry (HRMS/MS) data, MS databases search (e.g., Metlin and HMDB) and data described in the literature. When available, reference standards were used to confirm the identification of compounds by comparing their retention times and HRMS/MS data, also supporting the tentative identification of some structurally related compounds.

A total of forty-four phenolic compounds, belonging to nine different groups, were tentatively identified in rosemary infusions: seven hydroxybenzoic acids (1–4, 6, 7, and 12), seven hydroxycinnamic acids (9–11, 13, 16, 18, and 24), one flavan-3-ol (15), one flavanone (25), thirteen flavones (17, 19–23, 26–30, 34, and 37), eleven phenolic diterpenes (31–33, 35, 36, 39–44), one hydroxybenzaldehyde (5), two coumarins (8 and 14) and one pyranochromanone (38). Table 2 summarizes the identified compounds, as well as their chromatographic characteristics and HRMS data.

Table 3 presents a quantitative approach of the tentatively identified compounds. As previously mentioned, whenever available, the identified compounds were quantified using reference standards, whereas tentatively annotated compounds were semi-quantified using reference standards with structural similarity. The results are expressed as ng of compound/mL of infusion. Table 3 also contains the accumulated amounts of the different classes and subclasses of compounds in rosemary infusions, which was quite similar between all commercial brands: phenolic diterpenes constituted the main group of compounds, followed by hydroxycinnamic acids and flavan-3-ols groups. A color code ranging from high concentration (dark color) to low concentration (light color) allow us to follow the amount of each compound in the analyzed infusions. In addition, Figure 1 presents the total ion chromatograms (TIC) of the different infusions, where the similarities between them can be noticed.

Table 3. Quantification of compounds detected in rosemary infusions by UHPLC-ESI-QTOF-MS (ng of compound/mL of infusion).

