Characterization of phenolic compounds in Helichrysum melaleucum by high-performance liquid chromatography with on-line ultraviolet and mass spectrometry detection.

Helicrysum melaleucum is a medicinal plant traditionally used in the islands of the Macaronesia region for the treatment of respiratory diseases. In this work, the phenolic compounds of Helicrysum melaleucum plants collected in different geographical locations of Madeira Island and their morphological parts (total aerial parts, leaves, flowers and stems) were extracted and analyzed separately by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-DAD/ESI-MS(n)). A total of 68 compounds were characterized based mainly on their UV and mass spectra. These included derivatives of O-glycosylated flavonoids (flavonol and flavones type), quinic acid, caffeic acid, lignans and polyphenols. The flowers were found to be the morphological part with higher variety of phenolic compounds. The large differences in the phenolic composition of plants collected from different geographical locations allowed the identification of a few components, such as pinoresinol and methoxylated flavone derivatives, likely to be useful as geographical markers. Also, these results promote further comparison of the bioactivities of the different samples analyzed. This paper marks the first report on the chemical analysis of Helichrysum melaleucum species.

There are more than 500 species of Helichrysum genus distributed around the world. Plants of this genus have been found to possess several biological activities, such as antimicrobial, antiallergic, antioxidant, anti-inflammatory, cough relief, cold and wounds. 1 In Madeira Archipelago (Portugal) there are some Helichrysum species used in traditional medicine. Several of them are imported and four are endemic species. Helichrysum melaleucum Rchb. Ex Holl is one of these endemic subspecies and, according to folk medicine, the leaves and the flowers heads are used for the treatment of bronchitis and pharingitis while infusions of the flowers are used as cardiotonic and cough relief remedy. 2 This particular plant only grows on the north coast of Madeira Island. It is very common in locations near the sea and rare in high altitude locations (up to 1200 m).
The biological activities of Helichrysum plants have been attributed to several classes of compounds such as flavonoids, a-pyrones, coumarins and terpenoids, detected in different morphological parts of the plant. 3 To our knowledge, there is no report establishing a relation between the phenolic composition of Helichrysum melaleucum and its biological activities. In previous work by our group, the phenolic composition of Helichrysum devium was established using a high-performance liquid chromatography diode-array detection/electrospray ionization tandem mass spectrometry (HPLC-DAD/ESI-MS n ) method. The most abundant phenolic compounds were found to be hydroxycinnamic derivatives, in particular quinic acid derivatives. 4 Quinic acids were also found in other Helichrysum plants showing strong antioxidant activity. 1 Phenolic compounds are a large class of low molecular weight secondary plant metabolites, which are fundamental for plant normal development and an important key in their defence mechanisms. The great interest in this class of compounds is a result of their important biological activities such as antioxidant activity, protection against cancer, cardiovascular and neurodegenerative diseases. They can also be used as natural antioxidants in food processing in order to prevent lipid peroxidation. 5 The main classes of phenolic compounds are phenolic acids and flavonoids. The major subclasses of flavonoids are flavonols, flavones, isoflavones, flavanones, catechins, aurones, anthocyanins and chalcones.
In plant cells, flavonoids usually occur as glycosides and are divided into two groups, according to the site of glycoside substitution on the flavonoid structure: O-glycosides) and C-glycosides. Besides glycosylation, flavonoids can occur in more modified forms, due to additional hydroxylation, methylation and acetylation. 6 Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with UV diode-array detector (DAD) is widely used for separation and detection of phenolic compounds from complex samples including natural sources like plant extracts. Coupling a tandem mass spectrometry detector (LC/MS/MS) with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) has proved to be a powerful tool for the unequivocal characterization and structural identification of phenolic compounds.
The use of ESI as ionization source operating in the negative mode has proved to be more efficient and selective for the detection of phenolic compounds like flavonoids glycosides. 6,7 Also, it allows for the detection of minor components, difficult to detect by other means. With multistage tandem mass spectrometry (MS n ) analysis, it is possible to obtain structural information and also to exclude the presence of interferences, which is not possible with UV detection.
In this paper, a HPLC-DAD/ESI-MS n method was used to separate and identify the phenolic compounds present in the methanolic extracts of Helichrysum melaleucum. Given the use of different morphological parts of this plant (leaves, flowers, stems and total aerial parts) for different medicinal purposes, it is important to perform a screening of their phenolic profile. In addition, a comparison of the phenolic composition was made for plants collected in different geographical environments: São Vicente (located at sea level, just across the road from the beach) and Fajã da Nogueira (about 1000 m altitude).
This report is the first exhaustive study on the phenolic composition of methanolic extracts of different morphological parts from Helichrysum melaleucum.

