Mode Electrospray Ionization Mass Spectrometry Research Articles

Simultaneous quantification of free and glucuronidated cannabinoids in human urine by liquid chromatography tandem mass spectrometry

BackgroundCannabis is the most commonly abused drug of abuse and is commonly quantified during urine drug testing. We are conducting a controlled drug administration study investigating efficacy of urinary cannabinoid glucuronide metabolites for documenting recency of cannabis intake and for determining stability of urinary cannabinoids. MethodsA liquid chromatography tandem mass spectrometry method was developed and validated quantifying Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol, cannabinol, THC-glucuronide and THCCOOH-glucuronide in 0.5ml human urine via supported-liquid extraction. Chromatography was performed on an Ultra Biphenyl column with a gradient of 10mmol/l ammonium acetate, pH 6.15 and 15% methanol in acetonitrile at 0.4ml/min. Analytes were monitored by positive and negative mode electrospray ionization and multiple reaction monitoring mass spectrometry. ResultsLinear ranges were 0.5–50ng/ml for THC-glucuronide, 1–100ng/ml for THCCOOH, 11-OH-THC and cannabidiol, 2–100ng/ml for THC and cannabinol, and 5–500ng/ml for THCCOOH-glucuronide (R2>0.99). Mean extraction efficiencies were 34–73% with analytical recovery (bias) 80.5–118.0% and total imprecision 3.0–10.2% coefficient of variation. ConclusionThis method simultaneously quantifies urinary cannabinoids and phase II glucuronide metabolites, and enables evaluation of urinary cannabinoid glucuronides for documenting recency of cannabis intake and cannabinoid stability. The assay is applicable for routine urine cannabinoid testing.

Simultaneous quantification of nicotine, cotinine, trans-3′-hydroxycotinine, norcotinine and mecamylamine in human urine by liquid chromatography–tandem mass spectrometry

BackgroundMecamylamine is a nicotine antagonist under investigation in combination with nicotine replacement for smoking treatment. MethodsA simple, rapid and reliable liquid chromatography tandem mass spectrometry (LCMSMS) method was developed and validated for quantifying nicotine, cotinine, trans-3′-hydroxycotinine, norcotinine and mecamylamine in human urine. Chromatography was performed on a Synergi PolarRP column with a gradient of 0.1% formic acid and 0.1% formic acid in acetonitrile at 0.25ml/min with an 8-min total runtime. Analytes were monitored by positive mode electrospray ionization and multiple reaction monitoring mass spectrometry. ResultsLinear dynamic ranges were 1–500ng/ml for nicotine and norcotinine, 0.5–500ng/ml for trans-3′-hydroxycotinine, 0.2–500ng/ml for cotinine, and 0.1–100ng/ml for mecamylamine; correlation coefficients were consistently greater than 0.99, and all calibrator concentrations were within 20% of target. Extensive endogenous and exogenous interferences were evaluated. At 3 concentrations spanning the linear dynamic range of the assay, mean extraction efficiencies from urine were 55.1–109.1% with analytical recovery (bias) 82.0–118.7% and total imprecision of 0.7–9.1%. Analytes were stable for 24h at room temperature, 72h at 4°C, 72h in autosampler at 15°C and after three freeze/thaw cycles. ConclusionThis method is useful for monitoring mecamylamine, nicotine and nicotine metabolites in smoking cessation and other clinical nicotine research.

Hydride abstraction in positive-ion electrospray interface: oxidation of 1,4-dihydropyridines in electrospray ionization mass spectrometry

During the positive-ion mode electrospray ionization mass spectrometry analysis of 1,4-diphenyl-3-benzoyl-1,4-dihydropyridines with methanol as the solvent, the major ion generated is one mass unit lower than the analyte, which can be rationalized as the oxidative aromatization of 1,4-dihydropyridines to pyridine cations.

