Identification of weak and strong organic acids in atmospheric aerosols by capillary electrophoresis/mass spectrometry and ultra-high-resolution fourier transform ion cyclotron resonance mass spectrometry

A novel approach using a combination of capillary electrophoresis/mass spectrometry (CE/MS) and off-line Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) revealed the structural details of acidic constituents of atmospheric organic aerosol. Both techniques utilized electrospray ionization (ESI), a soft ionization method, to facilitate the analysis of complex mixtures of organic compounds. CE/ESI-MS using an UltraTrol LN-precoated capillary and acidic background electrolytes at different pH values (2.5 and 4.7) was used to differentiate between weak (carboxylic) and strong (sulfonic) organic acids. On the basis of the electrophoretic mobility, m/z constraints from CE/ESI(-)-MS, and elemental composition information retrieved from off-line FTICR-MS, a variety of aliphatic and aromatic carboxylic acids (CHO-bearing molecules), nitrogen-containing carboxylic acids (CHON-bearing molecules), organosulfates (CHOS-bearing molecules), and (nitrooxy)organosulfates (CHONS-bearing molecules) were tentatively identified in the Oasis-HLB-extracted urban PM(2.5) (particulate matter with an aerodynamic diameter of <2.5 μm). The chemical known/unknown structures of detected compounds were confirmed by the semiempirical Offord model (effective mobility linearly correlated to Z/M(2/3)). The majorities of the identified compounds are products of atmospheric reactions and are known contributors to secondary organic aerosols.     

Chip-based capillary electrophoresis/mass spectrometry determination of carnitines in human urine

A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system is described for the CE separation and on-line electrospray detection of carnitine and selected acylcarnitines from mixtures of analytical standards as well as extracts of fortified human urine. Chip-based CE/MS experiments in two different laboratories were carried out using a triple-quadrupole mass spectrometer and a quadrupole time-of-flight (QTOF) mass spectrometer, respectively. The glass chips used with both systems were comparably equipped with a microfabricated capillary electrophoresis (CE) channel but with different electrosprayers. The quadrupole chip-based CE/MS experiments employed a miniature coupled microsprayer, which allowed coupling of the microelectrospray process via a micro liquid junction at the exit of the CE capillary channel. Selected ion monitoring (SIM) CE/MS experiments were employed for all of the quadrupole CE/MS work. The QTOF CE/MS full-scan single MS and MS/MS experiments were carried out in another laboratory using accurate mass measurement TOF mass spectrometry techniques. The electrospray process that was employed with the QTOF system differed in that an inserted nanoelectrospray capillary needle was carefully affixed into a flat-bottomed hole that was aligned with the CE channel exit orifice. SIM CE/MS using the described quadrupole system provided acceptable ion current electropherograms from fmole levels from analytical standard solutions of carnitine and acylcarnitines that were manually injected (loaded) onto the chip. In addition, the corresponding electropherograms for human urine fortified with the target carnitine and acylcarnitines at a 10-20 microg/mL (35-124 microM) level were obtained via SIM CE/MS techniques. The measured CE separation efficiency for the SIM CE/MS electropherograms was determined to be 2860 plates (peak width at half-height method or N = 5.54(T/WO.5(2)), and carnitine and three acylcarnitines were separated in less than 48 s. In contrast, using quadrupole-TOF technologies, the same samples could be diluted by a factor of 2-4 to obtain a comparable detector response for the target compounds. In the full-scan, single mass analyzer mode (m/z 150-500), the CE separation efficiency was measured to be 2600 plates, but mass measurement accuracy was less than 5.0 ppm for the quaternary cations. In the CE/MS/MS mode, full-scan collision-induced dissociation (CID) mass spectra were obtained with a mass accuracy of < or =10 ppm for the higher mass ions and < or =27 ppm for the lower mass product ions. These results demonstrate the feasibility for on-chip CE separation and electrospray mass spectrometric detection for these important compounds in synthetic mixtures, as well as in human urine extracts.     

On-column sample enrichment for capillary electrophoresis sheathless electrospray ionization mass spectrometry: evaluation for peptide analysis and protein identification

Although several designs have been advanced for coupling sample enrichment devices to a sheathless electrospray ionization-mass spectrometry (MS) interface on a capillary electrophoresis (CE) column, most of these approaches suffer from difficulties in fabrication, and the CE separation efficiency is degraded as a result of the presence of coupling sleeves. We have developed a design that offers significant improvements in terms of ease of fabrication, durability, and maintenance of the integrity of the CE-separated analyte zones. Capillaries with different inside and outside diameters were evaluated to optimize the performance of the CE-MS system, resulting in a mass limit of detection of 500 amol for tandem MS analysis of a standard peptide using a 20-microm-i.d. capillary. The improved design incorporates an efficient method to preconcentrate a sample directly within the CE capillary followed by its electrophoretic separation and detection using a true zero dead-volume sheathless CE-MS interface. Testing of this novel CE-MS system showed its ability to characterize proteomic samples such as protein digests, in-gel-digested proteins, and hydrophobic peptides as well as to quantitate ICAT-labeled peptides.     