Peak	Compound	Std *	Concentration (ng/mL of Infusion)
			A	B	C	D
1	Dihydroxybenzoic acid hexoside	A	343.3 ± 1.8 ab	223.0 ± 23.5 b	364.3 ± 64.9 ab	456.1 ± 5.4 a
2	Hydroxydimethoxybenzoic acid	B	516.4 ± 198.5 b	1269.9 ± 32.4 a	1190.9 ± 6.3 a	1134.3 ± 35.0 a
3	Hydroxymethoxybenzoic acid (I)	C	489.2 ± 82.6 a	487.1 ± 89.8 a	524.6 ± 79.5 a	544.9 ± 47.5 a
4	Protocatechuic acid	A	325.7 ± 46.8 a	458.5 ± 91.5 a	325.9 ± 54.8 a	456.3 ± 36.3 a
5	Dihydroxybenzaldehyde	A	1125.6 ± 37.2 a	851.8 ± 88.9 a	452.7 ± 76.4 b	436.5 ± 12.7 b
6	Hydroxybenzoic acid-O-hexoside	D	62.3 ± 1.2 a	33.8 ± 5.3 a	51.3 ± 8.3 a	59.4 ± 2.3 a
7	4-hydroxybenzoic acid	D	228.8 ± 6.4 a	165.9 ± 18.4 a	216.2 ± 50.5 a	257.9 ± 7.1 a
8	Dihydroxycoumarin glucoside (I)	E	n.d.	n.d.	29.7 ± 4.9	n.d.
9	Caffeic acid-O-hexoside	F	189.6 ± 52.6 a	134.8 ± 14.3 a	126.5 ± 10.2 a	119.0 ± 9.9 a
10	Coumaric acid hexoside	E	65.5 ± 0.6 a	63.8 ± 4.0 a	88.3 ± 10.1 a	89.5 ± 6.9 a
11	Caffeoylquinic acid	G	322.2 ± 81.5 a	361.5 ± 79.7 a	841.3 ± 52.2 a	711.9 ± 148.6 a
12	Hydroxymethoxybenzoic acid (II)	C	423.9 ± 32.8 a	300.9 ± 15.0 a	448.2 ± 85.4 a	494.1 ± 24.4 a
13	Caffeic acid	F	97.0 ± 23.0 b	460.5 ± 93.1 a	298.7 ± 6.8 ab	144.4 ± 12.4 b
14	Dihydroxycoumarin glucoside (II)	E	n.d.	n.d.	60.6 ± 36.6	n.d.
15	Gallocatechin	H	15,352.1 ± 831.4 a	10,574.4 ± 1057.5 a	13,920.5 ± 1968.0 a	16,253.0 ± 577.3 a
16	p-Coumaric acid	E	157.1 ± 9.0 ab	151.4 ± 8.6 b	125.6 ± 3.0 b	187.3 ± 5.4 a
17	Hydroxyluteolin/Quercetin-O-hexoside	I	97.7 ± 14.2 a	126.7 ± 13.3 a	139.8 ± 1.6 a	131.8 ± 9.2 a
18	Rosmarinic acid-O-hexoside	J	1050.2 ± 279.2 a	834.7 ± 200.3 a	1047.5 ± 99.2 a	917.1 ± 96.2 a
19	Luteolin-O-rutinoside	K	679.4 ± 20.9 a	370.3 ± 83.3 ab	348.5 ± 66.1 b	532.5 ± 28.8 ab
20	Luteolin/Scutellarein-O-glucuronide (I)	I	1164.0 ± 37.9 a	627.2 ± 86.8 b	591.3 ± 123.1 b	893.9 ± 41.2 ab
21	Luteolin-7-O-glucoside	I	248.4 ± 8.7 a	243.9 ± 12.7 a	205.0 ± 8.7 ab	184.6 ± 3.4 b
22	Nepitrin	I	2492.5 ± 14.8 a	1804.6 ± 70.6 b	1765.4 ± 124.4 b	2061.6 ± 40.8 b
23	Apigenin-O-rutinoside	K	136.4 ± 1.2 a	65.0 ± 8.4 b	53.4 ± 11.4 b	63.3 ± 1.7 b
24	Rosmarinic acid	J	47,541.6 ± 10,449.0 a	42,038.5 ± 4795.7 a	50,352.6 ± 3949.0 a	46,825.6 ± 77.7 a
25	Hesperidin	L	964.8 ± 58.4 a	869.2 ± 3.3 a	713.9 ± 84.5 a	859.9 ± 36.7 a
26	Homoplantaginin	I	1729.9 ± 22.5 a	875.1 ± 69.8 b	900.4 ± 108.0 b	1011.2 ± 19.5 b
27	Luteolin/Scutellarein-O-glucuronide (II)	I	1288.3 ± 277.3 a	1204.7 ± 14.1 a	725.7 ± 157.2 a	1434.7 ± 22.5 a
28	Tetrahydroxyflavone-O-acetylglucuronide (I)	I	301.3 ± 39.7 a	224.6 ± 9.7 a	178.8 ± 39.0 a	311.6 ± 22.1 a
29	Tetrahydroxyflavone-O-acetylglucuronide (II)	I	267.7 ± 48.0 a	253.7 ± 0.6 a	162.0 ± 35.5 a	310.2 ± 12.1 a
30	Tetrahydroxyflavone-O-acetylglucuronide (III)	I	223.9 ± 47.5 a	244.7 ± 7.9 a	139.2 ± 23.8 a	253.8 ± 2.4 a
31	(Epi)(iso)Rosmanol (I)	M	802.7 ± 258.7 b	12,960.9 ± 1928.9 a	15,272.0 ± 922.7 a	15,975.9 ± 1377.5 a
32	Rosmanol derivative	M	4941.4 ± 334.6 a	3604.9 ± 1072.5 a	6538.5 ± 1189.6 a	6249.1 ± 578.8 a
33	(Epi)(iso)Rosmanol (II)	M	28,364.3 ± 194.3 a	21,466.6 ± 3748.7 a	24,849.2 ± 98.3 a	22,977.6 ± 1608.3 a
34	Cirsimaritin	N	32.1 ± 2.3 a	32.1 ± 6.1 a	43.6 ± 5.5 a	41.1 ± 5.0 a
35	(Epi)(iso)Rosmanol (III)	M	13,647.7 ± 2013.1 a	2698.7 ± 267.9 b	5604.5 ± 456.7 b	5888.5 ± 235.3 b
36	Carnosic acid isomer	M	1185.6 ± 55.0 a	832.9 ± 63.0 b	602.7 ± 52.3 b	810.8 ± 37.1 b
37	Genkwanin	O	0.7 ± 0.2 a	1.7 ± 0.8 a	1.8 ± 0.3 a	1.1 ± 0.2 a
38	Isocalolongic acid	M	1225.6 ± 145.0 b	9520.2 ± 1416.1 a	11,964.3 ± 511.7 a	7809.5 ± 1487.5 a
39	Rosmadial/Safficinolide (I)	M	10,479.4 ± 2532.0 a	733.9 ± 57.8 b	1358.8 ± 208.3 b	2384.1 ± 6.5 b
40	Epirosmanol methyl ether	M	999.0 ± 30.4 a	126.5 ± 8.6 b	278.2 ± 53.3 b	826.5 ± 0.1 a
41	Carnosol	M	12,227.1 ± 2277.0 c	23,022.8 ± 260.8 b	34,392.3 ± 410.0 a	27,136.5 ± 2089.0 ab
42	Rosmadial/Safficinolide (II)	M	1506.4 ± 125.5 a	588.3 ± 18.2 b	1707.7 ± 200.5 a	1095.7 ± 38.7 ab
43	Rosmaridiphenol	M	707.7 ± 121.2 a	235.7 ± 37.9 b	546.2 ± 88.9 ab	407.9 ± 5.5 ab
44	Carnosic acid	M	90.1 ± 38.2 b	456.7 ± 38.1 a	580.4 ± 22.3 a	183.0 ± 8.7 b
Total			154,094.7	141,602.1	180,129.0	168,924.0
Total Phenolic acids			51,813	46,984	56,002	52,398
Total Hydroxybenzoic acids			2390	2939	3121	3403
Total Hydroxycinnamic acids			49,423	44,046	52,881	48,995
Total Flavonoids			24,979	17,518	19,889	24,344
Total Flavan-3-ols			15,352	10,574	13,921	16,253
Total Flavones			8662	6074	5255	7231
Total Flavanones			965	869	714	860
Total Phenolic diterpenes			74,952	66,728	91,731	83,936
Total Hydroxybenzaldehydes			1126	852	453	437
Total Coumarins			n.d.	n.d.	90	n.d.
Total Pyranochromanones			1226	9520	11,964	7810

