Liquid chromatographic/electrospray ionization tandem mass spectrometric study of the phenolic composition of cocoa (Theobroma cacao)

Liquid chromatography coupled with ionspray mass spectrometry in the tandem mode (LC/MS/MS) with negative ion detection was used for the identification of a variety of phenolic compounds in a cocoa sample. Gradient elution with water and acetonitrile, both containing 0.1% HCOOH, was used. Standard solutions of 31 phenolic compounds, including benzoic and cinnamic acids and flavonoid compounds, were studied in the negative ion mode using MS/MS product ion scans. At low collisional activation, the deprotonated molecule [M - H](-) was observed for all the compounds studied. For cinnamic and benzoic acids, losses of CO(2) or formation of [M - CH(3)](-*) in the case of methoxylated compounds were observed. However, for flavonol and flavone glycosides, the spectra present both the deprotonated molecule [M - H](-) of the glycoside and the ion corresponding to the deprotonated aglycone [A - H](-). The latter ion is formed by loss of the rhamnose, glucose, galactose or arabinose residue from the glycosides. Different fragmentation patterns were observed in MS/MS experiments for flavone-C-glycosides which showed fragmentation in the sugar part. Fragmentation of aglycones provided characteristic ions for each family of flavonoids. The optimum LC/MS/MS conditions were applied to the characterization of a cocoa sample that had been subjected to an extraction/clean-up procedure which involved chromatography on Sephadex LH20 and thin-layer chromatographic monitoring. In addition to compounds described in the literature, such as epicatechin and catechin, quercetin, isoquercitrin (quercetin-3-O-glucoside) and quercetin-3-O-arabinose, other compounds were identified for the first time in cocoa samples, such as hyperoside (quercetin-3-O-galactoside), naringenin, luteolin, apigenin and some O-glucosides and C-glucosides of these compounds.     

Liquid chromatography/electrospray ionization mass spectrometry for the characterization of twenty-three flavonoids in the extract of Dalbergia odorifera

A method incorporating high-performance liquid chromatography (HPLC) with electrospray ionization and tandem mass spectrometry, with parallel analysis by HPLC with UV detection using a diode-array detector, was developed for the qualitative characterization of flavonoids in D. odorifera. Twenty-three flavonoids, including six isoflavones, six neoflavones, four isoflavanones, three flavanones, two chalcones, one isoflavanonol and one pterocarpan, were unambiguously identified by comparing their retention times, UV and MS spectra with those of authentic compounds. Furthermore, the collision-induced dissociations of the [M-H]- ions were studied to clarify the MS behavior of the different types of flavonoids. In negative ion ESI-MS all the flavonoids yielded prominent [M-H]- ions in the first order mass spectra. Fragments involving losses of CH3*, H2O, CO, C2H2O, and CO2 were observed in the MS/MS spectra. Each of the seven types of flavonoid showed characteristic MS/MS fragmentation patterns. The isoflavanones, flavanones and chalcones were observed to undergo retro-Diels-Alder fragmentations. The spectra of almost all the neoflavonoids unexpectedly exhibited only [M-H-CH3]-* radical anions as base peaks without any further fragmentation. Substitution positions also remarkably influenced the fragmentation behavior, which could assist in distinction among the flavonoid isomers. The fragmentation rules deduced here could aid in the characterization of other flavonoids of these types.     

Characterization of acylated flavonoid-O-glycosides and methoxylated flavonoids from Tagetes maxima by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Liquid chromatography coupled to negative electrospray ionization (ESI) tandem mass spectrometry (MS/MS) employing a triple quadrupole mass spectrometer was used in the structural determination of acylated flavonoid-O-glycosides and methoxylated flavonoids occurring in Tagetes maxima. The compounds were identified by experiments in full scan mode (MS), and tandem mass experiments (MS/MS) of precursor ion scan, product ion scan, and neutral loss scan modes. In order to characterize the aglycones of the flavonoid glycosides, in-source fragmentation of the deprotonated molecule [M-H]- followed by product ion scan of the resulting aglycone [A-H]- were performed. This combined approach allowed the identification of 51 phenolic compounds, including flavonoid-O-glycosides acylated with galloyl, protocatechuoyl, coumaroyl or caffeoyl groups, methoxylated flavonoids, and hydroxycinnamic acid and phenolic acid derivatives, none of them previously reported in Tagetes maxima.     

