Determination of B-group vitamins in Turkish honey using ultra-performance liquid chromatography with electrospray ionization coupled to tandem mass spectrometry

A rapid and sensitive ultra-performance liquid chromatography–tandem mass spectrometry method with electrospray ionization (UPLC–ESI–MS/MS) for analysis of B-group vitamins in honey has been presented. Aim of this study is the characterization of different types of Turkish honeys according to B-group vitamins. Vitamins were determined in 17 different types of Turkish honey samples by UPLC–ESI–MS/MS. Heather honey samples were distinguished among the studied honeys with the richest vitamin content with 286.10?mg/kg, and it is followed by sunflower honey and thyme honey with the total vitamin contents of 206.01 and 163.27?mg/kg, respectively. The presence of vitamin B₁ (thiamine), vitamin B₂ (riboflavin), vitamin B₃ (nicotinamide, B₃N and nicotinic acid, B₃H), vitamin B₆ (pyridoxine), and vitamin B₉ (folic acid) was detected in all the honey samples analyzed. Moreover, vitamin B₅ (pantothenic acid) was observed in most of them. Vitamin B₁₂ (cyanocobalamin) and vitamin B₇ (biotine) were not detected in the studied honey samples. Turkish honey samples showed efficacious vitamin content for the consumers.     

Quantitative profiling of biomarkers related to B‐vitamin status,tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass spectrometry

Vitamins B₂ and B₆ serve as cofactors in enzymatic reactions involved in tryptophan and homocysteine metabolism. Plasma concentrations of these vitamins and amino acids are related to smoking and inflammation, and correlate with other markers of immune activation. Large‐scale studies of these relations have been hampered by lack of suitable analytical methods. The assay described includes riboflavin, five vitamin B₆ forms (pyridoxal 5′‐phosphate, pyridoxal, 4‐pyridoxic acid, pyridoxine and pyridoxamine), tryptophan and six tryptophan metabolites (kynurenine, kynurenic acid, anthranilic acid, 3‐hydroxykynurenine, xanthurenic acid and 3‐hydroxyanthranilic acid), cystathionine, neopterin and cotinine. Trichloroacetic acid containing 13 isotope‐labelled internal standards was added to 60 µL of plasma, the mixture was centrifuged, and the resulting supernatant used for analysis. The analytes were separated within 5 min on a stable‐bond C8 column by a gradient‐type mobile phase containing acetonitrile, heptafluorobutyric acid and high concentration (650 mmol/L) of acetic acid, and detected using electrospray ionization tandem mass spectrometry (ESI‐MS/MS). The mobile phase ensured sufficient separation and high ionization efficiency of all analytes. Recoveries were 75–123% and within‐day and between‐day coefficients of variance (CVs) were 2.5–9.5% and 5.4–16.9%, respectively. Limits of detection ranged from 0.05 to 7 nmol/L. The method enables quantification of endogenous plasma concentrations of 16 analytes related to B‐vitamin status and inflammation, and may prove useful in large‐scale epidemiological studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid separation and identification of major constituents in Pseudolarix kaempferi by ultra‐performance liquid chromatography coupled with electrospray and quadrupole time‐of‐flight tandem mass spectrometry

A rapid and reliable method based on ultra‐performance liquid chromatography (UPLC) coupled with photodiode‐array detection (PDA) and electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (ESI‐Q‐TOF‐MS/MS) has been developed for separation and identification of major constituents in extracts of root bark of Pseudolarix kaempferi Gordon (PKG). Identification of the constituents was carried out by interpretation of their retention times, UV absorption spectra, MS and MS/MS spectra, as well as the data provided by authentic standards and literatures. A total of 20 components were separated in only 8.0 min on a small particle size C₁₈ column (1.7 µm). These components included nine diterpene acids, seven glycosides and four triterpenoids, among which pseudolaric acid C‐O‐β‐D‐glucopyranoside and pseudolaric acid C₂‐O‐β‐D‐glucopyranoside were separated and identified for the first time in this study. Furthermore, the fragmentation patterns of the three types of compounds were elucidated for the first time. This established UPLC‐PDA/Q‐TOF‐MS/MS method is reliable and effective for the separation and identification of the 20 compounds and will be useful for quality control of the crude materials of Pseudolarix kaempferi Gordon and their related preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural characterization and identification of iridoid glycosides,saponins, phenolic acids and flavonoids in Flos Lonicerae Japonicae by a fast liquid chromatography method with diode‐array detection and time‐of‐flight mass spectrometry

