Negative Electrospray Ionization Tandem Mass Research Articles

Comparison of negative ion electrospray mass spectra measured by seven tandem mass analyzers towards library formation

A library of negative ion electrospray ionization mass spectra and tandem mass spectra (MS/MS) of sulfonated dyes has been developed for fast identification purposes. The uniform protocol has been elaborated and applied to the measurements of more than 50 anionic dyes. Three collision energies are selected in our protocol which ensures that at least one of them provides a suitable ratio of product ions to the precursor ion. The robustness is investigated with altered values of tuning parameters (e.g. the pressure of the nebulizing gas, the temperature and the flow rate of drying gas, and the mobile phase composition). The results of the inter-laboratory comparison of product ion mass spectra recorded on seven different tandem mass spectrometers (three ion traps, two triple quadrupoles and two hybrid quadrupole time of flight instruments) are presented for four representative anionic dyes--azo dye Acid Red 118, anthraquinone dye Acid Violet 43, triphenylmethane dye Acid Blue 1 and Al(III) metal-complex azo dye. The fragmentation patterns are almost identical for all tandem mass analyzers, only the ratios of product ions differ somewhat which confirms the possibility of spectra transfer among different mass analyzers with the goal of library formation.

Negative electrospray ionization tandem mass spectrometric investigation of ent‐kaurane diterpenoids from the genus Isodon

ent-Kaurane diterpenoids are a class of natural compounds isolated from genus Isodon, which have been found to have important bioactivities. Negative electrospray ionization tandem mass spectrometry ((-)ESI-MS(n)) was used to investigate the fragmentation pattern of C-20-nonoxygenated ent-kauranes and two subtypes of C-20-oxygenated ent-kauranes by using an ion trap instrument and accurate mass measurement on an ESI-Q-time-of-flight (TOF) mass spectrometer. The analysis revealed that loss of CH(2)O or CO(2) is the predominant process for 7, 20-epoxy and 7, 20 : 14, 20-diepoxy subgroup of C-20-oxygenated ent-kauranes. In addition, compounds of C-20-nonoxygenated ent-kauranes with a conserved core structure but different substituent groups, such as a hydroxyl, aldehyde, carboxyl, and acetyl moiety, resulted in diagnostic product ions through losses of H(2)O, CO, CO(2), and AcOH, respectively. This work clearly demonstrates the utility of tandem mass spectrometry for studies on the rationalization of the diagnostic fragmentation of a series of compounds from two main types of the ent-kaurane diterpenoids.

Comparison of flavonoid profiles of Agauria salicifolia (Ericaceae) by liquid chromatography-UV diode array detection–electrospray ionisation mass spectrometry

Liquid chromatography (LC) coupled to negative electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) was used for the rapid and sensitive identification of flavonoid compounds in Agauria salicifolia. The leaf flavonoid content in individual of A. salicifolia originating from population with contrasted ecogeographical situation and morphological characteristics was found to be variable qualitatively and highly variable quantitatively. Identification of the compounds was carried out by interpretation of UV, MS and MS/MS spectra. Fourteen flavonoids were identified, all of which had not previously been reported in Agauria spp. Two flavonol- O-glucuronides were found to differentiate the two populations.

Quantitative profiling of epoxyeicosatrienoic, hydroxyeicosatetraenoic, and dihydroxyeicosatetraenoic acids in human intrauterine tissues using liquid chromatography/electrospray ionization tandem mass spectrometry

A reversed-phase liquid chromatography negative ion electrospray tandem mass spectrometry (LC/ESI–MS/MS) method was developed and validated to quantify a range of physiologically relevant eicosanoids, including 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs); 5-, 8-, 9-, 12-, and 15-hydroxyeicosatetraenoic acids (HETEs), and 5,6-, 8,15-, and 12,20-dihydroxyeicosatetraenoic acids (DiHETEs) in human intrauterine tissues. A solid-phase extraction method was employed to extract the eicosanoids, and gradient LC separation was performed on a Kromasil C 18 column. Mass spectrometric detection was performed by multiple reaction monitoring over a 31-min run time. The calibration curves were linear over the range of 4–400 pmol/g tissue, and the intra- and interday precision and accuracy were within a coefficient of variation of 2.0 to 27.4% and 4.6 to 17.9%, respectively. The lower limit of quantitation was 1.0 pmol/g tissue. The method was applied successfully to the characterization and quantitation of eicosanoids in the different compartments of human intrauterine tissues. Our results demonstrate significantly greater amounts of HETEs than of either the EETs or DiHETEs ( P < 0.001), irrespective of tissue type. Specifically, the metabolite 12-HETE was significantly more abundant ( P < 0.001) than all other HETEs. Of the EET metabolites, 5,6-EET predominated ( P < 0.001). A significant negative correlation between EETs and HETEs for all tissues ( ρ = −0.390, P < 0.001) was identified, implying a biological feedback mechanism between these two arachidonate metabolite classes.

