Selective and sensitive detection of chromium(VI) in waters using electrospray ionization mass spectrometry.

From 2000 through 2011, there were 14 criminal cases of violations of the Clean Water Act involving the discharge of chromium, a toxic heavy metal, into drinking and surface water sources. As chromium(VI), a potential carcinogen present in the environment, represents a significant safety concern, it is currently the subject of an EPA health risk assessment. Therefore, sensitive and selective detection of this species is highly desired. This study reports the analysis of chromium(VI) in water samples by electrospray ionization mass spectrometry (ESI-MS) following its reduction and complexation with ammonium pyrrolidinedithiocarbamate (APDC). The reduction and subsequent complexation produce a characteristic [Cr(III)O]-PDC complex which can be detected as a protonated ion of m/z 507 in the positive ion mode. The detection is selective to chromium(VI) under acidic pH, even in the presence of chromium(III) and other metal ions, providing high specificity. Different water samples were examined, including deionized, tap, and river waters, and sensitive detection was achieved. In the case of deionized water, quantification over the concentration range of 3.7 to 148ppb gave an excellent correlation coefficient of 0.9904 using the enhanced MS mode scan. Using the single-reaction monitoring (SRM) mode (monitoring the characteristic fragmentation of m/z 507 to m/z 360), the limit of detection (LOD) was found to be 0.25ppb. The LOD of chromium(VI) for both tap and river water samples was determined to be 2.0ppb. A preconcentration strategy using simple vacuum evaporation of the aqueous sample was shown to further improve the ESI signal by 15 fold. This method, with high sensitivity and selectivity, should provide a timely solution for the real-world analysis of toxic chromium(VI).

Validation of LC-MS/MS methods for quantitative analysis of kynurenine pathway metabolites in human plasma and cerebrospinal fluid.

Background: Dysregulation of the kynurenine metabolic pathway has been reported in several neurological conditions. Methods & results: Sensitive and selective LC-MS/MS methods have been validated for six kynurenine pathway metabolites in human cerebrospinal fluid and plasma. For each matrix, we validated three methods - one for the simultaneous determination of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxy-kynurenine (four-analyte assay), one for quinolinic acid and one for tryptophan - using stable-isotopically labeled internal standards. The dynamic range and quantitation limits were based on endogenous concentrations for each analyte. Conclusion: The use of validated methods for kynurenine pathway metabolites in human cerebrospinal fluid and plasma will provide definitive information in neurological diseases.

Signal and Charge Enhancement for Protein Analysis by Liquid Chromatography-Mass Spectrometry with Desorption Electrospray Ionization.

We recently reported the use of desorption electrospray ionization (DESI) as a novel interface to couple high-performance liquid chromatography (HPLC) with mass spectrometry (MS) (Chem. Commun. 2011, 47, 4171). One of the benefits of such an interface is that post-column derivatization of separated analytes can be integrated with ionization via a "reactive" DESI approach in which a derivatizing reagent is doped into the spray solvent. The reactive DESI interface allows analyte desorption/ionization from the end of the chromatographic column with prompt MS detection; a short time delay of ~20 ms was demonstrated. In this study, we extended this application by "supercharging" proteins following HPLC separation using a DESI spray solvent containing supercharging reagents, m-nitrobenzyl alcohol (m-NBA) or sulfolane. Proteins (insulin, ubiquitin, lysozyme and α-lactalbumin) eluted out of the LC column can be supercharged with the protein charge state distributions (CSDs) significantly increased (to higher charge), which would be advantageous for subsequent top-down MS analysis of proteins. Interestingly, supercharging combined with reactive DESI enhances tolerance towards trifluoroacetic acid (TFA), which is known to be a superior additive in the mobile phase for premium peptide/protein chromatographic separation but has severe signal suppression effects for conventional electrospray ionization (ESI). In comparison to electrosonic spray ionization (ESSI), a variant form of ESI, the sensitivity of protein analysis using LC/DESI-MS with the mobile phase containing TFA can be improved by up to 70-fold for lysozyme and α-lactalbumin by including m-NBA in the DESI spray solvent. Presumably, by reducing TFA dissociation in the droplet, supercharging agents lower trifluoroacetate anion concentrations and concomitantly reduce ion pairing to analyte cationic sites. The reduced ion pairing therefore decreases the TFA signal suppression effect. The supercharging capability and the reduction of TFA signal suppression suggest that LC/DESI-MS is a valuable method for protein analysis.

Development of submillisecond time-resolved mass spectrometry using desorption electrospray ionization.