A fast liquid chromatography method with diode‐array detection (DAD) and time‐of‐flight mass spectrometry (TOF‐MS) has been developed for analysis of constituents in Flos Lonicerae Japonicae (FLJ), a traditional Chinese medicine derived from the flower bud of Lonicera japonica. The chromatographic analytical time decreased to 25 min without sacrificing resolution using a column packed with 1.8‐µm porous particles (4.6 × 50 mm), three times faster than the performance of conventional 5.0‐µm columns (4.6 × 150 mm). Four major groups of compounds previously isolated from FLJ were structurally characterized by DAD‐TOF‐MS: iridoid glycosides showed maximum UV absorption at 240 nm; phenolic acids at 217, 242, and 326 nm; flavonoids at 255 and 355 nm; while saponins had no absorption. In electrospray ionization (ESI)‐TOF‐MS experiments, elimination of a glucose unit (162 Da), and successive losses of H₂O, CH₃OH and CO, were generally observed in iridoid glycosides; saponins were characterized by a series of identical aglycone ions; phenolic acids typically generated a base peak at [M–H–caffeoyl]^? by loss of a caffeic acid unit (162 Da) and several marked quinic acid moiety ions; cleavage of the glycosidic bond (loss of 162 or 308 Da), subsequent losses of H₂O, CO, RDA and C‐ring fragmentation were the most possible fragmentation pathways for flavonoids. By accurate mass measurements within 4 ppm error for each molecular ion and subsequent fragment ions, as well as the ‘full mass spectral’ information of TOF‐MS, a total of 41 compounds including 13 iridoid glycosides, 11 phenolic acids, 7 saponins, and 10 flavonoids were identified in a methanolic extract of FLJ. Copyright © 2009 John Wiley & Sons, Ltd.     

Fusaricidins from Paenibacillus polymyxa M‐1, a family of lipohexapeptides of unusual complexity—a mass spectrometric study

Paenibacillus polymyxa are rhizobacteria with a high potential to produce natural compounds of biotechnological and medical interest. Main products of P . polymyxa are fusaricidins, a large family of antifungal lipopeptides with a 15‐guanidino‐3‐hydroxypentadecanoic acid (GHPD) as fatty acid side chain. We use the P . polymyxa strain M‐1 as a model organism for the exploration of the biosynthetic potential of these rhizobacteria. Using matrix‐assisted laser‐desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) about 40 new fusaricidins were detected which were fractionated by reversed‐phase (rp) HPLC. Their structure was determined by MALDI‐LIFT‐TOF/TOF fragment analysis. The dominant fragment in the product ion spectra of fusaricidins appeared at m /z 256.3, 284.3 and 312.4, respectively, indicating variations in their fatty acid part. Two new subfamilies of fusaricidins were introduced which contain guanidino‐3‐hydroxyhepta‐ and nonadecanoic acid as fatty acid constituents. Apparently, the end‐standing guanidine group is not modified as shown by direct infusion nano‐electrospray ionization mass spectrometry (nano‐ESI MS). The results of this study suggest that advanced mass spectrometry is the method of choice for investigating natural compounds of unusual diversity, like fusaricidins. Copyright © 2016 John Wiley & Sons, Ltd.     