Positive and Negative Ion Electrospray Tandem Mass Spectrometry (ESI MS/MS) of Boc-Protected Peptides Containing Repeats of L-Ala-γ 4Caa/γ 4Caa-L-Ala: Differentiation of Some Positional Isomeric Peptides

High-resolution electrospray ionization (ESI) quadrupole time-of-flight and ion trap tandem mass spectrometry has been used to distinguish the positional isomers of a new class of N-blocked hybrid peptides containing repeats of the amino acids, L-Ala-gamma(4)Caa ((l))/gamma(4)Caa((l))-L-Ala [Caa((l)) = Carbo (lyxose) amino acid, derived from D-mannose]. Both MS/MS and MS(3) of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, Boc-group, and the side-chain. It is interesting to observe that the abundant y(n)(+) ions are formed when the corresponding amide -NH does not participate in the helical structures in solution phase and relatively low abundance y(n)(+) ions resulted when the amide -NH involves in the H-bonding. Thus, it was possible to identify the amide -NH hydrogens that participate in the helical structures through H-bonding in solution phase. Further, negative ion ESI MS/MS has also been found to be useful for differentiating these isomeric peptide acids.

Liquid–liquid extraction of strongly protein bound BMS-299897 from human plasma and cerebrospinal fluid, followed by high-performance liquid chromatography/tandem mass spectrometry

BMS-299897 is a γ-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Liquid–liquid extraction (LLE), chromatographic/tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for the quantitation of BMS-299897 in human plasma and cerebrospinal fluid (CSF). Both methods utilized 13C 6-BMS-299897, the stable label isotope analog, as the internal standard. For the human plasma extraction method, two incubation steps were required after the addition of 5 mM ammonium acetate and the internal standard in acetonitrile to release the analyte bound to proteins prior to LLE with toluene. For the human CSF extraction method, after the addition of 0.5 N HCl and the internal standard, CSF samples were extracted with toluene and no incubation was required. The organic layers obtained from both extraction methods were removed and evaporated to dryness. The residues were reconstituted and injected into the LC/MS/MS system. Chromatographic separation was achieved isocratically on a MetaChem C18 Hypersil BDS column (2.0 mm × 50 mm, 3 μm). The mobile phase contained 10 mM ammonium acetate pH 5 and acetonitrile. Detection was by negative ion electrospray tandem mass spectrometry. The standard curves ranged from 1 to 1000 ng/ml for human plasma and 0.25–100 ng/ml for human CSF. Both standard curves were fitted to a 1/ x weighted quadratic regression model. For both methods, the intra-assay precision was within 8.2% CV, the inter-assay precision was within 5.4% CV, and assay accuracy was within ±7.4% of the nominal values. The validation and sample analysis results demonstrated that both methods had acceptable precision and accuracy across the calibration ranges.

Linkage and Branch Determination of N-linked Oligosaccharides Using Sequential Degradation/Closed-Ring Chromophore Labeling/Negative Ion Trap Mass Spectrometry

A method based on sequential degradation, p-aminobenzoic ethyl ester (ABEE) closed-ring labeling, and negative ion electrospray ionization tandem mass spectrometry is presented for the study of linkage and branch determination for N-linked oligosaccharides. Closed-ring labeling provides greater linkage information than the more popular open-ring reductive amination approach. In addition, after high-performance liquid chromatography (HPLC) separation, closed-ring labeling allows for regeneration of the underivatized oligosaccharide, a requirement for alkaline sequential degradation. The analytical scheme presented here uses HPLC separation of closed-ring labeled oligosaccharides to resolve the mixture into individual forms that undergo subsequent structural analysis by negative ion tandem mass spectrometry. To facilitate complete structural analysis, particularly for larger sugars, the closed-ring labels are removed and the sugars are sequentially degraded by controlled alkaline hydrolysis. It is noteworthy that for sugars containing sialic acid moieties, a protecting group must be used to stabilize sialic acid groups during sequential alkaline degradation. This described approach was applied to two high mannose oligosaccharides M5G2, M6G2 cleaved from the ribonuclease B and a complex oligosaccharide A2 cleaved from transferrin.

