Identification and quantitation of despropionyl-bezitramide in postmortem samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry.

A sensitive and specific method for the determination and quantitation of (despropionyl) bezitramide in postmortem samples using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is presented. The method is the result from a simple methodological transfer of a liquid chromatographic method with fluorescence detection (LC-FL) previously developed in our laboratory. A liquid-liquid back-extraction procedure using n-hexane isoamyl alcohol (93:7, v/v) as the extraction solvent is performed for a basic sample cleanup. N-Methyldespropionyl bezitramide is used as the internal standard. Chromatographic separation of the analytes of interest is achieved on a Hypersil ODS 5-micron column, using a 80:20 (v/v) mixture of 1.0 mM ammonium acetate and methanol/acetonitrile (50:50, v/v) and 1.0 mM ammonium acetate as the mobile phase. To obtain as high a sensitivity and selectivity as possible, a selected reaction-monitoring mass spectrometric technique is applied. In addition, low-energy collisional-activated dissociation (CAD) product ion spectra are recorded for a few samples. Calibration graphs are prepared for blood and urine, and good linearity is achieved over a concentration range of 1-150 ng/mL. The intra- and interassay coefficients of variation (CV%) for the analysis of quality control samples at 10 and 50 ng/mL concentration levels do not exceed 10.2% and percent of targets are within 12.1%. Postmortem samples (blood, urine, stomach contents, bile, liver, and kidney) from three fatalities, all suspected victims of drug overdoses, are analyzed, and the results are reported. The results obtained with LC-ESI-MS/MS are in close agreement with those obtained using the LC-FL method. Moreover, the isolates' identity and structure are confirmed by the CAD product ion spectra, thus allowing to make unequivocal conclusions about the prior intake of bezitramide by the three subjects.     

Molecular resolution and fragmentation of fulvic acid by electrospray ionization/multistage tandem mass spectrometry

Molecular weight distributions of fulvic acid from the Suwannee River, Georgia, were investigated by electrospray ionization/quadrupole mass spectrometry (ESI/ QMS), and fragmentation pathways of specific fulvic acid masses were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry (ESI/MST/ MS). ESI/QMS studies of the free acid form of low molecular weight poly(carboxylic acid) standards in 75% methanol/25% water mobile phase found that negative ion detection gave the optimum generation of parent ions that can be used for molecular weight determinations. However, experiments with poly(acrylic acid) mixtures and specific high molecular weight standards found multiply charged negative ions that gave a low bias to molecular mass distributions. The number of negative charges on a molecule is dependent on the distance between charges. ESI/MST/MS of model compounds found characteristic water loss from alcohol dehydration and anhydride formation, as well as CO2 loss from decarboxylation, and CO loss from ester structures. Application of these fragmentation pathways to specific masses of fulvic acid isolated and fragmented by ESI/MST/MS is indicative of specific structures that can serve as a basis for future structural confirmation after these hypothesized structures are synthesized.     

Analysis of keratan sulfate oligosaccharides by electrospray ionization tandem mass spectrometry

Keratan sulfate (KS) is a glycosaminoglycan consisting of repeating disaccharide units composed of alternating residues of d-galactose and N-acetyl-d-glucosamine linked beta-(1-4) and beta-(1-3), respectively. In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to identify keratan sulfate oligosaccharides. Two nonsulfated disaccharide isomers and two monosulfated disaccharide isomers were distinguished through MS/MS. In MS(1) spectra of multiply sulfated KS oligosaccharides, the charge state of the most abundant molecular ion equals the number of sulfates. Subsequent MS(2) and MS(3) spectra of mono-, di-, tri-, and tetrasulfated KS oligosaccharides and sialylated tetrasaccharides reveal diagnostic ions that can be used as fingerprint maps to identify unknown KS oligosaccharides. Based on the pattern of fragment ions, the compositions of an oligosaccharide mixture from shark cartilage KS and of two enzyme digests of bovine corneal KS were determined directly, without prior isolation of individual oligosaccharides by HPLC or other methods.     

Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry

A method for the determination of underivatized amino acids based on capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) is described. To analyze free amino acids simultaneously a low acidic pH condition was used to confer positive charge on whole amino acids. The choice of the electrolyte and its concentration influenced resolution and peak shape of the amino acids, and 1 M formic acid was selected as the optimal electrolyte. Meanwhile, the sheath liquid composition had a significant effect on sensitivity and the highest sensitivity was obtained when 5 mM ammonium acetate in 50% (v/v) methanol-water was used. Protonated amino acids were roughly separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow electrospray ionization interface. Under the optimized conditions, 19 free amino acids normally found in proteins and several physiological amino acids were well determined in less than 17 min. The detection limits for basic amino acids were between 0.3 and 1.1 mumol/L and for acidic and low molecular weight amino acids were less than 6.0 mumol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. This method is simple, rapid, and selective compared with conventional techniques and could be readily applied to the analysis of free amino acids in soy sauce.     

Elucidation of double bond position in unsaturated lipids by ozone electrospray ionization mass spectrometry

The position(s) of carbon-carbon double bonds within lipids can dramatically affect their structure and reactivity and thus has a direct bearing on biological function. Commonly employed mass spectrometric approaches to the characterization of complex lipids, however, fail to localize sites of unsaturation within the molecular structure and thus cannot distinguish naturally occurring regioisomers. In a recent communication [Thomas, M. C.; Mitchell, T. W.; Blanksby, S. J. J. Am. Chem. Soc. 2006, 128, 58-59], we have presented a new technique for the elucidation of double bond position in glycerophospholipids using ozone-induced fragmentation within the source of a conventional electrospray ionization mass spectrometer. Here we report the on-line analysis, using ozone electrospray mass spectrometry (OzESI-MS), of a broad range of common unsaturated lipids including acidic and neutral glycerophospholipids, sphingomyelins, and triacylglycerols. All lipids analyzed are found to form a pair of chemically induced fragment ions diagnostic of the position of each double bond(s) regardless of the polarity, the number of charges, or the adduct ion (e.g., [M - H](-), [M - 2H](2-), [M + H](+), [M + Na](+), [M + NH(4)](+)). The ability of OzESI-MS to distinguish lipids that differ only in the position of the double bonds is demonstrated using the glycerophosphocholine standards, GPCho(9Z-18:1/9Z-18:1) and GPCho(6Z-18:1/6Z-18:1). While these regioisomers cannot be differentiated by their conventional tandem mass spectra, the OzESI-MS spectra reveal abundant fragment ions of distinctive mass-to-charge ratio (m/z). The approach is found to be sufficiently robust to be used in conjunction with the m/z 184 precursor ion scans commonly employed for the identification of phosphocholine-containing lipids in shotgun lipidomic analyses. This tandem OzESI-MS approach was used, in conjunction with conventional tandem mass spectral analysis, for the structural characterization of an unknown sphingolipid in a crude lipid extract obtained from a human lens. The OzESI-MS data confirm the presence of two regioisomers, namely, SM(d18:0/15Z-24:1) and SM(d18:0/17Z-24:1), and suggest the possible presence of a third isomer, SM(d18:0/19Z-24:1), in lower abundance. The data presented herein demonstrate that OzESI-MS is a broadly applicable, on-line approach for structure determination and, when used in conjunction with established tandem mass spectrometric methods, can provide near complete structural characterization of a range of important lipid classes. As such, OzESI-MS may provide important new insight into the molecular diversity of naturally occurring lipids.     

Enhancement of amino acid detection and quantification by electrospray ionization mass spectrometry

A new strategy for amino acid analysis is reported involving derivatization with an N-hydroxysuccinimide ester of N-alkylnicotinic acid (Cn-NA-NHS) followed by reversed-phase chromatography and electrospray ionization mass spectrometry (RPC-MS). Detection sensitivity increased as the N-alkyl chain length of the nicotinic acid derivatizing agent was increased from 1 to 4. N-Acylation of amino acids with the Cn-NA-NHS reagents in water produced a stable product in roughly 1 min using a 4-fold molar excess of derivatizing agent in 0.1 M sodium borate buffer at pH values ranging from 8.5 to 10. Some O-acylation of tyrosine was also observed, but the product hydrolyzed within a few minutes at pH 10. The cystine product also degraded slowly over the course of a few days from reduction of the disulfide bond to form cysteine. The retention time of Cn-NA derivatized amino acids was lengthened in reversed-phase chromatography to the extent that polar amino acids were retained beyond the solvent peak, particularly in the cases of the C3-NA and C4-NA derivatives. Complete resolution of 18 amino acids was achieved in 28 min using the C4-NA-NHS reagent. Compared to N-acylation with benzoic acid, derivatization with C4-NA-NHS increased MS detection sensitivity 6-80-fold. This was attributed to the surfactant properties of the Cn-NA-NHS reagents. The quaternary amine increased the charge on amino acid conjugates while the presence of an adjacent alkyl chain further increased ionization efficiency by apparently enhancing amino acid migration to the surface of electrospray droplets. Further modification of the Cn-NA-NHS reagents with deuterium was used to prepare coded sets of derivatizing agents. These coding agents were used to differentially code samples and after mixing carry out comparative concentration measurements between samples using extracted ion chromatograms to estimate relative peak areas of derivatized amino acids.     

