Mass Spectra Of Products Research Articles

Using a triple‐quadrupole mass spectrometer in accurate mass mode and an ion correlation program to identify compounds

Atomic masses and isotopic abundances are independent and complementary properties for discriminating among ion compositions. The number of possible ion compositions is greatly reduced by accurately measuring exact masses of monoisotopic ions and the relative isotopic abundances (RIAs) of the ions greater in mass by +1 Da and +2 Da. When both properties are measured, a mass error limit of 6-10 mDa (< 31 ppm at 320 Da) and an RIA error limit of 10% are generally adequate for determining unique ion compositions for precursor and fragment ions produced from small molecules (less than 320 Da in this study). 'Inherent interferences', i.e., mass peaks seen in the product ion mass spectrum of the monoisotopic [M+H]+ ion of an analyte that are -2, -1, +1, or +2 Da different in mass from monoisotopic fragment ion masses, distort measured RIAs. This problem is overcome using an ion correlation program to compare the numbers of atoms of each element in a precursor ion to the sum of those in each fragment ion and its corresponding neutral loss. Synergy occurs when accurate measurement of only one pair of +1 Da and +2 Da RIAs for the precursor ion or a fragment ion rejects all but one possible ion composition for that ion, thereby indirectly rejecting all but one fragment ion-neutral loss combination for other exact masses. A triple-quadrupole mass spectrometer with accurate mass capability, using atmospheric pressure chemical ionization (APCI), was used to measure masses and RIAs of precursor and fragment ions. Nine chemicals were investigated as simulated unknowns. Mass accuracy and RIA accuracy were sufficient to determine unique compositions for all precursor ions and all but two of 40 fragment ions, and the two corresponding neutral losses. Interrogation of the chemical literature provided between one and three possible compounds for each of the nine analytes. This approach for identifying compounds compensates for the lack of commercial ESI and APCI mass spectral libraries, which precludes making tentative identifications based on spectral matches.

Submicromolar Concentrations of Palmitoyl-CoA Specifically Thioesterify Cysteine 244 in Glyceraldehyde-3-phosphate Dehydrogenase Inhibiting Enzyme Activity: A Novel Mechanism Potentially Underlying Fatty Acid Induced Insulin Resistance

The accumulation of fatty acids and their metabolites results in insulin resistance and reduced glucose utilization through a variety of complex mechanisms that remain incompletely understood. Herein, we demonstrate that submicromolar concentrations of palmitoyl-CoA inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) enzyme activity through the covalent thioesterification of palmitate to GAPDH. First, incubation of GAPDH with palmitoyl-CoA (0.5-5 microM) resulted in the dramatic concentration-dependent inhibition of GAPDH enzyme activity. Second, incubation of GAPDH with [(14)C]palmitoyl-CoA followed by SDS-PAGE and autoradiography identified a covalently radiolabeled adduct present at approximately 35 kDa with a stoichiometry of one molecule of palmitoyl-CoA per GAPDH tetramer. Third, mass spectrometric analyses of intact GAPDH treated with palmitoyl-CoA demonstrated the covalent addition of palmitate to the GAPDH protein. Fourth, trypsinolysis of the modified protein revealed that the peptide (232)VPTPNVSVVDLTRC*R(245) was covalently modified. Fifth, the site of palmitoylation was demonstrated to be Cys-244 by analyses of product ion mass spectra. These assignments were further substantiated using different molecular species of acyl-CoAs resulting in the anticipated changes in both the masses of adduct ions and their fragmentation patterns. Sixth, GAPDH palmitoylation was demonstrated to facilitate the translocation of GAPDH to either lipid vesicles or naturally occurring biologic membranes. Since the hallmark of lipotoxicity is the accumulation of fatty acids and their acyl-CoA metabolites in excess of a cell's ability to appropriately metabolize them, these results identify a novel mechanism potentially contributing to the insulin resistance, reduced glucose utilization, and maladaptive metabolic alterations underlying the lipotoxic state.