Solvent Molecules Undergo Homolytic Cleavage and Radical Recombination Processes during Negative-Mode Electrospray Ionization: Adduct Formation with Antimony(III)-Tartrate Dianion

Negative-ionization mode electrospray ionization-mass spectrometry (ESI-MS) analysis of antimony(III)-tartrate in frequently used solvent systems, ACN/H(2)O and MeOH/H(2)O, revealed that the antimony(III)-tartrate dianion associates to solvent reaction products generated by radical formation and their subsequent recombination during the negative-mode electrospray process. A systematic increase and decrease in negative spray capillary voltage (SCV) from normal operational voltage ranges of a conventional quadrupole ion trap instrument during these analyses showed initially unobserved adduct ions to correspondingly increase and diminish in relative ion intensity. The identity of the adducted species, including products such as H(2)O(2), NCCH(2)CH(2)CN, and CH(2)(OH)(2), were confirmed by performing similar experiments with deuterated and nondeuterated solvent mixtures. Relative intensity dependence of these adducted ions was monitored as the volume composition of each solvent system was changed. It was clearly observed that the relative intensity of {[Sb(2)-tar(2)][H-O-O-H]}(2-) and {[Sb(2)-tar(2)][NC-CH(2)-CH(2)-CN]}(2-) adduct ions increased with the volume percent of H(2)O and CH(3)CN, respectively. Similarly, an increase in volume percent of CH(3)OH increased the relative intensity of {[Sb(2)-tar(2)][H-O-CH(2)-O-H]}(2-) adducted ions. On the basis of this evidence, it was proposed that homolytic cleavage of C-H bonds for CH(3)CN and CH(3)OH molecules, and O-H bonds for H(2)O molecules, produces a series of radicals during negative-ionization mode ESI, and subsequent self-recombination or cross-recombination of these radicals then occurs to form the neutral solvent products, which are observed in the mass spectra as [Sb(2)-tar(2)]-adducted ionic species. These findings provide new insight into processes, which are relevant to understanding the mechanism of electrospray ionization, a widely used technique.

Evaluation of tetracationic salts as gas‐phase ion‐pairing agents for the detection of trivalent anions in positive mode electrospray ionization mass spectrometry

In previous studies, new electrospray ionization mass spectrometry (ESI-MS) approaches were developed for the highly sensitive detection of singly and doubly charged anions in positive mode ESI-MS by using specially synthesized dicationic and tricationic ion-pairing agents, respectively. By detecting the positively charged ion complex in the positive mode, limits of detection (LODs) for the anions can be lowered by several magnitudes. In this work, we used eighteen newly synthesized tetracationic ion-pairing agents, constructed with different geometries, linkages and cation moieties, for the detection of eighteen triply charged anions of different structural motifs. The LODs for these anions were from ten to several thousand times lower in the positive selective ion monitoring (SIM) mode than in the negative mode. These tetracationic agents also were shown to be useful for the detection of -1 and -2 anions. In addition, the LODs for -3 anions can be further lowered by monitoring the product fragments of the ion-pairing complexes in the single reaction monitoring (SRM) mode.

Nitrogenous disinfection byproducts formation and nitrogen origin exploration during chloramination of nitrogenous organic compounds

Formation of nitrogenous disinfection by-products (N-DBPs) of cyanogen chloride (CNCl), dichloroacetonitrile (DCAN) and chloropicrin was evaluated during chloramination of several selected groups of nitrogenous organic (organic-N) compounds, including α-amino acids, amines, dipeptides, purines, and pyrimidines, The intermediates generated, reaction pathways, and nitrogen origin in N-DBPs were explored as well. CNCl was observed in chloramination of all tested organic-N compounds, with glycine giving the highest yields. DCAN was formed during chloramination of glutamic acid, cytosine, cysteine, and tryptophan. Chloramination of most organic-N compounds except for cysteine and glutamic acid generated chloropicrin. Aldehydes and nitriles were identified as the intermediates by negative mode electrospray ionization mass spectrometry during reactions of NH 2Cl and organic-N compounds. Labeled 15N-monochloramine ( 15NH 2Cl) techniques showed that nitrogen in N-DBPs may originate from both NH 2Cl and organic-N compounds and the nitrogen partition percentages vary as functions of reactants and pH.

Sensitivity of positive ion mode electrospray ionization mass spectrometry (ESI MS) in the analysis of purine bases in ESI MS and on-line electrochemistry ESI MS (EC/ESI MS)

A cone-shaped MS inlet and on-line electrochemistry (EC) were used to enhance the ionization efficiency in electrospray ionization mass spectrometry (ESI MS) of purine bases. A pathway of positive ion mode ESI may involve oxidation of purine bases, guanine, adenine, xanthine and hypoxanthine, by 1e −, 1H + processes. The electrospray process generates dimers of purine bases that are detected in ESI MS as protonated ions, except for xanthine, for which a protonated radical dimer is detected. Thus electrochemical oxidation of purine bases during ESI may generate reactive radicals that can subsequently dimerize. Dimer formation is facilitated in ESI MS when the carrier solution pH is high. The positive ion mode ESI MS ionization is consistent with the reactivity of the bases toward oxidation. Furthermore, the formation of the protonated ions, and Na + and K + adducts of the bases, expected in positive ion ESI MS, are observed. In addition, unusual H-bonding of purine bases guanine and xanthine is confirmed by ESI MS. Application of low EC voltage to the on-line EC cell in EC/ESI MS improves the sensitivity and correlates with the decrease of the intensity of the dimers, possibly as a result of their further oxidation.