Identification and characterization of conjugated fatty acid methyl esters of mixed double bond geometry by acetonitrile chemical ionization tandem mass spectrometry

Fatty acids with conjugated double bonds have attracted great interest because of their reported potent bioactivities. However, there are currently no rapid methods for their structural characterization. We report here a convenient mass spectrometry-based strategy to establish double bond geometry by analysis of collisional dissociation products of cis/trans and trans/cis conjugated linoleic acids (CLAs), as methyl esters, and to distinguish CLAs from homoallylic (methylene-interrupted) fatty acids in a single-stage mass spectrum. A series of CLA standards with double bond positions 6,8; 7,9; 8,10; 9,11; 10,12; 11,13; 12,14; and 13,15, with all four possible geometries (cis/trans; trans/cis; cis/cis; trans/trans) were analyzed. The m/z 54 (1-methyleneimino)-1-ethenylium ion, generated by self-reaction of acetonitrile under chemical ionization conditions, reacts with unsaturated fatty acids to yield an [M + 54]+ ion, which decomposes in the single-stage mass spectrum by loss of neutral methanol to form [M + 54 - 32]+. The ratio of [M + 54]+/[M + 54 - 32]+ in the single-stage mass spectra of CLA isomers is 1 order of magnitude less than for homoallylic diene FAME. Collisional dissociation of the [M + 54]+ ion yields two diagnostic ions that contain the alpha- and omega-carbon atoms and is characteristic of double bond position in the analyte. The fragment vinylic to the trans double bond is significantly more abundant than that for the cis double bond, revealing double bond geometry. The ratio of alpha to we diagnostic ion abundances is >4.8 for cis/trans isomers, <0.5 for trans/cis isomers, and 0.7-3.2 for cis/cis and trans/trans isomers. This method provides a rapid alternative to conventional conjugated fatty acid analysis and, together with complementary elution time information provided by gas chromatography, enables rapid, positive identification of double bond position and geometry in most CLA FAME.     

Sequencing of sulfated oligosaccharides from mucins by liquid chromatography and electrospray ionization tandem mass spectrometry

As part of a strategy for profiling diverse mixtures of sulfated mucin-derived oligosaccharides, liquid chromatography coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the negative ion mode has been explored. Two mixtures of sulfated oligosaccharide alditols from porcine stomach and large intestine were analyzed by straight phase chromatography using an amino-bonded column connected to a Q-TOF instrument. Nine sulfated mucin-derived oligosaccharide alditols from porcine stomach underwent extensive fragmentation allowing determination of their sequence. The fragmentation generated primary, secondary, and tertiary fragment ions informative for the elucidation of the saccharide sequence and localization of the sulfate group. From a single chromatographic analysis, the sequences of 28 different sulfated mucin oligosaccharide alditols purified from porcine large intestine were elucidated, revealing information concerning prominent core sequences and terminal blood group-type epitopes. Analysis of these two sulfated oligosaccharide mixtures demonstrated the usefulness of HPLC-ESI-MS/MS: the on-line separation of multiple isomeric suffated oligosaccharides as present in biological samples, informative fragmentation allowing the identification of the sequence of nonderivatized oligosaccharides, and a sensitivity sufficient for the analysis of quantities as obtained from natural sources.     

Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer

The present study reports a procedure developed for the identification of SDS-polyacrylamide gel electrophoretically separated proteins using an electrospray ionization quadrupole time-of-flight mass spectrometer (Q-TOF MS) equipped with pressurized sample introduction. It is based on in-gel digestion of the proteins without previous reduction/alkylation and on the capability of the Q-TOF MS to provide data suitable for peptide mass fingerprinting database searches and for tandem mass spectrometry (MS/MS) database searches (sequence tags). Omitting the reduction/alkylation step reduces sample contamination and sample loss, resulting in increased sensitivity. Omitting this step can leave disulfide-connected peptides in the analyte that can lead to misleading or ambiguous results from the peptide mass fingerprinting database search. This uncertainty, however, is overcome by MS/MS analysis of the peptides. Furthermore, the two complementary MS approaches increase the accuracy of the assignment of the unknown protein. This procedure is thus, highly sensitive, accurate, and rapid. In combination with pressurized nanospray sample introduction, it is suitable for automated sample handling. Here, we apply this approach to identify protein contaminants observed during the purification of the yeast DNA mismatch repair protein Mlh 1.     

Structural analysis of polymer end groups by electrospray ionization high-energy collision-induced dissociation tandem mass spectrometry

Chemical structures of polymer end groups play an important role in determining the functional properties of a polymeric system. We present a mass spectrometric method for determining end group structures. Polymeric ions are produced by electrospray ionization (ESI), and they are subject to source fragmentation in the ESI interface region to produce low-mass fragment ions. A series of source-fragment ions containing various numbers of monomer units are selected for high-energy collision-induced dissociation (CID) in a sector/time-of-flight tandem mass spectrometer. It is shown that high-energy CID spectra of source-induced fragment ions are very informative for end group structure characterization. By comparing the CID spectra of fragment ions with those of known chemicals, it is possible to unambiguously identify the end group structures. The utility of this technique is illustrated for the analysis of two poly(ethylene glycol)-based slow-releasing drugs where detailed structural characterization is of significance for drug formulation, quality control, and regulatory approval. Practical issues related to the application of this method are discussed.     

Quantitative thin-layer chromatography/mass spectrometry analysis of caffeine using a surface sampling probe electrospray ionization tandem mass spectrometry system

Quantitative determination of caffeine on reversed-phase C8 thin-layer chromatography plates using a surface sampling electrospray ionization system with tandem mass spectrometry detection is reported. The thin-layer chromatography/electrospray tandem mass spectrometry method employed a deuterium-labeled caffeine internal standard and selected reaction monitoring detection. Up to nine parallel caffeine bands on a single plate were sampled in a single surface scanning experiment requiring 35 min at a surface scan rate of 44 mum/s. A reversed-phase HPLC/UV caffeine assay was developed in parallel to assess the mass spectrometry method performance. Limits of detection for the HPLC/UV and thin-layer chromatography/electrospray tandem mass spectrometry methods determined from the calibration curve statistics were 0.20 ng injected (0.50 muL) and 1.0 ng spotted on the plate, respectively. Spike recoveries with standards and real samples ranged between 97 and 106% for both methods. The caffeine content of three diet soft drinks (Diet Coke, Diet Cherry Coke, Diet Pepsi) and three diet sport drinks (Diet Turbo Tea, Speed Stack Grape, Speed Stack Fruit Punch) was measured. The HPLC/UV and mass spectrometry determinations were in general agreement, and these values were consistent with the quoted values for two of the three diet colas. In the case of Diet Cherry Coke and the diet sports drinks, the determined caffeine amounts using both methods were consistently higher (by approximately 8% or more) than the literature values.     

Identification and quantitation of changes in the platelet activating factor family of glycerophospholipids over the course of neuronal differentiation by high-performance liquid chromatography electrospray ionization tandem mass spectrometry

Glycerophospholipids are important structural lipids in membranes with changes associated with progressive neurodegenerative disorders such as Alzheimer disease. Synthesis of the platelet activating factor (PAF) glycerophospholipid subclass is implicated in the control of neuronal differentiation and death. In this article, we combine nanoflow HPLC and mass spectrometry to screen, identify, and quantitate changes in glycerophospholipid subspecies, specifically PAF family members, over the course of neuronal differentiation. Furthermore, precursor ion scans for fragments characteristic of PAF phosphocholine family members and the standard additions of PAF subspecies were combined to perform absolute quantitation of PAF lipids in undifferentiated and differentiated PC12 cells. Surprisingly, a marked asymmetry was detected in the two predominant PAF species (C16:0, C18:0) over the course of differentiation. These results describe a new technique for the sensitive analysis of lipids combining nanoflow HPLC, ESI-MS, and precursor ion scan. Limits of detection of as little as 2 pg of PAF and LPC were obtained, and analysis of the lipidome of as little as 70,000 cells was performed on this system. Furthermore, application to the PC12 model identified a quantifiable difference between PAF molecular species produced over the course of neuronal differentiation.     

