Showing papers in "Rapid Communications in Mass Spectrometry in 2002"

A new linear ion trap mass spectrometer

Abstract: Characteristics of mass selective axial ion ejection from a linear quadrupole ion trap in the presence of an auxiliary quadrupole field are described. Ion ejection is shown to occur through coupling of radial and axial motion in the exit fringing fields of the linear ion trap. The coupling is efficient and can result in extraction of as much as 20% of the trapped ions. This, together with the very high trapping efficiencies, can yield high sensitivity mass spectral responses. The experimental apparatus is based on the ion path of a triple quadrupole mass spectrometer allowing either the q2 collision cell or the final mass analysis quadrupole to be used as the linear trap. Space charge induced distortions of the mass resolved features while using the pressurized q2 linear ion trap occur at approximately the same ion density as reported for conventional three-dimensional ion traps. These distortions are, however, much reduced for the lower pressure linear trap possibly owing to the proposed axial ejection mechanism that leads to ion ejection only for ions of considerable radial amplitude. RF heating due to the high ejection q-value and the low collision frequency may also contribute. Two hybrid RF/DC quadrupole-linear ion trap instruments are described that provide high sensitivity product ion scanning while operated in the linear ion trap mode while also retaining all conventional triple quadrupole scan modes such as precursor ion and neutral loss scan modes. Copyright © 2002 John Wiley & Sons, Ltd.

Metabonomics: the use of electrospray mass spectrometry coupled to reversed‐phase liquid chromatography shows potential for the screening of rat urine in drug development

Abstract: The application of liquid chromatography/mass spectrometry (LC/MS) followed by principal components analysis (PCA) has been successfully applied to the screening of rat urine following the administration of three candidate pharmaceuticals. With this methodology it was possible to differentiate the control samples from the dosed samples and to identify the components of the mass spectrum responsible for the separation. These data clearly show that LC/MS is a viable alternative, or complementary, technique to proton NMR for metabonomics applications in drug discovery and development.

Amino acid residue specific stable isotope labeling for quantitative proteomics

Abstract: Various stable isotope labeling (SIL) techniques have recently emerged to improve the efficiency and accuracy of protein quantitation by mass spectrometry (MS). We have developed a mass-tagging strategy to incorporate stable isotope tagged amino acids into cellular proteins in a residue-specific manner during cell growth. In this study, we further extend this residue-specific SIL approach to the accurate quantitation of protein abundances in different cell populations. For proteins whose expression levels are the same in cells grown in the normal and labeled media, the relative areas of the normal (light) and labeled (heavy) isotopic peaks are linearly correlated with the cells mixing ratios. This approach was first used to determine the effect of the zinc-responsive transcription factor Zap1 on the yeast proteome. Ten protein spots from a PAGE gel were chosen randomly and their differential protein expression levels in wild-type and zap1Δ cells were readily determined by the isotopic ratio. Methionine synthase (Met6) was identified to be up-regulated more than four times in the zap1Δ mutant strain whereas the expression level of other nine proteins remained unchanged. Further, we applied this strategy to study the cellular response to radiation in human skin fibroblast cells. Analyzing one protein band randomly selected from SDS-PAGE, the expression level of a novel protein was found to increase two-fold in response to radiation whereas the expression level of a control protein remained unchanged. This strategy is generally applicable using any particular type of amino acid as the labeling precursors for accurate quantitation of protein relative abundances. Published in 2002 by John Wiley & Sons, Ltd.

Dual parallel electrospray ionization and atmospheric pressure chemical ionization mass spectrometry (MS), MS/MS and MS/MS/MS for the analysis of triacylglycerols and triacylglycerol oxidation products.

