Automation of a surface sampling probe/electrospray mass spectrometry system

An image analysis automation concept and the associated software (HandsFree TLC/MS) were developed to control the surface sampling probe-to-surface distance during operation of a surface sampling electrospray system. This automation system enables both "hands-free" formation of the liquid microjunction used to sample material from the surface and hands-free reoptimization of the microjunction thickness during a surface scan to achieve a fully automated surface sampling system. The image analysis concept and the practical implementation of the monitoring and automated adjustment of the sampling probe-to-surface distance (i.e., liquid microjunction thickness) are presented. The added capabilities for the preexisting surface sampling electrospray system afforded through this software control are illustrated by an example of automated scanning of multiple development lanes on a reversed-phase C8 TLC plate and by imaging inked lettering on a paper surface. The post data acquisition processing and data display aspects of the software package are also discussed.     

Direct plant tissue analysis and imprint imaging by desorption electrospray ionization mass spectrometry

The ambient mass spectrometry technique, desorption electrospray ionization mass spectrometry (DESI-MS), is applied for the rapid identification and spatially resolved relative quantification of chlorophyll degradation products in complex senescent plant tissue matrixes. Polyfunctionalized nonfluorescent chlorophyll catabolites (NCCs), the "final" products of the chlorophyll degradation pathway, are detected directly from leaf tissues within seconds and structurally characterized by tandem mass spectrometry (MS/MS) and reactive-DESI experiments performed in situ. The sensitivity of DESI-MS analysis of these compounds from degreening leaves is enhanced by the introduction of an imprinting technique. Porous polytetrafluoroethylene (PTFE) is used as a substrate for imprinting the leaves, resulting in increased signal intensities compared with those obtained from direct leaf tissue analysis. This imprinting technique is used further to perform two-dimensional (2D) imaging mass spectrometry by DESI, producing well-resolved images of the spatial distribution of NCCs in senescent leaf tissues.     

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.     

Evaluation of opiate separation by high-resolution electrospray ionization-ion mobility spectrometry/mass spectrometry

The separation of opiates and the primary metabolites was evaluated with ESI-IMS/MS. Seven opiate molecules were analyzed, and spectra were shown for each compound. The IMS separation of two isomers (morphine and norcodeine) was shown with baseline separation. Differences in the mobilities were found for the nonacetylated, monoacetylated, and biacetylated compounds. In this study, two primary findings are reported. First, IMS can easily separate metabolic isomers, and second, the two-dimensional separation capability of IMS/MS can be employed to confidently identify and separate both the opiates and metabolites. Although previous IMS studies have shown the separation of isomers, this is the first example to show the capability of IMS to separate metabolic isomers (within 70 s), a significant advantage in high-throughput screening for pharmacokinetic studies. Second, the monoacetylated and biacetylated compounds were found to form more compact ions for the sodium adducts in comparison to the protonated molecular ions. On the basis of the mobilities, information on structures and conformation can be deduced when sodium and protonated ions are compared.     

Direct determination of levoglucosan at the picogram per milliliter level in Antarctic ice by high-performance liquid chromatography/electrospray ionization triple quadrupole mass spectrometry

A method for the direct determination of levoglucosan at the picogram per milliliter level in less than 1 mL of Antarctic ice has been developed. Chemical analysis is performed by high-performance liquid chromatography with triple quadrupole tandem mass spectrometric detection. Levoglucosan, a specific molecular marker for biomass burning, is identified by negative ion electrospray mass spectrometry using m/z 161/113, 161/101, 161/85, and 161/71 as monitoring ion transitions. Contamination problems were carefully taken into account by adopting ultraclean procedures during sampling and sample pretreatment phases. The limit of detection is 3 pg mL(-1) (0.3 pg absolute amount injected); the repeatability ranges between 20% and 50% at a concentration of 20 and 9 pg mL(-1), respectively. This methodology allowed the direct determination of levoglucosan in a 1 mL sample of Antarctic ice with concentration ranges between 4 and 30 pg mL(-1). To our knowledge these are the first levoglucosan concentrations reported for Antarctic ice.     