The effect of temperature on the stability of compounds used as UV‐MALDI‐MS matrix: 2,5‐dihydroxybenzoic acid, 2,4,6‐trihydroxyacetophenone, α‐cyano‐4‐hydroxycinnamic acid, 3,5‐dimethoxy‐4‐hydroxycinnamic acid,nor‐harmane and harmane

The thermal stability of several commonly used crystalline matrix‐assisted ultraviolet laser desorption/ionization mass spectrometry (UV‐MALDI‐MS) matrices, 2,5‐dihydroxybenzoic acid (gentisic acid; GA), 2,4,6‐trihydroxyacetophenone (THA), α‐cyano‐4‐hydroxycinnamic acid (CHC), 3,5‐dimethoxy‐4‐hydroxycinnamic acid (sinapinic acid; SA), 9H‐pirido[3,4‐b]indole (nor‐harmane; nor‐Ho), 1‐methyl‐9H‐pirido[3,4‐b]indole (harmane; Ho), perchlorate of nor‐harmanonium ([nor‐Ho + H]⁺) and perchlorate of harmanonium ([Ho + H]⁺) was studied by heating them at their melting point and characterizing the remaining material by using different MS techniques [electron ionization mass spectrometry (EI‐MS), ultraviolet laserdesorption/ionization‐time‐of‐flight‐mass spectrometry (UV‐LDI‐TOF‐MS) and electrospray ionization‐time‐of‐flight‐mass spectrometry (ESI‐TOF‐MS)] as well as by thin layer chromatography analysis (TLC), electronic spectroscopy (UV‐absorption, fluorescence emission and excitation spectroscopy) and ¹H nuclear magnetic resonance spectroscopy (¹H‐NMR). In general, all compounds, except for CHC and SA, remained unchanged after fusion. CHC showed loss of CO₂, yielding the trans‐/cis‐4‐hydroxyphenylacrilonitrile mixture. This mixture was unambiguously characterized by MS and ¹H‐NMR spectroscopy, and its sublimation capability was demonstrated. These results explain the well‐known cluster formation, fading (vanishing) and further recovering of CHC when used as a matrix in UV‐MALDI‐MS. Commercial SA (SA 98%; trans‐SA/cis‐SA 5 : 1) showed mainly cis‐ to‐trans thermal isomerization and, with very poor yield, loss of CO₂, yielding (3′,5′‐dimethoxy‐4′‐hydroxyphenyl)‐1‐ethene as the decarboxilated product. These thermal conversions would not drastically affect its behavior as a UV‐MALDI matrix as happens in the case of CHC. Complementary studies of the photochemical stability of these matrices in solid state were also conducted. Copyright © 2008 John Wiley & Sons, Ltd.     

Urinary metabolomics analysis reveals the effect of volatile oil from Angelica sinensis on LPS‐induced inflammation rats

Lipopolysaccharide (LPS)‐induced inflammation occurs commonly and volatile oil from Angelica sinensis (VOAS) can be used as an anti‐inflammatory agent. The molecular mechanisms that allow the anti‐inflammatory factors to be expressed are still unknown. In this paper, we applied gas chromatography–mass spectrometry (GC–MS) and high‐performance liquid chromatography–time‐of‐flight mass spectrometry (LC‐Q/TOF–MS) based on a metabolomics platform coupled with a network approach to analyze urine samples in three groups of rats: one with LPS‐induced inflammation (MI); one with intervention with VOAS; and normal controls (NC). Our study found definite metabolic footprints of inflammation and showed that all three groups of rats, MI, intervention with VOAS and NC have distinct metabolic profiles in urine. The concentrations of 48 metabolites differed significantly among the three groups. The metabolites in urine were screened by the GC–MS and LC‐Q/TOF–MS methods. The significantly changed metabolites (p < 0.05, variable importance in projection > 1.5) between MI, NC and VOAS were included in the metabolic networks. Finally, hub metabolites were screened, including glycine, arachidonic acid, l ‐glutamate, pyruvate and succinate, which have high values of degree (k). the Results suggest that disorders of glycine, arachidonic acid, l ‐glutamate, pyruvate and succinate metabolism might play an important part in the predisposition and development of LPS‐induced inflammation. By applying metabolomics with network methods, the mechanisms of diseases are clearly elucidated.     