Abstract: Two mass spectrometers, in parallel, were employed simultaneously for analysis of triacylglycerols in canola oil, for analysis of triolein oxidation products, and for analysis of triacylglycerol positional isomers separated using reversed-phase high-performance liquid chromatography. A triple quadrupole mass spectrometer was interfaced via an atmospheric pressure chemical ionization (APCI) interface to two reversed-phase liquid chromatographic columns in series. An ion trap mass spectrometer was coupled to the same two columns using an electrospray ionization (ESI) interface, with ammonium formate added as electrolyte. Electrospray ionization mass spectrometry (ESI-MS) under these conditions produced abundant ammonium adduct ions from triacylglycerols, which were then fragmented to produce MS/MS spectra and then fragmented further to produce MS/MS/MS spectra. ESI-MS/MS of the ammoniated adduct ions gave product ion mass spectra which were similar to mass spectra obtained by APCI-MS. ESI-MS/MS produced diacylglycerol fragment ions, and additional fragmentation (MS/MS/MS) produced [RCO](+) (acylium) ions, [RCOO+58](+) ions, and other related ions which allowed assignment of individual acyl chain identities. APCI-MS of triacylglycerol oxidation products produced spectra like those reported previously using APCI-MS. APCI-MS/MS produced ions related to individual fatty acid chains. ESI-MS of triacylglycerol oxidation products produced abundant ammonium adduct ions, even for those molecules which previously produced little or no intact molecular ions under APCI-MS conditions. Fragmentation (MS/MS) of the [M+NH(4)](+) ions produced results similar to those obtained by APCI-MS. Further fragmentation (MS/MS/MS) of the diacylglycerol fragments of oxidation products provided information on the oxidized individual fatty acyl chains. ESI-MS and APCI-MS were found to be complementary techniques, which together contributed to a better understanding of the identities of the products formed by oxidation of triacylglycerols.

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

Abstract: 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.

Triplex and quadruplex DNA structures studied by electrospray mass spectrometry.

Abstract: DNA triplex and quadruplex structures have been successfully detected by electrospray ionization mass spectrometry (ESI-MS). Circular dichroism and UV-melting experiments show that these structures are stable in 150 mM ammonium acetate at pH 7 for the quadruplexes and pH 5.5 for the triplexes. The studied quadruplexes were the tetramer [d(TGGGGT)](4), the dimer [d(GGGGTTTTGGGG)](2), and the intramolecular folded strand dGGG(TTAGGG)(3), which is an analog of the human telomeric sequence. The absence of sodium contamination allowed demonstration of the specific inclusion of n - 1 ammonium cations in the quadruplex structures, where n is the number of consecutive G-tetrads. We also detected the complexes between the quadruplexes and the quadruplex-specific drug mesoporphyrin IX. MS/MS spectra of [d(TGGGGT)](4) and the complex with the drug are also reported. As the drug does not displace the ammonium cations, one can conclude that the drug binds at the exterior of the tetrads, and not between them. For the triplex structure the ESI-MS spectra show the detection of the specific triplex, at m/z values typically higher than those typically observed for duplex species. Upon MS/MS the antigene strand, which is bound into the major groove of the duplex, separates from the triplex. This is the same dissociation pathway as in solution. To our knowledge this is the first report of a triplex DNA structure by electrospray mass spectrometry.

Technical considerations for RNA-based stable isotope probing: an approach to associating microbial diversity with microbial community function.

Abstract: An ongoing challenge within microbial ecology is the development of methodologies that attribute microbial community functions to microbial diversity. One approach, involving the incorporation of stable isotopes from labelled tracer compounds into biological signature molecules (biomarkers), may overcome this current limitation. To examine the potential of RNA as the biomarker in stable isotope probing we have generated a series of atom % 13C-enriched RNA samples through exploitation of the anabolic abilities of a phenol-degrading environmental isolate. Isotope ratio mass spectrometry was used to determine the atom % 13C of each RNA sample (ca. 1–100%). The corresponding buoyant density (1.755–1.795 g mL−1) was determined by equilibrium density gradient centrifugation and agarose gel electrophoresis. This empirically defined relationship between the atom % 13C of RNA and its buoyant density suggests ribonucleic acids with atom % 13C enrichments greater than 10% can be isolated by equilibrium density centrifugation. The processing and analysis of isolated RNA by reverse transcription polymerase chain reaction, denaturing gradient gel electrophoresis, cloning and sequencing are discussed. The RNA-based stable isotope probing protocol presented here will find particular utility in assessing the roles of microbial community members in the biodegradation of natural and anthropogenic xenobiotic compounds. Copyright © 2002 John Wiley & Sons, Ltd.

Stable isotope (13C, 15N and 34S) analysis of the hair of modern humans and their domestic animals.