Analysis of minor flavonoids in Piper hostmannianum var. berbicense using liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

The fragmentations of hydroxylated flavanones, chalcones and dihydrochalcones were investigated by direct loop injection using an ion trap mass spectrometry equipped with atmospheric pressure chemical ionization (APCI) probe. Some of them have been isolated from the leaves of Piper hostmannianum var. berbicense and standards were used to confirm their fragmentation behaviour. In negative ion mode, fragmentations of these three types of flavonoids revealed specific diagnostic ions which allowed us to identify aglycones in a crude plant extract. The major fragment ion obtained in MS/MS experiment for methoxylated chalcones is the neutral loss of a methyl radical whereas a H(2)O molecule is lost in the case of methoxylated dihydrochalcones. Methoxylated chalcones and flavanones isomers could be differentiated by the relative intensity ratio of [M-H-CH(3)]*(-) and [M-H-C(2)H(2)O](-) ions. Based on UV and MS data, a decision tree that includes UV lambda(max) absorptions and MS/MS diagnostic ions was built in order to obtain structural information of unknown compounds present in the extract. This tree was used to identify flavonoids in the ethyl acetate extract of P. hostmannianum var. berbicense leaves after analysis by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionization ion trap multistage mass spectrometry. A total of 11 flavonoids were tentatively characterized based on the MS fragmentations pattern observed in MS(n) experiments.     

Electrospray tandem mass spectrometry of 2H-chromenes

Several 2H-chromenes derived from carbazoles were analyzed by electrospray tandem mass spectrometry. The 2H-chromenes constitute an important class of compounds that exhibit photochromic activity. The fragmentation pathways of the protonated molecular species [M+H]+ were studied, and main fragmentation pathways of these compounds were identified. Fragmentation pathways of [M+D]+ ions were also studied in order to obtain information about the location of the ionizing proton or deuteron. It was found that the proton is not preferentially located on the nitrogen atom. The charge is preferentially located as a tertiary carbocation, resulting from the uptake of the proton (or deuteron) by the zwitterionic open structure of the chromenes. The major fragmentation occurred by cleavage of the gamma-bond relative to the carbocation center, leading to a fragment at m/z 191 (C5H11+ or C14H9N+), which are the most abundant fragment ions for almost all compounds. The presence of substituents in the chromene ring does not change this behavior. Other observed common fragmentation pathways included loss of CH3* (15 Da), loss of CO (28 Da), combined loss of CO and CH3 (43 Da), and loss of the phenyl ring via combined loss of C6H4 and CH3* (-91 Da) and combined loss of C6H6 and CO (-106 Da).     

MALDI‐MS of flavonoids: a systematic investigation of ionization and in‐source dissociation mechanisms

Matrix assisted laser desorption ionization (MALDI) is a technique widely employed in the analysis of proteins and peptides, and nowadays it has also been applied to small molecules. There is little significant information regarding the in‐source dissociation processes on MALDI for natural products. Twenty‐six flavonoids (flavanones, flavones and flavonols) were analyzed by MALDI using different methods (with different matrices) and without matrix to comprehend the in‐source reactions and establish good analysis methods for these compounds. Depending on the class, structure and the laser intensity applied, methoxylated flavonoid aglycones can eliminate methyl radicals (˙CH₃) in the source, such as flavonols, but lithium 2,4‐dihydroxybenzoate matrix suppresses the ˙CH₃ eliminations and retro‐Diels–Alder cleavages in the source. All of the flavonoid O‐glycosides evaluated herein eliminated the sugar in source, even in the presence of the matrix, and its product radical ions ([M‐H‐sugar]^?˙) were observed in the negative mode. The flavone C‐glycosides suffered intense dissociation, which was reduced by the addition of a matrix and the application of low laser intensity, mainly in the negative mode. Depending on the hydroxyl substituents, the [M‐H‐H]^?˙ ion was observed with variable relative intensity in the spectra. Copyright © 2015 John Wiley & Sons, Ltd.     