EXPERIMENTAL Chemical and standards
HPLC grade acetonitrile (CH 3 CN) (Lab-Scan, 99%), ultrapure water (Milli-Q, Waters) and formic acid (analytical grade) were used for mobile phase preparation in the LC/MS analysis. The methanol used for extraction of Helichrysum devium was AR grade, purchased from Fisher. Eluents prepared for LC/MS analysis were additionally filtered through 0.45 mm (Millipore) membranes.

Plant material and sample preparation
Samples of Helichrysum melaleucum were collected in the wild in two different locations of the north cost of Madeira: São Vicente and Fajã da Nogueira. The plant material collected in São Vicente consisted of total aerial parts and individually separated leaves, flowers and stems. The amount of plant material collected at Fajã da Nogueira was very small, so only the total aerial parts were analyzed. The plants were authenticated by taxonomist Fátima Rocha and a voucher was deposited in the Madeira Botanical Garden herbarium collection.
Dried and powdered plant material (100 g) was exhaustively extracted by maceration with methanol (1 L), at room temperature for 24 h.
In all cases the solutions were filtered and concentrated to dryness under reduced pressure in a rotary evaporator (408C). Stock solutions with concentrations (m/v) of 5 mg/ mL were prepared by dissolving dried extract in initial HPLC mobile phase (CH 3 CN/H 2 O (20:80)).
These solutions were filtered through 0.45 mm micropore membranes prior to use and 10 mL were injected for LC-DAD/ESI-MS n analysis. Three independent assays were performed for each sample.

Liquid chromatography
The HPLC analysis was performed on a Dionex ultimate 3000 series instrument coupled to a binary pump, a diode-array detector (DAD), an autosampler and a column compartment. The wavelength range was set at 210-520 nm and was monitored at 280 nm. Samples were separated on a Phenomenex Gemini C 18 column (5 mm, 250 Â 3.0 mm i.d.; Phenomenex) with a sample injection volume of 10 mL. The mobile phase consisted of CH 3 CN (A) and water/formic acid (100:0.1, v/v) (B). A gradient program was used as follows: 20% A (0 min), 25% A (10 min), 25% A (20 min), 50% A (40 min), 100% A (42-47 min), 20% A (49-55 min). The mobile phase flow rate was 0.4 mL/min; the chromatogram was recorded at 280 nm and spectral data for all peaks were accumulated in the range of 190-400 nm. Column temperature was controlled at 308C.

Mass spectrometry
For LC/ESI-MS n analysis, a model 6000 ion trap mass spectrometer (Bruker Esquire, Bremen, Germany) fitted with an ESI source was used. Data acquisition and processing were performed using Esquire control software. Negative ion mass spectra of the column eluate were recorded in the range m/z 100-1000 at a scan speed of 13000 Da/s. Highpurity nitrogen (N 2 ) was used both as drying gas at a flow of 10.0 mL/min and as a nebulizing gas at a pressure of 50 psi. The nebulizer temperature was set at 3658C and a potential of þ4500 V was used on the capillary. Ultra-high-purity helium (He) was used as collision gas at a pressure of 1 Â 10 À5 mbar and the collision energy was set at 40 V.
The acquisition of MS n data was made in auto MS n mode, with an isolation width of 4.0 m/z. For MS n analysis, the mass spectrometer was scanned from 10 to 1000 m/z with a fragmentation amplitude of 1.0 V (MS n up to MS 4 ) and two precursor ions.

RESULTS AND DISCUSSION
As mentioned before, Helichrysum melaleucum was collected in the wild in two different geographical locations. To simplify the identification of extracts, samples from São Vicente were denominated as SV and the one from Fajã da Nogueira as FN.
It must be mentioned that in Fajã da Nogueira, located at 1000 m altitude, H. melaleucum is a rare specimen and it was not possible to collect enough plant material to separate into different morphological parts, as it was done for the plants collected in São Vicente.
The base peak chromatogram (BPC) profiles of the methanolic extracts from plants from SV and FN are shown in Figs. 1 and 2. To a large extent, the compounds could be well separated and no relevant variation was observed in the three independent assays performed for each sample.
When a reference pure standard was available, the compound was identified by comparing the HPLC retention time, UV and mass spectra with those obtained for the standard. However, since the access to pure reference compounds was limited and the characterization of several compounds detected based only on information provided by the UV spectra is not possible, the structures of unknown compounds were proposed based mainly on the MS n fragmentation pattern.