Abstract: Relationships between dietary status and recent migration were examined by delta(13)C, delta(15)N and delta(34)S analysis of hair samples from 43 modern humans living in a rural community in SW England. The isotopic content of 38 'local' hair samples was compared with that of five recently arrived individuals (from Canada, Chile, Germany and the USA). Hair samples from domestic animals (i.e. mainly cats, dogs, cows and horses) were analysed to examine the difference in delta(13)C, delta(15)N and delta(34)S values between herbivores and carnivores. Generally, modern human hair data from the triple stable isotope (delta(13)C, delta(15)N and delta(34)S) provided enough information to confirm the dietary status and origin of the individual subjects. The dietary intake was generally reflected in the animal hair delta(15)N and delta(13)C values, i.e. highest in the carnivores (cats). However, a non-local origin of food sources given to domesticated omnivores (i.e. dogs) was suggested by their hair delta(34)S values.

9-Aminoacridine as a matrix for negative mode matrix-assisted laser desorption/ionization

Abstract: 9-Aminoacridine (9AA) is introduced as a matrix for negative mode matrix-assisted laser desorption/ionization (MALDI) While most traditional MALDI matrices have been acidic in character, 9AA is a moderately strong base The mechanism by which 9AA brings about ionization in the negative mode appears to involve abstraction of a labile proton in an acid/base reaction Most MALDI matrices readily donate protons; 9AA readily accepts them leading to the formation of [M − H]− species 9AA was first examined as a matrix for low molecular weight compounds having acidic protons, such as phenols, carboxylic acids, sulfonates, amines and alcohols Also studied were larger molecules frequently analyzed by MALDI including oligonucleotides, oligoamides and proteins Spectra obtained from 9AA were compared with those recorded using more traditional matrices such as 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (α-CHCA) Copyright © 2002 John Wiley & Sons, Ltd

Detection of norbolethone, an anabolic steroid never marketed, in athletes' urine

Abstract: Norbolethone (13-ethyl-17-hydroxy-18,19-dinor-17α-pregn-4-en-3-one) is a 19-nor anabolic steroid first synthesized in 1966. During the 1960s it was administered to humans in efficacy studies concerned with short stature and underweight conditions. It has never been reported by doping control laboratories. Norbolethone was identified in two urine samples from one athlete by matching the mass spectra and chromatographic retention times with those of a reference standard. The samples also contained at least one likely metabolite. The samples were also unusual because the concentrations of endogenous steroids were exceptionally low. Since norbolethone is not known to be marketed by any pharmaceutical company, a clandestine source of norbolethone may exist. Copyright © 2002 John Wiley & Sons, Ltd.

Combined quantum chemical and RRKM modeling of the main fragmentation pathways of protonated GGG. II. Formation of b2, y1, and y2 ions

Abstract: Quantum chemical and RRKM calculations were performed on protonated GGG in order to determine the atomic details of the main fragmentation pathways leading to formation of b 2 ,y 1 , and y 2 ions. Formation of y 1 ions on the 'diketopiperazine' pathway is initiated from relatively high-energy C-terminal amide nitrogen protonated species for which the N-terminal amide bond is in the cis isomerization state. The reaction goes through a transition structure which is only slightly less favored than the reactive configuration itself. RRKM calculations indicate that this reaction is extremely fast as soon as the fragmenting species have more internal energy than the reaction threshold. The calculated energetics suggests that y 1 ions are formed on the 'diketopiperazine' pathway with a non-negligible (6-10 kcal/mol) reverse activation barrier. Investigation of species occurring during the formation of b 2 ions having an oxazolone structure indicates that y 1 ions can be formed also from intermediates previously thought to result in only b 2 ions. As the first step of the 'b x -y z ' pathway proposed here the extra proton must reach the nitrogen of the C-terminal amide bond. Attack of the N-terminal amide oxygen on the carbon center of the C-terminal amide bond results in formation of the oxazolone ring while the detaching G leaves the precursor ion. Under low-energy collision conditions the complex of protonated 2-aminomethyl-5-oxazolone and G can rearrange to form a proton-bonded dimer of these species. In such circumstances the extra proton is shared by the two monomers and dissociation of the dimer will be determined by the thermochemistry involved. Based on the 'b x -y z ' pathway one can easily explain the linear relationship between the logarithm of the y 1 /b 2 ion abundance ratio and the proton affinity of the C-terminal amino acid substituent for the series of H-Gly-Gly-Xxx-OH tripeptides where Xxx was varied (Morgan DG, Bursey MM. Org. Mass. Spectrom. 1994; 29: 354). The calculated energetics indicates that both y 1 and b 2 ions are formed with no reverse activation barrier on the 'b x -y z ' pathway.

Simultaneous separation of 17 inorganic and organic arsenic compounds in marine biota by means of high-performance liquid chromatography/inductively coupled plasma mass spectrometry.