Structural characterization of flavonol 3,7-di-O-glycosides and determination of the glycosylation position by using negative ion electrospray ionization tandem mass spectrometry

Flavonol 3,7-di-O-glycosides were investigated by negative ion electrospray ionization tandem mass spectrometry using a quadrupole linear ion trap (LIT) mass spectrometer. The results indicate that the fragmentation behavior of flavonol 3,7-di-O-glycosides is substantially different from that of their isomeric mono-O-diglycosides. In order to characterize a flavonoid as a flavonol 3,7-di-O-glycoside, both [Y3(0) - H]-* and [Y(0) - 2H]- ions should be present in [M - H]- product ion spectrum. The MS(3) product ion spectra of Y3(0)-, [Y3(0) - H]-* and Y7(0)- ions generated from the [M - H]- ion provide sufficient structural information for the determination of glycosylation position. Furthermore, the glycosylation positions are determined by comparing the relative abundances of Y3(0)- and Y7(0)- ions and their specific fragmentation patterns with those of flavonol mono-O-glycosides. In addition, a [Y3(0) - H]-* ion formed by the homolytic cleavage of 3-O glycosidic bond with high abundance points to 3-O glycosylation, while a [Y(0) - 2H]- ion formed by the elimination of the two sugar residues is consistent with glycosylation at both the 3-O and 7-O positions. Investigation of negative ion ESI-MS(2) and MS(3) spectra of flavonol O-glycosides allows their rapid characterization as flavonol 3,7-di-O-glycoside and their differentiation from isomeric mono-O-diglycosides, and also enables their direct analysis in crude plant extracts.     

Fragmentation study of iridoid glucosides through positive and negative electrospray ionization, collision-induced dissociation and tandem mass spectrometry

Mass spectrometric methodology based on the combined use of positive and negative electrospray ionization, collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS) has been applied to the mass spectral study of a series of six naturally occurring iridoids through in-source fragmentation of the protonated [M+H]+, deprotonated [M--H]- and sodiated [M+Na]+ ions. This led to the unambiguous determination of the molecular masses of the studied compounds and allowed CID spectra of the molecular ions to be obtained. Valuable structural information regarding the nature of both the glycoside and the aglycone moiety was thus obtained. Glycosidic cleavage and ring cleavages of both aglycone and sugar moieties were the major fragmentation pathways observed during CID, where the losses of small molecules, the cinnamoyl and the cinnamate parts were also observed. The formation of the ionized aglycones, sugars and their product ions was thus obtained giving information on their basic skeleton. The protonated, i.e. [M+H]+ and deprotonated [M--H]-, ions were found to fragment mainly by glycosidic cleavages. MS/MS spectra of the [M+Na]+ ions gave complementary information for the structural characterization of the studied compounds. Unlike the dissociation of protonated molecular ions, that of sodiated molecules also provided sodiated sugar fragments where the C0+ fragment corresponding to the glucose ion was obtained as base peak for all the studied compounds.     

