High-performance Liquid Chromatography-electrospray Ionization Mass Research Articles

Liquid chromatographic–electrospray mass spectrometric study of the phthalides of Angelica sinensis and chemical changes of Z-ligustilide

High-performance liquid chromatography–electrospray ionization–mass spectrometry has been applied to analyze the chemical constituents of Danggui (the rhizome of Angelica sinensis) and to study chemical changes of Z-ligustilide. Twelve phthalides were unambiguously identified as senkyunolide I ( 3), senkyunolide H ( 4), sedanenolide ( 8), butylphthalide ( 9), E-ligustilide ( 13), Z-ligustilide ( 14), Z-butylidenephthalide ( 15), Z, Z′-6.8′,7.3′-diligustilide ( 16), angelicide ( 17), levistolide A ( 18), Z-ligustilide dimer E-232 ( 19) and Z, Z′-3.3′,8.8′-diligustilide ( 20) in Danggui extract. The existence of 12 other phthalides ( 2, 5– 7, 11, 12, 22– 27), ferulic acid ( 1) and coniferyl ferulate ( 10) in Danggui extract has also been demonstrated. Phthalides 3, 4, 16– 18 and 20 were determined to be the products from chemical change of Z-ligustilide. This is the first report of the existence of 16 compounds ( 2– 8, 10– 12, 20, 22– 25 and 27) in Danggui extract.

Determination of Diethylpyrocarbonate-Modified Amino Acid Residues in α1-Acid Glycoprotein by High-Performance Liquid Chromatography Electrospray Ionization–Mass Spectrometry and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight–Mass Spectrometry

The chemical modification reagent diethylpyrocarbonate (DEPC) was used to modify α1-acid glycoprotein (orosomucoid, OMD) under various conditions. The extents of DEPC modification of the histidine and tyrosine residues were followed by UV spectrophotometry. The resulting modified OMD was analyzed using enzyme digestion, reverse-phase HPLC, electrospray ionization–mass spectrometry (ESI/MS), and matrix-assisted laser desorption ionization time-of-flight–mass spectrometry (MALDI-TOF/MS). The inherent problem of instability of DEPC-modified histidine residues was overcome by adjusting the time scale of the postreaction processing of modified OMD. There were observed differences in reactivity of histidine 97 and histidine 100 that were consistent throughout the pH range 6–8. Furthermore, several lysine residues were modified and the amount of modification increased over the pH range 6–8. These experiments show that HPLC–ESI/MS and MALDI-TOF/MS analysis coupled with enzyme digestion provide the necessary information to describe the reaction of DEPC with OMD. In addition, the results provide the carbethoxy-histidine stability and histidine reactivity information of DEPC-modified OMD necessary for the design of experiments to characterize the drug binding properties of OMD.

Selective analysis of the herbicides glyphosate and aminomethylphosphonic acid in water by on-line solid-phase extraction–high-performance liquid chromatography–electrospray ionization mass spectrometry

A fully automated on-line solid-phase extraction–high-performance liquid chromatography–electrospray ionization mass spectrometry (SPE-HPLC–ESI-MS–MS) method was developed for the determination of glyphosate and its degradation product aminomethylphosphonic acid (AMPA), in the concentration range of 0.05 to 3 μg/l in various types of water. Derivatisation of glyphosate and AMPA directly in the native aqueous sample with 9-fluorenyl methoxycarbonyl chloride allowed the preconcentration by SPE and HPLC separation. Identification and quantification based on the constant ratio of three selected ions for each compound (precursor ion and two product-ions) together with the retention time proved to be a very selective procedure for the reliable determination of these pesticides. Although developed for drinking water and surface water, the selectivity of this method also allows their determination in waste water. Moreover, the structurally similar herbicide glufosinate could also be determined using the same derivatisation procedure and analytical method. Validation of the HPLC–MS method for glyphosate and AMPA showed detection limits as low as 0.03 μg/l. Recoveries of 96% and relative standard deviations for repeatability <8.4% were determined at 0.2 concentration.

Characterization of water-soluble components of slurries using solid-phase microextraction coupled to liquid chromatography-mass spectrometry

Water-soluble components from sludge and sediments were sampled and analyzed with solid-phase microextraction (SPME) and high-performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC–ESI–MS). The extraction, desorption, and detection conditions were examined with Carbowax-coated SPME fibers for a number of selected carboxylic acids, phthalates, and surfactants. The maximum recoveries of the defined compounds were obtained for extraction times ranging from 1 to 15 h (phthalates and surfactants) with a desorption time of 2 min for a of 80:20 (v/v) methanol–ethanol mixture. The reference compounds were detected in the low-ppb range. The main components of analyzed sludge and sediment samples were identified as phthalates, fatty acids, nonionic surfactants, chlorinated phenols, and carbohydrate derivatives. Additional information for compound identification was obtained using different spray potentials for the mass spectrometric ionization. Switching from positive to negative ionization modes changed the selectivity and sensitivity of detection, thus allowing the detection of substances of different nature. © 1998 John Wiley & Sons, Inc. J Micro Sep 10: 225–234, 1998