Characterization of rat liver microsomal and hepatocytal metabolites of brevetoxins by liquid chromatography–electrospray tandem mass spectrometry

Brevetoxins are natural neurotoxins that are produced by "red tide" algae. This class of compounds can cause neurotoxic shellfish poisoning and other health problems. Brevetoxin-2 is the most abundant among the nine brevetoxins that have been characterized, whereas brevetoxin-1 is the most toxic. In this study, brevetoxin-1 and brevetoxin-2 were incubated with rat liver hepatocytes and rat liver microsomes, respectively. After clean-up steps were taken to remove the proteins, samples were analyzed by liquid chromatography (LC) coupled with electrospray mass spectrometry (LC-MS). After incubation of brevetoxin-1, two metabolites were found: brevetoxin-1-M1 (molecular weight = 900 Da), and brevetoxin-1-M2 (molecular weight = 884 Da). The increase in molecular weight combined with evidence from tandem mass spectrometry showing an increased tendency for loss of water molecules, along with considerations of established precedents for chemical transformations led to the conclusion that brevetoxin-1-M1 was formed by converting one double bond in the E or F ring of brevetoxin-1 into a diol. The second metabolite (brevetoxin-1-M2) is proposed to be a hydrolysis product of brevetoxin-1 involving opening of the lactone ring with the addition of a water molecule. The incubation study of the other starting compound, brevetoxin-2, found two metabolites in the LC-ES-MS selected ion chromatogram. Brevetoxin-2-M1 (molecular weight = 912 Da) gave a large [M-H]- peak at m/z 911, and its product ion mass spectrum allowed the deduction that this metabolite was the hydrolysis product of brevetoxin-2 involving conversion of the lactone to a carboxylic acid and an alcohol. The second metabolite (brevetoxin-2-M2, molecular weight = 896 Da) was deduced to have the same structure as that of brevetoxin-3 based on identical chromatographic retention times and similar mass spectra as those obtained for a brevetoxin-3 standard.

Mono‐ and diiodocyclophosphamide as possible internal standards for cyclophosphamide quantification: characterization by ion trap multi‐stage mass spectrometry and effects of iodine‐chlorine substitution on the fragmentation pattern

Hospital personnel involved in antineoplastic drug preparation and administration to patients are exposed to large amounts of these drugs. Labour legislation indicates the necessity of planning monitoring strategies aimed at prevention and/or reduction of drug exposure. Monitoring strategies consist of quantitative determinations of indicators, present in environmental and biological matrices. Among the antineoplastic drugs widely used, cyclophosphamide (CP) has been identified as a suitable indicator of potential exposure to mixtures of antineoplastic drugs. Many literature methods for quantitative analysis of CP involve either liquid (LC) or gas chromatography (GC) with mass spectrometry (MS), both of which require use of a suitable internal standard. The present work focuses on the synthesis of mono- and diiodocyclophosphamide (CPI and CPI(2)) to be used as internal standard. These compounds were analyzed by GC/EI-MS/MS and LC/ESI-MS(n) using ion trap mass spectrometry. The product ion mass spectra are interpreted in terms of proposed structures of fragment ions. Iodine-chlorine substitution resulted in a weakening of the carbon-halogen bond with a noteworthy influence on the ion fragmentation processes. The proposed suitability of CPI and CPI(2) as internal standards was based on similarities to CP as regards ionization and fragmentation processes. The results obtained suggest that CPI could be used as internal standard for CP quantification by LC/ESI-MS/MS, and CPI(2) for GC/EI-MS/MS analyses.

Reduction of ferricytochrome c by tyrosyltyrosylphenylalanine

Cytochrome c (cyt c) was reduced by a tyrosine-containing peptide, tyrosyltyrosylphenylalanine (TyrTyrPhe), at pH 6.0-8.0, while tyrosinol or tyrosyltyrosine (TyrTyr) could not reduce cyt c effectively under the same condition. Cyt c was reduced at high peptide concentration, whereas the reaction did not occur effectively at low concentration. The reaction rate varied with time owing to a decrease in the TyrTyrPhe concentration and the production of tyrosine derivatives during the reaction. The initial rate constants were 2.4 x 10(-4) and 8.1 x 10(-4) s(-1) at pH 7.0 and 8.0, respectively, for the reaction with 1.0 mM TyrTyrPhe in 10 mM phosphate buffer at 15 degrees C. The reciprocal initial rate constant (1/k(int)) increased linearly against the reciprocal peptide concentration and against the linear proton concentration, whereas logk(int) decreased linearly against the root of the ionic strength. These results show that deprotonated (TyrTyrPhe)(-), presumably deprotonated at a tyrosine site, reduces cyt c by formation of an electrostatic complex. No significant difference in the reaction rate was observed between the reaction under nitrogen and oxygen atmospheres. From the matrix-assisted laser desorption ionization time-of-flight mass spectra of the reaction products, formation of a quinone and other tyrosine derivatives of the peptide was supported. These products should have been produced from a tyrosyl radical. We interpret the results that a cyt c(ox)/(TyrTyrPhe)(-)right harpoon over left harpooncyt c(red)/(TyrTyrPhe)(*) equilibrium is formed, which is usually shifted to the left. This equilibrium may shift to the right by reaction of the produced tyrosyl radical with the tyrosine sites of unreacted TyrTyrPhe peptides.