Mass spectral characterization of C-glycosidic flavonoids isolated from a medicinal plant (Passiflora incarnata)

The four major C-glycosidic flavonoids isolated from Passiflora incarnata were identified as schaftoside, isoschaftoside, isovetexin-2'-O-glucopyranoside and isoorientin-2'-O-glucopyranoside on the basis of mass spectral and 13C NMR data. The daughter ion spectra of [M + H]+ ions of schaftoside and isoschaftoside showed differences for the [M + H - 104]+ ions, which could be rationalized by hydrogen bonding effects. In the negative-ion mode, pronounced differences were found for the [M - H - 90]- and [M - H - 120]- ions, formed by prevalent fragmentation in the C-6-linked sugar moiety. With respect to isovitexin-2'-O-beta-glucopyranoside and isoorientin-2'-O-beta-glucopyranoside, the daughter ion spectra of both the [M + H]+ and [M - H]- ions provided evidence for a 1----2 linkage in the diglucosidic moiety. Support for C-6 glucosylation was obtained by recording the daughter ion spectra of [M - H - 162]- ions, which were in good agreement with that obtained for [M - H]- ions of isovitexin.     

Determination of phenolic compounds in rose hip (Rosa canina) using liquid chromatography coupled to electrospray ionisation tandem mass spectrometry and diode-array detection

Liquid chromatography coupled with negative and positive electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) and diode-array detection (DAD) was used for determination of phenols in rose hip (Rosa canina) extract. ESI mass spectra of the chromatographically separated phenols gave the molecular weight of the compounds through prominent [M - H](-) ions for most of the compounds and M(+) ions for the anthocyanins. Collision induced dissociation (CID) of the [M - H](-) (or M(+)) precursor ions yielded product ions which determined the molecular weight of the aglycones. In-source fragmentation followed by CID of the resulting deprotonated aglycone ([A - H](-)) provided product ions for the identification of the unconjugated phenols. The identification was based on comparison with product ion spectra of commercial standards. UV-diode-array spectra were used for identity confirmation. This combined approach allowed the identification in rose hip extract of an anthocyanin, i.e. cyanidin-3-O-glucoside, several glycosides of quercetin and glycosides of taxifolin and eriodictyol. Phloridzin was identified, and several conjugates of methyl gallate were also found, one of which was tentatively identified as methyl gallate-rutinoside. Catechin and quercetin were found as the aglycones in the extract.     

Characterization of fifty-one flavonoids in a Chinese herbal prescription Longdan Xiegan Decoction by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry and photodiode array detection

High-performance liquid chromatography coupled to electrospray ionization (ESI) tandem mass spectrometry and photodiode array detection (HPLC-DAD-ESI-MS(n)) was developed to identify and characterize the flavonoids in a Chinese formulated preparation, Longdan Xiegan Decoction (LXD). In total, fifty-one flavonoids (27 flavones, 10 flavanones, 7 chalcones, 5 flavonols and 2 isoflavones) were characterized. Eighteen compounds among them including a newly detected flavonoid, naringin, from the ingredient herbs, were unambiguously determined by comparing the retention times (t(R)), UV spectral data and mass fragmentation behaviors with those of the reference compounds. Another thirty-three compounds were tentatively identified by referencing to the reported data of their UV and MS spectra. The ESI-MS/MS fragmentation behavior of flavones (OMe-substituted, O-glycosides, C-glycosides), chalcones, flavonols and their appropriate characteristic pathways were proposed. In negative ion ESI-MS all the flavonoids yielded prominent [M--H](-) ions in the first order mass spectra. Fragmentation with a loss of mass of 15 Da (CH(3)), 18 Da (H(2)O), 28 Da (CO), 44 Da (CO(2)), 56 Da (2CO) and the residues of glucose and glucuronic acid observed in the MS/MS spectra were useful for aiding the structural identification of the flavonoids investigated.     