Analysis of the traditional medicine YiGan San by the fragmentation patterns of cadambine indole alkaloids using HPLC coupled with high‐resolution MS

YiGan San (YGS) has long been used in traditional Japanese and Chinese folk medicine and serves as a potent and novel therapeutic agent to treat Alzheimer's disease. In the present study, a rapid and sensitive method based on HPLC coupled with diode‐array detection and quadrupole TOF MS (Q‐TOF‐MS) was designed to reveal the chemical constituents of YGS. Thirty‐six compounds were identified and assigned in YGS, including 14 alkaloids, nine γ‐lactones, six flavonoids, three triterpenoid saponinares, two small molecular organic acids, and two other types of compounds. In addition, the accurate fragment weight and MS/MS fragmentation reactions of a subtype indole alkaloid in Uncariae ramulus cum uncis were summarized for the first time to realize rapid identification without reference substances. For the first time, 11 major constituents were comprehensively quantified with a HPLC coupled with triple‐quadrupole MS method. A three‐section switch was used to realize such multicomponent identification. The contents of saikosaponin B₂ and isoliquiritin, which produce anti‐inflammatory and antidepressant‐like effects, were extremely different, up to 700 times, in two sources of YGS. The developed qualitative and quantitative method was proved to be precise, accurate, and reproducible.     

MALDI‐TOF MS characterization of dihydroxy telechelic polyisobutylene

MALDI‐TOF MS characterizations of dihydroxy telechelic polyisobutylene is reported. Dichloro telechelic polyisobutylene (Cl—PIB—Cl) was synthesized by means of living cationic polymerization using p‐dicumyl chloride/BCl₃/DMSO initiating systems. The resulting polymer was functionalized by polymer analogous reactions to yield dihydroxy telechelic polyisobutylene (HO—PIB—OH). It was then investigated by MALDI‐TOF MS in the cation mode using 1,8‐dihidroxy‐9(10H)‐anthracenone (dithranol)/CF₃COOAg matrix. The MALDI TOF MS spectra showed an increase in mass by 56 amu units attributed to the isobutylene monomer increment. The endgroups of HO—PIB—OH were determined. A good agreement was also found between the calculated isotope distributions and the isotope distributions determined by means of MALDI.     

Direct characterization of aqueous extract of Hibiscus sabdariffa using HPLC with diode array detection coupled to ESI and ion trap MS

The phenolic fraction and other polar compounds of the Hibiscus sabdariffa were separated and identified by HPLC with diode array detection coupled to electrospray TOF and IT tandem MS (DAD‐HPLC‐ESI‐TOF‐MS and IT‐MS). The H. sabdariffa aqueous extract was filtered and directly injected into the LC system. The analysis of the compounds was carried out by RP HPLC coupled to DAD and TOF‐MS in order to obtain molecular formula and exact mass. Posterior analyses with IT‐MS were performed and the fragmentation pattern and confirmation of the structures were achieved. The H. sabdariffa samples were successfully analyzed in positive and negative ionization modes with two optimized linear gradients. In positive mode, the two most representative anthocyanins and other compounds were identified whereas the phenolic fraction, hydroxycitric acid and its lactone were identified using the negative ionization mode.     

Direct identification of trypanosomatids by matrix‐assisted laser desorption ionization–time of flight mass spectrometry (DIT MALDI‐TOF MS)