Kinetics of secondary ion complexes in O 2[rad]+CH 3CN and Xe [rad]+CH 3CN systems : Formation of nonprotonated ion complexes of acetonitrile

A detailed kinetic study for the production of secondary and ternary ions in O 2 + (Xe +)–CH 3CN systems was made by applying the SIFDT techniques. “Nonprotonated complexes” (cation radical dimers or higher aggregates) of acetonitrile were present in the mass spectra of product ions. The formation of nonprotonated dimers of acetonitrile ions is ascribed to switching reactions of the ion complexes [O 2 +CH 3CN] and [Xe +CH 3CN] with acetonitrile. Mathematical procedures for the treatment of kinetic dependences, in which the rate constants of the studied reactions have been determined, are described. Energy and pressure dependences of the rate constants are presented and discussed.

The development of an algorithm for the mass spectral interpretation of phosphoproteins

Extended from the peptide mapping method (Fenyo, D., Curr. Opin. Biotechnol. 2000, 11, 391-395) the proposed algorithm takes the mass information of a precursor ion to reconstruct all possible phosphorylated peptide sequences. The mass spectra of product ions from the corresponding precursor ion is then used and compared to the reconstructed sequences to deduce the most probable phosphoprotein. The proposed algorithm also predicts all possible combinations of phosphopeptides, which may serve as a clue for designing proper phosphorylation experiments to validate the existence of these peptides and the corresponding proteins.

Functional Demonstration of Reverse Transsulfuration in the Mycobacterium tuberculosis Complex Reveals That Methionine Is the Preferred Sulfur Source for Pathogenic Mycobacteria

Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.

A tandem mass spectrometric study of saccharides at high mass resolution

Nine monosaccharides and four disaccharides were mass analyzed using a quadrupole time-of-flight tandem mass spectrometer combined with an electrospray ionization source. Product ion mass spectra of deprotonated, protonated, and sodiated saccharides were observed and were compared within each group of saccharides. Each of the deprotonated pentoses, hexoses and disaccharides yielded a significantly different product ion mass spectrum with the exception of alpha-lactose and beta-lactose. The disaccharides alpha- and beta-lactose differ only at the glycosidic linkage. Product ion mass spectra of protonated and sodiated alpha- and beta-lactose were indistinguishable also.

A mass spectrometric study of glucose, sucrose, and fructose using an inductively coupled plasma and electrospray ionization

Large polyatomic ions were observed in inductively coupled plasma mass spectra of solutions containing glucose, fructose, and sucrose, whereas mass spectra of solutions of acetic acid and ethanol obtained under similar operating conditions showed that these polyatomic ions were either totally absent or were observed at much lower intensities. These results suggest that sugar molecules, in particular, have an ability to survive the plasma, giving rise to polyatomic ions. Interferences on several isotopes were observed previously in a mass spectrometric study of diluted Canadian icewine; the interferences were attributed to the high sugar content in the solution, and not the ethanol content, demonstrating the need for careful consideration of the sample matrix for quantitative analysis. Product ion mass spectra of the sugar solutions were acquired by electrospray ionization tandem quadrupole time-of-flight mass spectrometry to better understand the composition and fragmentation of the sugar molecules. Fragmentation schemes for the formation of polyatomic ions from the sugar molecules and observed by inductively coupled plasma mass spectrometry (ICP-MS) are proposed.

Tandem Time-of-Flight (TOF/TOF) Mass Spectrometry and Proteomics.