A preliminary analysis of the UV spectra of all compounds allowed the identification of hydroxycinnamic acid derivatives and flavonoid derivatives. The first group showed characteristic absorption bands at 230-240 and 320-340 nm, together with a shoulder around 300-310 nm. In the group of flavonoids, flavonol glycosylated derivatives showed two maximum absorptions at 250-270 and 320-360 nm. Flavones showed two absorptions at 230-250 and 330-360 nm.
More than 68 different compounds were detected and 55 of them were characterized. Their MS n fragmentation ions and UV absorptions are presented in Tables 1 and 2 and their chemical structures are given in Fig. 3.
Most of the detected phenolic compounds gave deprotonated molecular ions, [M-H] -, of high abundance that allowed for MS n analysis. Usually, the base peak in a full MS spectrum was assigned as the [M-H]ion.
When two or more isomers were detected, their identification was made based on previous reports of Helichrysum species, comparison of HPLC retention times and relative intensity of characteristics fragments. 4 Compounds were numbered by their order of elution since some of them were not found in all samples.

Identification of flavonoids
The occurrence of flavonoids in Helichrysum species has been reported previously 4 with aglycones belonging to two subtypes, flavonols and flavones. In this study, several flavonoids were detected in their glycosylated form and/or esterified with acyl groups. Free aglycones were found in trace amounts in some samples.
MS n fragmentation of the ion [M-H]gave the deprotonated aglycone ion (Y À 0 ) by the loss of the sugar residue. The nature of the glycoside groups was identified based on the neutral losses of hexoside, caffeoyl, rhamnoside, coumaroyl and malonyl moieties (À162, À162, À146, À146 and À86 Da, respectively).
The nomenclature proposed by Ma et al. 8 for MS n fragment ions of flavonoids was adopted in this work. For free aglycones, the i,j Aand i,j Blabels correspond to ions containing intact A-and B-rings, respectively, in which i and j indicate the C-ring bonds that have been broken. For conjugated aglycones, Y À 0 is used to refer to the aglycone fragment [M-H-glycoside] -.
Compounds 4 (retention time (t R ) ¼ 4.0 min) and 8 (t R ¼ 5.9 min) were only detected in the SV flowers methanolic extract. Compound 4 (t R ¼ 4.0 min) displayed a [M-H]ion at m/z 609 which easily loses two residues of 162 Da each, by MS n fragmentation, forming fragment ions at m/z 447 (base peak) and 285 (ca. 30% of base peak). The fragmentation of the ion at m/z 285 gave a fragment ion at m/z 255 (loss of 30 Da, [Y À 0 -CH 2 OH] -) which is characteristic of kaempferol (comparison made with a standard solution of kaempferol). The two fragments with 162 Da could be attributed either to a caffeoylhexoside or a dihexoside residue. However, a fragment ion at m/z 323 was not observed, indicating the presence of a caffeoylhexoside residue; moreover, it is known that acylated flavonoids appear at higher retention time. Based on these assumptions, the two 162 Da residues were identified as being hexoside residues.
Given that the MS 2 spectrum base peak does not correspond to the deprotonated aglycone ion and based on the rules described by Ablajan et al. 9 it is clear that the two hexoside residues are not linked in the same position of the aglycone skeleton. It is known that for flavonols, like kaempferol, the 3-OH and 7-OH positions are the more favoured positions for glycosylation to occur. Thus, compound 4 was identified as kaempferol-3,7-O-dihexoside. Compound 8 (t R ¼ 5.9 min) gave a [M-H]ion at m/z 651. In the MS 2 spectrum a fragment ion was observed at m/z 447, due to the loss of 206 Da, which indicates a hexoside residue (162 Da) linked to an acetyl group (44 Da). The MS 3 spectrum of the ion at m/z 447 gave a fragment ion at m/z at 284 as base peak and a low intensity ion at m/z 285 (18.3% of the base peak). The further MS n fragmentation gave a fragment ion at m/z 255, consistent with the MS n data of kaempferol. As mentioned above, the favoured glycosylation positions for kaempferol are 3-OH and 7-OH. In the MS 3 spectrum, the aglycone radical ion is more abundant than the deprotonated aglycone ion, which corresponds to an aglycone substituted in position 3-OH. 10 Therefore, compound 8 was identified as kaempferol-3-O-hexoside-7-O-acetylhexoside.
In the FN total aerial parts methanolic extract, at a retention time of 9.8 min, there are two overlapping peaks (compounds 15 and 16). However, their deprotonated molecular ions, [M-H] -, could be clearly identified and presented enough intensity to be subjected to further MS n fragmentation.
