Determination of small drug molecules by capillary electrophoresis-atmospheric pressure ionization mass spectrometry.

Capillary electrophoresis (CE) separations are reported for sulfonamides and benzodiazepines in an uncoated fused-silica capillary. The capillary column exit was connected to a liquid junction-ion spray interface combination coupled to an atmospheric pressure ionization (API) triple quadrupole mass spectrometric (MS) system. On-line UV detection occurred 20 cm from the inlet of the capillary and with the API mass spectrometer (CE-API-MS) after the entire length of the capillary (100 cm). The separations were made using volatile buffers composed of ammonium acetate (15-20 mM) with 15-20% of methanol to facilitate ionization under electrospray conditions. This study showed that the major metabolite of flurazepam in man, N-1-hydroxyethylflurazepam, could be detected and characterized in human urine by CE-UV-MS following the administration of a single oral dose of 30 mg of flurazepam dihydrochloride. The presence of additional flurazepam metabolites in human urine was observed by using the system, suggesting that a combination of UV with MS detection should be useful for metabolic studies. In addition to molecular weight determination of compounds, structural information may be obtained by utilizing online tandem mass spectrometry (CE-UV-MS-MS). This was demonstrated for sulfamethazine where the protonated molecule species was transmitted into the collision cell of the tandem triple quadrupole mass spectrometer. Collision-induced dissociation of the protonated sulfamethazine molecule yielded structural information characteristic of the sulfa drug following the on-column injection of 2 pmol of sulfamethazine.     

Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides

The successful coupling of capillary electrochromatography (CEC) to an ion trap mass spectrometer via a nanoelectrospray interface (nESI) is described. Using a conductively coated tip butted to the end of a CEC column, it was possible to obtain a stable spray without any sheath liquid being employed. Selected small peptides were separated with CEC columns (100 microm i.d./25 cm long) packed with 3 microm Hypersil C8 or C18 bonded silica particles with an eluent composed of ammonium acetate/acetonitrile. Peptide mixtures of desmopressin, peptide A, oxytocin, carbetocin and [Met(5)]-enkephalin were detected in the mid-attomole range, which is the lowest amount analyzed using CEC combined with MS detection. It was also observed that sensitivity can be compromised at higher separation voltages. We demonstrate that CEC/nESI-MS, at the current stage of development, represents one of the most sensitive systems for peptide analysis.     

Capillary isotachophoresis with UV and tandem mass spectrometric detection for peptides and proteins

The application of capillary isotachophoresis (CITP) and combined CITP-mass spectrometry (MS) for peptides and proteins is demonstrated. Separation of simple peptide mixtures, as well as enzymatic digets of proteins, is also reported using CITP with UV detection. The potential utility of CITP for proteins is demonstrated. Initial studies of combined CITP-MS of enzymatic digests is also demonstrated, showing the potential for rapid sequence determination.     

Capillary supercritical fluid chromatography with simultaneous flame ionization and mass spectrometric detection

A simple interface between a capillary supercritical fluid chromatograph and an Extranuclear Simulscan mass spectrometer is described. The SFC column is directly inserted into the ion source through the existing GC-interface. The system is equipped with a splitting device which allows simultaneous EI/MS and flame ionization detection when CO₂ is used as the supercritical phase. The effect of source temperature and pressure on CO₂ clustering was studied for optimization of source conditions. The performance of the system was evaluated with a series of model compounds and standard mixtures.     

Sensitivity considerations for large molecule detection by capillary electrophoresis-electrospray ionization mass spectrometry

The use of the electrospray ionization (ESI) method for interfacing capillary electrophoresis with mass spectrometry (CE-MS) is particularly well suited for the analysis of large molecules due to the multiple charging phenomenon. While ionization efficiency is very high, the available ion current is dispersed over more peaks so that the maximum peak intensity obtainable declines significantly for large molecules. Sensitivity with ESI can be improved by operation at very low flow-rates, an ideal situation for CE-MS. These and other considerations related to sensitivity are illustrated using ESI-MS measurements for cytochrome c.     