Characterization of archaeological beeswax by electron ionization and electrospray ionization mass spectrometry

To better detect and identify beeswax in ancient organic residues from archaeological remains, we developed a new analytical methodology consisting of the analysis of (i) the trimethylsilylated organic extract by GC/MS and (ii) the crude extract by ESI-MS. Selective scanning modes, such as SIM or MRM, permit separate quantification of each chemical family (fatty acids, monoesters, monohydroxyesters, and diesters) and allow an improvement in sensitivity and selectivity, allowing the crude extract to be treated without further purification. GC/MS (SIM) was revealed to be a powerful method for the detection of components, with a detection limit down to a total lipid extract in the range of approximately 50 ng in a complex matix, such as archaeological degraded material, whereas ESI-MS/MS is instead used for the detection of nonvolatile biomarkers. Identification by GC/MS (SIM) and ESI-MS/ MS (MRM) of more than 50 biomarkers of beeswax in an Etruscan cup at the parts-per-million level provides the first evidence for the use of this material by the Etruscans as fuel or as a waterproof coating for ceramics.     

Identification of protein ubiquitylation by electrospray ionization tandem mass spectrometric analysis of sulfonated tryptic peptides

We report here the application of electrospray ionization tandem mass spectrometry for the characterization of protein ubiquitylation, an important posttranslational modification of cellular proteins. Trypsin digestion of ubiquitin-conjugated proteins produces diglycine branched peptides containing the modification sites. Chemical derivatization by N-terminal sulfonation was carried out on several model peptides for the formation of a characteristic fragmentation pattern in their MS/MS analysis. The fragmentation of derivatized singly charged peptides results in a product ion distribution similar to that already observed by MALDI-TOF MS/MS. Signature fragments distinguished the diglycine branched peptides from other modified and unmodified peptides, while the sequencing product ions reveal the amino acid sequence and the location of the ubiquitylation site. Doubly charged peptide derivatives fragment in a somewhat different manner, but several fragments characteristic to diglycine branched peptides were observed under low collision energy conditions. These signature peaks can also be used to identify peptides containing ubiquitylation sites. In addition, a marker ion corresponding to a glycine-modified lysine residue produced by high-energy fragmentation provides useful information for identity verification. The method is demonstrated by the analysis of three ubiquitin-conjugated proteins using LC/MS/MS.     

Preview: a program for surveying shotgun proteomics tandem mass spectrometry data

Database search programs for peptide identification by tandem mass spectrometry ask their users to set various parameters, including precursor and fragment mass tolerances, digestion specificity, and allowed types of modifications. Even proteomics experts with detailed knowledge of their samples may find it difficult to make these choices without significant investigation, and poor choices can lead to missed identifications and misleading results. Here we describe a program called Preview that analyzes a set of mass spectra for mass errors, digestion specificity, and known and unknown modifications, thereby facilitating parameter selection. Moreover, Preview optionally recalibrates mass over charge measurements, leading to further improvement in identification results. In a study of Bruton's tyrosine kinase, we find that the use of Preview improved the number of confidently identified mass spectra and phosphorylation sites by about 50%.     

Detection and partial structure elucidation of basic taxoids from Taxus wallichiana by electrospray ionization tandem mass spectrometry

A sensitive method for the detection and characterization of basic taxoids from the ethyl acetate extract of Taxus wallichiana has been described. A combined analysis of the fragmentation spectra of 3 purified standard basic taxoids and the substructure analysis of 139 previously reported taxoids provided information on typical primary and secondary product ions that are generated by CID mass spectrometry of basic taxoids. Precursor-scan analysis of selected product ions allowed for the detection of 57 basic taxoids from the ethyl acetate extract of T. wallichiana, 45 of which have not been reported. The method describe in this paper provides a fast method for the "dereplication" of natural products. The mass spectrometric data derived by this method was sufficient for the partial structure elucidation of novel basic taxoids. The method presented in this paper can be easily adapted into a high-throughput screening protocol for the identification and characterization of bioactive natural products.     