Compound 15 displayed a [M-H]ion at m/z 493 and its MS 2 spectrum showed the aglycone ion (Y À 0 ) at m/z 331, as base peak, suggesting the presence of a hexoside residue. This ion, under MS n fragmentation, gave a radical fragment [Y À 0 -CH 3 ] .at m/z 316, consistent with literature data for mearnsetin. 11 Compound 16  It is known that, despite the fact that any of the hydroxyl groups of the flavonoid aglycone can be glycosylated, certain positions are favoured. For flavonols the 3-OH and 7-OH positions are regular glycosylation sites. 6 Even so, based only on MS n data of these two compounds, neither the nature of the hexoside residue nor the sugar linkage position to the aglycone could be determined. Thus, compounds 15 and 16 were preliminary characterized as mearnsetin-O-hexoside and quercetin-O-hexoside, respectively. They were also      found only in the total aerial parts, leaves and flowers methanolic extracts of H. melaleucum from SV. Quercetin-Ohexoside was detected in the flower extracts, while mearnsetin-O-hexoside was identified in the total aerial parts and leaves extracts.
Compound 17 (t R ¼ 10.6 min) was tentatively identified as isorhamnetin-O-hexoside. This compound gave a deprotonated molecular ion [M-H]at m/z 477 and its MS 2 spectrum showed an aglycone ion (Y À 0 ) at m/z 315 due to the loss of 162 Da, suggesting the presence of a hexoside residue. MS n fragmentation of the ion at m/z 315 was very similar to that of isorhamnetin reported in our previous studies on Helichrysum devium, where this compound was detected only in the flowers extract. 4,11,12 For Helichrysum melaleucum, the compound was not detected in the stems extract, but was present in all the other extracts analyzed.
Compound 25 (t R ¼ 14.0 min) showed a [M-H]ion at m/z 461 and its MS 2 spectrum exhibited a fragment ion at m/z 299 as base peak, suggesting the presence of a hexoside moiety (loss of 162 Da). A weak ion at m/z 446 was also detected (ca. 50% of base peak) which corresponds to the loss of a methyl group (15 Da) from the [M-H]ion. This ion at m/z 299 corresponds to the aglycone ion (Y À 0 ) which under MS 3 fragmentation easily lost a methyl group (15 Da), producing a fragment ion at m/z 284 (Fig. 4).
The MS n fragmentation of the ion at m/z 284 yielded several fragments at m/z 228 2 -CO] -); and 167 ( 1,3 A -), originating from a RDA reaction (Scheme 1). According to these data, the aglycone was identified as being hispidulin, a 6-methoxyflavone. 13 For flavones like hispidulin, the 7-OH position is the most regular and common glycosylation site. 6 Therefore, compound 25 was identified as hispidulin-7-O-hexoside. This compound was detected in SV and FN total aerial parts and also in the SV leaves extract.
Compound 29 (t R ¼ 16.6 min) exhibited a [M-H]ion at m/z 489 which, under fragmentation, eliminated a neutral fragment of 204 Da forming the aglycone ion (Y À 0 ) at m/z 285. MS n fragmentation of this ion gave the characteristic fragments of kaempferol (m/z 257, 255 and 229). The loss of 204 Da can be associated with an acetylhexoside moiety. The linkage position of this moiety is difficult to establish only based on MS n data, but it is known that flavonols glycosylated at the 3-OH position present a radical aglycone ion ([Y À 0 -H] -) with a high relative abundance. 10 Nevertheless, this radical fragment was not detected in order to confirm the 3-OH position and compound 29 was classified as kaempferol-O-acetylhexoside; it was only detected in SV flowers extract.
Compound 39 (t R ¼ 20.9 min) yielded a [M-H]ion at m/z 609. The MS 2 spectrum of this ion showed a fragment ion at m/z 285, as base peak, due to the loss of 324 Da, and also a fragment ion at m/z 447 (loss of 162 Da). According to this, it is possible to infer that there is a combined loss of two residues of 162 Da.
The fragment ion at m/z 285 corresponds to the aglycone ion (Y À 0 ) and its MS 3 spectrum showed ions at m/z 229 ([Y À 0 -2CO]), 151 ( 1,3 A -) and, as base peak, a fragment ion at m/z 257 ([Y À 0 -CO] -). These RDA fragments are consistent with those found for a standard solution of kaempferol, as mentioned before.
Since the MS 2 spectrum base peak ion (m/z 285) corresponds to the aglycone ion, the two substituent groups must be attached to the same kaempferol hydroxyl group. Further evidence for this type of substitution is that the fragments [Y 3 0 -H] -. and [Y 0 -2H] -, characteristic ions for di-Oglycosides, were not detected. 9 The two substituent groups of kaempferol can be either a moiety composed of two hexosides residues or one hexoside residue esterified with a caffeoyl group. The last hypothesis was confirmed by the presence of a fragment ion at m/z 323 (ca. 44% of base peak) assigned as [caffeoylhexose-H]and a [caffeic acid-H]ion at m/z 179; 12 the long retention time is also an indication of the presence of an acyl group, rather than a dihexoside group.