Atmospheric pressure ionization mass spectrometry

Atmospheric pressure ionization has been used to great effect by mass spectrometrists in widely diverse fields. The aim of this brief review is to set down the advantages of API/MS to fundamental and analytical chemists alike through discussion of the basic processes of ionisation and through its application.     

Capillary electrophoretic bioanalysis of therapeutically active peptides with UV and mass spectrometric detection after on-capillary preconcentration.

An earlier developed capillary electrophoresis (CE) system with an on-capillary adsorptive phase is investigated for its suitability to quantitate low concentrations of angiotensin II and gonadorelin in plasma. An off-line solid-phase extraction is used for sample preparation. The on-line preconcentration CE system allows multiple capillary volumes of sample solution to be injected, increasing the concentration sensitivity of CE with 3-4 orders of magnitude. Furthermore, possible influence of matrix salts can be ruled out by employing a rinsing step after sample application. Using short-wavelength UV detection, reproducibility and linearity in the low nanomolar range were satisfactory. The capillary could be efficiently regenerated using a programmed between-run rinsing procedure, allowing 20-30 large injections of sample extracts. Coating of the capillary improved the robustness of the method. Mass spectrometric detection via a previously reported sheathless interface increased the selectivity and sensitivity substantially. Recommendations are provided for the sample preparation process, the most critical part of the system. Further purification of the sample is required to allow the loading of larger sample volumes and to optimize the system's robustness.     

C-terminal sequencing of peptides using electrospray ionization mass spectrometry.

A low-flow reactor is described for the on-line monitoring of peptides digested with carboxypeptidase P by electrospray ionization. Two peptides were analyzed using this technique: glucagon (average MW 3482.8 Da), and apomyoglobin (average MW 16,951.5). Both peptides gave interpretable results. The first 19 amino acids of glucagon were successfully sequenced. Apomyoglobin yielded sequence information to the 30th amino acid with some gaps. At 300 nL/min, 50% of the first 30 amino acids were sequenced and at 1 microL/min, 67% of the first 30 amino acids were observed.     

Characterization of dielectric barrier electrospray ionization for mass spectrometric detection

A novel electrospray interface is presented which induces an electric field by dielectric polarization through a non-conductive barrier. Therefore, a square-wave high-voltage signal is applied. This technique allows mass spectrometric measurements in the positive as well as in the negative mass spectrometry mode without changing the polarity of the potential applied, and it decreases the risk of undesired discharges, induced by high electric currents. The applicability of this technique is demonstrated by mass spectrometric determination of reserpine.     

Capillary electrophoresis-atmospheric pressure chemical ionization-mass spectrometry using an orthogonal interface: Set-up and system parameters

The feasibility of atmospheric pressure chemical ionization (APCI) as an alternative ionization technique for capillary electrophoresis-mass spectrometry (CE-MS) was investigated using a grounded sheath-flow CE-MS sprayer and an orthogonal APCI source. Infusion experiments indicated that highest analyte signals were achieved when the sprayer tip was in close vicinity of the vaporizer entrance. The APCI-MS set-up enabled detection of basic, neutral, and acidic compounds, whereas apolar and ionic compounds could not be detected. In the positive ion mode, analytes could be detected in the entire transfer voltage range (0–5 kV), whereas highest signal intensities were observed when the corona discharge current was between 1000 and 2000 nA. In the negative ion mode, the transfer voltage typically was 500 V and the optimum corona discharge current was 6000 nA. Analyte signals were raised with increasing nebulizing gas pressure, but the pressure was limited to 25 psi to avoid siphoning and current drops. Signal intensities appeared to be optimal and constant over a wide range of sheath liquid flow rate (5–25 μL/min) and vaporizer temperature (200–350 °C). APCI-MS signals were unaffected by the composition of the background electrolyte (BGE), even when it contained sodium phosphate and sodium dodecyl sulfate (SDS). Consequently, BGE composition, sheath-liquid flow rate, and vaporizer temperature can be optimized with respect to the CE separation without affecting the APCI-MS response. The analysis of a mixture of basic compounds and a steroid using volatile and nonvolatile BGEs further demonstrates the feasibility of CE-APCI-MS. Detection limits (S/N = 3) were 1. 6–10 μM injected concentrations.     