Characterization of polyphosphates by electrospray mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was applied for the characterization of inorganic polyphosphates [orthophosphate, pyrophosphate, tripolyphosphate, trimetaphosphate, and tetrapolyphosphatel. The high selectivity of ESI-MS allows the detection of different polyphosphate species without preseparation by ion chromatography or capillary electrophoresis. Furthermore, ESI-MS does not require the incorporation of UV-absorbing chromophores into the analytical method for the detection of phosphates, unlike conventional UV-chromatographic methods. Limits of detection by ESI-MS were estimated to range from approximately 1 to 10 ng/mL. The quantification of polyphosphate samples as single-component and multicomponent mixtures was investigated. Linear signal response for single-component samples ranged from the limit of detection to approximately 10 microg/mL Quantification of polyphosphate in streamwater is demonstrated using the standard addition method. The effect of multi-polyphosphate components and salts on signal response was also studied. For concentrations less than 2.0 microg/mL, signal response from a tetrapolyphosphate sample was comparable to those obtained from tetrapolyphosphate-tripolyphosphate mixtures. Signal response obtained from tetrapolyphosphate in the presence of tripolyphosphate or NH4NO3 at higher concentrations (approximately 50 microg/mL and 35 microg/mL, respectively) was significantly lower relative to single-component standards (approximately 40%-70%).     

Sequence determination of sulfated carrageenan-derived oligosaccharides by high-sensitivity negative-ion electrospray tandem mass spectrometry

Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation is assessed for sequence determination of multiply sulfated oligosaccharide fragments of carrageenan obtained from partial depolymerization of the polysaccharides by either enzymatic digestion or mild acid hydrolysis. Carrageenan oligosaccharides with homogeneous disaccharide compositions were used to establish their fragmentation pattern, which was then applied to sequence determination of unusual oligosaccharides with either "hybrid" biose compositions or odd-numbered residues. As sulfate groups are labile, sulfate loss during collision-induced association was prevented by sodium adduction. The product ion spectra of [M - Na]- (where M represents the sodium salt of oligosaccharides) feature an extensive series of B- and C-type glycosidic cleavages, whereas the Y-type cleavage occurs mainly at the sulfated residues. The assignment of reducing or nonreducing terminal fragments was assisted by oligosaccharide reduction and the product ion spectra of the derived alditols. Due to the anionic nature of the sulfate present, high-sensitivity detection (1-5 pmol, using a hexasaccharide as an example) was obtained.     

Direct monitoring of heat-stressed biopolymers with temperature-controlled electrospray ionization mass spectrometry

The ability to monitor protein aggregation at the molecular level is critical for progress in many areas of life sciences ranging from understanding mechanisms of amyloidosis and etiology of conformational diseases to development of safe and efficient biopharmaceutical products. Despite the spectacular progress in understanding the mechanisms of protein aggregation in recent years, many aspects of the aggregating proteins behavior remain unclear because of the extreme difficulty in tracking evolution of these notoriously complex and heterogeneous systems. Here, we introduce a mass spectrometry-based methodology that allows the early stages of heat-induced aggregation to be studied by monitoring both conformational changes and formation of oligomers as a function of temperature. The new approach allows biopolymer behavior (both reversible and irreversible processes) to be monitored in a wide temperature range. Validation of the methodology is carried out by comparing temperature profiles of model proteins and nucleic acids deduced from mass spectrometry measurements and differential scanning calorimetry. Application of the methodology to study heat-induced aggregation of human glucocerebrosidase unequivocally links loss of conformational fidelity to formation of soluble oligomers, which serve as precursors to aggregation.     

A method for the determination of binding constants by electrospray ionization mass spectrometry

A new method for the determination of binding constants using electrospray ionization mass spectrometry is presented. The intensity of a reference complex with a known log K value is monitored before and after addition of a second host or guest. On the basis of the change in intensity of the reference complex and extrapolation from a calibration curve, the log K value is then derived for the complex of interest using a set of simultaneous equilibrium equations. Binding constants of several crown ether-alkali metal cation complexes that were previously studied were determined to validate this strategy. Log K values for complexes involving dibenzo-16-crown-5 and its sym-oxyacetate derivative with Na+ or K+ were also determined.     

