Analysis of phenolic compounds from different morphological parts of Helichrysum devium by liquid chromatography with on-line UV and electrospray ionization mass spectrometric detection.

A simple and rapid method has been used for the screening and identification of the main phenolic compounds from Helichrysum devium using high-performance liquid chromatography with on-line UV and electrospray ionization mass spectrometric detection (LC-DAD/ESI-MS(n)). The total aerial parts and different morphological parts of the plant, namely leaves, flowers and stems, were analyzed separately. A total of 34 compounds present in the methanolic extract from Helichrysum devium were identified or tentatively characterized based on their UV and mass spectra and retention times. Three of these compounds were positively identified by comparison with reference standards. The phenolic compounds included derivatives of quinic acid, O-glycosylated flavonoids, a caffeic acid derivative and a protocatechuic acid derivative. The characteristic loss of 206 Da from malonylcaffeoyl quinic acid was used to confirm the malonyl linkage to the caffeoyl group. This contribution presents one of the first reports on the analysis of phenolic compounds from Helichrysum devium using LC-DAD/ESI-MS(n) and highlights the prominence of quinic acid derivatives as the main group of phenolic compounds present in these extracts. We also provide evidence that the methanolic extract from the flowers was significantly more complex when compared to that of other morphological parts.

Plants of the genus Helichrysum belong to the Asteraceae family, a name originating from the Greek words helios (sun) and chrysos (gold) that reflect the attractive yellow flowers displayed by several species of these genus. 1 This genus comprises more than 500 species mainly distributed in South Africa, although many endemic species can be found in southern Europe, south-west Asia, southern India, Sri Lanka and Australia. Several studies performed on Helichrysum species showed that they have a wide range of biological activities, such as antimicrobial, anti-inflammatory, antiallergic, relief abdominal pain, heart burn, cough, cold and wounds. 2 In Madeira Archipelago (Portugal) there are four endemic species together with several imported species of Helichrysum that are mostly used in horticulture and in folk medicine, especially in rural areas. Helichrysum devium Johns., the subject of this investigation, is one of those endemic subspecies that is used in folk medicine against respiratory diseases, such as bronchitis and pharyngitis. This plant faced near extinction due to massive collection of wild specimens. Fortunately, a successful programme of green house reproduction has facilitated its re-introduction in its natural habitat on the rocky slopes of the south-east cost of the island of Madeira.
The pharmacological activities of Helichrysum plants have been associated to several classes of compounds such as flavonoids, phloroglucinols, a-pyrones, coumarins and terpenoids which have been previously described. 3 Previous studies have reported the occurrence of quinic acid derivatives esterified with one to three residues of caffeic acid. 2,4 A few studies using analysis by liquid chromatography with diode-array detection coupled with mass spectrometric detection (LC-DAD/MS n ) also described the characterization of phenolic compounds from Helichrysum species. Carini et al. 2 studied Helichrysum stoechas and found the presence of some phenolic compounds, namely caffeoylquinic acid and flavonol derivatives, with potent antioxidant properties.
Phenolic compounds are a class of low molecular weight compounds which are secondary metabolites synthesized by the plants during normal development and in response to stress conditions like infection, wounding and UV radiation. 5 These compounds are not only associated with the colour, flavour and taste in many plants, but are also reported to have valuable medicinal properties such as protection against cancer, cardiovascular and neurodegenerative dis-eases. 6 For these reasons, many studies have been performed in order to identify and characterize phenolic compounds from natural sources.
The main classes of phenolic compounds are flavonoids and phenolic acids (e.g. hydroxybenzoic and hydroxycinnamic acids).
Flavonoid conjugates represent a very large and diverse group of phenolic compounds with similar structure having a common C6-C3-C6 flavone skeleton. 7 In cell plants, flavonoids may occur in modified forms corresponding to additional hydroxylation, methylation and/or glycosylation. It is also possible to have aromatic and aliphatic acids, sulfate, prenyl, methylenedioxyl or acyl groups also attached to the flavonoid skeleton or its glycoside moieties. Flavonoid glycosides are the most common phenolic compounds and are divided according to the site of the flavonoid aglycone where the sugar moiety is attached. O-Glycosides have glycoside groups connected to hydroxyl groups while in the C-glycosides the sugar bond connects the carbon atoms in ring A.