Since the aglycone radical ion was not detected, it is possible to infer that the aglycone is not substituted at the 3-OH position. Thus, compound 39 was classified as kaempferol-O-caffeoylhexoside. It was detected only in the FN total aerial parts extract and in the SV flowers extract.
Another two compounds, 40 (t R ¼ 21.5 min) and 44 (t R ¼ 23.8 min), with a [M-H]ion at m/z 609, were identified in the FN total aerial parts extract, although they have a different MS n fragmentation pathway of that found for compound 39, which gave also a deprotonated molecular ion The aglycone was identified as kaempferol based on the principal RDA reaction fragment ions. Full characterization of these compounds was achieved by comparison of the MS n fragmentation behaviour with that described in our previous work with similar compounds. 4 Thus, compounds 51 and 53 were identified as kaempferol 7-O-coumaroylhexoside and kaempferol 4 0 -O-coumaroylhexoside, respectively. However, the occurrence of these two compounds is not the same in all extracts. For example, compound 51 was identified in all extracts with the exception of the SV total aerial parts and stems extracts. Compound 53 was detected in the FN and SV total aerial parts and SV flowers extracts.
Compound 54 (t R ¼ 30.1 min) exhibited a [M-H]ion at m/z 491 and was only detected in the SV leaves extract. Its MS 2 fragmentation produced a fragment ion at m/z 329, probably due to the loss of a hexoside residue (162 Da). The sequential MS n fragmentation allowed the identification of two losses of 15 Da each, due to the presence of two methoxyl groups. This fragmentation behaviour is consistent with that described before for 3 0 ,4 0 -dihydroxy-5,6-dimethoxy-7-Ohexoside flavone. 15 Compound 56 (t R ¼ 30.6 min) was only found in the FN total aerial parts extract and showed a [M-H]ion at m/z 475 ( Fig. 5(a)). The MS 2 fragmentation of this ion produced a base peak at m/z 313, attributed to the loss of 162 Da, suggesting the presence of a hexoside residue. MS n fragmentation of the ion at m/z 313 gave ions at m/z 298 and 283, due to two consecutive losses of 15 Da, probably due to two methyl groups. Based on the MS n data it was possible to identify a flavone skeleton.
This compound exhibited a low-intensity but clear band I absorption at 322 nm ( Fig. 5(b)) which is characteristic of flavones (band I at 315-322 nm) with 6-oxygenation but without B-ring oxygenation. 7 Furthermore, the band II at 267 nm, with a bathochromic shift, is also characteristic of a flavones with a 7-hydroxylated and 6,8-methoxylated A-ring. 7  forming the ion at m/z 447. The nature of this fragment could not be determined. The MS 3 spectrum of the ion at m/z 447 gave a fragment ion at m/z 285, as base peak, suggesting the presence of a hexoside residue (162 Da). The ion at m/z 285 corresponds to the aglycone ion (Y À 0 ) and its fragmentation gave RDA characteristic fragments of kaempferol at m/z 257 and 255. So, compound 61 was identified as a kaempferol-Ohexoside derivative, probably substituted with an acyl group which will explain the long retention time.  149 ( 1,4 B þ 2H). This fragmentation behaviour matches that of a standard solution of apigenin and agrees with the literature data for this compound. 4 This compound was only found in the FN total aerial parts and in SV flowers extract. Normally, the presence of free aglycones indicates the presence of their glycosylated forms, but no glycosylated apigenin was detected.
The peak that occurs at 34.9 min showed two intense ions at m/z 547 (base peak) and 343 (84.3% of base peak). MS n fragmentation of the ion at m/z 547 gave the aglycone ion (Y À 0 ) at m/z 343 (loss of 204 Da), probably combined loss of a hexoside (162 Da) and an acetyl group (42 Da).
The 204 Da residue can be located in two -OH positions: 5-OH and 2 0 -OH. It is well known that 5-O-glycosides are rare for compounds with a carbonyl group at position 4, since the 5-OH group participates in hydrogen bonding with the adjacent 4-C¼O group. So, compound 66 was identified as being 5-hydroxy-7,8,6 0 -trimethoxy-2 0 -hexoside (acetyl) flavone.

Hydroxycinnamic derivatives
It was possible to detect a total of 20 hydroxycinnamic acid derivatives in the five analyzed samples from Helichrysum melaleucum. For all compounds, the deprotonated molecular ion, [M-H] -, was formed with sufficient intensity to undergo MS n fragmentation. The loss of the substituent groups is always referred in respect of this ion.