A dual reflectron tandem time-of-flight (TOF/TOF) mass spectrometer reported in 1993 gave rise to the invention and development of the curved-field reflectron (CFR) for focusing product ions. The CFR is used in this case as the second mass analyzer in a tandem instrument (based on the Kratos AXIMA CFR) in which the first mass analyzer is a linear TOF that focuses ions by pulsed extraction. Because ions can be focused over a broad range of kinetic energies, deceleration of precursor ions and/or reacceleration of product ions is not required. Thus, product ions produced by post-source processes (laser induced dissociation or LID, metastable decomposition and opportunistic collisions) are recorded in the product ion mass spectra at the same times as their isomass ions produced by collision induced dissociation (CID). In general both LID and CID product ion mass spectra are very similar, producing primarily b-series and y-series ions, though there is some preference for fragmentation at weaker bonds such as those at proline or aspartic acid residues. The tandem mass spectrometer has been used to determine the acetylation sites for a histone acetyl transferase (HAT) protein. A novel and improved method for derivatizing tryptic fragments by N-terminal sulfonation produces almost exclusively y-series ions, and has been used to determine protein ubiquitination. The tandem mass spectrometer has also been used to identify potential biomarkers associated with heart failure, in particular that fraction containing albumin that is generally removed from serum samples to permit protein biomarker analysis. Analysis of the unfractionated serum, the albuminome, and the depleted serum is also carried out using surface-enhanced laser desorption/ionization (SELDI) and the high molecular weight proteins are monitored by using a Comet macromizer TOF mass spectrometer with a very high mass cryocooled detector.

Packed capillary liquid chromatography–electrospray ionization (tandem) mass spectrometry of mustard hydrolysis products in soil

A packed capillary liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed and applied to the identification of mustard hydrolysis products in aqueous extracts of soil. In the first application the LC-ESI-MS/MS method was used to identify thiodiglycol and nine longer chain diols in soil samples taken at different locations and depths from a former mustard storage site as part of an ongoing environmental assessment. Aqueous extracts of the soil samples were analysed by LC-ESI-MS/MS using a quadrupole/time-of-flight tandem mass spectrometer operating with a resolution of 9000. High resolution product mass spectra were acquired for thiodiglycol, the hydrolysis product of mustard and nine other sulfur containing diols, including five longer chain diols that could not be identified during prior LC-ESI-MS analyses. The high resolution LC-ESI-MS/MS method was also incorporated into an analytical approach designed to provide rapid chemical warfare agent identification in cases where the chemical and/or biological warfare agent content of a sample is unknown. A sample handling method involving aqueous extraction of the soil sample in biocontainment level 3 (BL-3), followed by autoclave sterilization of the aqueous extract was developed. Once sterilized, the container and aqueous extract can then be safely manipulated outside of BL-3 in the analytical laboratories and may be analysed for the presence or absence of chemical warfare agents, their hydrolysis products or related compounds by LC-ESI-MS/MS.

Packed capillary liquid chromatography–electrospray ionization (tandem) mass spectrometry of mustard hydrolysis products in soil

A packed capillary liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) method was developed and applied to the identification of mustard hydrolysis products in aqueous extracts of soil. In the first application the LC–ESI-MS/MS method was used to identify thiodiglycol and nine longer chain diols in soil samples taken at different locations and depths from a former mustard storage site as part of an ongoing environmental assessment. Aqueous extracts of the soil samples were analysed by LC–ESI-MS/MS using a quadrupole/time-of-flight tandem mass spectrometer operating with a resolution of 9000. High resolution product mass spectra were acquired for thiodiglycol, the hydrolysis product of mustard and nine other sulfur containing diols, including five longer chain diols that could not be identified during prior LC–ESI-MS analyses. The high resolution LC–ESI-MS/MS method was also incorporated into an analytical approach designed to provide rapid chemical warfare agent identification in cases where the chemical and/or biological warfare agent content of a sample is unknown. A sample handling method involving aqueous extraction of the soil sample in biocontainment level 3 (BL-3), followed by autoclave sterilization of the aqueous extract was developed. Once sterilized, the container and aqueous extract can then be safely manipulated outside of BL-3 in the analytical laboratories and may be analysed for the presence or absence of chemical warfare agents, their hydrolysis products or related compounds by LC–ESI-MS/MS.

Direct assignment of positional isomers by mass spectrometry: ortho, meta and para acyl and amidyl anilines and phenols and derivatives

AbstractA direct MS/MS method for the ortho, meta or para configuration assignment of any single molecule that forms reference ions upon ionization and dissociation is demonstrated. Gas‐phase structure diagnostic ion–molecule reactions with acetonitrile are shown to distinguish the isomeric 2‐, 3‐ and 4‐hydroxybenzoyl cations and the 2‐ from the 3‐ and 4‐aminobenzoyl cations. These reference ions, which display indistinguishable 15 eV collision‐induced dissociation product ion mass spectra, react with acetonitrile to yield characteristic ratios of product ions, most particularly for the 2‐isomers. The reactivity of the 2‐benzoyl cations is the most characteristic since the ortho configuration allows for [4+ + 2] polar cycloaddition that yields relatively stable heterocycles in N‐protonated forms. Distinction of the reference isomeric 2‐, 3‐ and 4‐hydroxy‐ and aminobenzoyl cations permits, therefore, partially or completely, direct ‘MS‐only’ positional assignment of either ortho, meta or para configuration for any single molecule that forms such reference ions upon ionization and dissociation. This “class‐universal” method for direct MS assignment of a single positional isomer should therefore be applicable to many members of the homologous series of isomeric ortho, meta and para acyl and amidyl anilines and phenols and derivatives. Such molecules dissociate, or are likely to dissociate, after or during ionization processes to form the reference and structurally diagnostic ortho, meta or para hydroxy‐ or aminobenzoyl cations. Copyright © 2004 John Wiley &amp; Sons, Ltd.