Since phenolic compounds are usually found as complex mixtures in plant extracts, efficient and selective analytical methods are required to analyze them. Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) has proved to be a very powerful tool for the analysis of these compounds. According to several studies, using either APCI or ESI, the negative ionization mode typically provides enhanced sensitivity and yields complementary information. However, detection in the positive ion mode provides useful structural information for MS/MS characterization. A comparison between ESI and APCI indicated that the latter ionization mode is favoured for the analysis of phenolic compounds of plant extracts due to its higher ionization efficiency and selectivity for these compounds. [8][9][10] The mass spectra of flavonoids obtained with quadrupole and ion-trap instruments are typically very similar, even though differences in the relative abundances of fragment and adduct ions have been noted. 11 The aim of this work was to characterize by LC-DAD/ESI-MS n the main phenolic compounds present in the methanolic extracts of Helichrysum devium. Since the use of this plant in folk medicine shows variation in activity depending on the parts of the plant used (flowers only, leaves only or total aerial parts), it is important to evaluate the distribution of phenolic compounds in different morphological parts and correlate their contribution to the biological activity. As part of this study we used negative ion mode in LC/MS and LC/ MS n analysis. A total of 34 compounds were identified or tentatively characterized, including flavonoids and quinic acid derivatives. This work represents a first detailed analysis on the distribution and characterization of these bioactive compounds from the different parts of the plant.
HPLC grade acetonitrile (CH 3 CN) (Lab-Scan, 99%) and ultra-pure water (Milli-Q, Waters) were used for all analysis. The methanol used for extraction of Helichrysum devium was AR grade, purchased from Fisher. Eluents prepared for LC/ MS analysis (formic acid 0.1%, v/v) were additionally filtered through a 0.45 mm membrane (Millipore).

Sample preparation
Samples of Helichrysum devium were collected in the wild and identified by taxonomist Fátima Rocha and a voucher was deposited in the Madeira Botanical Garden Herbarium collection. Dried and powdered plant material (total aerial parts, 100 g) was exhaustively extracted by maceration with methanol (1 L), at room temperature for 24 h, yielding 8.64 g of dry extract.
For assessment of morphological parts, the leaves, flowers and stems of the plant were collected, dried and powdered separately. Each sample was extracted sequentially with four solvents of increasing polarity (n-hexane, chloroform, ethyl acetate and methanol), 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). At this stage only methanolic extracts were used for the LC-DAD/ESI-MS n analysis. Stock solutions with concentrations (w/v) of 5 mg/mL were prepared by dissolving dried extract in initial HPLC mobile phase (ACN/ H 2 O, 20:80).
These solutions were filtered through 0.45 mm micropore membranes prior to use and volumes of 10 mL were injected for LC-DAD/ESI-MS n analysis. Three independent assays were performed for each sample.

LC conditions
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 acetonitrile (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 spectrometric conditions
For LC/ESI-MS 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 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
Three independent assays were performed for the analysis of the methanolic extracts from Helichrysum devium by LC-DAD/ESI-MS n and no relevant variation were observed that can be related to the nature of detected fragments and their relative intensities.
The base peak chromatogram profiles of the four methanolic extracts under analysis are shown in Fig. 1. As can be seen, the majority of compounds could be well separated.
Whenever possible, the HPLC retention time, UV and mass spectra of detected compounds were compared with reference standards. Because only a few reference compounds were available, structures of unknown compounds were characterized based mainly on their own MS n fragmentation behaviour, on retention times and on studies of their UV spectra.
The UV profile and spectral similarities were useful characteristics for the establishment of classes of detected compounds. The hydroxycinnamic acid derivatives showed two maximum absorption bands at 230-240 nm and 320-330 nm, together with a shoulder around 300-310 nm. Flavonols and their glycosides exhibited two maximum absorptions at 250-270 nm and 320-360 nm, derived from the aglycone A and B rings, respectively. Peaks corresponding to flavone glycosides showed three absorptions at 210-230 nm, 250-280 nm and 330-350 nm.