Compositional analysis of glycosaminoglycans by electrospray mass spectrometry

The purpose of this work is to analyze glycosaminoglycans (GAGs) directly from complex mixtures without the need to purify individual components. Novel conditions for negative ion electrospray MS of chondroitin sulfate (CS) oligosaccharides are described in which sodium adduction and fragmentation are avoided. Differentiation between positional sulfation isomers is demonstrated for CS disaccharides, and a selected reaction monitoring scheme is used to quantify sulfation isomers in disaccharides liberated from decorin and biglycan. A size exclusion chromatography LC/MS method is shown to be effective for compositional analysis of longer CS oligosaccharides. The SEC step serves to simplify the composition of GAGs entering the mass spectrometer at any time, thus allowing the masses of the constituent molecules to be extracted. Mass spectrometric detection produces far more information than conventional UV or fluorescent detectors and allows the monosaccharide composition of individual components to be determined.     

Laser desorption/in situ chemical ionization aerosol mass spectrometry for monitoring tributyl phosphate on the surface of environmental particles

The possibility of using real-time aerosol mass spectrometry (RTAMS) for the detection of surface-adsorbed tributyl phosphate (TBP) as an alkali metal adduct has been investigated. Environmental particles contain variable amounts of easily ionizable alkali metals. During laser desorption of surface-adsorbed TBP molecules, Na+ and K+ ions are generated by the interaction of the laser radiation with the particle's material. The alkali metal ions serve as in situ chemical ionization reagents of the neutral analyte molecules. The effect of laser fluence on the signal intensities of the potassium ion and cationized TBP was also studied. The best performance of the instrument was observed with laser fluences that produce high abundances of K+ but low abundances of ions from the particle's bulk material. The relatively low laser fluence, necessary to produce potassium ions, prevents the excessive fragmentation of the analyte. The instrument is capable of real-time monitoring of submonolayer coverage of TBP on the surface of micron-sized particles.     

Extraction of HNO3 with solutions of zirconium salt of dibutyl hydrogen phosphate in 30% tributyl phosphate and in xylene

Zirconium salt of dibutyl hydrogen phosphate (ZS HDBP) dissolved in xylene or 30% tributyl phosphate (TBP) + xylene mixture extracts noticeable amounts of HNO₃ at its low concentrations in the aqueous phase, when extraction of HNO₃ with straight HDBP itself is not observed. IR data show that DBP^? and NO ₃ ^? ions compete for coordination to Zr, which also adds from 2 to 4 HDBP molecules. With a further increase in the HDBP concentration, the HDBP and HNO₃ molecules compete for coordination in the outer coordination sphere of zirconium. Inflections in the S-shaped dependence of the HNO₃ concentration in the organic phase on the [Zr]:[HDBP] molar ratio lie within the interval from 1:8 to 1:12, which corresponds to filling of the Zr outer sphere with HDBP molecules. In the presence of TBP, these curves become smooth and have no apparent inflection.     

Direct determination of the peptide content in microspheres by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A quantitative determination of peptides incorporated into poly(d,l-lactide-co-glycolide) microspheres by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was accomplished in a single step without pretreatment for extracting the peptide from the microsphere. The conventional extraction methods often underestimate the actual amount of peptide because of incomplete extraction from the microspheres or loss during the procedures. In this study, the microspheres dissolved in acetonitrile containing 0.1% trifluoroacetic acid were mixed with matrix solution containing the internal standard, and the peptide content was directly determined by MALDI-TOF MS. The drug content values determined by MALDI-TOF MS in both the leuprolide- and salmon calcitonin-incorporated microspheres were closer to the theoretical contents than those determined by the conventional extraction method. This method using MALDI-TOF MS could be a good alternative to time-consuming and less-accurate conventional methods.     

Secondary electrospray ionization ion mobility spectrometry/mass spectrometry of illicit drugs

A secondary electrospray ionization (SESI) method was developed as a nonradioactive ionization source for ion mobility spectrometry (IMS). This SESI method relied on the gas-phase interaction between charged particles created by electrospray ionization (ESI) and neutral gaseous sample molecules. Mass spectrometry (MS) was used as the detection method after ion mobility separation for ion identification. Preliminary investigations focussed on understanding the ionization process of SESI. The performance of ESI-IMS and SESI-IMS for illicit drug detection was evaluated by determining the analytical figures of merit. In general, SESI had a higher ionization efficiency for small volatile molecules compared with the electrospray method. The potential of developing a universal interface for both GC- and LC-MS with an addition stage of mobility separation was demonstrated.     