Abstract: A method using high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICP-MS) has been developed to determine inorganic arsenic (arsenite, arsenate) along with organic arsenic compounds (monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, arsenocholine, trimethylarsine oxide, tetramethylarsonium ion and several arsenosugars) in fish, mussel, oyster and marine algae samples. The species were extracted by means of a methanol/water mixture and a dispersion unit in 2 min, with extraction efficiencies ranging from 83 to 107% in the different organisms. Up to 17 different species were determined within 15 min on an anion-exchange column, using a nitric acid gradient and an ion-pairing reagent. As all species are shown in one chromatogram, a clear overview of arsenic distribution patterns in different marine organisms is given. Arsenobetaine is the major compound in marine animals whereas arsenosugars and arsenate are dominant in marine algae. The method was validated with CRM DORM-2 (dogfish muscle). Concentrations were within the certified limits and low detection limits of 8 ng g(-1) (arsenite) to 50 ng g(-1) (arsenate) were obtained.

Delta13C and delta18O isotopic composition of CaCO3 measured by continuous flow isotope ratio mass spectrometry: statistical evaluation and verification by application to Devils Hole core DH-11 calcite.

Abstract: A new method was developed to analyze the stable carbon and oxygen isotope ratios of small samples (400 +/- 20 micro g) of calcium carbonate. This new method streamlines the classical phosphoric acid/calcium carbonate (H(3)PO(4)/CaCO(3)) reaction method by making use of a recently available Thermoquest-Finnigan GasBench II preparation device and a Delta Plus XL continuous flow isotope ratio mass spectrometer. Conditions for which the H(3)PO(4)/CaCO(3) reaction produced reproducible and accurate results with minimal error had to be determined. When the acid/carbonate reaction temperature was kept at 26 degrees C and the reaction time was between 24 and 54 h, the precision of the carbon and oxygen isotope ratios for pooled samples from three reference standard materials was

N-Terminal peptide labeling strategy for incorporation of isotopic tags: a method for the determination of site-specific absolute phosphorylation stoichiometry.

Abstract: Determining the phosphorylation stoichiometry at specific sites in a phosphoprotein is a very challenging task. We describe here a novel mass spectrometry based method that is capable of measuring the absolute phosphorylation stoichiometry at specific sites without the need for specific internal standards, phospho-site antibodies or radioactivity. The method is based on a gentle chemical labeling strategy which specifically and differentially labels the N-terminus of all peptides in a sample with either a D(5)- or D(0)-propionyl group and measures the ratio of the abundance of the D(5)/D(0) peptide pairs simultaneously using mass spectrometry. Using matrix-assisted laser desorption/ionization (MALDI), the method can measure absolute stoichiometry to within at least 10% and can be applied to both in vitro and in vivo phosphorylated peptides and proteins. Furthermore, this method can potentially be applied to the quantitative study of other types of protein post-translational modifications, and the profiling of protein expression on the proteome level.

Intramolecular cross-linking experiments on cytochrome c and ribonuclease A using an isotope multiplet method.

Abstract: Mass spectral analysis of tryptic digests of cross-linked proteins offers considerable promise as a simple technique to probe protein structure and study protein-protein interactions. We describe the use of a 1:1 mixture of isotopically labeled and unlabeled cross-linkers, disuccinimidyladipate (DSA) and dimethyladipimidate (DMA), to enhance visualization of cross-linked peptides in a tryptic digest. Optimized intramolecular reactions of cytochrome c and ribonuclease A (RNase A) with DSA yielded an average of two cross-links per protein molecule. After digestion of the cross-linked cytochrome c with trypsin and analysis by liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization (MALDI), eight modified peptides, five cross-linked and two end-capped, were detected by virtue of their doublet character. An eighth modified peptide's identity remained ambiguous because of its inability to fragment. The lysine-lysine distance constraints obtained are discussed in the context of the known NMR and X-ray structures of cytochrome c. Analysis of cross-linked RNase A by LC/MS and MALDI yielded nine modified peptides, four of which were modified twice, as indicated by the isotopic triplets. Although seven of these peptides contained cross-links, few distance constraints were gained due to the fact that the cross-linked products were variations of modification of the same three lysine residues.

Determination of selected sulfonamide antibiotics and trimethoprim in manure by electrospray and atmospheric pressure chemical ionization tandem mass spectrometry.