The structures were further and more fully characterized based on their MS n fragmentation behaviour. MS n fragmentation ions of the compounds detected in all extracts are presented in Tables 1-4 and their chemical structures are shown in Fig. 2.
An essential step in the LC-DAD/ESI-MS n analysis was to determine the molecular weight of each detected compound. Most of the phenolic compounds gave deprotonated molecular ions [M-H] À of high abundance, which allowed them to undergo MS n analysis. Usually, the most abundant peak in a full MS spectrum was assigned to [M-H] À and this assignment was more consistent if adduct ions and dimers were present. 12 Among the identified compounds, there were hydroxycinnamic acids, flavonoids (flavonol and flavone type), caffeic acid and a protocatechuic acid derivative.
Identification will be presented in the next subsections, grouping the compounds by the nature of the respective aglycones. Compounds were numbered by their order of elution and this numeration was kept identical for all samples. Some of the compounds were present in all analyzed extracts while some were absent from one or more morphological parts.

Identification of hydroxycinnamic acid derivatives
In this work several hydroxycinnamic acid derivatives were identified by LC-DAD/ESI-MS n experiments and their chemical structures and identification are presented in Fig. 2 and Table 5, respectively. The deprotonated molecular ion ([M-H] À ) was abundantly produced under the MS n conditions for all hydroxycinnamic acid derivatives and the loss of the substitution groups is always referred to in respect to this ion.
Mono-, di-and tricaffeoylquinic acids (1,4,5,9,10,11,21) Compound 1 occurred at a retention time of 3.1 min and exhibited a [M-H] À ion at m/z 191. Its MS 2 fragmentation produced a [M-H-CO-2H 2 O] À ion at m/z 127 as base peak; a [M-H-H 2 O] À ion at m/z 173 was also observed. Compound 1 was identified as quinic acid, taking into account its MS n fragmentation pattern and literature data. 13 It was reported previously 14 that the linkage position of acyl groups on quinic acid could be determined by the analysis of the [M-H] À ion MS 2 fragmentation. In general, the [quinic acid-H] À ion at m/z 191 appears as the base peak when the acyl group is linked to the 3-OH or 5-OH position; these two isomers can be further differentiated since the [caffeic acid-H] À ion at m/z 179 is more significant for 3-OH compounds. When the acyl group is connected to 4-OH, the [quinic acid-H 2 O-H] À ion at m/z 173 will appear as the base peak. 14,15 Identification of the detected quinic acid derivatives was performed based on these assumptions and by using the hierarchical key for the identification by LC/MS n of caffeoylquinic and dicaffeoylquinic acids derivatives proposed by Clifford et al. 16 Compound 4 (t R ¼ 5.0 min) was unequivocally identified as 5-O-caffeoylquinic acid (chlorogenic acid) by comparison of the retention time and mass spectra with those of a reference standard. This compound displayed a [M-H] À ion at m/z 353, and its MS 2 spectrum gave a [quinic acid-H] À ion at m/z 191 as the base peak and a [caffeic acid-H] À ion at m/z 179 (weak ion, ca. 3% of the base peak). The occurrence of 5-O-caffeoylquinic acid in plants of the Helichrysum genus is very common. 2 In addition to the monocaffeoylquinic acid, several dicaffeoylquinic acid (diCQA) isomers and a tricaffeoylquinic acid (triCQA) were identified in Helichrysum devium.
Compounds 5, 9, 10 and 11 all gave molecular ions [M-H] À at m/z 515; their fragmentation in MS 2 spectra gave, as the base peak, a [M-H-162] À ion at m/z 353, indicating the presence of more than one caffeoyl group linked to different hydroxyl groups.
However, their MS 3 and MS 4 spectra of the m/z 353 ions were significantly different. The ion at m/z 191 was observed as the base peak for compound 5, 10 and 11, but the ion at m/z 173 was the base peak for compound 9 which, as mentioned above, indicates the presence of a 4-OH-substituted quinic acid.