The linkage position of acyl groups on the quinic acid structure can be established based on the main fragment ions from MS n fragmentation of [M-H]ions. Acyl groups linked to the 4-OH position gave a [caffeic acid-H]ion at m/z 173 as base peak. When the acyl group is connected to the 3-OH or 5-OH position, the [quinic acid-H]ion at m/z 191 appears as the base peak and the [caffeic acid-H]ion at m/z 179 is more significant for 3-OH compounds. 16 The quinic acid derivatives found were identified based on these assumptions and on the hierarchical key for the identification by LC/MS n of quinic acid derivatives proposed by Clifford et al. 16 Compound 2 (t R ¼ 3.  (Tables 1 and 2) these compounds were assigned as 1,3-O-dicaffeoylquinic acid (9), 3,4-O-dicaffeoylquinic acid (21), 1,5-O-dicaffeoylquinic acid (22) and 3,5-O-dicaffeoylquinic acid(23). The full explanation concerning the characterization of these isomers is given in our previous work. 4 These four compounds were found in the SV and FN total aerial parts extract and compound 9 was detected in all extracts. The occurrence of the other compounds in the other extracts is variable.
Compounds 19 (t R ¼ 11.4 min) and 20 (t R ¼ 11.8 min) yielded a [M-H]ion at m/z 547. Their MS 2 spectra showed a fragment ion at m/z 353, as base peak, and an intense fragment ion at m/z 515 (ca. 80% of base peak). MS n fragmentation of the ion at m/z 353 gave common fragments to those obtained for caffeoylquinic acid fragmentation. For example, the MS 3 spectrum displayed fragment ions at m/z 191 (base peak) and 179 (<10% of base peak), which indicates a quinic acid substituted at position 1-OH or 5-OH. 16 This conclusion was achieved taking into account the presence of weak fragment ions characteristic of that compound, namely the MS 2 ion at m/z 335 (ca. 4% of base peak) and the MS 3 ion (ca. 2% of base peak). Nevertheless, based only on MS n data, it was not possible to completely identify the structures of these two compounds. Thus, compounds 19 and 20 were tentatively characterized as a 1,5-O-dicaffeoylquinic acid derivatives. Compound 19 was found in SV and FN total aerial parts and compound 20 was only detected in the FN total aerial parts extract.
Compound 14 (t R ¼ 8.6 min) was only detected in the SV leaves methanolic extract and it displayed a [M-H]ion at m/z 677. In the MS 2 spectrum, a loss of 162 Da, probably a hexoside residue, was observed forming a base peak at m/z 515, which is characteristic for dicaffeoylquinic acid derivatives. However, the further MS n fragmentation led to a fragmentation behaviour very different from those isomers. For example, in the MS 3 spectrum the most intense peak was a fragment ion at m/z 323 (loss of 192 Da) and in the MS 4 spectrum the base peak corresponds to a fragment ion at m/z 161 (loss of 162 Da). However, despite the fact that common fragments of dicaffeoylquinic acids were detected, compound 14 was not completely characterized being assigned as a dicaffeoylquinic acid hexoside.
Compound 59 (t R ¼ 32.3 min) gave a [M-H]ion at m/z 681. Fragmentation of this ion gave fragment ions at m/z 353 (base peak); 515 (67.0% of base peak); and 191 (12.0% of base peak), which led to the identification of a dicaffeoylquinic acid derivative. However, based only on these MS n data it was not possible to fully characterize compound 59 that was only detected in the SV total aerial parts extract.  16 5-CQA is more hydrophobic than 1-CQA, so 5-CQA derivatives should appear at a lower retention time than 1-CQA derivatives. Based only on MS n data the linkage position of the coumaroyl group could not be determined. Therefore, compounds 31 and 37 were characterized as coumaroyl 5-O-caffeoyl quinic acid and coumaroyl 1-O-caffeoyl quinic acid, respectively.

Coumaroylcaffeoylquinic acid
For compound 32 (t R ¼ 17.5 min), the ion at m/z 499 easily lost a caffeoyl moiety (162 Da) to form in the MS 2 spectrum a base peak ion at m/z 337. The MS 3 spectrum was similar to that described above for 5-O-p-coumaroylquinic acid (compound 13). The caffeoyl group must therefore be linked to the 4-OH position of quinic acid since an intense fragment ion at m/z 173 was detected in the MS 3 spectrum. It is known that the residues connected to the 5-OH position are more easily lost than those at the 4-OH position; however, that situation was not observed for this compound. Therefore, compound 32 was identified as 4-O-caffeoyl-5-O-p-coumaroylquinic acid.