Abstract: A method for the determination of sulfonamides and trimethoprim in the complex matrix liquid manure has been developed using reversed-phase liquid chromatography and atmospheric pressure chemical ionization (APCI) tandem mass spectrometry. A comparison was made between electrospray and atmospheric pressure chemical ionization. APCI proved to be more robust and less sensitive to matrix effects. High-performance liquid chromatographic (HPLC) separation of the analytes was achieved in less than 7 min. The compounds were extracted with ethyl acetate and the extracts were cleaned up by solid-phase extraction on an aminopropyl column. Recoveries were not dependent on the concentration level. The mean recoveries were as follows: trimethoprim 79.0%, sulfadiazine 80.5%, N(4)-acetylsulfadiazine 91.0%, sulfamerazine 78.6%, sulfadimidine 77.2% and sulfamethoxazole 82.8%. Linearity was established over a concentration range of 5 to 5000 microg/kg with correlation coefficients greater than 0.99. The method had a limit of quantitation (LOQ) of 5 microg/kg manure.

Oxygen isotope corrections for online δ34S analysis

Abstract: Elemental analyzers have been successfully coupled to stable-isotope-ratio mass spectrometers for online measurements of the δ34S isotopic composition of plants, animals and soils. We found that the online technology for automated δ34S isotopic determinations did not yield reproducible oxygen isotopic compositions in the SO2 produced, and as a result calculated δ34S values were often 1–3‰ too high versus their correct values, particularly for plant and animal samples with high C/S ratio. Here we provide empirical and analytical methods for correcting the S isotope values for oxygen isotope variations, and further detail a new SO2-SiO2 buffering method that minimizes detrimental oxygen isotope variations in SO2. Published in 2002 by John Wiley & Sons, Ltd.

New algorithms for processing and peak detection in liquid chromatography/mass spectrometry data.

Abstract: Two new algorithms for automated processing of liquid chromatography/mass spectrometry (LC/MS) data are presented. These algorithms were developed from an analysis of the noise and artifact distribution in such data. The noise distribution was analyzed by preparing histograms of the signal intensity in LC/MS data. These histograms are well fit by a sum of two normal distributions in the log scale. One new algorithm, median filtering, provides increased performance compared to averaging adjacent scans in removing noise that is not normally distributed in the linear scale. Another new algorithm, vectorized peak detection, provides increased robustness with respect to variation in the noise and artifact distribution compared to methods based on determining an intensity threshold for the entire dataset. Vectorized peak detection also permits the incorporation of existing algorithms for peak detection in ion chromatograms and/or mass spectra. The application of these methods to LC/MS spectra of complex biological samples is described.

Improved method for the simultaneous determination of d4T, 3TC and ddl intracellular phosphorylated anabolites in human peripheral-blood mononuclear cells using high-performance liquid chromatography/tandem mass spectrometry.

Abstract: There is still a need for direct determination (i.e. without dephosphorylation) of nucleoside reverse transcriptase inhibitor (NRTI) triphosphorylated nucleotides in the peripheral-blood mononuclear cells (PBMCs) of HIV-positive patients. The objective of this paper was first to improve our previously described direct liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for stavudine triphosphate (d4T-TP). Preparation of PBMCs was modified to reduce degradation of d4T-TP during cell preparation and to simplify this step for routine use in clinical units. The performance of several HPLC columns was compared in order to improve the stability of peak shape over time. The SMT C(18) column was replaced by a Supelcogel ODP-50, thereby reducing two-fold the concentration of the first standard. Various internal standards were compared to optimize peak shape and remove an interfering peak in LC. 2-Chloroadenosine 5prime prime or minute-triphosphate was chosen as the most appropriate internal standard. Substitution of the narrowbore column by a microbore column (150 x 0.32 mm) is also presented and discussed. Secondly, this improved method was successfully applied to the simultaneous determination of d4T-TP, dideoxyadenosine triphosphate (ddA-TP) and lamivudine triphosphate (3TC-TP) in PBMCs, which is useful in view of the common use of NRTI combinations. The method was subsequently applied to clinical samples from HIV-positive patients receiving antiretroviral therapy containing d4T, ddl and/or 3TC. This method can be used simply and routinely on approximately 200 samples per week, using commercially available instruments and with a simple cell lysis as sample treatment.