According to the literature, 15 it is possible to distinguish the 3,4-diCQA from the 4,5-diCQA since the two isomers differ in the intensity of the MS 2 'dehydrated' ion at m/z 335 . For 3,4-diCQA, the peak at m/z 335 is more intense ($15% of base peak). In contrast, for 4,5-diCQA this ion is barely detectable (<5% of base peak). The MS 2 spectrum of compound 9 exhibited a secondary ion at m/z 335 ($13% of base peak), thus compound 9 was plausibly identified as 3,4-O-dicaffeoylquinic acid.
It has been reported that 3,4-O-dicaffeoylquinic acid is more easily eluted from the reversed-phase column when compared with 3,5-O-dicaffeoylquinic acid. Based on this information and comparing its MS n spectra and fragment intensities with the literature data, 15 compound 11 was identified as 3,5-O-dicaffeoylquinic acid.
Compound 21 appeared at a retention time (t R ) of 29.2 min and displayed a [M-H] À ion at m/z 677, easily losing a caffeoyl moiety (162 Da) to form a base peak ion at m/z 515 in the MS 2 spectrum. MS 3 and MS 4 spectra were identical with those described above for 3,4-diCQA (compound 9). So, it can be inferred that compound 21 is either 1,3,4-triCQA or 3,4,5-triCQA. As, in general, CQAs with a larger number of free equatorial hydroxyl groups in the quinic acid residue are more hydrophilic than those with a larger number of free axial hydroxyl groups, 16 the long retention time of this compound suggests a hydrophobic compound. Thus, compound 21 was identified as 3,4,5-triCQA.
All compounds mentioned above were found in all analyzed methanolic extracts.
Malonylcaffeoylquinic acid (12,13,14) Compounds   For all compounds, the MS 2 fragmentation of the deprotonated molecular ion led to the formation of an ion at m/z 395 (base peak) due to the loss of 206 Da (acetylcaffeoyl). Based on the occurrence of this fragment, it is possible to deduce that the malonyl group is attached to one caffeoyl group instead of being linked to the quinic acid structure. To our knowledge, this is the first time that this linkage is described for malonylcaffeoylquinic acid derivatives.
The base peak in all the MS 3 spectra was a [M-H-44-162-162] À ion at m/z 233 assigned to acetylquinic acid, as previously described by Zhang et al. 17 This acetylation can stabilize the ring structure of quinic acid, which was confirmed by the non-observation of ions corresponding to ring fragmentation.
The malonyl group should be attached to the caffeoyl group at the 3-OH position of the quinic acid structures. This evidence is supported by fragmentation of the ion at m/z 395, where a fragment at m/z 173 ($25%) is observed. This ion is due to the loss of a caffeoyl group linked to the 4-OH position.
As already mentioned, compounds 12 and 14 showed a similar fragmentation pattern when compared to compound 13, but it was not possible to fragment the [M-H-86] À ion in order to establish the exact position where the caffeoyl moieties are attached. However, the occurrence of an ion at m/z 173 as the base peak in the MS 4 spectrum indicates the presence of a 4-OH linkage position in the quinic acid structure.
According to the rules for diCQA, 15 it was assumed that malonylcaffeoylquinic acid isomers have the same order of elution. So, accepting that 3,4-diCQA is more easily eluted from the reversed-phase column than 4,5-diCQA, compounds 12, 13 and 14 were identified as malonyl-1,4-Odicaffeoylquinic acid, malonyl-3,4-O-dicaffeoylquinic acid and malonyl-4,5-O-dicaffeoylquinic acid, respectively. These three compounds were detected in all extracts, with the exception of compound 12 which was not detected in the flowers extract.
Caffeoylferuloylquinic acid (16,26) It was possible to identify two feruloylquinic acid derivatives (compound 16 and 26) in the four analyzed extracts. Identification of compound 16 was tentatively made by referring to the hierarchical key developed by Clifford et al. 15 Since the MS 3 spectrum displayed an ion at m/z 191 as the base peak, this compound should be a 3-OH-or 5-OHsubstituted quinic acid. If it was a 3-OH-substituted compound, the peak abundance at m/z 179 should be above 50% of the base peak, which it is not observed in this case, so compound 16 was plausibly identified as 1-O-caffeoyl-5-Oferuoylquinic acid.