Porous Polymer Monolith Assisted Electrospray

Coupling low-flow analytical separation instrumentation such as capillary electrophoresis, capillary electrochromatography, nano-HPLC, and microfluidic-based devices with electrospray ionization mass spectrometry has yielded powerful analytical tools. However, conventional coupling methodologies such as nanospray suffer from limitations including poor conductive coating robustness, constant clogging, complicated fabrication processes, and incompatibility with large flow rate regimes. This study demonstrates that robust nanospray emitters can be fabricated through the formation and utilization of a porous polymer monolith (PPM) at the end of a fused-silica capillary. Stable electrosprays can be produced from capillaries (75-100-microm i.d.) at a variety of flow rates (50-1000 nL/min) without the need to taper the capillaries by etching or pulling. The PPM is photopatterned to be present only near the capillary exit aperture using conditions that generate pore sizes similar to those seen with nanospray tips. The porous nature of the PPM aids in developing a stable electrospray generating a single clearly visible Taylor cone at relatively high flow rates while at low flow rates (<100 nL/min) a mist, presumably from multiple small Taylor cones, develops. The hydrophobic nature of the PPM should limit problems with band broadening associated with droplet spreading at the capillary exit, while the multiple flow paths inherent in the PPM minimize clogging problems associated with conventional nanospray emitters. Total ion current traces for a constant infusion of standard PPG and cytochrome c solutions are very stable with deviations ranging from only 3 to 8%. The PPM-assisted electrospray produces mass spectra with excellent signal-to-noise ratios from only a few femtomoles of material.

In vitro and in vivo metabolism of pyronaridine characterized by low-energy collision-induced dissociation mass spectrometry with electrospray ionization.

The in vitro and in vivo metabolism of pyronaridine, an antimalarial agent, was investigated in rats and humans. In vitro incubation of pyronaridine with rat and human liver microsomes resulted in the formation of 11 metabolites, with pyronaridine quinoneimine (M3) as the major metabolite. The structures of pyronaridine metabolites were characterized on the basis of the product ion mass spectra obtained under low-energy collision-induced dissociation (CID) ion trap mass spectrometry. Both pyronaridine (m/z 518) and M3 (m/z 516) produced the same product ion (m/z 447). These results could be explained by the characteristic neutral loss of a 69 Da fragment from M3 via gamma-H rearrangement and 1,7 sigmatropic shift, whereas the neutral loss of a 71 Da fragment from the pyronaridine occurred by charge site-initiated heterolytic cleavage. These fragmentations were further supported by the tandem mass spectrum of D(3)-pyronaridine. Other metabolites generated in the microsomal incubations were carbonylated, hydroxylated and O-demethylated derivatives. Pyronaridine and its metabolites were detected in both feces and urine after intraperitoneal administration to rats. The in vivo metabolic profile in rats was different from the in vitro profile. M3, a chemically reactive quinonimine, was not detected whereas O-demethylated derivatives (M14, M15, M16, and M19) were identified in fecal and urinary extracts. The role of quinoneimine metabolites in pyronaridine-caused toxicity should be further evaluated, although these metabolites or their conjugates were not detected in urine and feces.

One-year routine application of a new method based on liquid chromatography–tandem mass spectrometry to the analysis of 16 multiclass pesticides in vegetable samples