For each commercial brand, the concentrations are expressed as mean ± standard deviation (ng/mL of infusion) of the infusions prepared in triplicate. Within each line, different letters represent significant differences between commercial brands, at p < 0.05. n.d., not detected. *, Reference standard used for quantification or semi-quantification: A: Protocatechuic acid, B: Syringic acid, C: Vanillic acid, D: 4-hydroxybenzoic acid, E: p-Coumaric acid, F: Caffeic acid, G: Chlorogenic acid, H: Catechin, I: Luteolin-7-O-glucoside, J: Rosmarinic acid, K: Rutin, L: Hesperidin, M: Carnosic acid, N: Hesperetin, O: Genkwanin.

Considering the chemical characterization of the compounds detected in rosemary infusions, their tentative identification will be discussed by classes or subclasses. In relation to hydroxybenzoic acids, most of the compounds identified are structurally related with each other: compounds 1 and 6 were tentatively identified as hexosides of compounds 4 (protocatechuic acid) and 7 (4-hydroxybenzoic acid), respectively, because of the loss of a hexose moiety (162 Da) which generated fragments ions at m/z 153 and 137, and which correspond to their respective aglycones. Moreover, the typical fragments at m/z 109 and 93, also detected in compounds 4 and 7, respectively, and originated due to loss of CO₂ from the respective precursor ions (153 and 137) also helped to better elucidate the identification of compounds 1 and 6. As far as we know, compound 1 (a dihydroxybenzoic acid hexoside) is being identified for the first time in rosemary infusions. Similarly to compounds 4 and 7, compound 3 (hydroxymethoxybenzoic acid) also loses a CO₂ moiety (44 Da) from the corresponding precursor ion, characteristic of hydroxybenzoic acid derivatives ^[20]. Moreover, it also presents a fragment ion at m/z 108, which is commonly found in MS/MS databases of hydroxymethoxybenzoic acid isomers, and which was also detected for compound 12, another hydroxymethoxybenzoic acid isomer. Compound 2 (hydroxydimethoxybenzoic acid), unlike others, generate a base peak at m/z 135 through the successive loss of a water molecule and a CO₂ molecule ([M − H − H₂O − CO₂]⁻) and a m/z 123 [M − H − C₃H₆O₂]⁻ fragment, which seems to result from the loss of a methoxyethenol group.

The only hydroxybenzaldehyde derivative found in rosemary infusions (compound 5) was identified as dihydroxybenzaldehyde. The pattern of fragmentation was quite similar to MS spectra available on databases, which facilitated the identification of the compound. The more relevant fragment ions at m/z 108 and 119 resulted, from the loss of the aldehyde moiety and of a water molecule, respectively. As far as we know, this is the first time that dihydroxybenzaldehyde is being described for rosemary infusions.