Determination of drug binding sites to proteins by electrospray ionisation mass spectrometry: the interaction of cisplatin with transferrin

Abstract: Cisplatin, cis-[PtCl2(NH3)2], is known to bind to human serum transferrin, but the binding site remains a matter of some debate. Electrospray ionisation mass spectrometry has been used to characterise the interaction of cisplatin with transferrin. The studies indicate that cisplatin initially docks with, and subsequently bonds covalently to, the hydroxyl functional group of threonine 457, with the loss of HCl affording a transferrin-O-PtCl(NH3)2 adduct.

A rapid and precise method for sampling and determining the oxygen isotope ratio of atmospheric water vapor

Abstract: A quantitative method for cryogenically sampling atmospheric water vapor on the temporal scale of 10 to 15 min in the field or laboratory is described. The sample apparatus is lightweight, affordable, and easy to assemble. The method allows for H2O:CO2 equilibration within the same sampling tubes and hence increases turnaround time for δ18O analysis. Quantitative analysis in the laboratory showed recovery of a vaporized, known, 18O water standard to 0.2‰ precision. Copyright © 2002 John Wiley & Sons, Ltd.

Identification of proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing as a prefractionation step followed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation mass spectrometry

Abstract: Cerebrospinal fluid (CSF) is in close proximity to the brain and changes in the protein composition of CSF may be indicative of altered brain protein expression in neurodegenerative disorders. Analysis of brain-specific proteins in CSF is complicated by the fact that most CSF proteins are derived from the plasma and tend to obscure less abundant proteins. By adopting a prefractionation step prior to two-dimensional gel electrophoresis (2-DE), less abundant proteins are enriched and can be detected in complex proteomes such as CSF. We have developed a method in which liquid-phase isoelectric focusing (IEF) is used to prefractionate individual CSF samples; selected IEF fractions are then analysed on SYPRO-Ruby-stained 2-D gels, with final protein identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). To optimise the focusing of the protein spots on the 2-D gel, the ampholyte concentration in liquid-phase IEF was minimised and the focusing time in the first dimension was increased. When comparing 2-D gels from individual prefractionated and unfractionated CSF samples it is evident that individual protein spots are larger and contain more protein after prefractionation of CSF. Generally, more protein spots were also detected in the 2-D gels from prefractionated CSF compared with direct 2-DE separations of CSF. Several proteins, including cystatin C, IgM-kappa, hemopexin, acetyl-coenzyme A carboxylase-alpha, and alpha-1-acid glycoprotein, were identified in prefractionated CSF but not in unfractionated CSF. Low abundant forms of posttranslationally modified proteins, e.g. alpha-1-acid glycoprotein and alpha-2-HS glycoprotein, can be enriched, thus better resolved and detected on the 2-D gel. Liquid-phase IEF, as a prefractionation step prior to 2-DE, reduce sample complexity, facilitate detection of less abundant protein components, increases the protein loads and the protein amount in each gel spot for MALDI-MS analysis.

Generic method for on‐line extraction of drug substances in the presence of biological matrices using turbulent flow chromatography

Abstract: The use of liquid chromatography/mass spectrometry (LC/MS) to quantify drugs in biological matrices has been well established over the last decade. Extremely fast LC/MS methods are commonplace in the pharmaceutical industry for high-throughput Absorption, Distribution, Metabolism and Excretion (ADME) screening. However, to truly take full advantage of high-throughput ADME screening, a generic method is needed that eliminates the need to develop a new method for each new compound being screened. New developments in the stationary phase of turbulent flow columns has allowed us to develop an on-line biological sample cleanup method that is suitable for over 99% of the compounds in the Cephalon database.

High-speed gradient parallel liquid chromatography/tandem mass spectrometry with fully automated sample preparation for bioanalysis: 30 seconds per sample from plasma.

Abstract: In this work, a high-throughput and high-performance bioanalytical system is described that is capable of extracting and analyzing 1152 plasma samples within 10 hours. A Zymark track robot system interfaced with a Tecan Genesis liquid handler was used for simultaneous solid-phase extraction of four 96-well plates in a fully automated fashion. The extracted plasma samples were injected onto four parallel monolithic columns for separation via a four-injector autosampler. The use of monolithic columns allowed for fast and well-resolved separations at a considerably higher flow rate without generating significant column backpressure. This resulted in a total chromatographic run cycle time of 2 min on each 4.6 x 100 mm column using gradient elution. The effluent from the four columns was directed to a triple quadrupole mass spectrometer equipped with an indexed four-probe electrospray ionization source (Micromass MUX interface). Hence, sample extraction, separation, and detection were all performed in a four-channel parallel format that resulted in an overall throughput of about 30 s per sample from plasma. The performance of this system was evaluated by extracting and by analyzing twelve 96-well plates (1152) of human plasma samples spiked with oxazepam at different concentrations. The relative standard deviation (RSD) of analyte sensitivity (slope of calibration curve) across the four channels and across the 12 plates was 5.2 and 6.8%, respectively. An average extraction recovery of 77.6% with a RSD of 7.7% and an average matrix effect of 0.95 with a RSD of 5.2% were achieved using these generic extraction and separation conditions. The good separation efficiency provided by this system allowed for rapid method development of an assay quantifying the drug candidate and its close structural analog metabolite. The method was cross-validated with a conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay.