Compound 45 (t R ¼ 24.9 min) exhibited a [M-H]ion at m/z 483. The MS 2 spectrum gave a fragment ion at m/z 337, which corresponds to the loss of 146 Da. A second loss of 146 Da was observed in the MS 3 spectrum forming the fragment ion at m/z 191. Comparing these results with literature data 18,19 it is possible to infer that compound 45 is a di-p-coumaroylquinic acid.
According to Clifford et al., 18 if the fragmentation of the ion at m/z 337 leads to a fragment ion at m/z 191, the linkage position of the p-coumaroyl group should be assigned to the 5-OH group. This type of fragmentation was observed for compound 13. The other p-coumaroyl group should be connected to the 1-OH position, which is more easily expelled forming the 5-O-p-coumaroylquinic acid residue. Therefore, compound 45 was identified as 1,5-di-O-pcoumaroylquinic acid.
Compound 38 (t R ¼ 20.4 min) showed a [M-H]ion at m/z 819 and was only detected in the SV leaves extract. The MS 2 spectrum showed a fragment ion at m/z 517 (loss of 302 Da) and the base peak in the MS 3 spectrum corresponds to a fragment ion at m/z 337 (loss of 150 Da). Fragmentation of this ion at m/z 337 gave characteristic ions of 5-p-coumaroylquinic acid. The available MS n data were not sufficient to identify the other residues. So, compound 38 was assigned as a 5-O-pcoumaroylquinic acid derivative.
Compound 5 yielded a [M-H]ion at m/z 341 and its MS 2 spectrum showed a base peak at m/z 179, resulting from the loss of 162 Da, which indicates the presence of a hexoside residue. The ion at m/z 179 is formed probably due to the presence of a caffeic acid residue. With no further information and comparing with literature data, 14 where the same fragmentation pattern was observed, compound 5 was assigned as a caffeic acid O-hexoside.
Compound 1 originated a [M-H]ion at m/z 683 as base peak, and an intense fragment ion at m/z 341. By means of MS 2 fragmentation, it was possible to deduce that the ion at m/z 683 is a dimer of the ion at m/z 341. MS n fragmentation of the MS 2 ion, at m/z 341, led to the identification of a similar pattern to compound 5.
Another caffeic acid hexoside derivative (compound 12) was found at a retention time of 8.0 min in the SV leaves extract. This compound showed a [M-H]ion at m/z 533. The base peak in the MS 2 spectrum is a fragment ion at m/z 371, due to the loss of 162 Da (hexoside moiety). The sequential MS n fragmentation and the detection of fragment ions at m/z 353 and 179 led to the identification of a caffeic acid residue.
Five more caffeic acid derivatives were found in Helichrysum melaleucum extracts. They gave very different MS n patterns but all had in common the fragment ion at m/z 179 [caffeic acid-H] -. Compound 7 (t R ¼ 5.5 min) exhibited a [M-H]ion at m/z 481. The MS 2 spectrum gave a fragment ion at m/z 445 due to the loss of 36 Da. In the MS 3 spectrum, the base peak is a fragment ion at m/z 221, but it showed also an intense fragment ion at m/z 179 (83.2% of base peak). In the MS 4 experiment only this ion was fragmented forming a fragment ion at m/z 101. Based on MS n data compound 7 was tentatively characterized as a caffeic acid derivative.
Compound 11 (t R ¼ 7.9 min) showed a [M-H]ion at m/z 367 and its fragmentation produced the fragment ion at m/z 179 as base peak. The MS 3 spectrum displayed a fragment ion at m/z 135 which corresponds to a loss of 44 Da (probably decarboxylation). 14 Compound 34 (t R ¼ 18.4 min) exhibited a [M-H]ion at m/z 625. The MS n experiments gave fragment ions at m/z 473, 341 and 179. This behaviour is similar to that described previously 4 for a caffeic acid derivative.
Compound 41 (t R ¼ 21.7 min) presented a [M-H]ion at m/z 529 and easily lost a 162 Da moiety (probably a hexoside) to form a base peak ion at m/z 367 in the MS 2 spectrum. The presence of this ion indicates a feruoylquinic residue, but with the MS n fragmentation the presence of a ferulic acid could not be confirmed. However, the base peak at m/z 179 in the MS 3 spectrum corresponds to a [caffeoyl-H]ion indicating that compound 41 is also a caffeic acid derivative.
Compound 48 (t R ¼ 26.9 min) exhibited a [M-H]ion at m/z 425 and the occurrence of a fragment ion at m/z 179, as base peak in the MS 3 spectrum, led to the identification of a caffeic acid derivative. Due to the long retention time of compound 48, it is possibly conjugated with another hydrophobic group.