CHCA-modified Au nanoparticles for laser desorption ionization mass spectrometric analysis of peptides

Gold nanoparticles (AuNPs) have been studied as a potential solid-state matrix for laser desorption/ionization mass spectrometry (LDI-MS) but the efficiency in ionization remains low. In this report, AuNPs are capped by a self-assembled monolayer of cysteamine and modified with α-cyano-4-hydroxycinnanic acid (CHCA) for effective MALDI measurements. CHCA-terminated AuNPs offer marked improvement on peptide ionization compared with citrate-capped or cysteamine-capped AuNPs. The coating also effectively suppresses formation of Au cluster ions and analyte fragment ions, leading to cleaner mass spectra. Addition of glycerol and citric acid to the peptide/AuNPs sample further improves the performance of these AuNPs for LDI-MS analysis. Glycerol appears to enhance the dispersion of AuNPs in sample spots, increasing the sample ionization and shot-to-shot reproducibility, while citric acid serves as an external proton donor, providing high production of protonated analyte ions and reducing fragmentation of peptides on the nanoparticle-based surface. Optimal ratios of citric acid, glycerol, and AuNPs in sample solution have been systematically studied. A more than 10-fold increase for desorption ionization of peptides can be achieved by combining 5% glycerol and 20 mM citric acid with the CHCA-terminated AuNPs. The applicability of the CHCA-AuNPs for LDI-MS analysis of protein digests has also been demonstrated. This work shows the potential of AuNPs for SALDI-MS analysis, and the improvement with chemical functionalization, controlled dispersion, and use of an effective proton donor.     

Comparison of atmospheric pressure chemical ionization and electrospray ionization mass spectrometry for the detection of lignans from sesame seeds

In sesame seeds, high concentrations of lignans are present. When these lignans are fermented in the human colon, a range of structurally different lignans is formed. A good liquid chromatography/mass spectrometry (LC/MS) protocol for the analysis of lignans in complex mixtures is lacking. In order to develop such a protocol, electrospray ionization (ESI)-MS and atmospheric pressure chemical ionization (APCI)-MS, both in the positive and negative ionization mode, were compared. An extract from defatted sesame meal was analyzed by APCI-MS and ESI-MS, before and after deglucosylation. APCI-MS was found to be a more generic method than ESI-MS because lignans, especially sesamolin, sesamin and pinoresinol, were better detected by APCI-MS than by ESI-MS. Positive and negative ionization modes had to be combined in order to detect all lignans in a bacterial culture grown on aglyconic, acid-treated lignans from sesame oil and defatted sesame meal. Lignans with methylenedioxy-bridged furanofuran structures mostly lack phenolic hydroxyl groups and were, therefore, optimally detected in positive ionization mode. Dibenzylbutadiene lignans, which were formed during fermentation, carry hydroxyl groups and were better detected in negative ionization mode.     

Electrospray ionization tandem mass spectrometric and electron impact mass spectrometric characterization of mycosporine-like amino acids

Positive-ion mass spectral fragmentations of seven mycosporine-like amino acids (MAAs) are reported and discussed. The MAAs studied are small compounds composed of a cycloheximine ring substituted with amino acid or amino alcohol units. Techniques used include electron impact (EI) and electrospray ionization (ESI) with tandem mass spectrometry (MS/MS). ESI-MS/MS showed unusual small radical losses, generally resulting from the loss of a methyl group with the exception of shinorine and porphyra for which the initial losses were 30 and 44 Da, respectively. As expected from structural similarities, porphyra, shinorine and palythinol displayed similar fragmentation patterns, while palythenic acid and palythene fragmented in a similar manner. Overall, the ESI-MS/MS fragmentations at m/z <200 exhibited a distinctive pattern for all seven MAAs with characteristic ions at m/z 137, 168, 186, and 197 or 199. Several ions were observed for each of the MAAs analyzed, and together provide a useful and potentially diagnostic pattern for identification of MAAs and as an aid in structure elucidation of novel MAAs. For GC/EI-MS analysis, trimethylsilyl (TMS) derivatives were made. The EI-MS fragmentation patterns of TMS-MAAs showed many features typical of TMS-derivatized alpha-amines. The precursor TMS-MAA ion was not detected, but a [M-90](+ radical) ion was the highest-mass intense peak observed for palythine, palythinol and shinorine, while palythene gave a [M-116](+ radical) ion. Besides determining the number of acidic hydrogens, EI-MS of TMS-derivatized MAAs will aid in structure elucidation of novel MAAs.     