Accurate and rapid determination of trypanosomatids is essential in epidemiological surveillance and therapeutic studies. Matrix‐assisted laser desorption ionization/time of flight mass spectrometry (MALDI‐TOF MS) has been shown to be a useful and powerful technique to identify bacteria, fungi, metazoa and human intact cells with applications in clinical settings. Here, we developed and optimized a MALDI‐TOF MS method to profile trypanosomatids. trypanosomatid cells were deposited on a MALDI target plate followed by addition of matrix solution. The plate was then subjected to MALDI‐TOF MS measurement to create reference mass spectra library and unknown samples were identified by pattern matching using the BioTyper software tool. Several m/z peaks reproducibly and uniquely identified trypanosomatids species showing the potentials of direct identification of trypanosomatids by MALDI‐TOF MS. Moreover, this method discriminated different life stages of Trypanosoma cruzi, epimastigote and bloodstream trypomastigote and Trypanosoma brucei, procyclic and bloodstream. T. cruzi Discrete Typing Units (DTUs) were also discriminated in three clades. However, it was not possible to achieve enough resolution and software‐assisted identification at the strain level. Overall, this study shows the importance of MALDI‐TOF MS for the direct identification of trypanosomatids and opens new avenues for mass spectrometry‐based detection of parasites in biofluids. Copyright © 2016 John Wiley & Sons, Ltd.     

Rapid characterization of triterpene saponins from Conyza blinii by liquid chromatography coupled with mass spectrometry

Conyza blinii Le'vl is a medicinal herb used for the treatment of inflammation in Chinese folk medicine. Its major bioactive constituents are triterpene saponins, most of which contain 6–8 sugar residues. In this report, electrospray ionization tandem mass spectrometry fragmentation behaviors of bisdesmosidic triterpene saponins (conyzasaponin A, B, and C) were studied in both positive and negative ion modes with an ion‐trap mass spectrometer. In full scan mass spectrometry, these saponins gave predominant [M–H]^? and [M+Na]⁺ ions, which determined the molecular weights. In tandem mass spectrometry (MSⁿ, n = 2–4), the [M–H]^? and [M+Na]⁺ ions yielded fragments [Y_0α–H]^? and [B_α+Na]⁺, which were diagnostic for the structures of the triterpene skeleton and sugar chains. The structural elucidation was approved by accurate mass data using IT‐TOF‐MS. An interpretation guideline based on MSⁿ (n = 2–4) diagnostic ions was proposed in order to elucidate the chemical structures of unknown triterpene saponins in C. blinii extract. The saponins in C. blinii were separated by liquid chromatography with a methanol/acetonitrile/water solvent system, and then analyzed by ion‐trap and IT‐TOF mass spectrometers. Based on the interpretation guideline, a total of 35 triterpenoid saponins were tentatively identified. Among them, 15 saponins had been previously reported, and the other 20 saponins were reported from Conyza species for the first time. This study indicates that LC/MS is a powerful technology for the rapid characterization of complicated saponins in herbal extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Ultrasensitive Determination of Vitamin B12 Using Disposable Graphite Screen‐Printed Electrodes and Anodic Adsorptive Voltammetry

A facile, rapid and ultra‐sensitive method for the determination of vitamin B₁₂ (cyanocobalamin) at the sub‐nanomolar concentration range by using low‐cost, disposable graphite screen‐printed electrodes is described. The method is based on the cathodic preconcentration of square planar vitamin B_12s, as occurred due to the electro reduction of Co(III) center in vitamin B_12a to Co(I), at ?1.3 V versus Ag/AgCl/3 M KCl for 40 s. Then, an anodic square wave scan was applied and the height of the peak appeared at ca. ?0.73 V versus Ag/AgCl/3 M KCl, due to the oxidation of Co(I) to Co(II) in the adsorbed molecule, was related to the concentration of the vitamin B₁₂ in the sample. EDTA was found to serve as a key‐component of the electrolyte by eliminating the background signal caused by metal cations impurities contained in the electrolyte (0.1 M phosphate buffer in 0.1 M KCl, pH 3). It also blocks trace metals contained in real samples, thus eliminating their interference effect. The method was optimized to various working parameters and under the selected conditions the calibration curve was linear over the range 1×10^?10–8×10^?9 mol L^?1 vitamin B₁₂ (R²=0.994), while the limit of detection for a signal‐to‐noise ratio of 3 (7×10^?11 mol L^?1 vitamin B₁₂) is the lowest value of any reported in the literature for the electrochemical determination of vitamin B₁₂. The sensors were successfully applied to the determination of vitamin B₁₂ in pharmaceutical products.     