Laser-Induced Hydrogen Radical Removal in UV MALDI-MS Allows for the Differentiation of Flavonoid Monoglycoside Isomers

Negative-ion matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra and tandem mass spectra of flavonoid mono-O-glycosides showed the irregular signals that were 1 and/or 2 Da smaller than the parent deprotonated molecules ([M – H]^–) and the sugar-unit lost fragment ions ([M – Sugar – H]^–). The 1 and/or 2 Da mass shifts are generated with the removing of a neutral hydrogen radical (H*), and/or with the homolytic cleavage of the glycosidic bond, such as [M – H* – H]^–, [M – Sugar – H* – H]^–, and [M – Sugar – 2H* – H]^–. It was revealed that the hydrogen radical removes from the phenolic hydroxy groups on the flavonoids, not from the sugar moiety, because the flavonoid backbones themselves absorb the laser. The glycosyl positions depend on the extent of the hydrogen radical removals and that of the homolytic cleavage of the glycosidic bonds. Flavonoid mono-glycoside isomers were distinguished according to their TOF MS and tandem mass spectra.

Characterisation via electrospray ionisation multistage mass spectrometry of three related series of nitrido technetium complexes containing phosphinothiolate and dithiocarbamate ligands

Nine nitrido technetium compounds comprising bis-substituted Tc(N)(PS)(2) (1-4) (PS = bidentate phosphinothiolate ligands) and Tc(N)(dtc)(2) (5, 6) derivatives (dtc = bidentate dithiocarbamate), and mixed-ligand Tc(N)(PS)(dtc) (7-9) species, were subjected to electrospray ionisation mass spectrometry and MS(n) experiments. Bis-substituted phosphinothiolato complexes 1-4 lead to the straightforward formation of dinuclear species reasonably originating from proton bound dimers. These dinuclear species do not show, under collisionally induced fragmentation processes, the formation of monomeric units but cleavages related to the ligand framework, thereby proving the high stability of the [Tc--H(+)--Tc] bond. Bis-dithiocarbamate compounds 5 and 6 show, instead, abundant [M+H](+), [M+Na](+) and [2M+Na](+) ions, and their collisionally induced fragmentations are highly favoured with cleavages related to the C--N and C--S bonds. During these processes, the coordination of a water molecule to [MH-L](+) product ions is observed, as proved by the collisionally induced H(2)O loss detected for this species. Mixed-ligand compounds 7 and 8 show the protonated molecules and Na(+)-cationised ions with fragmentation processes related to the dithiocarbamate moiety. This behaviour indicates that coordination of ether- and ester-substituted dithiocarbamates to the [Tc [triple chemical bond] N] group is weaker than that of phosphinothiolates. Conversely, diethyldithiocarbamate inserted in mixed complex 9 enhances both C--N and Tc--S bonds, and fragmentation processes suggest that metal-phosphinothiolate and metal-dithiocarbamate show comparable strength.     

Analysis of homoisoflavonoids in <Emphasis Type="Italic">Ophiopogon japonicus</Emphasis> by HPLC-DAD-ESI-MS

The homoisoflavonoids in Ophiopogon japonicus (Thunb.) Ker-Gawler were analyzed by high-performance liquid chromatography-diode array detection-electrospray ion trap tandem mass spectrometry (HPLC-DAD-ESI-MSn). Homoisoflavonoids gave prominent [M - H]- ions by electrospray ionization monitored in the negative ion mode. They could be classified into two types depending on the fragmentation behavior of their [M - H]- ions in the ion trap mass analyzer. The [M - H]- ions of homoisoflavonoids with a saturated C2-3 bond underwent C3-9 bond cleavage to lose the B-ring, which was followed by the loss of a molecule of CO. The [M - H]- ions of homoisoflavonoids with a C2-3 double bond usually eliminated a CO molecule first, and then underwent the cleavage of C3-9 or C9-1' bonds. For homoisoflavonoids with a C-6 formyl group, however, the neutral loss of CO was the first fragmentation step; the presence of a methoxyl group at C-8 could lead to the cleavage of C-ring. No retro Diels-Alder (RDA) fragmentation characteristic for normal flavonoids was observed. The above fragmentation rules were reported for the first time, and were implemented for the analysis of homoisoflavonoids in O. japonicus. The CHCl3-MeOH extract was separated on a Zorbax Extend-C18 column, eluting with a acetonitrile-0.3% acetic acid gradient. A total of 18 homoisoflavonoids, including seven new minor constituents, were identified or tentatively characterized based on the UV spectra and tandem mass spectra of the HPLC peaks.     