Generation of multiply charged peptides and proteins by radio frequency acoustic desorption and ionization for mass spectrometric detection

The design and implementation of a radio frequency acoustic desorption ionization (RADIO) source has been demonstrated for the analysis of multiply charged peptides and proteins. One μL aliquots of melittin, BNP-32, and ubiquitin (∼1 μg of analyte) were deposited onto a quartz crystal microbalance (QCM) electrode before radio frequency actuation for desorption. Continuous electrospray parallel to/above the sampling surface enabled the ionization of desorbed species. Detection by a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer confirmed the intact and dissociated species observed during MS and MS/MS experiments, respectively.     

Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography-electrospray ionization mass spectrometry and capillary electrophoresis-electrospray ionization mass spectrometry of proteins. II. Capillary electrophoresis.

Peptides and proteins were separated by capillary electrophoresis (CE) in fused-silica capillaries coated with an irreversibly adsorbed monolayer of derivatized polystyrene nanoparticles. Whereas phosphate buffer, pH 3.10, enabled the highly efficient separation of basic proteins with plate counts up to 1,400,000 m-1, volatile buffer components such as formic acid or acetic acid titrated with ammonia to the desired pH had to be used for the direct coupling of CE with electrospray ionization mass spectrometry (ESI-MS). Compared to 40 mM phosphoric acid-sodium hydroxide, pH 3.10, a background electrolyte containing 125 mM formic acid-ammonia, pH 4.00, was shown to yield equivalent separation efficiency. Investigation of the influence of buffered electrolytes on the ESI-MS signal of lysozyme at pH 2.70-4.00 showed that the charge state distribution shifted to lower charge states at higher pH with a concomitant five-fold decrease in signal intensity of the most abundant signal. The presence of trifluoroacetic acid in the background electrolyte greatly increased the level of baseline noise and completely inhibited the observation of any mass signals related to proteins. Full scan spectra could be acquired from 50-500 fmol amounts of proteins during coupled CE-ESI-MS utilizing 100-125 mM formic acid-ammonia, pH 3.10. However, compared to UV detection, considerable band broadening is observed with ESI-MS detection which is mainly attributed to column overloading, band spreading in the interface, and scanning data acquisition. Finally, the major whey proteins beta-lactoglobulin A, beta-lactoglobulin B, and alpha-lactalbumin were identified in a whey drink by comparison of molecular masses determined by CE-ESI-MS to molecular masses calculated from the amino acid sequence.     

Detection mass bias in atmospheric pressure ionization mass spectrometry

A previously uncharacterized source of detection mass bias is shown to be associated with atmospheric pressure ionization mass spectrometry (APIMS), and is attributed to a mass dependence in the sampling of ions from the supersonic free jet expansion of gas emerging from the ion source. The halide ions Cl ^?, Br^?, and I^? are shown to be transported from the ion source aperture to a quadrupole mass filter with efficiencies that increase linearly with increasing mass of the ion. While the polyatomic anions SF ₆ ^- and C₇F ₁₄ ^- are detected with even greater efficiencies than would be expected for monatomic anions of the same mass, this additional sensitivity to the polyatomic anions is thought to be related to ion loss processes occurring within the ion source. The experimental conditions under which these mass bias effects can be minimized or enhanced in APIMS are described.     

Design considerations for high speed quantitative mass spectrometry with MALDI ionization

A MALDI ion source on a triple quadrupole mass spectrometer constructed for the purpose of obtaining high speed quantitative measurements on drugs and other low molecular weight compounds is described. Particular attention is given to the ion generation and transport phenomena that affect analysis speed, throughput, and practical instrument robustness. In this regard parameters that affect desorption speed, beam spreading, ion flight times, sensitivity, signal-to-noise, ion fragmentation, sample carry-over, and instrument contamination are examined and experimental results are provided. MALDI and electrospray sensitivity is compared, to provide a practical frame of reference.