Mass Spectrometry of Small Bimetal Monolayer-Protected Clusters

Monolayer-protected clusters were prepared by procedures like those yielding Au25L18 (where L=-SCH2CH2Ph=-SC2Ph) but using, instead, mixtures of Au and Pd salts, as starting materials, with the intent of creating and characterizing Au25-xMxL18 clusters. Isolation of small nanoparticle product followed by partial ligand exchange to introduce thiolated poly(ethylene glycol) (SPEG=-S(CH2CH2O)5CH3) into the nanoparticle ligand shell enabled characterization of the Au25-xMxL18 content by positive mode electrospray ionization mass spectrometry (ESI-MS). For synthetic feed mole ratios of Au:Pd of 9:1 and 13:12, electrospray spectra of the PEGylated MPCs showed that the reaction and isolation produce a mixture of Au25(SC2Ph)18 and a mono-Pd nanoparticle Au24Pd(SC2Ph)18. A higher proportion of the mono-Pd nanoparticle is produced by the 13:12 mole ratio, and also when the thiol:metal ratio was lowered, according to ESI-MS and MALDI-TOF-MS. As the nanoparticle mixture is enriched, by solvent fractionations, in Au24Pd(SC2Ph)18 relative to Au25(SC2Ph)18, the distinctive optical and electrochemical signatures of Au25(SC2Ph)18 are replaced by Au24Pd(SC2Ph)18 nanoparticle responses, which are very different, even though only one Au atom is replaced by a Pd atom.

Quantification of Almond Skin Polyphenols by Liquid Chromatography‐Mass Spectrometry

Reverse phase HPLC coupled to negative mode electrospray ionization (ESI) mass spectrometry (MS) was used to quantify 16 flavonoids and 2 phenolic acids from almond skin extracts. Calibration curves of standard compounds were run daily and daidzein was used as an internal standard. The inter-day relative standard deviation (RSD) of standard curve slopes ranged from 13% to 25% of the mean. On column (OC) limits of detection (LOD) for polyphenols ranged from 0.013 to 1.4 pmol, and flavonoid glycosides had a 7-fold greater sensitivity than aglycones. Limits of quantification were 0.043 to 2.7 pmol OC, with a mean of 0.58 pmol flavonoid OC. Mean inter-day RSD of polyphenols in almond skin extract was 6.8% with a range of 4% to 11%, and intra-day RSD was 2.4%. Liquid nitrogen (LN(2)) or hot water (HW) blanching was used to facilitate removal of the almond skins prior to extraction using assisted solvent extraction (ASE) or steeping with acidified aqueous methanol. Recovery of polyphenols was greatest in HW blanched almond extracts with a mean value of 2.1 mg/g skin. ASE and steeping extracted equivalent polyphenols, although ASE of LN(2) blanched skins yielded 52% more aglycones and 23% less flavonoid glycosides. However, the extraction methods did not alter flavonoid profile of HW blanched almond skins. The recovery of polyphenolic components that were spiked into almond skins before the steeping extraction was 97% on a mass basis. This LC-MS method presents a reliable means of quantifying almond polyphenols.

One‐Electron Oxidation and Sensitivity of Uric Acid in On‐Line Electrochemistry and in Electrospray Ionization Mass Spectrometry