Regarding the coumarins class, two compounds with the same deprotonated molecular ions were identified as dihydroxycoumarin hexoside I (compound 8) and dihydroxycoumarin hexoside II (compound 14). These compounds, apparently detected for the first time in rosemary infusions (only in brand C), presented the same base peak at m/z 177 as a result of the neutral loss of a hexose moiety (162). Further characterization of these compounds is required, as they may correspond to various dihydroxycoumarin hexosides, such as esculin, cichoriin, or daphnin, already found in other plants and with reported biological properties ^{[25][26][27][28][29][30]}. For example, esculin has been proved to inhibit lipid peroxidation and to scavenge hydroxyradicals, as well as to possess anti-inflammatory activity and gastroprotective effects ^[26][29]. Interestingly, all of these properties are also described for rosemary and, to some extent, might be associated with these compounds (even though they have been found in quite low quantities).

As observed for hydroxybenzoic acids, identified hydroxycinnamic acids are structurally related to each other. For compounds 9 and 10, the loss of a hexose moiety (162 Da) leads to fragment ions at m/z 179 and 163, together with their correspondingly characteristic fragment ions at m/z 135 and 119, both resulted from the loss of a CO₂ moiety, which allowed their identification as hexosides of the compounds 13 (caffeic acid) and 16 (p-coumaric acid). As far as we know, coumaric acid hexoside (compound 10) was detected for the first time in rosemary. Compound 11 was identified as caffeoylquinic acid according to its characteristic fragments at m/z 191 (quinic acid), m/z 179 (caffeic acid), and the characteristic m/z 173 (loss of a water from quinic acid) of 4-caffeoylquinic acid (cryptochlorogenic acid) where this fragment represents the base peak, and its identity was confirmed by its reference standard. The most relevant hydroxycinnamic acid found in rosemary infusions was rosmarinic acid (compound 24). This compound, which presented the highest concentrations in all infusions representing around 28–31% of the total compounds detected, is well described in literature as a major compound of the plant ^[1][2][5][9] and, in fact, is considered a “family marker” for the Lamiaceae family ^[31]. It is noteworthy that rosmarinic acid is endowed by numerous beneficial properties, such as antioxidant, antiulcerogenic, antidepressant, and anti-inflammatory properties and, therefore, is considered one of the main responsible for the effects reported for rosemary infusions ^[3][4][5][8]. Concerning its fragmentation pattern, as a caffeic acid derivative, rosmarinic acid presented, as described, a typical fragment ion at m/z 179 corresponding to caffeic acid, as well as two caffeic acid fragments at m/z 161 and 135, resulted from the loss of a water molecule and a CO₂ moiety, respectively. The fragment ion found at m/z 197 is also characteristic of rosmarinic acid and corresponds to the 2-hydroxy derivative of hydrocaffeic acid ^[20]. Additionally, a hexoside of rosmarinic acid was also detected in rosemary (compound 18), showing the characteristic fragments of rosmarinic acid and [M − H − C₆H₁₀O₅]⁻ and [M − H − C₉H₁₀O₅]⁻ ions—resultant from the loss of the hexose and the 2-hydroxy-hydrocaffeic acid moieties, respectively—in the MS/MS spectra.

Flavonoids represented one of the main groups of compounds found in rosemary infusions, although the results were a bit dissimilar between the three identified flavonoid subclasses. Despite being the only flavan-3-ol identified in the studied infusions, compound 15 represents around 7–10% of the total compounds detected, making the flavan-3-ol subclass the main flavonoid subclass detected. The MS/MS data of compound 15 showed fragment ions at m/z 97 and 225, which are well documented in literature and databases as gallocatechin. Gallocatechin is well described as one of the major flavonoids found in rosemary extracts ^[2][9][20].

Flavones’ subclass includes a total of thirteen compounds identified at relatively low abundance, accounting for about 3–6% of the total compounds detected in the different rosemary infusions. Compounds 17, 21, 22, and 26 were identified as hexosides of hydroxyluteolin/quercetin, luteolin, nepetin and hispidulin, respectively, because of the neutral loss of a hexose moiety (162 Da), which resulted in the respective aglycone. For compound 17, two different flavonoids (the flavone hydroxyluteolin-O-glucoside and the flavonol quercetin-O-glucoside) are documented to be present in rosemary ^[2][5][20]. Although it was not possible to completely establish the identification of compound 17 due to the lack of standards, this compound might correspond to an hexoside of the flavone hydroxyluteolin, as this compound did not contain a fragment ion at m/z 300, which is usually found for quercetin derivatives. The unambiguous identification of compound 21 was confirmed by comparing its retention time and MS/MS data with its corresponding reference standard. Compounds 19 and 23 were identified as rutinosides of luteolin and apigenin, as deducted from the neutral loss of 308 Da and the resulting fragment ions at m/z 285 and 269, respectively. Compound 19 also exhibited a characteristic fragment ion of luteolin, at m/z 151, that helped to confirm the identity of the compound.