Compound 26 (t R ¼ 21.9 min) yielded a different fragmentation behaviour when compared with compound 16. The Identification of flavonoids compounds (7,8,17,19,20,22,24,25,27,28,29, and 30) The present work led to the identification and characterization of a number of flavonoids with aglycones belonging to two subtypes: flavonols (quercetin, isorhamnetin and kaempferol) and flavones (luteolin and apigenin) (Fig. 2). Nearly all flavonoids were identified as glycosides contain- ing one or more sugar moieties and some were esterified with acyl groups. The MS n fragmentation of these phenolic compounds showed the deprotonated molecular ion ([M-H] À ) and the deprotonated aglycone ion (Y À 0 ) as a result of the loss of the sugar residue. The presence of hexoside, rhamnose, malonyl and glucunoride moieties was characterized by neutral losses of 162, 146, 146 and 176 Da, respectively. The flavonoid fragment ions were designated according to the nomenclature proposed by Ma et al. 18 (Fig. 4). For free aglycones, the i,j A À and i,j B À labels correspond to ions containing intact A-and B-rings, respectively, in which i and j indicate the C-ring bonds that have been broken (Fig. 4). For conjugated aglycones, Y À 0 is used to refer to the aglycone fragment [M-H-glycoside] À .
Most of the identified flavonoids were exclusively detected in the flowers extract (7,8,17,19,24,25,27,28,29 [19][20][21] Comparing these MS n data with the fragmentation of a standard quercetin solution (data not showed) it is possible to observe that they are very similar and so quercetin should be the aglycone of compound 7.
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. 10 Even so, based only on MS n data, neither the nature of the hexoside residue nor the sugar linkage position to the aglycone could be determined. Thus, compound 7 was preliminary characterized as a quercetin-O-hexose. For both compounds the MS 3 spectra gave a base peak ion at m/z 301, corresponding to the deprotaned aglycone (Y 0 À ), due to the loss of an hexoside residue: the corresponding aglycone radical ion [Y À 0 -H] À at m/z 300 (< 30% of the base peak) was also observed.
Flavonols substituted at 3-OH position should present relative high intensity aglycone radical fragment sometimes higher than the Y À 0 ion. 23 Such a pattern was not observed for compounds 17 and 27; thus the glycosylation site cannot be surely confirmed. As mentioned above, either a malonyl or a rhamnosyl group could be attached to the hexoside residue but, based only on the MS n data, it is hard to clearly make the attribution of either to compound 17 or 27. However, it has been reported that, generally, flavonoid glycosides esterified The fragmentation showed a loss of 44 Da, which indicates a decarboxylation from a dicarboxylic acid linked to the flavonoid glycoside. The MS 3 spectrum showed a base peak ion at m/z 505 (Y 7À 0 ) originating from the loss of a hexoside moiety (162 Da) and also a very intense peak at m/z 301 (Y 3À 0 ) (Fig. 5).
This type of fragmentation (Scheme 2), in which the loss of a sugar unit gives the most abundant base peak different from the base peak of the aglycone, indicates that there is a glycosylation in more than one phenolic hydroxyl group of the aglycone. 25 The fragmentation of the ion at m/z 505 yielded the aglycone fragment ion at m/z 301, by the loss of 204 Da from the decarboxylated malonyl group linked to the The glycosylation sites were established attending to the guidelines presented by Ablajan et al. 9 In the MS 4 spectrum, the intensity of the fragment Y À 0 is higher than that of fragment [Y À 0 -H] À , which implies a cleavage of an hexoside group at the 3-OH position. Therefore, the first sugaraglycone bond to cleave is at the 7-OH position.
The exact location of the malonyl group on the hexoside part is difficult to define on the basis of obtained MS n data, but it appears to be predominantly located at the 6-position of a hexoside moiety. 10 According to these MS n data, compound 20 was plausible identified as quercetin-7-Ohexoside-3-O-(malonyl)hexoside.