AbstractThe sensitivity of detection of uric acid (H2U) in positive ion mode electrospray ionization mass spectrometry (ESI MS) was enhanced by uric acid oxidation during electrospray ionization. With a carrier solution of pH 6.3>pKa1=5.4 of H2U, protonated unoxidized uric acid [H2U+H]+ (m/z 169) was detected together with the protonated uric acid dimer [2H2U+H]+ (m/z 337). The dimer likely forms by 1e− oxidation of urate (HU−) followed by rapid radical dimerization. A covalent structure of the dimer was verified by H/D exchange experiments. Efficiency of 2e−, 2H+ oxidation of uric acid is low during ESI in pH 6.3 carrier solution and improves when a low on‐line electrochemical cell voltage is floated on the high voltage of the ES in on‐line electrochemistry ESI MS (EC/ESI MS). The intensity of the uric acid dimer decreases with an increase in the low applied voltage. In a carrier solution with 0.1 M KOH, pH 12.7>pKa2=9.8 of H2U, allantoin (Allnt) (MW 158.04), the final 2e−, 2H+ oxidation product of uric acid, was detected as a potassium complex [K(Allnt)+K]+ (m/z 235) and the [2H2U+H]+ dimer was not detected. In direct ESI MS analysis of 1000‐fold diluted urine [NaHU+H]+ (pKsp NaHU=4.6) was detected in 40/60 (vol%) water/methanol, 1 mM NH4Ac, pH ca. 6.3 carrier solution. A new configuration of the ESI MS instrument with a cone‐shaped capillary inlet significantly enhanced sensitivity in ESI and EC/ESI MS measurements of uric acid.

Antioxidant Pathways and One‐Electron Oxidation of Dopamine and Cysteine in Electrospray and On‐Line Electrochemistry Electrospray Ionization Mass Spectrometry

AbstractDopamine [DA]+ (m/z 154), DA dimer [2DA‐H]+ (m/z 307) and DA quinone [DAQ]+ (m/z 152) are detected in positive ion mode electrospray ionization mass spectrometry (ESI MS) of dopamine in 50/1/49 (vol%) water/acetic acid/methanol. H/D exchange experiments support a covalent structure of DA dimer. Thus, ESI of DA may involve 1e−, 1H+ oxidation processes followed by rapid radical dimerization. The DA quinone signal is low in ESI MS, which indicates a low efficiency of the 2e−, 2H+ oxidation reaction. On‐line electrochemistry ESI MS (EC/ESI MS) with low electrochemical cell voltage floated on high ES voltage increases electrospray current and improves sensitivity for DA. The DA quinone signal increases and DA dimer signal decreases. A new configuration of the ESI MS instrument with a cone‐shaped capillary inlet significantly enhanced sensitivity of ESI and EC/ESI MS measurements. A DA quinone‐cysteine adduct [DAQ+Cys]+ was detected in solutions of DA with cysteine (Cys). ESI MS and EC/ESI MS indicate formation of the DA quinone‐cysteine adduct by 1e− pathway. Oxidation pathways in ESI MS are relevant to biological reactivity of DA and Cys.

Influence of salt bridge interactions on the gas-phase stability of DNA/peptide complexes

Negative ion mode electrospray ionization mass spectrometry was used to study DNA duplexes–peptide interaction. In the present study, we show that peptides that contain two adjacent basic residues interact noncovalently with DNA single strand or duplex. Fragmentation of the complexes between peptides containing basic residues and DNA were studied under collisions and showed unexpected dissociation pathways, as previously reported for peptide–peptide interactions. The binary complexes are dissociated either along fragmentation of the covalent bonds of the peptide backbone and/or along the single DNA strand backbone cleavage without disruption of noncovalent interaction, which demonstrates the strong binding of peptide to the DNA strand. Sequential MS/MS and MS n were further performed on ternary complexes formed between duplexes and peptides to investigate the nature of interaction. The CID spectra showed as major pathway the disruption of the noncovalent interactions and the formation of binary complexes and single-strand ions, directed by the nucleic acid gas-phase acidity. Indeed, a preferential formation of complexes with thymidine containing single strands is observed. An alternative pathway is also detected, in which complexes are dissociated along the covalent bond of the peptide and/or DNA according to the basicity. Our experimental data suggest the presence of strong salt bridge interactions between DNA and peptides containing basic residues.

Quantitative analysis of total retronecine esters-type pyrrolizidine alkaloids in plant by high performance liquid chromatography

Pyrrolizidine alkaloids (PAs) are alkaloids which typically contain a necine (7-hydroxy-1-hydroxymethyl-6,7-dihydro-5H-pyrrolizidine) base unit, and they can be found in one third of the higher plants around the world. They are hepatotoxic, mutagenic and carcinogenic and pose a threat to human health and safety. A specific, quick and sensitive method is therefore needed to detect and quantify the PAs sometimes in trace amount in herbs, tea or food products. Based on high performance liquid chromatography with prior derivatization of the alkaloids using o-chloranil and Ehrlich's reagent, we report an improved method for quantitative analysis of the total amount of retronecine esters-type pyrrolizidine alkaloids (RET-PAs) in a plant extract. The total quantitation of RET-PAs is achieved because of a common colored retronecine marker, a 7-ethoxy-1-ethoxylmethyl retronecine derivative, is produced with all the different RET-PAs during the derivatization reaction. The chemical identity of the common retronecine marker was characterized on-line by positive mode electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. The limit of detection using the improved method is 0.26 nmol mL −1 and the limit of quantitation is 0.79 nmol mL −1. The advantages of this method are much enhanced sensitivity in detection and quantitation, and, no restriction on the choice of RET-PA as a calibration standard. Application of the developed method to the quantitation of total RET esters-type PAs in Senecio scandens from different regions of China is also reported.