Sulfatide with short fatty acid dominates in astrocytes and neurons

Glycosphingolipids are located in cell membranes and the brain is especially enriched. We speculated that the subcellular location of glycosphingolipids depends on their fatty acid chain length because their sugar residues are constant, whereas fatty acid chain length can vary within the same molecule. To test this hypothesis we analysed the glycosphingolipid sulfatide, which is highly abundant in myelin and has mostly long fatty acids. We used a negative ion electrospray tandem mass spectrometry precursor ion scan to analyse the molecular species of sulfatide in cultured astrocytes and a mouse model of the human disease metachromatic leukodystrophy. In these arylsulfatase A (ASA)-deficient mice sulfatide accumulates intracellularly in neurons and astrocytes. Immunocytochemistry was also performed on cultured astrocytes and analysed using confocal laser scanning microscopy. Analyses of the molecular species showed that cultured astrocytes contained sulfatide with a predominance of stearic acid (C18), which was located in large intracellular vesicles throughout the cell body and along the processes. The same was seen in ASA-deficient mice, which accumulated a higher proportion (15 mol% compared with 8 mol% in control mice) of sulfatide with stearic acid. We conclude that the major fatty acid composition of sulfatide differs between white and grey matter, with neurons and astrocytes containing mostly short-chain fatty acids with an emphasis on stearic acid. Based on our results, we speculate that the fatty acid chain length of sulfatide might determine its intracellular (short chain) or extracellular (long chain) location and thereby its functions.

Rapid Quantitative Determination of L-FMAU-TP from Human Peripheral-Blood Mononuclear Cells of Hepatitis B Virus-Infected Patients Treated with L-FMAU by Ion-Pairing, Reverse-Phase, Liquid Chromatography/Electrospray Tandem Mass Spectrometry

The purpose of this study was to develop an analytical method for the determination of 2'-fluoro-5-methyl-beta-l-arabinofuranosyl uracil triphosphate (L-FMAU-TP) in human peripheral blood mononuclear cells (PBMCs), and its application in the determination of cellular levels of L-FMAU-TP in PBMCs isolated from patients treated with 2'-fluoro-5-methyl-beta-l-arabinofuranosyl uracil (L-FMAU). An ion-pairing liquid chromatography (IPC) method, coupled with negative ion electrospray ionization tandem mass spectrometry (ESI-MS/MS), was developed for the accurate and repeatable detection of L-FMAU-TP, with a limit of detection of 1.6 pmol/10 cells. The calibration curve for L-FMAU-TP was linear over the concentration range 1.6 to 80 pmol/10(6) cells. The intra- and inter-day precision was lower than 11.2%, and the accuracy was between 97.1 and 106.9%. When applied to the determination of L-FMAU-TP in PBMCs isolated from HBV-infected patients undergoing L-FMAU treatment, the levels reached a steady state concentration 4 weeks after daily single oral administration of 20 mg L-FMAU, and these levels were maintained for up to 12 weeks, but then decreased 12 weeks after drug cessation. The terminal half-life of L-FMAU-TP in PBMCs after drug cessation was estimated to be 15.6 days.