Characterization of water‐soluble components of slurries using solid‐phase microextraction coupled to liquid chromatography–mass spectrometry

Water-soluble components from sludge and sediments were sampled and analyzed with solid-phase microextraction (SPME) and high-performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC–ESI–MS). The extraction, desorption, and detection conditions were examined with Carbowax-coated SPME fibers for a number of selected carboxylic acids, phthalates, and surfactants. The maximum recoveries of the defined compounds were obtained for extraction times ranging from 1 to 15 h (phthalates and surfactants) with a desorption time of 2 min for a of 80:20 (v/v) methanol–ethanol mixture. The reference compounds were detected in the low-ppb range. The main components of analyzed sludge and sediment samples were identified as phthalates, fatty acids, nonionic surfactants, chlorinated phenols, and carbohydrate derivatives. Additional information for compound identification was obtained using different spray potentials for the mass spectrometric ionization. Switching from positive to negative ionization modes changed the selectivity and sensitivity of detection, thus allowing the detection of substances of different nature. © 1998 John Wiley & Sons, Inc. J Micro Sep 10: 225–234, 1998

New procedure for the use of high-performance liquid chromatography–electrospray ionization mass spectrometry for the analysis of nucleotides and oligonucleotides

A method is described which allows the combination of high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry for the analysis of nucleotides and oligonucleotides without compromising the performance of either technique. The essential feature of the method uses 1,1,1,3,3,3-hexafluoro-2-isopropanol as an additive to the HPLC mobile phase. This novel additive results in both good HPLC separation and efficient negative ion mode electrospray ionization. The method is demonstrated for oligonucleotides samples such as synthetic homopolymers of thymidine (PolyT), of fragments based on the pBR322 plasmid sequence and analysis of phosphorothioate ester antisense oligonucleotides. The differentiation of phosphorylation states of natural nucleotides as well as nucleotide analogues, such as ziduvodine (AZT), is also demonstrated.

Identification and mechanism of formation of potentially genotoxic metabolites of tamoxifen: study by LC-MS/MS

On-line high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI MS) and tandem mass spectrometry (MS/MS) have been applied to the study of tamoxifen metabolism in liver microsomes and to the identification of potentially genotoxic metabolites. The results showed that the hydroxylated derivatives, including 4-hydroxytamoxifen and α-hydroxytamoxifen are detoxication metabolites, while arene oxides, their free radical precursors or metabolic intermediates, are the most probable species involved in DNA-adduct formation.

Electrospray ionization-tandem mass spectrometry analysis of peptides derived by enzymatic digestion of oxidized globin subunits: An improved method to determine amino acid substitution in the hemoglobin “core”

The determination of an amino acid substitution in the "core" region of abnormal hemoglobin is technically difficult and is time- and labor-intensive both by conventional and by mass spectrometry techniques. Underivatized subunits cleaved with trypsin and lysyl endopeptidase were analyzed by high-performance liquid chromatography-electrospray ionizationmass spectrometry. The core peptides showed a series of multiply charged ions of homo- and heterodimers. Abnormal peptides in the core region could be detected in dimeric form. The sequence of core peptides was determined by product ion spectra of the peptides from oxidized globin digested with trypsin and lysyl endopeptidase. Oxidation of cysteine residues to cysteic acids in the core region resulted in the strong promotion of y-series ions by product ion analysis with a tryptic peptide from apoprotein B-100 as previously reported by Burlet, Yang, and Gaskell (J. Am. Soc. Mass Spectrom. 1992, 3, 337-344). These techniques were used to prove that substitution of an unstable hemoglobin, known as Hb Santa Ana (β88 leucine → proline), occurred in a patient with congenital hemolytic anemia. The tandem mass spectrometry analysis with oxidized globin digested with trypsin and lysyl endopeptidase offers a novel method to detect substitutions in the core region of hemoglobin.

Study of phenformin metabolism in rat liver microsomes by HPLC, CE and on-line HPLC-electrospray ionization mass spectrometry.

Methods for the analysis of phenformin and its metabolite by high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESIMS) are developed. The effects of pH, buffer concentration and proportion of organic modifier on the retention of the compounds in HPLC have been studied. The optimum condition was used for the separation and identification of phenformin and its metabolite in microsomal metabolism by HPLC-ESIMS. A simple CE method is also described for the separation of these compounds. Optimum incubation conditions and cofactor requirements for the formation of 4-hydroxyphenformin by microsomal preparations of rat liver were determined. A linear response in the formation of product was found with increasing concentrations of protein and up to 15 min incubation. High concentrations of phenformin inhibited its metabolite formation, and K(m) was 4 microM.