Reaction kinetics studied by mass spectrometry (MS) has previously been limited to millisecond time resolution. This paper presents the development of a submillisecond time-resolved mass spectrometric method for fast reaction kinetic study, based on the capability of desorption electrospray ionization (DESI) for direct and fast ionization of a high-speed liquid jet stream. The principle underlying this methodology is that two reactant solutions undergo rapid mixing to produce a free liquid jet which is ionized by DESI at different positions corresponding to different reaction times. Due to the high velocity of the liquid jet, high time resolution can be achieved. In this study, the fast reduction reaction of 2, 6-dichlorophenolindophenol (DCIP) and L-ascorbic acid (L-AA) was chosen as an example to demonstrate this concept, and the reaction rate constant was successfully measured with an unprecedented time resolution of 300 µs. The good agreement of the measured value of (116 ± 3) s(-1) with that measured by the stopped-flow optical method (105 ± 2) s(-1) validates the feasibility of such a DESI-MS approach. Unlike classical spectroscopic techniques that require either chromophoric substrates or labeling, MS is a general detector with high chemical specificity. Therefore, this time-resolved DESI-MS method should find wide applications in fast (bio)chemical reaction investigations.

Direct ionization of large proteins and protein complexes by desorption electrospray ionization-mass spectrometry.

Desorption electrospray ionization-mass spectrometry (DESI-MS) has advantages for rapid sample analysis with little or no sample pretreatment, but performance for large biomolecules has not been demonstrated. In this study, liquid sample DESI, an extended version of DESI used for analysis of liquid samples, was shown to have capabilities for direct ionization of large noncovalent protein complexes (>45 kDa) and proteins (up to 150 kDa). Protein complex ions (e.g., superoxide dismutase, enolase, and hemoglobin) desorbed from solution by liquid sample DESI were measured intact, indicating the capability of DESI for preserving weak noncovalent interactions. Doping the DESI spray solvent with supercharging reagents resulted in protein complex ions having increased multiple charging without complex dissociation. Ion mobility measurements of model protein cytochrome c showed that the supercharging reagent favored the more compact conformation for the lower charged protein ions. Liquid sample DESI of hydrophobic peptide gramicidin D suggests that the ionization mechanism involves a droplet pick-up mixing process. Measurement of liquid samples significantly extends the mass range of DESI-MS, allowing the analysis of high-mass proteins such as 150 kDa immunoglobulin G (IgG) and thus represents the largest protein successfully ionized by DESI to date.

Direct analysis of liquid samples by desorption electrospray ionization-mass spectrometry (DESI-MS).

Desorption electrospray ionization-mass spectrometry (DESI-MS) was evaluated for the direct analysis of liquid samples. Several interesting results were found. First, in contrast to the previous DESI analysis of dried solid samples that was limited to proteins with MW < or = 25 kDa (Anal. Chem. 2007, 79, 3514), bovine serum albumin (BSA, 66 kDa) was successfully ionized from solutions by DESI with observation of corresponding multiply charged ions. Second, direct DESI analysis of protein tryptic digest solutions without chromatographic separation, sample clean-up, and the sample drying step was demonstrated, providing reasonably good sequence coverage of 52% to 97%. Third, direct analysis of biofluids such as an undiluted urine sample without sample pretreatment is possible, emphasizing the high tolerance of DESI with salt. These results suggest that a charged droplet pick-up mechanism is responsible for desorption and ionization of liquid samples by DESI. Also, unlike in electrospray ionization (ESI), inhibition of electrochemical reduction in the negative ion mode was observed for liquid sample DESI. In addition, reactive DESI can be performed with ion/ion reactions of Zn(II) complexes for the selective binding of phosphoserine in the presence of serine. DESI experiment can also be carried out directly to liquid samples flowing out of a pumped syringe needle tip, allowing rapid analysis. Furthermore, on-line coupling of electrochemical cell with DESI-MS was demonstrated, in which perylene radical cations generated in the cell were successfully transferred to the gas-phase for MS detection by DESI. This study extended the scope of DESI-MS applications, which could have potentials in bioanalytical and forensic analysis.

The impact of hemolysis on stability of N-desethyloxybutynin in human plasma.

Aim: Stability must be evaluated before quantitation of drugs or metabolites concentrations in biological matrices. We reported a case study where instability of a drug metabolite was mediated by hemolysis. Materials & methods: The instability of both enantiomers of N-desethyloxybutynin was observed in hemolyzed plasma stored at -20°C. The investigations indicated that heme-mediated oxidation converted the metabolite to its N-oxide. Storing samples under lower temperature (-50°C or below) or treatment with the antioxidant ascorbic acid stabilized the metabolite. Conclusion: The evaluation of the stability of some analytes in a hemolyzed sample is crucial as it may negatively impact incurred sample reanalysis or pharmacokinetic profiles on highly hemolyzed samples.

Bioanalysis of emixustat (ACU-4429) in whole blood collected with volumetric absorptive microsampling by LC-MS/MS.

BACKGROUND: A method to quantify emixustat (an investigational drug agent) in human blood collected using volumetric absorptive microsampling (VAMS) could be more practical for sample collection at sites with limited facilities for processing and storage of plasma. METHODS: A LC-MS/MS method was developed and evaluated for accuracy and precision, linearity, carryover, selectivity, recovery, matrix effects, hematocrit effects and stability. RESULTS: Core validation parameters met acceptance criteria within the normal ranges of hematocrit levels for adults (30-55%). Stability of emixustat in blood collected with and without anticoagulant (NaF/KOx) on the VAMS device at ambient, refrigerated and frozen conditions was established. CONCLUSION: The method has been validated and is suitable for the bioanalysis of emixustat in human blood collected by VAMS.