Like hexosides and rutinosides, glucuronide compounds can be readily identified based on the neutral loss of the glucuronide moiety (176 Da), which results in the corresponding aglycones ([M − H − 176]⁻). Thus, compounds 20 and 27 detected in rosemary infusions were identified as glucuronides of two isomers (luteolin and scutellarein), both already described for rosemary. Compounds 28, 29, and 30 were identified as three tetrahydroxyflavone-O-acetylglucuronide isomers based on the neutral loss of an acetylglucuronide moiety (218 Da), leading to a fragment ion at m/z 285, corresponding to various possible tetrahydroxyflavone (e.g., luteolin and scutelarrein). These compounds could be assigned as luteolin-O-acetylglucuronide isomers, based on data reported in the literature ^[2][5][9]. More specifically, according to Borras-Linares et al. (2014), compounds 28 and 29 could be identified as luteolin-3′-O-(2″-O-acetyl)-β-d-glucuronide isomers I and II based on the presence of fragment ions at m/z 285 and 399 which correspond to [M − H − C₈H₁₀O₇]⁻ and [M − H − C₃H₄O₄]⁻, whereas compound 30 could be identified as luteolin-3′-O-(3″-O-acetyl)-β-d-glucuronide or as luteolin-3′-O-(4″-O-acetyl)-β-d-glucuronide based on fragment ions at m/z 285 and 443, which are common to both isomers, and corresponding to acetylglucuronide and acetyl moieties, respectively ^[2]. Notwithstanding, their unambiguous identity cannot be confirmed by the analytical method employed in this work, and this is why they were generically identified as tetrahydroxyflavone-O-acetylglucuronide isomers. In turn, compounds 34 and 37 were identified as methoxyflavones’ derivatives, due to the characteristic neutral loss of methyl groups (15 Da), and, according to data reported for rosemary ^{[2][5][9][20]}, they were assigned as cirsimaritin and genkwanin. Finally, compound 25, the only flavanone identified, was assigned as hesperidin, as deducted from the loss of a rutinoside moiety, as well as based on data found in the literature search ^[2][5][9], and its identity was then confirmed with the reference standard of the compound.

The phenolic diterpenes identified in this work represent the most abundant group detected in these infusions, in accordance with other studies about rosemary phytochemical composition ^[2][9][21]. A total of eleven phenolic diterpenes have been detected, two of which (compound 33 and 41) stood out particularly due to their high concentration. Compound 33, together with compounds 31 and 35, were identified as (epi)(iso)rosmanol. These three isomeric forms (rosmanol, epirosmanol and episorosmanol) are equally described in the literature for rosemary ^[2][5][9] and cannot be distinguished by the analytical method employed in this work, exhibiting characteristic fragment ions at m/z 301 and 283 resulted, respectively, from the loss of a CO₂ molecule ([M − H − CO₂]⁻), followed by the loss of a water molecule ([M − H − CO₂ − H₂O]⁻). Similar neutral losses were found for compound 32, a rosmanol derivative that shows the base peak at m/z 359—an m/z equal to the deprotonated molecular ion of compound 40, which was previously described by Borrás-Linares et al. (2014) as epirosmanol methyl ether ^[2]. Compound 41 (carnosol), whose presence in rosemary is already well documented in several studies, show the characteristic base peak detected at m/z 285, which results from the loss of a CO₂ molecule ([M − H − CO₂]⁻). The same loss was also observed for compound 44, which was identified as carnosic acid, as well as for its isomer (compound 36). This phenolic diterpene is highly reported as the strongest antioxidant compound and one of the main compounds in rosemary ^{[1][2][5][9][20][21]}, although it was found in the studied infusions at very low levels. This can be explained due to its instability in the presence of oxygen and heat, leading to degradation into other phenolic diterpenes, such as carnosol, rosmanol, epirosmanol, epiisorosmanol, and rosmadial, which were found in high amounts in the studied samples ^[9]. Compounds 39 and 42 presented the same deprotonated molecular ions and might correspond to different isomeric forms already described in the literature ^[2][9][20]. The fragment ions found in each compound at m/z 299 and 315 from losses of propyl and ethylene moieties, respectively, are typical fragments of rosmadial and safficinolide. Compound 43, characterized by the loss of a methyl group from the precursor ion, was identified as rosmaridiphenol as reported in literature and databases ^[2][9].

Finally, a compound that does not belong to any of the compound families discussed above, was identified as isocalolongic acid (compound 38)—a pyranochromanone with reported bioactive potential, previously identified in plants of the genus Calophyllum ^[32][33]—based on the positive match of MS/MS data in MS databases. To the best of our knowledge, this compound in rosemary is being described for the first time.