Electrospray ionization mass spectrometry fingerprinting of propolis of native Brazilian stingless bees

Stingless bees are found in many tropical and subtropical regions of the word. The knowledge of the composition of their propolis as well as the plants that are visited as sources of resins is therefore of prime importance. Here the negative ion mode electrospray ionization mass spectrometry [ESI(-)-MS] fingerprints of propolis from various species of native stingless bees from different regions in Brazil are compared to determine their composition patterns. The correlation among the propolis samples was investigated via chemometric analysis.

Quantification of Diacylglycerol Species from Cellular Extracts by Electrospray Ionization Mass Spectrometry Using a Linear Regression Algorithm

Diacylglycerols (DAGs) play significant roles in both intermediate metabolism and signal transduction. These lipid species are second messengers involved in modulating a plethora of cellular processes. Evaluation of DAG species concentrations has been hampered by the lack of a reliable method for molecular species analysis within a complex mixture of cellular lipids. We describe a new method for quantitative analysis of DAG species from complex biological extracts based on positive mode electrospray ionization mass spectrometry without prior derivatization. Quantification is achieved using internal standards and calibration curves constructed by spiking cell extracts with different concentrations of DAG species containing various acyl chain lengths and degrees of unsaturation. The new mass spectral data processing algorithm incorporates a multiple linear regression model including a factor accountable for possible interactions between experimental preparations and the slope of the curve for the standards, allowing the examinations of the effects of sample origin conditions (such as cell types, phenotypes, etc.) and instrument variability on this slope. Internal standards provide a basis for quantification of 28 DAG molecular species detected in RAW 264.7 cells after stimulation of a G-protein coupled receptor with platelet activating factor. This method displays excellent reproducibility over the established range of concentrations with variations of < or =10% and is highly sensitive with a detection limit of 0.1-0.4 pmol/microL depending upon acyl chain composition. We have shown differential effects on various DAGs in response to a ligand which illustrates the importance of examining lipids at the molecular species level rather than as a single homogeneous entity.

Electrospray Ionization Mass Spectra of Amino Acid Methyl Ester 5′-Phosphoramidates of 2′, 3′-Isopropylideneuridine

Amino acid methyl ester phosphates were synthesized and determined by using positive-ion mode electrospray ionization mass spectrometry (ESIMS) in combination with multistage tandem mass spectrometry. The fragmentation pathways were investigated, and it was observed that most fragment ions contained the phosphoryl group. It was interesting to observe that the fragmentation pathways of the protonated molecule show some differences when compared with those of the sodium ion adduct. The methoxy group of amino acid methyl ester can migrate from the carbonyl group to the phosphoryl group in the sodium ion adduct.

Purification and characterization of the central segment of prothymosin‐α: methodology for handling highly acidic peptides

Prothymosin-alpha is a highly acidic protein consisting of 110 amino acids. The central segment of this protein, residues 51-89, is thought to be involved in metal binding which may be necessary for its physiological function. To carry out studies of this peptide, this central segment was synthesized in a linear fashion using Fmoc-based methods on rink amide MBHA resin. However, this peptide could not be purified with the typical straightforward approach of RP HPLC followed by negative mode electrospray ionization mass spectrometry (ESI-MS). This was attributed to the high proportion of acidic residues: 26 out of the 39 residues are aspartic and glutamic acids. The acidity of the peptide prevented retention on the RP HPLC column. Additionally, the ability of the highly negatively charged peptide to retain sodium ions prevented molecular weight determination with ESI-MS. A systematic approach to the purification of this highly acidic peptide was undertaken. Ultimately, strong anion exchange chromatography was used to purify the peptide. Extensive desalting using dialysis was required prior to ESI-MS, and the choice of the buffer proved to be critical. In the end, a purification method was devised that yielded a highly purified peptide and is readily compatible with analysis by ESI-MS.