An analytical procedure has been developed for determining a group of 16 multiclass pesticides most commonly used in crop protection. The extraction step is performed with a mixture of ethyl acetate and sodium sulphate, in the presence of 6.5 M NaOH. After evaporation, a final extract containing 1 mg sample/ml extract, is obtained in methanol. Analysis of the methanolic extract, without additional clean-up steps, is performed by liquid chromatography–electrospray ionisation-tandem mass spectrometry combining positive and negative ion mode. The analytical performance of the method has been validated for three different matrices: pepper, lettuce and aubergine. Mean recoveries obtained were between 70 and 110% in most of the cases with a precision of <28%. Linearity of response over 2 orders of magnitude was demonstrated ( r ≥ 0.99) with limits of detection ≤0.01 mg/kg in all the cases. No significant matrix effects were observed with the exception of triflumizol that presented a drastic decrease in response as a consequence of an ion formation suppression effect in the presence of pepper matrix. The method has been applied to the analysis of 560 vegetable samples, as a part of the monitoring programme of the Association of Producers and Exporters of Fruits and Vegetables of Almerı́a (COEXPHAL). The unambiguous confirmation of the positive findings by comparison of the product ion mass spectra of the peaks in samples and standards, demonstrated the applicability of the method in routine analysis.

The identification of three human metabolites of a peptide-doxorubicin conjugate using HPLC-MS-MS in positive and negative ionization modes.

A peptide-doxorubicin conjugate (I) is a drug candidate that has been evaluated for the treatment of prostate cancer. During the high-performance liquid chromatographic (HPLC)-fluorescence analysis of clinical samples for compound I and its two known metabolites [doxorubicin (II) and leucine-doxorubicin (III)], additional metabolites are observed in postdose human plasma extracts. Using neutral loss, precursor, and product ion tandem mass spectrometric (MS-MS) experiments, two of these metabolites are identified as doxorubicinol (IV) and leucine-doxorubicinol (V), the active 13-hydroxy metabolites of doxorubicin and leucine-doxorubicin, respectively. A third metabolite, 7-deoxydoxorubicinol aglycone (VI), is detected using single-ion monitoring at m/z 399 in the negative ionization mode. The product ion mass spectrum of this metabolite contains a major fragment at m/z 351, resulting from the loss of water and formaldehyde from the pseudomolecular ion. An HPLC-MS-MS method for simultaneous analysis of II, III, IV, V, and VI is developed utilizing gradient HPLC with a combination of positive/negative ionization MS in the multiple reaction monitoring mode and monitoring the appropriate MS-MS transitions. Using this methodology, rat, dog, and human plasma metabolite profiles are compared and found to be qualitatively similar. Simultaneous fluorescence and MS detection experiments confirm that the peaks observed in the HPLC-fluorescence chromatograms of plasma extracts correspond to each of the five metabolites (II-VI).

Reproducible product-ion tandem mass spectra on various liquid chromatography/mass spectrometry instruments for the development of spectral libraries.

The results of the comparison of product-ion tandem mass (MS/MS) spectra recorded on three ion trap mass spectrometers, a triple quadrupole mass spectrometer and a Fourier transform ion cyclotron resonance mass spectrometer are reported. The spectra were recorded in accordance with a simple experimental protocol, which involved the collision-induced dissociation (CID) attenuation of the abundance of the [M+H]+ ion to between 10 and 50% of its original abundance. The degree of similarity between the spectra from four of the mass spectrometers was calculated off-line by comparing the five most abundant ions from the spectrum on each instrument. A percentage fit value (20% for each ion that matches) was calculated by comparing each spectrum against the spectra recorded for the same compound on each instrument. The percentage of the inter-library pairwise comparisons (total = 434) that matched to > or = 60% ranged from 64-89%, depending on the instrument pair. A blind trial was also undertaken using five unknown compounds resulting in 1670 pairwise comparisons with the library entries. The blind trial produced no false positives and correct identifications in all cases. The results of the study have established the basis for the construction of a transferable product-ion MS/MS library.

Simultaneous quantitative analysis of isobars by tandem mass spectrometry from unresolved chromatographic peaks.

A method was developed for the simultaneous quantitation of isobars from unresolved chromatographic peaks. The method is based on differences in branching ratios of ion abundances in their tandem mass spectra and an assumption that the product ion mass spectra of a mixture can be considered as a linear combination of the spectra of individual constituents. We present analytical equations and a matrix-based approach for deconvoluting the concentration of individual components from the total peak intensity for two and three isobars and also a matrix-based generalization to any number of compounds. The feasibility of the simultaneous analysis of mixtures containing two compounds was assessed. The approach was evaluated for the analysis of structural isomers of methylmalonic and succinic acids in human plasma and urine samples for a group of 270 samples. The linear regression equation, standard error and correlation coefficient for the agreement with a traditional method utilizing chromatographic separation of the isomers were y = 0.999x - 0.005, 0.024 micro mol l(-1), and 0.985, respectively. The utility of a spectral contrast angle as a predictor of analysis feasibility was evaluated.