Compound 22 (t R ¼ 30.3 min) exhibited a [M-H] À ion at m/z 629 and was identified as being a quercetin-O-hexoside derivative based on the MS n fragmentation. The MS 2 spectrum showed a base peak ion at m/z 463, which corresponds to the loss of 166 Da (this fragment could not be identified based in the available data). The fragmentation of the ion at m/z 463 led to the formation of the same fragment ions detected for compound 7.
Compounds 29 (t R ¼ 28.3 min) and 30 (t R ¼ 29.7 min) exhibited a very similar MS n pattern and gave a molecular ion [M-H] À at m/z 593. Their MS 2 spectra contained a base peak ion [M-H-146-162] À at m/z 285 and a [M-H-146] À ion at m/z 447 ($10% of the base peak). As already known, 26 the neutral loss of 146 Da is characteristic of a coumaroyl group which was confirmed by the formation of a [coumaroylhexose-H] À ion at m/z 307. According to these considerations compounds 29 and 30 were preliminarily characterized as acylated flavonoid glycosides.
The peak at m/z 285 corresponds to the aglycone (Y 0 À -H) and its MS n spectra showed a (Y À 0 -H-CO) ion at m/z 257, a (Y 0 À -2CO) ion at m/z 229 and, as base peak, an ion at m/z 151 ( 1,3 A À ), produced from a RDA reaction. 14 These RDA fragments are consistent with those found for a standard solution of kaempferol (MS n fragmentation data not shown).
Theoretically, any of the kaempferol hydroxyl groups can be glycosylated, although certain positions are favoured: the 3-OH and 7-OH are the most common glycosylated positions.
As stated before, for flavonols glycosylated at the 3-OH position, the relative abundance of radical aglycone ion ([Y À 0 H] À. ) is very pronounced. 23 However, this radical fragment was detected for both compounds but with a very low relative intensity ($4% of the base peak). So, glycosylation at the 3-OH position is not evident, leaving the 7-OH and 4 0 -OH positions as the most probable sites of glycosylation for these compounds. The 5-OH position is also available but 5-O-glycosides are very rare for compounds with a carbonyl at position 4, since the 5-OH group participates in hydrogen bonds with the adjacent 4-carbonyl group. 10 As already mentioned, compounds 29 and 30 have an acyl group in their structures, but the exact location of the acyl group on the hexoside moiety is difficult to define based only on MS n data. Acyl groups are predominantly located at the 6-position of a hexoside moiety, 21 but only when a 0,4 X fragment is present in the spectrum can the location at the 6-position be confirmed, which did not happen in this particular case.
With no further information, it was assumed that compounds 29 and 30 are kaempferol 7-O-coumaroylhexoside and kaempferol 4 0 -O-coumaroylhexoside. . This compound was identified as luteolin by comparison of its MS n fragmentation pattern with that of a reference standard (data not shown) and literature data. 19 Compound 24 (t R ¼ 11.0 min) exhibited a [M-H] À ion at m/z 461. When submitted to further fragmentation this ion readily eliminated a glucuronic acid residue (observed by the loss of 176 Da) to produce the deprotonated aglycone ion Y 0 À at m/z 285. The glucuronic acid residue was confirmed by the MS 2 ions at m/z 357 and 327. The MS 3 spectrum of the aglycone ion gave fragments at m/z 243, 217, 199 and 175, characteristic ions of luteolin as described above. The favoured substitution position for flavones, like luteolin, is Identification of a protocatechuic and caffeic acid derivatives (18, 15,  Fragmentation of the ion at m/z 237 gave an ion at m/z 153 that could possibly be from a protocatechuic acid unit. 28 However, the intensity of this fragment was not enough to perform further fragmentation in order to confirm the presence of protocatechuic acid. Compound 18 was thus speculatively classified as a protocatechuic acid derivative; it is present in trace amounts only in the total plant extract. Compound 15 (t R ¼ 18.2 min) was identified as a caffeic acid derivative, based on the MS n pattern of fragmentation. It showed a [M-H] À ion at m/z 625 which when fragmented led to the formation of a product ion at m/z 473 (loss of 152 Da). Further fragmentation of this ion produced a MS 3 spectrum with a base peak at m/z 341 that corresponds to the loss of 132 Da, probably resulting from neutral loss of a pentose (arabinose, xylose or apiose) or a tartaric acid unit. The ion at m/z 341 has already been assigned to caffeic acid hexoside, which was confirmed by the fragment ion at m/z 179 [caffeic acid-H] À obtained in the MS 4 spectrum. 24 It is noteworthy that this compound was not detected in the flowers extract but was present in all the other morphological parts.