Identification of Isoflavonoids in Several Kudzu Samples by High-Performance Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry

Pueraria lobata is a rich source of isoflavonoids. The detection and identification of isoflavonoid components from Pueraria radix (RP), callus and cell cultures, is very important for the safest and most effective use of kudzu as a medicinal plant, and for the studies on quantitative analysis and secondary metabolism of isoflavonoids in vitro cultures. Liquid chromatography is coupled with negative and positive electrospray ionization (ESI) tandem mass spectrometry (MS-MS), and photodiode array detection is used to characterize and detect isoflavonoids in root, callus, and cell samples of P. lobata. Characteristic product ions of aglycones, O-glucosides, and C-glucosides were obtained from the full-scan ESI-MS chromatography of the major peaks and the MS-MS spectra of the protonated ions. Five major components of puerarin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, and daidzein are detected and identified from the methanolic extract of P. lobata callus cultures. The major isoflavonoid components of P. lobata cell suspension cultures are identified as puerarin, daidzin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, genistein-8-C-glucoside-6"-O-malonylester, and daidzein, on the basis of ESI-MS and MS-MS spectra analysis. Likewise, puerarin, daidzin, genistein-6"-O-malonylester, 3'-methoxypuerarin, and daidzein are detected and identified from RP. Of those isoflavonoid components detected, daidzin-6"-O-acetylester is a new isoflavonoid glucoside and is for the first time detected from P. lobata cultures in vitro.

Analysis of Chain and Blood Group Type and Branching Pattern of Sialylated Oligosaccharides by Negative Ion Electrospray Tandem Mass Spectrometry

We previously reported sequence determination of neutral oligosaccharides by negative ion electrospray tandem mass spectrometry on a quadrupole-orthogonal time-of-flight instrument with high sensitivity and without the need of derivatization. In the present report, we extend our strategies to sialylated oligosaccharides for analysis of chain and blood group types together with branching patterns. A main feature in the negative ion mass spectrometry approach is the unique double glycosidic cleavage induced by 3-glycosidic substitution, producing characteristic D-type fragments which can be used to distinguish the type 1 and type 2 chains, the blood group related Lewis determinants, 3,6-disubstituted core branching patterns, and to assign the structural details of each of the branches. Twenty mono- and disialylated linear and branched oligosaccharides were used for the investigation, and the sensitivity achieved is in the femtomole range. To demonstrate the efficacy of the strategy, we have determined a novel complex disialylated and monofucosylated tridecasaccharide that is based on the lacto-N-decaose core. The structure and sequence assignment was corroborated by methylation analysis and 1H NMR spectroscopy.

Separation and characterization of phenolic compounds in argan fruit pulp using liquid chromatography–negative electrospray ionization tandem mass spectroscopy

Liquid chromatography (LC) coupled to negative electrospray ionization (ESI) tandem mass spectrometry (MS/MS) was used for the sensitive identification of phenolic compounds in argan fruit pulp. Sixteen compounds were identified, mainly flavonoid glycosides and flavonoid aglycons.

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.

Phosphate Group-Driven Fragmentation of Multiply Charged Phosphopeptide Anions. Improved Recognition of Peptides Phosphorylated at Serine, Threonine, or Tyrosine by Negative Ion Electrospray Tandem Mass Spectrometry

The nanoelectrospray product ion spectra of multiply charged phosphopeptide anions reveal the occurrence of phosphate-specific high-mass fragment ions of the type [M - nH - 79](n-1)-. These so far unrecognized fragments, which are observed for phosphoserine-, phosphothreonine-, and phosphotyrosine-containing peptides, are the counterparts of the established inorganic phosphopeptide marker ion found at m/z 79 = [PO3]-. The high-mass marker ions are formed with high efficiency at moderate collision offset values and are particularly useful for sensitive recognition of pSer-, pThr-, and pTyr-peptides due to the low background level in MS/MS spectra at m/z values above those of the precursor ions. By virtue of this feature, the detection of the new phosphorylation-specific fragment ions appears to be more sensitive than the detection of the low-mass phosphate marker ion at m/z 79, where a higher interference by nonspecific background signals is generally observed. The number of phosphate groups within a phosphopeptide can also be estimated on the basis of the [M - nH - 79](n-1)- ions, since these exhibit an effective, sequential neutral loss of H3PO4 of the residing phosphate groups. A mechanistic explanation for the formation of the [M - nH - 79](n-1)- ions from multiply charged phosphopeptides is given. The high-mass marker ions are proposed to originate from phosphopeptide anions, which carry two negative charges located at the phosphate group. A new search tool denominated "variable m/z gain analysis", which utilizes these newly recognized high-mass fragments for spotting of phosphopeptides in a negative ion parent scan, is proposed. The findings strengthen the value of negative ion ESI-MS/MS for analysis of protein phosphorylation.