Internal glucose residue loss in protonated O-diglycosyl flavonoids upon low-energy collision-induced dissociation

The low-energy collision-induced dissociation of protonated flavonoid O-diglycosides, i.e., flavonoid O-rutinosides and O-neohesperidosides, containing different aglycone types has been studied. The results indicate that the unusual [M + H - 162]+ ion formed by internal glucose residue loss, which in a previous study was shown to be a rearrangement ion, is strongly dependent upon the aglycone type. For 7-O-diglycosides, the internal glucose loss is very pronounced for aglycones of the flavanone type, but is completely absent for aglycones of the flavone and flavonol types. Internal glucose residue loss was found to correspond to a minor fragmentation pathway for flavonol 3-O-diglycosides. A plausible mechanism is proposed based on proton mobilization from the aglycone to the disaccharidic part of the flavonoid O-diglycosides which is supported by theoretical calculations and model building.     

Structural characterization of flavonoid glycosides by collisionally activated dissociation of metal complexes

Collisionally activated dissociation is used for structural characterization of a series of flavonoid glycosides. Dissociation of transition metal/flavonoid binary complexes of the type [MII(L - H+)]+ and transition metal/2,2'-bipyridine/flavonoid ternary complexes of the type [MII(L - H+)bpy]+ give fragmentation patterns that are complementary and more diagnostic than those of the protonated, deprotonated, or sodium-cationized flavonoids. Analysis of fragmentation patterns of the [MII(L - H+)bpy]- complexes permits determination of the disaccharide as a rutinose or neohesperidose and the relative placement of the disaccharide (i.e., 3 vs. 7 positions).     

Mass spectra of trimethylsilyl derivatives of naturally occurring flavonoid aglycones and chalcones

Electron impact mass spectra of the trimethylsilyl derivatives of a series of flavonoid aglycones and chalcones are reported. The spectra show prominent ions arising from fragmentation of the trimethylsilyl (TMS) groups. Inter-actions between adjacent TMS groups, and between TMS groups in the 3- or 5-position (6′-position for the chalcones) and the C-ring carbonyl, yield structurally significant ions. Few fragments associated with the retro-Diels-Alder cleavage of the C-ring characteristic of the underivatized compounds, are observed. The TMS derivatives thus provide complementary information for the identification of flavonoid aglycones and chalcones in biological systems.     

Analysis of lipid hydroperoxides and long-chain conjugated keto acids by negative ion electrospray mass spectrometry

Lipid hydroperoxides are important products of enzymatic processes and autooxidation products of polyunsaturated fatty acids. Analysis of such compounds has proved difficult in the past, but negative ion electrospray ionization mass spectrometry was found to be suitable for direct analysis. Abundant [M - H]⁻ ions were observed in full scan mode for hydroperoxyeicosatetraenoic (HPETE), hydroperoxyoctadecenoic acid isomers, and 5,12-diHPETE. Loss of water was observed for all species. Collisional activation and tandem mass spectrometry generated unique and characteristic spectra that shared some common features such as loss of small neutral molecules. More importantly, fragment ions that were indicative of the position of the hydroperoxide were observed. Collision-induced decomposition (CID) of [M - H₂O]⁻ for the HPETE isomers was found to be virtually identical to the CID mass spectra of the [M - H]⁻ anions from corresponding keto-eicosatetraenoic acids, which suggests that the hydroperoxide anions decompose via a dehydration intermediate that resembles the keto acid molecular anion. Cleavage of the double bond allylic to the hydroperoxide formed structurally characteristic ions at m/z 129 from 5-HPETE, m/z 153 from 12-HPETE, and m/z 113 from 15-HPETE. Charge-driven allylic fragmentation led to formation of m/z 203 from 5-HPETE, m/z 179 from 12-HPETE, and m/z 219 from 15-HPETE. Mechanisms consistent with the decomposition of stable isotope analogues are proposed for the formation of these and other characteristic ions. These specific decompositions can be used in multiple reaction monitoring to measure picomolar concentrations of hydroperoxides by direct high performance liquid chromatography tandem mass spectrometry.     