Optimization of a liquid chromatography method based on simultaneous electrospray ionization mass spectrometric and ultraviolet photodiode array detection for analysis of flavonoid glycosides.

Abstract: Different reversed-phase liquid chromatography (LC) columns of conventional dimensions were coupled to an ultraviolet photodiode array detector (UV-DAD) and a magnetic sector-type spectrometer, equipped with an electrospray ionization (ESI) source, by a laboratory-made flow splitter. A mixture of three flavonoid-O-glycosides was employed to examine the effects of the solvent composition, the flow rate, the stationary phase, the pH and the organic acid added, on the chromatographic separation, the UV-DAD detection, the ESI process and the entire LC system with ESI-MS and UV-DAD detection. In the positive ion mode, methanol containing 1% acetic acid was by far the most sensitive in ESI-MS analysis, whereas an acetonitrile/water mobile phase containing 0.5% formic acid was proved to give the best sensitivity in LC/ESI-MS/UV-DAD analysis. In the negative ion mode, the highest sensitivity was obtained with a mobile phase containing 0.1% formic acid, while addition of bases decreased the sensititvity. The optimal flow rate was higher in negative ESI (20-50 micro L/min) than in positive ESI (5 micro L/min), and the percentage of organic phase had an influence on the sensitivity of ESI-MS detection. With regard to the selection of a suitable C(18) reversed-phase LC column, a column which is well end-capped is to be preferred, because residual silanol groups appear to impair the separation of flavonoid glycosides. The optimized LC/ESI-MS/UV-DAD method was applied to a commercial Crataegus extract, which is used in phytomedicine to treat cardiovascular problems and is known to be rich in flavonoids. It is demonstrated how UV spectra and first-order ESI mass spectra allow a fast characterization of flavonoids, even if reference compounds are not available or at hand.

Direct surface analysis of fungal species by matrix-assisted laser desorption/ionization mass spectrometry.

Abstract: In this study various methods of sample preparation and matrices were investigated to determine optimum collection and analysis criteria for fungal analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Intact spores and/or hyphae of Aspergillus niger, Rhizopus oryzae, Trichoderma reesei and Phanerochaete chrysosporium were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The fungal samples were applied to the MALDI sample target as untreated, sonicated, or acid/heat treated samples, or blotted directly from the fungal culture with double-stick tape. Ferulic acid or sinapinic acid matrix solution was layered over the dried samples and analyzed by MALDI-MS. Statistical analysis showed that simply using double-stick tape to collect and transfer to a MALDI sample plate typically worked as well as the other preparation methods, and required the least sample handling.

Differentiation of vegetable oils by mass spectrometry combined with statistical analysis.

Abstract: The main triacylglycerol (TAG) composition of different plant oils (almond, avocado, corn germ, grape seed, linseed, mustard seed, olive, peanut, pumpkin seed, sesame seed, soybean, sunflower, walnut and wheat germ) were analyzed using two different mass spectrometric techniques: HPLC/APCI-MS (high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry) and MALDI-TOFMS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry). Linear discriminant analysis (LDA) as a multivariate mathematical statistical method was successfully used to distinguish different plant oils based on their relative TAG composition. With LDA analysis of either APCI-MS or MALDI-MS data, the classification among the almond, avocado, grape seed, linseed, mustard seed, olive, sesame seed and soybean oil samples was 100% correct. In both cases only 6 different oil samples from a total of 73 were not classified correctly. Copyright © 2002 John Wiley & Sons, Ltd.

Simultaneous determination of a drug candidate and its metabolite in rat plasma samples using ultrafast monolithic column high-performance liquid chromatography/tandem mass spectrometry.