Differentiation of Boc‐ α,β‐ and β,α‐peptides and a pair of diastereomeric β,α‐dipeptides by positive and negative ion electrospray tandem mass spectrometry (ESI‐MS/MS)

Positive and negative ion electrospray ionization (ESI) tandem mass spectral study of a new series of hybrid peptides, viz, BocN-alpha,beta-peptides and BocN-beta,alpha-peptides, synthesized from C-linked carbo-beta3-amino acids [Caa (S)] and L-Ala has been carried out. The alpha,beta-peptides have been differentiated from beta,alpha-peptides by the collision-induced dissociation (CID) of [M + H]+ and [M - H]- ions in positive and negative ion ESI-MS respectively. The fragment ion [M + H - C(CH3)3 + H]+ formed from [M + H]+ ions by the loss of 2-methyl-prop-2-ene in alpha,beta-peptides with L-Ala at the N-terminus is insignificant or totally absent for beta,alpha-peptides which have the Caa (S) at N-terminus. The fragment ion [M - H-C(CH3)3OH - HNCO]- formed from [M - H]- of beta,alpha-peptide acids is totally absent for alpha,beta-peptide acids. This has been attributed to the absence of the beta-methylene group in alpha,beta-peptides, and the participation of the beta-methylene group in the loss of HNCO in beta,alpha-peptide acids is confirmed by the deuteration experiments. The CID of [M + H-Boc + H]+ ions of these peptides also produce characteristic fragmentation. In the CID spectra of alpha,beta-peptides, the b(n)+ ions and the resulting y(n)+ ions occur at a mass difference of 243 and 71 Da corresponding to the successive losses of Caa and L-Ala, whereas a mass difference of 71 and 243 Da is observed for beta,alpha-peptides. In contrast to the CID of protonated peptides, the CID of [M - H]- ions of the alpha,beta- and beta,alpha-peptide acids do not give b(n)- ions and show abundant z(n) (-) ions. Further, a pair of diastereomeric dipeptide esters and acids have been distinguished by the CID of [M + H]+ ions. The loss of 2-methyl-prop-2-ene is more pronounced for Boc-NH-Caa(R)-D-Ala-OCH3 (21) and Boc-NH-Caa(R)-D-Ala-OH (23) with Caa (R) at the N-terminus, whereas it is totally absent for Boc-NH-Caa (S)-D-Ala-OCH3 (22) and Boc-NH-Caa(S)-D-Ala-OH (24) peptides, which have Caa (S) at the N-terminus. Thus, on the basis of our previous and present studies, we propose that the CID of [M + H]+ ions provides a simple and useful method for distinguishing the configuration of Caa (S or R) at the N-terminus of BocN-carbo beta,alpha- and beta,beta-dipeptides.

31P NMR and Genetic Analysis Establish hinT as the Only Escherchia coli Purine Nucleoside Phosphoramidase and as Essential for Growth under High Salt Conditions

Eukaryotic cells encode AMP-lysine (AMP-N-epsilon-(N-alpha-acetyl lysine methyl ester) 5'-phosphoramidate) hydrolases related to the rabbit histidine triad nucleotide-binding protein 1 (Hint1) sequence. Bacterial and archaeal cells have Hint homologs annotated in a variety of ways, but the enzymes have not been characterized, nor have phenotypes been described due to loss of enzymatic activity. We developed a quantitative (31)P NMR assay to determine whether Escherichia coli possesses an adenosine phosphoramidase activity. Indeed, soluble lysates prepared from wild-type laboratory E. coli exhibited activity on the model substrate adenosine 5'-monophosphoramidate (AMP-NH(2)). The E. coli Hint homolog, which had been comprehensively designated ycfF and is here named hinT, was cloned, overexpressed, purified, and characterized with respect to purine nucleoside phosphoramidate substrates. Bacterial hinT was several times more active than human or rabbit Hint1 on five model substrates. In addition, bacterial and mammalian enzymes preferred guanosine versus adenosine phosphoramidates as substrates. Analysis of the lysates from a constructed hinT knock-out strain of E. coli demonstrated that all of the cellular purine nucleoside phosphoramidase activity is due to hinT. Physiological analysis of this mutant revealed that the loss of hinT results in failure to grow in media containing 0.75 m KCl, 0.9 m NaCl, 0.5 m NaOAc, or 10 mm MnCl(2). Thus, cation-resistant bacterial cell growth may be dependent on the hydrolysis of adenylylated and/or guanylylated phosphoramidate substrates by hinT.