Interactions and Encapsulation of Vitamins C,B3, and B6 with Dendrimers in Water

Titrations of commercial diaminobutane (DAB) and polyamidoamine (PAMAM) dendrimers by vitamins C (ascorbic acid, AA), B₃ (nicotinic acid), and B₆ (pyridoxine) were monitored by ¹H NMR spectroscopy using the chemical shifts of both dendrimer and vitamin protons and analyzed by comparison with the titration of propylamine. Quaternarizations of the terminal primary amino groups and intradendritic tertiary amino groups, which are nearly quantitative with vitamin C, were characterized by more or less sharp variations (Δδ) of the ¹H chemical shift (δ) at the equivalence points. The peripheral primary amino groups of the DAB dendrimers were quaternarized first, but not selectively, whereas a sharp chemical‐shift variation was recorded for the inner methylene protons near the tertiary amines, thereby indicating encapsulation, when all the dendritic amines were quaternarized. With DAB‐G5‐64‐NH₂, some excess acid is required to protonate the inner amino groups, presumably because of basicity decrease due to excess charge repulsion. On the other hand, this selectivity was not observed with PAMAM dendrimers. The special case of the titration of the dendrimers by vitamin B₆ indicates only dominant supramolecular hydrogen‐bonding interactions and no quaternarization, with core amino groups being privileged, which indicates the strong tendency to encapsulate vitamins. With vitamin B₃, a carboxylic acid, titration of DAB‐G3‐16‐NH₂ shows that only six peripheral amino groups are protonated on average, even with excess vitamin B₃, because protonation is all the more difficult due to increased charge repulsion, as positive charges accumulate around the dendrimer. Inner amino groups interact with this vitamin, however, thus indicating encapsulation presumably with supramolecular hydrogen bonding without much charge transfer.     

Pharmacokinetic studies of soyalkaloid A from Portulaca oleracea L. using ultra high‐performance liquid chromatography electrospray ionization quadrupole–time of flight mass spectrometry and its antioxidant activity

Soyalkaloid A was isolated from Portulaca oleracea L. for the first time in our laboratory and then a rapid and sensitive ultra‐high‐performance liquid chromatography electrospray ionization quadrupole–time of flight mass spectrometry (UHPLC–ESI–Q–TOF/MS) method with hesperidin as internal standard (IS) was developed and validated to investigate the pharmacokinetics of soyalkaloid A in rats after oral and intravenous administrations. The analysis was achieved on an Agilent Zorbax Eclipse Plus C₁₈ Column (2.1 × 50 mm, 1.8 μm) by elution with acetonitrile and water (containing 0.1% formic acid), at a flow rate of 0.3 mL/min. The MS analysis was performed in the positive ion mode with monitored ion m/z 227.0814 [M + H]⁺ and 611.1971 [M + H]⁺ for soyalkaloid A and IS, respectively. The linear range was established over the concentration range 7.5–6000 ng/mL (r = 0.9951). The intra‐ and inter‐assay accuracy and precision were between ?4.86‐4.49 and 1.93–9.66, respectively. The lower limits of detection and quantitation observed were 2.1 and 7.4 ng/mL, respectively. The rapid, sensitive and specific UHPLC–ESI–Q–TOF/MS method was successfully applied to a pharmacokinetic study of soyalkaloid A. Moreover, its antioxidant was studied via a 1,1‐diphenyl‐2‐picryl‐hydrazyl radical scavenging assay, the IC₅₀ value being 20.73 ± 0.51 μM.     