Other compounds
Three other compounds that do not belong to the subclasses presented above were also identified.
Compound 10 (t R ¼ 7.2 min) was only detected in the SV leaves methanolic extract. This compound exhibited a [M-H]ion at m/z 463 and its fragmentation by MS 2 experiments showed a loss of 162 Da, probably due to a hexoside residue, forming a fragment ion at m/z 301. Further MS n fragmentation of this ion gave intense ions at m/z 283, 257 and 229, which are similar to those obtained for a standard solution of ellagic acid and described in literature data. 20 Ellagic acid belongs to the polyphenols, more precisely to hydroxyben-zoic acids that are commonly O-glycosylated. Hence, compound 10 was assigned as ellagic acid-O-hexoside.
Compound 18 (t R ¼ 11.1 min) belongs to the class of lignans and was only found in the FN total aerial parts extract. This compound exhibited a [M-H]ion at m/z 519. The MS 2 spectrum of this ion showed a fragment at m/z 357, indicating the loss of 162 Da, probably a hexoside moiety. The MS 3 spectrum of the ion at m/z 357 exhibited, as base peak, a fragment ion at m/z 151 that is assigned as a cleavage of a tetrahydrofuran ring. 12 In addition, fragment ions at m/z 342 and 327 were observed, indicative of successive losses of 15 Da from methyl groups. Based on these MS n data compound 18 was identified as pinoresinol-4-O-hexoside. It should be mentioned that natural furofuran lignans may exist as different stereoisomers but their configuration could not be assigned by MS n experiments.
Compound 43 (t R ¼ 22.8 min) was identified as ferulic acid. This compound exhibited a [M-H]ion at m/z 193 and its MS n fragmentation showed fragment ions at m/z 178 (loss 15 Da), 163 (loss 2 Â 15 Da) and 135 (loss 2 Â 15Da þ 28 Da). This fragmentation pattern matches the one observed for a standard solution of ferulic acid. The only extract where it was possible to find this compound was in SV flowers.
Compound 52 (t R ¼ 28.6 min) showed a [M-H]ion at m/z 409 and its MS 2 fragmentation gave a fragment ion at m/z 163 which indicates the presence of a coumaric acid moiety. The fragmentation of this ion at m/z 163 formed a fragment ion at m/z 119 (loss of 44 Da) and is similar to that of a standard solution of coumaric acid (MS n data not shown). However, based only on these data it was not possible to completely characterize compound 52, which was tentatively assigned as a coumaric acid derivative.
Compound 68 (t R ¼ 39.0 min) exhibited a [M-H]ion at m/z 329 and was only detected in the SV leaves methanolic extract. Under MS 2 fragmentation, the ion at m/z 329 gave a fragment ion at m/z 314 due to the loss of 15 Da. The MS 3 spectrum of this ion produced two very intense fragment ions at m/z 271 (base peak) and 299 (ca. 99.9% of base peak). Comparing these results with literature data, 21 compound 68 was identified as 1,2,6-trihydroxy-7,8-dimethoxy-3-methylanthraquinone.

Unknown compounds
Other peaks were detected although the elucidation of their structures based only on the MS n data was not completely achieved.
Compounds 36 (t R ¼ 19.8 min) and 42 (t R ¼ 21.9 min) exhibited a [M-H]ion at m/z 457. The MS n fragmentation behaviour was identical for both compounds. The MS 2 spectra showed a fragment ion at m/z 260 indicating the loss of 197 Da. Further fragmentation gave fragment ions at m/z 231 and 151. However, it was not possible to identify their structures.
Compound 67 (t R ¼ 37.0 min) gave a [M-H]ion at m/z 599 and was only detected in the SV leaves extract. In the MS 2 fragmentation, two successive losses of 162 Da were observed, probably due to hexoside residues. Nevertheless, with no other information available, it was not possible to identify the nature of this compound.

CONCLUSIONS
Phenolic compounds present in Helichrysum melaleucum were analyzed, for the first time to our knowledge, by a LC-DAD/ESI-MS n method. By the analysis of the different morphological parts of plants collected in São Vicente (SV) it is possible to conclude that the flowers extract revealed a larger number of compounds, most of them flavonoids substituted with glycosides and/or acyl groups.
A comparison was made for the total aerial parts methanolic extracts collected in different geographical locations. Plants collected at higher altitude, Fajã da Nogueira (FN), showed a much higher variety of phenolic compounds. Despite belonging to the same subspecies, the phenolic compositions of these two extracts were significantly different and some substances, such as pinoresinol (compound 18) and flavone derivatives (compounds 56 and 66), can be used as geographical markers.