Abstract: An ultrafast bioanalytical method using monolithic column high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) was evaluated for the simultaneous determination of a drug discovery compound and its metabolite in plasma. Baseline separation of the two compounds was achieved with run times of 24 or 30 s under isocratic or gradient conditions, respectively. The monolithic column HPLC/MS/MS system offers shorter chromatographic run times by increasing flow rate without sacrificing separation power for the drug candidate and its biotransformation product (metabolite). In this work, the necessity for adequate chromatographic resolution was demonstrated because the quantitative determination of the drug-related metabolism product was otherwise hampered by interference from the dosed drug compound. The chromatographic performance of a monolithic silica rod column as a function of HPLC flow rates was investigated with a mixture of the drug component and its synthetic metabolite. The assay reliability of the monolithic column HPLC/MS/MS system was checked for matrix ionization suppression using the post-column infusion technique. The proposed methods were successfully applied to the analysis of study rat plasma samples for the simultaneous quantitation of both the dosed drug and its metabolite. The analytical results obtained by the proposed monolithic column methods and the 'standard' silica particle-packed HPLC column method were in good agreement, within 10% error.

Liquid chromatography/tandem mass spectrometric bioanalysis using normal-phase columns with aqueous/organic mobile phases - a novel approach of eliminating evaporation and reconstitution steps in 96-well SPE.

Abstract: Bioanalytical methods using automated 96-well solid-phase extraction (SPE) and liquid chromatography with electrospray tandem mass spectrometry (LC/MS/MS) are widely used in the pharmaceutical industry. SPE methods typically require manual steps of drying of the eluates and reconstituting of the analytes with a suitable injection solvent possessing elution strength weaker than the mobile phase. In this study, we demonstrated a novel approach of eliminating these two steps in 96-well SPE by using normal-phase LC/MS/MS methods with low aqueous/high organic mobile phases, which consisted of 70-95% organic solvent, 5-30% water, and small amount of volatile acid or buffer. While the commonly used SPE elution solvents (i.e. acetonitrile and methanol) have stronger elution strength than a mobile phase on reversed-phase chromatography, they are weaker elution solvents than a mobile phase for normal-phase LC/MS/MS and therefore can be injected directly. Analytical methods for a range of polar pharmaceutical compounds, namely, omeprazole, metoprolol, fexofenadine, pseudoephedrine as well as rifampin and its metabolite 25-desacetyl-rifampin, in biological fluids, were developed and optimized based on the foregoing principles. As a result of the time saving, a batch of 96 samples could be processed in one hour. These bioanalytical LC/MS/MS methods were validated according to "Guidance for Industry - Bioanalytical Method Validation" recommended by the Food and Drug Administration (FDA) of the United States.

A method to identify and simultaneously determine the relative quantities of proteins isolated by gel electrophoresis

Abstract: Gel electrophoresis is often used for the primary analysis and purification of proteins, and peptide mapping by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a widely used technique for the rapid identification of unknown proteins. The identification is usually obtained by digesting the protein with an enzyme and matching the masses of the proteolytic peptides with those of each protein in a sequence database. Another important aspect in many proteomic experiments is the determination of the relative protein quantities (e.g. comparison between control and altered states). Usually, this is obtained by comparing the spot intensities of two independent gels. This procedure is time-consuming and not very accurate. Recently, several methodologies using isotope labeling of proteins for quantitative proteomic studies have been introduced (e.g. using ICAT reagents or growing cells in isotopically enriched nutrients). However, none of these methodologies is foolproof and there is still the need for simple and inexpensive alternatives for determining the relative quantities of proteins. Previously, we showed that a mixture of acrylamide and deuterated acrylamide could be used as cysteine alkylating reagent prior to electrophoresis, improving the coverage and the confidence of the protein identification procedure (Sechi S, Chait BT. Anal. Chem. 1998; 70: 5150). Here we show that a similar approach can be used to obtain relative quantitation at the femtomole level of proteins isolated by gel electrophoresis. Deuterated acrylamide is used to alkylate the cysteines in one sample and regular acrylamide is used to alkylate the cysteines in the second sample. The two samples are then mixed together in a 1:1 ratio and the relative protein quantities are determined from the ion intensity ratios of the two cysteine-containing peptides isotopic envelopes (regular/deuterated). The analysis of several proteins mixed in different ratios is reported showing that this approach can reliably be used for protein identification and quantification. Briefly, a simple and inexpensive method for quantifying and simultaneously identifying proteins isolated by gel electrophoresis using MALDI-MS is presented.