Direct electrospray ionization mass spectrometry quantitative analysis of sebacic and terephthalic acids in biodegradable polymers

A direct, rapid, and easy electrospray ionization mass spectrometry (ESI-MS) method to determine concentrations of sebacic acid (SA) and terephthalic acid (TA) residues in biodegradable copolymers was developed. Copolyester samples were synthesized from 1,4-butanediol and sebacic and terephthalic acids by melt polymerization. Extraction of monomers was performed in methanol. Their concentrations were determined by direct infusion ESI-MS, without chromatographic separation, using 1,12-dodecanedioic acid (DDA) as an internal standard. Calibration curves were obtained by plotting the ratio of the areas of the peaks relative to monomers and DDA standard as a function of their concentration ratio. We validated the method by determining the concentration of TA residue using both the ESI-MS protocol and high-performance liquid chromatography (HPLC) analysis with UV detection. The linearity range and the detection limit of this assay were 0.1-5.0 and 0.01 ppm for SA and 0.1-6.0 and 0.03 ppm for TA. This assay represents a useful alternative to conventional methods currently employed for acid quantification, resulting advantageous for its speed and high sensitivity.     

Sheathless capillary electrophoresis/electrospray mass spectrometry using a carbon-coated fused-silica capillary

A simple procedure was developed for preparing a carbon-coated fused-silica capillary for use in sheathless capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The tapered capillary tip was smeared with a marker pen before coating with carbon using a soft pencil. The layer from the ink of the marker pen was critical to the preparation of the carbon-coated capillary. The fabrication of a carbon-coated fused-silica capillary tip requires less than 1 min. The stability of this carbon-coated fused-silica capillary is examined, and its utility in on-line sheathless CE/ESI-MS is demonstrated with the separation of berberine, coptisine, and palmatine chlorides. Although the carbon-coated fused-silica capillary tip is not as rugged as a gold-coated capillary, it is durable enough for sheathless CE/ESI-MS applications. Moreover, it is easy to refurbish the column once the performance of the tip is degraded.     

Chiral analysis by electrospray ionization mass spectrometry/mass spectrometry. 2. Determination of enantiomeric excess of amino acids

The determination of enantiomeric excess (ee) of amino acids was achieved by investigating the collision-induced dissociation spectra of protonated trimers that were formed by electrospray ionization of amino acids in the presence of one of the following chiral selectors: L- or D-N-tert-butoxycarbonylphenylalanine, L- or D-N-tert-butoxycarbonylproline, and L- or D-N-tert-butoxycarbonyl-O-benzylserine. The protonated trimers were dissociated to form protonated dimers, and the observed dissociation efficiency r (i.e., the intensity ratio of protonated dimers to protonated trimers) for an enantiomeric mixture was found to be related to its ee value by the following equation: r = a + b/(c + ee), where a, b, and c were constants. A linear calibration plot was obtained by plotting r versus 1/(c + ee), where c was calculated with the MATLAB software, or by plotting 1/(r - r0) versus 1/ee, where r0 was the r value for the racemic mixture. The latter "two-reciprocal" method was more convenient for application. Another practical method for ee determination was the "three-point" method, whereby the ee of an unknown sample with a measured r value could be derived from the equation ee = 100?1/(rL - r0) - 1/(rD - r0)?/?2/(r - r0) - 1/(rL - r0) - 1/(rD - r0)?, with rL and rD being the r values for the enantiomerically pure L- and D-forms of the sample, respectively. A calibration plot was not required. The ee determination was achieved with acceptable precision even for the worst case of acceptable chiral recognition with a particular chiral selector, suggesting that the ee determination of all 19 common amino acids could be achieved by the present method. The ee of a histidine sample was determined both by the two-reciprocal method, giving an error of 0.2% ee (1.1% relative error) and consuming only approximately 5.3 nmol of sample, and by the three-point method, giving an error of 0.4% ee and consuming only approximately 2.3 nmol of sample. In the latter case, it took 27 min for the mass spectrometric measurements of the three calibration standards and an additional 9 min for the unknown sample. The direct ee determination of more than one amino acid in a mixture was also demonstrated in the study.