Both compounds 32 (t R ¼ 26.4 min) and 33 (t R ¼ 27.9 min) showed [M-H] À ions at m/z 583 and they have similar MS 2 spectra with a base peak at m/z 421, resulting from the neutral loss of 162 Da. However, the MS 3 and MS 4 spectra of these two compounds are quite different. For compound 32, the fragment ion at m/z 421 readily loses 162 Da to produce an ion at m/z 259, which when fragmented in MS 4 gave a peak at m/z 173. The nature of the aglycone could not be determined by these MS n results only; however, it is clear that there is successive loss of two residues of 162 Da, probably hexosides.
For compound 33, the MS 3 spectrum of the ion at m/z 421 exhibited a base peak at m/z 353 and several peaks with high relative intensity at m/z 335 (74.5%), 259 (72.5%), 179 (43.2%) and 173 (53.6%). The MS 4 spectrum of the fragment at m/z 353 exhibited as base peak a fragment at m/z 179 and a very intense peak at m/z 173 (95.7% of the base peak). The fragment ion at m/z 179 indicates the presence of a caffeic acid derivative but no other identification can be performed based on the available data. Therefore, compound 33 was characterized as a caffeic acid hexoside derivative.
Unidentified compounds (2,3,6,23,34) Other peaks were observed and denominated as compounds 2, 3, 6, 23 and 34. However, the elucidation of their structures based solely on MS n data has not been completely reached yet.
At a retention time of 7.5 min we observed an intense peak that exhibited a [M-H] À ion at m/z 429. The MS 2 spectrum showed an ion at m/z 393, resulting from the loss of 36 Da. MS n fragmentation gave ions at m/z 149 (loss of 244 Da) and 131 (loss of 18 Da due to a molecule of water). This peak was designated as compound 6 and showed three maximum absorptions at 230-245, 280-300 and 340 nm. Nevertheless, it was not possible to identify its structure. It must be mentioned that this compound was found in all plant extracts with the exception of the stems extract.
Compound 23 (t R ¼ 34.8 min) gave a [M-H] À ion at m/z 331 and additional fragmentation formed an ion at m/z 155 which corresponds to the loss of 176 Da (probably a glucuronide residue). The MS 3 and MS 4 spectra showed sequential losses of 15 Da that indicates the presence of methyl groups.

CONCLUSIONS
A simple and sensitive LC-DAD/ESI-MS n method has been used for the comprehensive separation and identification of phenolic compounds in different morphological parts of Helichrysum devium. Abundant [M-H] À ions were observed in ESI-MS n negative mode, and were used to identify molecular masses of the detected compounds. A total of 34 compounds found in the total aerial parts, leaves, flowers and stems were characterized or tentatively identified based on the MS n fragmentation behaviour, UV spectra and retention times. Positive identification was facilitated for three of these compounds using authentic standards.
Quinic acid derivatives were found to be the major constituents of Helichrysum devium extracts analyzed. A 206 Da neutral loss from [M-H] À ions of malonylcaffeoylquinic acid isomers was explored for the first time by our LC-DAD/ ESI-MS n method, and indicated that the malonyl group is attached to one caffeoyl group rather than being linked to the quinic acid structure.
The flowers extract revealed the presence of a much higher variety of phenolic compounds, namely flavonoids, most of them as glycosides and/or esterified with acyl groups. A large number of compounds were described for the first time in Helichrysum species using LC/MS n as an analytical tool. The antimicrobial and antioxidant properties of these extracts have been investigated and will be reported elsewhere.