Nonesterified fatty acid determination for functional lipidomics: comprehensive ultrahigh performance liquid chromatography-tandem mass spectrometry quantitation, qualification, and parameter prediction

Despite their central importance for lipid metabolism, straightforward quantitative methods for determination of nonesterified fatty acid (NEFA) species are still missing. The protocol presented here provides unbiased quantitation of plasma NEFA species by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Simple deproteination of plasma in organic solvent solution yields high accuracy, including both the unbound and initially protein-bound fractions, while avoiding interferences from hydrolysis of esterified fatty acids from other lipid classes. Sample preparation is fast and nonexpensive, hence well suited for automation and high-throughput applications. Separation of isotopologic NEFA is achieved using ultrahigh-performance liquid chromatography (UPLC) coupled to triple quadrupole LC-MS/MS detection. In combination with automated liquid handling, total assay time per sample is less than 15 min. The analytical spectrum extends beyond readily available NEFA standard compounds by a regression model predicting all the relevant analytical parameters (retention time, ion path settings, and response factor) of NEFA species based on chain length and number of double bonds. Detection of 50 NEFA species and accurate quantification of 36 NEFA species in human plasma is described, the highest numbers ever reported for a LC-MS application. Accuracy and precision are within widely accepted limits. The use of qualifier ions supports unequivocal analyte verification.     

Determination of cabergoline by electrospray ionization tandem mass spectrometry: picogram detection via column focusing sample introduction.

An electrospray ionization tandem mass spectrometric method was developed for low-picogram detection of an ergot alkaloid, cabergoline, in coyote plasma extracts. Cabergoline is under investigation as an abortifacient in canid species. Central to the successful development of this method was the ability to introduce relatively large sample volumes into the mass spectrometer. This was achieved by focusing the analyte on a conventional high-performance liquid chromatography guard column prior to elution into the spectrometer. Volumes up to at least 900 microL could be injected onto the guard column using a 100% aqueous mobile phase. Cabergoline retained on the column was eluted as a discreet band into the mass spectrometer by the rapid addition of methanol (30%) to the mobile phase. As compared to flow injection sample introduction, the ability to inject larger sample volumes led to a greatly lowered detection limit. Using this technique and a modification of a previously reported extraction procedure, cabergoline could be determined in coyote plasma at concentrations as low as 9 pg of cabergoline/mL of plasma.     

Organic chloramine analysis and free chlorine quantification by electrospray and atmospheric pressure chemical ionization tandem mass spectrometry

Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), together with tandem mass spectrometry (MSn), are used to study the mechanism of chlorination of amines and to develop a method for qualitative and quantitative determination of organic chloramines. Cyclohexylamine and 1,4-butanediamine (putrescine) are used as model compounds to investigate the mechanisms of the reactions between primary aliphatic amines and hypochlorous acid (aqueous Cl2). The chlorination products are identified and characterized by collision-induced dissociation (CID) and H/D exchange. Chlorination occurs by electrophilic addition of Cl+ and may be followed by HCl elimination, hydrolysis, or, in the case of diamines, amine elimination by intramolecular nucleophilic substitution. The relative rates of chlorination at amine and chloramine nitrogens are a function of pH and depend on the basicity of the amine. A novel method for active chlorine quantification using ESI or APCI mass spectrometry is suggested on the basis of the extent of chlorination of a sacrifical amine standard. This measurement has a limit of detection for N-chlorocyclohexylamine in the range of 0.1-10 microM, a linear dynamic range of 10(2)-10(3), and an accuracy of +/-10%, as determined for wastewater samples.     

Determination of dissolved metal species by electrospray ionization mass spectrometry

The distribution of metal species in solution was determined using flow injection electrospray ionization mass spectrometry. Complexes formed by selected metal ions with added organic ligands in 50:50 water/acetonitrile and 50:50 water/methanol under acidic, neutral, and basic conditions were detected using electrospray ionization conditions optimized to best represent solution-phase interactions. Metal species containing acetate, nitrate, and solvent molecules predominated in acidic solution but became less abundant at higher pH. Interactions between metal ions and added organic ligands became more selective with increasing pH, showing the expected preference of hard and soft ligands for metal ions of the corresponding type. Species distributions also tended toward larger complexes as pH increased. Overall ion yield was greater for aqueous acetonitrile than for aqueous methanol solutions; however, reduction of copper(II) in aqueous acetonitrile resulted in the detection of copper(I) complexes for certain ligands. Experimental results for copper(II) and 8-hydroxyquinoline in 50:50 water/methanol showed good agreement with aqueous speciation predicted using the thermodynamic equilibrium model MINEQL. Detection of neutral complexes was achieved by protonation, deprotonation, or electrochemical oxidation during electrospray.