Viscosalines B1,2 and E1,2: Challenging New 3‐Alkyl Pyridinium Alkaloids from the Marine Sponge Haliclona viscosa

Four new 3‐alkyl pyridinium alkaloids, the viscosalines B₁ ( 1 a ), B₂ ( 1 b ), E₁ ( 2 a ), and E₂ ( 2 b ), were isolated from the Arctic sponge Haliclona viscosa. The structure elucidation of these isomeric compounds was challenging due to ambiguous fragments that derive during “standard” mass spectrometric fragmentation experiments. The final structure elucidation relied on the use of a combination of synthesis, liquid chromatography, and mass spectrometry. Three different mass spectrometers were used to differentiate between the synthetic structural isomers: a time‐of‐flight (TOF) mass spectrometer and two ion‐trap mass spectrometers with different ion‐transfer technologies (i.e., skimmer versus funnel optics). Although at first none of the spectrometers returned spectra that permitted structure elucidation, all three mass spectrometers provided analysis that successfully differentiated between the isomers after thorough method optimization. The use of in‐source collision‐induced dissociation (CID) with the ion trap and TOF instrument returned the most interesting results. The mode of fragmentation of the viscosalines under different experimental conditions is described herein. After successful optimization of the mass spectrometric method applied, the chromatographic method was improved to distinguish the previously inseparable isomers. Finally, both the liquid chromatography and mass spectrometric methods were applied to the natural products and the results compared to those from the synthetic compounds.     

Phosphorylated serine and threonine residues promote site‐specific fragmentation of singly charged,arginine‐containing peptide ions

In order to investigate gas‐phase fragmentation reactions of phosphorylated peptide ions, matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass (MS/MS) spectra were recorded from synthetic phosphopeptides and from phosphopeptides isolated from natural sources. MALDI‐TOF/TOF (TOF: time‐of‐flight) spectra of synthetic arginine‐containing phosphopeptides revealed a significant increase of y ions resulting from bond cleavages on the C‐terminal side of phosphothreonine or phosphoserine. The same effect was found in ESI‐MS/MS spectra recorded from the singly charged but not from the doubly charged ions of these phosphopeptides. ESI‐MS/MS spectra of doubly charged phosphopeptides containing two arginine residues support the following general fragmentation rule: Increased amide bond cleavage on the C‐terminal side of phosphorylated serines or threonines mainly occurs in peptide ions which do not contain mobile protons. In MALDI‐TOF/TOF spectra of phosphopeptides displaying N‐terminal fragment ions, abundant b–H₃PO₄ ions resulting from the enhanced dissociation of the pSer/pThr–X bond were detected (X denotes amino acids). Cleavages at phosphoamino acids were found to be particularly predominant in spectra of phosphopeptides containing pSer/pThr–Pro bonds. A quantitative evaluation of a larger set of MALDI‐TOF/TOF spectra recorded from phosphopeptides indicated that phosphoserine residues in arginine‐containing peptides increase the signal intensities of the respective y ions by almost a factor of 3. A less pronounced cleavage‐enhancing effect was observed in some lysine‐containing phosphopeptides without arginine. The proposed peptide fragmentation pathways involve a nucleophilic attack by phosphate oxygen on the carbon center of the peptide backbone amide, which eventually leads to cleavage of the amide bond. Copyright © 2009 John Wiley & Sons, Ltd.     

Sequence determination of αs1‐casein isoforms from donkey by mass spectrometric methods

Four co‐eluting components, with experimentally measured M_r of 23 658, 23 786, 24 278 and 24 406 Da, were detected by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) analysis in the dephosphorylated casein fraction of a milk sample collected at middle lactation stage from an individual donkey belonging to the Ragusano breed. By coupling RP‐HPLC, two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE), enzymatic digestions, MALDI‐TOF MS and capillary RP‐HPLC/nano‐electrospray ionization tandem mass spectrometry (nESI‐MS/MS) analyses, the four components were identified as donkey's α_s1‐CNs and their sequences completely characterized, using the known mare's α_s1‐CN (GenBank Acc. No. AAK83668; M_r 23750.7 Da) as reference. The proteins with M_r of 23 786 and 23 658 Da differ in the presence of a glutamine residue at position 83 in the full‐length component and present the amino acid substitutions Q⁸→H and H¹¹⁵→Y with respect to the mare's α_s1‐CN. The other two components with M_r 24 406 and 24 278 Da, which also differ in the presence of a glutamine residue at position 88 in the full‐length component, show the insertion of the pentapeptide HTPRE between Leu³³ and the Glu³⁴. The two α_s1‐CNs bearing the pentapeptide insertion were named variants A (202 amino acids; M_r 24 406) and A₁ (201 amino acids; M_r 24 278), whereas the two α_s1‐CNs without the pentapeptide were named variants B (197 amino acids; M_r 23 786) and B₁ (196 amino acids; M_r 23 658). Copyright © 2009 John Wiley & Sons, Ltd.     