The study of protein conformation in solution via direct sampling by desorption electrospray ionization mass spectrometry.

The direct sampling feature of liquid sample desorption electrospray ionization (DESI) allows the ionization of liquid samples without adding acids/organic solvents (i.e., without sample pretreatment). As a result, it provides a new approach for probing protein conformation in solution. In this study, it has been observed that native protein ions are generated from proteins in water by DESI. Interestingly, the intensities of the resulting protein ions appear to be higher than those generated by ESI of the proteins in water or in ammonium acetate. For protein solutions that already contain acids/organic solvents, DESI can be used to investigate the influences of these denaturants on protein conformations and the obtained results are in good agreement with spectroscopic data. In addition, online monitoring of protein conformational changes by DESI is feasible; for instance, heat-induced unfolding of ubiquitin can be traced with DESI in water without influences of organic solvents/acids. This DESI method provides a new alternative tool for the study of protein conformation in solution.

The role of nitric-oxide synthase in the regulation of UVB light-induced phosphorylation of the alpha subunit of eukaryotic initiation factor 2.

UV light induces phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha) and inhibits global protein synthesis. Both eIF2 kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), have been shown to phosphorylate eIF2alpha in response to UV irradiation. However, the roles of PERK and GCN2 in UV-induced eIF2alpha phosphorylation are controversial. The one or more upstream signaling pathways that lead to the activation of PERK or GCN2 remain unknown. In this report we provide data showing that both PERK and GCN2 contribute to UV-induced eIF2alpha phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast cells. Reduction of expression of PERK or GCN2 by small interfering RNA decreases phosphorylation of eIF2alpha after UV irradiation. These data also show that nitric-oxide synthase (NOS)-mediated oxidative stress plays a role in regulation of eIF2alpha phosphorylation upon UV irradiation. Treating the cells with the broad NOS inhibitor N(G)-methyl-l-arginine, the free radical scavenger N-acetyl-l-cysteine, or the NOS substrate l-arginine partially inhibits UV-induced eIF2alpha phosphorylation. The results presented above led us to propose that NOS mediates UV-induced eIF2alpha phosphorylation by activation of both PERK and GCN2 via oxidative stress and l-arginine starvation signaling pathways.

Metal-organic frameworks constructed from 2,4,6-Tris(4-pyridyl)-1,3,5-triazine.

Five new metal-organic frameworks based on 2,4,6-tris(4-pyridyl)-1,3,5-triazine (tpt) ligand have been hydrothermally synthesized. Reaction of tpt and AgNO 3 in an acidic solution at 180 degrees C yields {[Ag(Htpt)(NO3)]NO(3).4H2O}n (1).Ag(I) is trigonally coordinated by two pyridyl nitrogen and one nitrato oxygen to form a 1D zigzag chain. Reaction of tpt with CuSO4 affords {[Cu2(tpt)2(SO4)2(H2O)2].4H2O}n (2). Copper(II) is bonded to two pyridyl nitrogen, two sulfato oxygen, and two water oxygen atoms to form an elongated octahedral geometry. Each H2O ligand bridges two copper(II), whereas sulfate bridges copper(II) via micro-1,3 and micro-1,1 fashions. The copper(II)-sulfate-H2O2D layers are linked by bidentate tpt to form a 3D polymeric structure. Reaction of Cu(SO4)2, tpt, and 1,2,4,5-benzenetetracarboxylic acid (H4btec) in the presence of piperidine gives [Cu(tpt)(H2btec)1/2]n (3). Copper(I) is located in a trigonal-pyramidal coordination environment and coordinated by three pyridyl nitrogen of tpt in a plane, whereas a carboxylate oxygen is coordinated to the copper(I) axially. The tpt-Cu forms a layer, and the layers are linked through H 2btec2- to form a 2D double-layered coordination polymer. Replacing CuSO4 with ZnI2 in the synthesis gives {[Zn(tpt)(btec)1/2].H2O}n (4). Zinc(II) is in a distorted tetrahedral geometry and linked through bidentate tpt and exotetradentate btec4- to form a 2D coordination grid. Reaction of tpt with CuCN leads to the assembly of a 3D metal-organic framework [Cu3(CN)3(tpt)]n (5). Copper(I) is trigonally coordinated by one pyridyl nitrogen and two cyanides to form an intriguing honeycomb architecture. Luminescence study shows that 1, 3, 4, and 5 have blue fluorescence, which can be assigned to be ligand-centered emissions. Thermal analysis shows that all of these complexes are quite stable, and especially for 4, the framework is stable up to 430 degrees C.