Application of high-performance liquid chromatography-electrospray ionization mass spectrometry and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry in combination with selective enzymatic modifications in the characterization of glycosylation patterns in single-chain plasminogen activator

The application of high-performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective enzymatic deglycosylation treatments is demonstrated in the analysis of glycosylation patterns in recombinant Desmodus salivary plasminogen activator, a heterogeneous glycoprotein. The sample was initially digested with a proteolytic enzyme (endoproteinase Lys-C) and then further treated with either PNGase F to remove N-linked carbohydrates or a combination of neuraminidase and O-glycosidase to remove sialic acid and O-linked carbohydrates. By comparison of the LC-ESI-MS peptide maps for the fully glycosylated and deglycosylated samples, it was possible to unambiguously identify the sites of N-linked glycosylation as well a number of N-linked glycopeptides. The O-linked glycopeptides, which are present at a low level (<1%), were not detected prior to the deglycosylation, nor could changes in peptide elution in the map following deglycosylation be correlated with potential O-linked glycosylation sites.

Signal enhancement for gradient reverse-phase high-performance liquid chromatography-electrospray ionization mass spectrometry analysis with trifluoroacetic and other strong acid modifiers by postcolumn addition of propionic acid and isopropanol

Trifluoroacetic acid (TFA) and other volatile strong acids, used as modifiers in reverse-phase high-performance liquid chromatography, cause signal suppression for basic compounds when analyzed by electrospray ionization mass spectrometry (ESI-MS). Evidence is presented that signal suppression is caused by strong ion pairing between the TFA anion and the protonated sample cation of basic sample molecules. The ion-pairing process "masks" the protonated sample cations from the ESI-MS electric fields by rendering them "neutral. " Weakly basic molecules are not suppressed by this process. The TFA signal suppression effect is independent from the well-known spray problem that electrospray has with highly aqueous solutions that contain TFA. This previously reported spray problem is caused by the high conductivity and surface tension of aqueous TFA solutions. A practical method to enhance the signal for most basic analytes in the presence of signal-suppressing volatile strong acids has been developed. The method employs postcolumn addition of a solution of 75% propionic acid and 25% isopropanol in a ratio 1:2 to the column flow. Signal enhancement is typically 10-50 times for peptides and other small basic molecules. Thus, peptide maps that use ESI-MS for detection can be performed at lower levels, with conventional columns, without the need to use capillary chromatography or reduced mass spectral resolution to achieve satisfactory sensitivity. The method may be used with similar results for heptafluorobutyric acid and hydrochloric acid. A mechanism for TFA signal suppression and signal enhancement by the foregoing method, is proposed.

Online High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry for the Determination of Brevetoxins in "Red Tide" Algae

On-line high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESMS) has been successfully applied to the separation and identification of brevetoxins associated with "red tide" algae. Brevetoxins are toxic polyethers produced by the marine dinoflagellate Gymnodinium breve. They are responsible for fish kills, and they pose certain health risks to humans. The LC-MS method employs reversed-phase microbore HPLC on a C18 column with a mobile phase consisting of 85:15 methanol/water, a flow rate of 8 microL/min, and a postcolumn split ratio of 3:1 (UV absorbance detector/mass spectrometer). A brevetoxin culture sample was found to contain at least six components, including two well-separated peaks corresponding to the brevetoxins PbTx-2 and PbTx-1, as well as several unknown compounds, including one with a molecular mass of 899 Da (possibly an isomer of PbTx-9). The brevetoxin molecules exhibited a high tendency to bind to alkali cations in positive ion ESMS. For standard PbTx-9, PbTx-2, and PbTx-1 brevetoxins analyzed on our LC-MS system, the detection limits (employing mass spectrometer scans of 100 m/z units) were determined to be less than 600 fmol, 1 pmol, and 50 fmol, respectively (S/N = 3); the total analysis time was about 35 min.

Characterization of the tryptic map of recombinant DNA derived tissue plasminogen activator by high-performance liquid chromatography-electrospray ionization mass spectrometry

A detailed tryptic map is presented for recombinant human tissue plasminogen activator (rt-PA). Electrospray ionization mass spectrometry is utilized as an on-line HPLC detector for tryptic mapping of this glycoprotein. The additional dimension provided by mass spectrometry gives considerably more detail about the complex tryptic map and significantly enhances the high-resolution chromatographic separation by distinguishing by mass any coeluting components. Through this improvement, the proline isomers of a tryptic peptide were observed eluting over a broad range of retention times. The glycopeptides of rt-PA are observed as well as any corresponding nonglycosylated peptides. In addition, the carbohydrate heterogeneity is readily observed, allowing analysis of the carbohydrate composition. The characteristic diagonal patterns formed by glycopeptides in a contour plot of the data allow rapid recognition of the glycopeptides.