Characterisation of interferon α‐2b by liquid chromatography and mass spectrometry techniques

Interferon α‐2b produced by Escherichia coli consists of 165 amino acids and contains two disulphide bonds; its purity was confirmed by LC‐UV (DAD)‐FLD and LC‐MS techniques. A C₄ column was used with UV detection at 214 nm; diode array detector (DAD) spectra were recorded from 200–400 nm and fluorescence detection was performed at specific wavelengths of trypthophan emission and excitation. Peptide mapping was performed with trypsin. Peptides produced by trypsin digestion were analysed by LC‐UV (DAD)‐FLD, LC‐MS, and LC‐MS/MS using a C₁₈ column. Amino acid sequence coverage was about 95%. UV spectra in the range from 200 nm to 400 nm, emission (Em) and excitation (Ex) spectra of each separated peptide were additionally compared with spectra of the same peptide produced by digestion of European Pharmacopaeia interferon α‐2b standard (spectral matching). The chromatogram of any interferon α‐2b (drug substance or certificated standard) sample produced in the same manner with the same amino acid composition should be similar to the chromatogram obtained by the method described in this paper. Molecular masses of peptides were obtained from MS experiments and MS/MS experiments gave additional structural information. The molecular mass of interferon α‐2b was obtained by MALDI‐TOF MS analysis in linear mode, with an accuracy comparable to the theoretical average mass ± 5 atomic mass units. The molecular mass was obtained from the deconvoluted ESI mass spectrum.     

Sodiation as a tool for enhancing the diagnostic value of MALDI‐TOF/TOF‐MS spectra of complex astaxanthin ester mixtures from Haematococcus pluvialis

The microalga Haematococcus pluvialis produces the pigment astaxanthin mainly in esterified form with a multitude of fatty acids, which results in a complex mixture of carotenol mono‐ and diesters. For rapid fingerprinting of these esters, matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF/TOF‐MS) might be an alternative to traditional chromatographic separation combined with MS. Investigation of ionization and fragmentation of astaxanthin mono‐ and diester palmitate standards in MALDI‐TOF/TOF‐MS showed that sodium adduct parent masses [M + Na]⁺ gave much simpler MS² spectra than radical / protonated [M]^+● / [M + H]⁺ parents. [M + Na]⁺ fragments yielded diagnostic polyene‐specific eliminations and fatty acid neutral losses, whereas [M]^+● / [M + H]⁺ fragmentation resulted in a multitude of non‐diagnostic daughters. For diesters, a benzonium fragment, formed by polyene elimination, was required for identification of the second fatty acid attached to the astaxanthin backbone. Parents were forced into [M + Na]⁺ ionization by addition of sodium acetate, and best signal‐to‐noise ratios were obtained in the 0.1 to 1.0 mM range. This method was applied to fingerprinting astaxanthin esters in a crude H. pluvialis extract. Prior to MALDI‐TOF/TOF‐MS, the extract was fractionated by normal phase Flash chromatography to obtain fractions enriched in mono‐ and diesters and to remove pheophytin a, which compromised monoester signals. All 12 types of all‐trans esterified esters found in LC were identified with MALDI‐TOF/TOF‐MS, with the exception of two minor monoesters. Copyright © 2013 John Wiley & Sons, Ltd.