Characterization of must and wine of six varieties of grapes by direct infusion electrospray ionization mass spectrometry

Samples of must derived from six different varieties of grapes taken during the fermentation process, as well as the respective wine samples directly after the end of the malolactic fermentation, were analyzed by direct infusion negative ion mode electrospray ionization mass spectrometry (ESI-MS). Diagnostic ions for must were different from those of wine samples, although small variations for each of the grape varieties were also detected. The addition of unfermented must or sugar to wine could also be clearly detected. The spectra were acquired in a few minutes per sample, indicating that ESI-MS can be used for high-throughput analysis of samples and should prove useful for quality control during and after the fermentation process.

Separation and Detection of Phosphorylated and Nonphosphorylated Peptides in Liquid Chromatography−Mass Spectrometry Using Monolithic Columns and Acidic or Alkaline Mobile Phases

Efficient chromatographic separation is a prerequisite for the sensitive analysis of complex peptide mixtures using liquid chromatography-mass spectrometry. This is especially true for the analysis of mixtures of unmodified and posttranslationally modified peptides, for example, phosphorylated peptides in the presence of their unmodified analogues. Applying monolithic capillary columns based on poly(styrene/divinylbenzene), the influence of acidic eluents based on trifluoroacetic and heptafluorobutyric acid as well as an alkaline eluent based on triethylamine-acetic acid (pH 9.2) on the separation of synthetic phosphopeptides was evaluated. Heptafluorobutyric acid offered the longest retention times and highest selectivities and, hence, the most effective separation. Application of the alkaline eluent in conjunction with detection in negative ion mode electrospray ionization mass spectrometry, on the other hand, allowed the detection of phosphorylated peptides with significantly lower limits of detection, as compared to acidic eluents in combination with detection in positive ion mode. Pairs of phosphorylated and nonphosphorylated synthetic peptides, ranging from 7- to 16-mers, as well as phosphorylated peptides form a tryptic protein digest could be separated both at acidic and alkaline pH. Utilizing a 60 x 0.20-mm-i.d. capillary column, the limit of detection in negative ion detection mode for a 4-fold phosphorylated peptide in a beta-casein digest was 10 fmol. Together with the capability for fast separation of protein digests, monolithic columns, thus, facilitate the effective and sensitive analysis of this important posttranslational modification.

Vanadium-Based, Extended Catalytic Lifetime Catechol Dioxygenases: Evidence for a Common Catalyst

In 1999, a catechol dioxygenase derived from a V-polyoxometalate was reported which was able to perform a record >100 000 total turnovers of 3,5-di-tert-butylcatechol oxygenation using O2 as the oxidant (Weiner, H.; Finke, R. G. J. Am. Chem. Soc. 1999, 121, 9831). An important goal is to better understand this and other vanadium-based catechol dioxygenases. Scrutiny of 11 literature reports of vanadium-based catechol dioxygenases yielded the insight that they all proceed with closely similar selectivities. This, in turn, led to a "common catalyst hypothesis" for the broad range of vanadium based catechol dioxygenase precatalysts presently known. The following three classes of V-based compounds, 10 complexes total, have been explored to test the common catalyst hypothesis: (i) six vanadium-based polyoxometalate precatalysts, (n-Bu4N)4H5PV14O42, (n-Bu4N)7SiW9V3O40, (n-Bu4N)5[(CH3CN)(x)Fe(II).SiW9V3O40], (n-Bu4N)9P2W15V3O62, (n-Bu4N)5Na2[(CH3CN)(x)Fe(II).P2W15V3O62], and (n-Bu4N)4H2-gamma-SiW10V2O40; (ii) three vanadium catecholate complexes, [V(V)O(DBSQ)(DTBC)]2, [Et3NH]2[V(IV)O(DBTC)2].2CH3OH, and [Na(CH3OH)2]2[V(V)(DTBC)3]2.4CH3OH (where DBSQ = 3,5-di-tert-butylsemiquinone anion and DTBC = 3,5-di-tert-butylcatecholate dianion), and (iii) simple VO(acac)2. Product selectivity studies, catalytic lifetime tests, electron paramagnetic resonance spectroscopy (EPR), negative ion mode electrospray ionization-mass spectrometry (negative ion ESI-MS), and kinetic studies provided compelling evidence for a common catalyst or catalyst resting state, namely, Pierpont's structurally characterized vanadyl semiquinone catecholate dimer complex, [VO(DBSQ)(DTBC)]2, formed from V-leaching from the precatalysts. The results provide a considerable simplification and unification of a previously disparate literature of V-based catechol dioxygenases.