Ultrafiltration tandem mass spectrometry of estrogens for characterization of structure and affinity for human estrogen receptors

Although hormone replacement therapy (HRT) is used by post-menopausal women for the relief of menopausal symptoms and the potential reduction of osteoporosis, HRT also increases their risk of Alzheimer's disease, stroke, breast cancer, and endometrial cancer. Since the majority of these effects are associated primarily with estrogen binding to only one of the estrogen receptors (ER), new assays are needed that can more efficiently evaluate ER-binding and identify ligands selective for ER-alpha and ER-beta. High performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) was combined with ultrafiltration as a new method to investigate the relative binding of compounds to the ERs and to evaluate the structures of these estrogens. Mixtures of estradiol and six equine estrogens, including equilin, equilenin, 8,9-dehydroestrone, and their 17beta-hydroxyl derivatives, were assayed simultaneously to determine their relative binding to human ER-alpha and ER-beta. Estrogens containing a 17beta-OH group were found to have higher relative affinities for the estrogen receptors than their ketone analogs. In addition, 17beta-EN showed selectivity for binding to ER-beta over ER-alpha. The results were compared to the IC50 values obtained by using a conventional radiolabled estradiol competitive binding assay. Finally, the utility of negative ion electrospray tandem mass spectrometry for the unambiguous identification of these estrogen isomers was investigated. Several characteristic recyclization pathways during tandem mass spectrometry were identified, which might be useful for distinguishing related estrogens.

Study of the reaction products of flavonols with 2,2‐diphenyl‐1‐picrylhydrazyl using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry

The products obtained after the reaction between flavonols and the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) in both methanol and acetonitrile were characterized using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) and NMR spectroscopy. The flavonols studied were quercetin, kaempferol and myricetin. In methanol, two reaction products of oxidized quercetin were identified using LC/ESI-MS/MS and NMR. Quercetin was oxidized through a transfer of two H-atoms to DPPH(*) and subsequently incorporated either two CH(3)OH molecules or one CH(3)OH- and one H(2)O molecule giving the products 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dimethoxy-2,3-dihydrochromen-4-one and 2-(3,4-dihydroxyphenyl)-3,3,5,7-tetrahydroxy-2-methoxy-2,3-dihydrochromen-4-one, respectively. LC/ESI-MS/MS analysis revealed that in methanol, kaempferol and myricetin also gave rise to methoxylated oxidation products similar to that identified for quercetin. Kaempferol, in addition, also exhibited products where a kaempferol radical, obtained by a transfer of one H-atom to DPPH(*), reacted with CH(3)OH through the addition of CH(3)O(*), yielding two isomeric products. When the reaction took place in acetonitrile, LC/ESI-MS/MS analysis showed that both quercetin and myricetin formed stable isomeric quinone products obtained by a transfer of two H-atoms to DPPH(*). In contrast, kaempferol formed two isomeric products where a kaempferol radical reacted with H(2)O through the addition of OH(*), i.e. similar to the reaction of kaempferol radicals with CH(3)OH.

Liquid chromatography-tandem mass spectrometry for the determination of lithospermic acid B in rat serum

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/ MS) method for the determination of lithospermic acid B (LSB) in rat serum was developed. LSB and internal standard, 7-hydroxy-3-phenyl-chromen-4-one (HPC) were extracted from rat serum with methyl-tert-butyl ether at acidic pH and analyzed on a Luna C8 column with the mobile phase of acetonitrile-ammonium formate (10 mM, pH 6.5) (50:50, v/v). The analytes were detected using a negative electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r2= 0.997) over the concentration range of 10.0-500 ng/mL. The coefficient of variation and relative error for intra- and interassay at three QC levels were 1.1 approximately 6.2% and -10.3 approximately -2.7%, respectively. The recovery of LSB from serum sample ranged from 73.2 to 79.5%, with that of HPC (internal standard) being 75.1 %. The lower limit of quantification for LSB was 10 ng/mL using 50 microL of serum sample.

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.