Electrospray mass spectrometry in the structural characterization of cephalosporins

Six cephalosporins of pharmacological interest, cephalexin, cephuroxime, cephazolin, cephoperazone sodium salt, cephatrizin free acid and cephonicid disodium salt, were analysed by electrospray mass spectrometry. [M - Na]- anions were produced in high yield in the case of cephalexin, cephuroxime, cephazolin and cephoperazone, leading to signals at least two orders of magnitude more intense than those related to [M + Na]+ cations observed in the positive ion mode. In cephatrizin, [M - H]- represented the most abundant species, whereas in cephonicid the [M - 2Na + H]- anions were easily produced. No fragment ions were detectable in the electrospray spectra of any of the compounds, and MSn turned out to be essential to draw the fragmentation patterns. Most of these patterns were related to the substituent of the 7-aminocephalosporin nucleus, suggesting that the nucleus itself is highly stable.     

Phenolic compounds of mountain tea from the Balkans: LC/DAD/ESI/MSn profile and content

Twenty-one samples of Sideritis species (S. scardica, S. raeseri, S. taurica, S. syriaca and S. perfoliata) from various locations on the Balkan Peninsula were evaluated for their chemical constituents. Chemical analyses were focused on secondary metabolites, particularly phenolic compounds, which have several roles in the plant physiological processes and have demonstrated significant health beneficial effects. The occurrence of hydroxycinnamic acids, phenylethanoid glycosides and flavonoids has been investigated in taxonomically related taxa of the genus Sideritis. A systematic method for phenolic compounds identification was developed using tandem mass spectrometry coupled to high performance liquid chromatography with diode array detection. Scanning for precursor ions of commonly found phenolics in Sideritis species using LC/MS11 with an ion trap instrument permitted the specific determination of hydroxycinnamic acid derivatives, and phenylethanoid and flavonoid glycosides. Further characterization of each phenolic compound was performed using MS/MS product-ion analysis and common-neutral-loss analysis. This on-line technique allowed identification of three hydroxycinnamic acid derivatives, eight phenylethanoid glycosides, and twenty-four flavonoid glycosides. All the taxa analysed produced very similar phenolic patterns characterized by the presence of 5-caffeoylquinic acid, lavandulifolioside, verbascoside, hypolaetin 7-O-[6'-O-acetyl]-allosyl(1-->2)glucoside, apigenin 7-(4"-p-coumaroylglucoside), 4'-O-methylisoscutellarein 7-O-[6'-O-acetyl]-allosyl(1-->2)glucoside, and minor amounts of isoverbascoside, apigenin 7-O-allosyl(1-->2)glucoside, isoscutellarein 7-O-allosyl-(1-->2)-[6"-O-acetyl]-glucoside, hypolaetin 7-O-allosyl-(1-->2)-[6"-O-acetyl]-glucoside and 4'-O-methylhypolaetin 7-O-[6'-O-acetyl]-allosyl-(1-->2)-[6"-O-acetyl]-glucoside. These results show that the investigated species are systematically very closely related. Phenylethanoid glycosides and flavonoid acetylglycosides are dominant and constitute 90% of the total phenolic compounds compared with hydroxycinnamic acid and flavonoid 7-O-glycosides. Principal component analysis (PCA) was performed for the nature and content of the different compounds to be correlated to the particular Sideritis species and also to the locations.