Analysis of inorganic species by capillary electrophoresis-mass spectrometry and ion exchange chromatography-mass spectrometry using an ion spray source

Mixtures of inorganic ions separated by capillary electrophoresis (CE) and ion exchange chromatography (IC) are detected by mass spectrometry (MS) using an ion spray atmospheric pressure ionization source. The selectable degree of ion-adduct declustering and molecular fragmentation in the MS interface region allows the system to be operated as an elemental analyzer or as a molecular detector suitable for oxidation state determinations. Both inorganic anions and cations (including alkalis, alkaline earths, transition metals, and lanthanides) are analyzed by CE-MS. A variety of CE separation buffers are evaluated for the cation analyses (e.g., creatinine, ammonium acetate, and tris[hydroxymethyl]aminomethane). Only one of the buffers (i.e., creatinine) can be used for CE-indirect UV detection. A CE capillary permanently coated with strong anion exchange sites and a pyromellitic acid buffer (suitable for indirect UV detection) is used for the inorganic anion separations. The coated column eliminates the need for buffer modifiers to reverse the flow in the capillary, which then reduces background noise and mass spectral complexity. The separation and detection of 13 inorganic anions are also accomplished by IC using an anion exchange column with a carbonate-bicarbonate mobile phase, on-line suppressed conductivity detection, and mass spectrometric detection.     

CE/electrospray ionization-MS analysis of underivatized d/l-amino acids and several small neurotransmitters at attomole levels through the use of 18-crown-6-tetracarboxylic acid as a complexation reagent/background electrolyte

A new capillary electrophoresis/mass spectrometry technique is introduced for attomole detection of primary amines (including several neurotransmitters), amino acids, and their d/l enantiomers in one run through the use of a complexation reagent while using only approximately 1 nL of sample. The technique uses underivatized amino acids in conjunction with an underivatized capillary, which significantly reduces cost and analysis time. It was found that when (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18-C-6-TCA, MW 440) was used as the background electrolyte/complexation reagent during the capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of underivatized amino acids, stable complexes were formed between the amino acids and the 18-C-6-TCA molecules. These complexes, which exhibited high ionization efficiencies, were detectable at attomole levels for most amino acids. The detection limits of the AA/18-C-6-TCA complexes were on the average more than 2 orders of magnitude lower than that of the free amino acids in solution. In addition to lower detection limits under CE/ESI-MS, a solution of 18-C-6-TCA in the concentration range of 5-30 mM provided high separation efficiency for mixtures of l-amino acids as well as mixtures of d/l-amino acids. By using a solution of 18-C-6-TCA as the background electrolyte in conjunction with an underivatized, 130-cm-long, 20-microm-i.d., 150-microm-o.d. fused-silica capillary and by monitoring the m/z range of the amino acid/18-C-6-TCA complexes (m/z 515-700), most of the standard amino acids and many of their enantiomers were separated and detected with high separation efficiency and high sensitivity (nanomolar concentration detection limits) in one run. The solutions of 18-C-6-TCA also worked well as the CE/ESI-MS BGE for low-level detection of several neurotransmitters and some of their d/l enantiomers as well as for the analysis of amino acids at endogenous levels in lysed red blood cells.     

On-line capillary electrophoresis/microelectrospray ionization-tandem mass spectrometry using an ion trap storage/time-of-flight mass spectrometer with SWIFT technology.

The development of a system capable of the speed required for on-line capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) of tryptic digests is described. The ion trap storage/reflectron time-of-flight (IT/reTOF) mass spectrometer is used as a nonscanning detector for rapid CE separation, where the peptides are ionized on-line using electrospray ionization (ESI). The ESI produced ions are stored in the ion trap and dc pulse injected into the reTOF-MS at a rate sufficient to maintain the separation achieved by CE. Using methodology generated by software and hardware developed in our lab, we can produce SWIFT (Stored Waveform Inverse Fourier Transform) ion isolation and TICKLE activation/fragmentation voltage waveforms to generate MS/MS at a rate as high as 10 Hz so that the MS/MS spectra can be optimized on even a 1-2 s eluting peak. In CE separations performed on tryptic digests of dogfish myelin basic protein (MBP) where eluting peaks 4-8 s wide are observed, it is demonstrated that an acquisition rate of 4 Hz provides > 20 spectra/peak and is more than sufficient to provide optimized MS/MS spectra of each of the eluting peaks in the electropherogram. The detailed structural analysis of dogfish MBP including several posttranslational modifications using CE-MS and CE-MS/MS is demonstrated using this method with < 10 fmol of material consumed.     

Capillary electrophoresis-fourier transform ion cyclotron resonance mass spectrometry for the identification of cationic metabolites via a pH-mediated stacking-transient isotachophoretic method

Capillary electrophoresis-mass spectrometry (CE-MS) is still widely regarded as an emerging tool in the field of metabolomics and metabolite profiling. A major reason for this is a reported lack of sensitivity of CE-MS when compared to gas chromatography-mass spectrometry GC/MS and liquid chromatography-mass spectrometry. The problems caused by the lack of sensitivity are exacerbated when CE is coupled to Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), due to the relatively low data acquisition rate of FT-ICR MS. Here, we demonstrate the use of an online CE sample preconcentration method that uses a combination of pH-mediated stacking and transient isotachophoresis, coupled with FT-ICR MS to improve the overall detection of cationic metabolites in the bacterium Desulfovibrio vulgaris Hildenborough. This method showed a significant increase in signal-to-noise ratio when compared to CE normal sample stacking, while providing good separation efficiency, reproducibility, and linearity. Detection limits for selected amino acids were between 0.1 and 2 microM. Furthermore, FT-ICR MS detection consistently demonstrated good mass resolution and sub-ppm mass accuracy.     

Heart-cutting two-dimensional capillary electrophoresis for the on-line purification and separation of derivatized amino acids

Heart-cutting two-dimensional (2D) capillary electrophoresis (CE) in a single capillary was used for analysis of derivatized amino acids. A mixture of 12 amino acids derivatized with UV-active benzyl 4-(3-(2-chloroethyl)-3-nitrosoureido)butylcarbamate label served as a model of a moderately complex sample due to the presence of numerous derivatization byproducts. The first step of the heart-cutting 2D approach was sample cleanup by capillary zone electrophoresis (CZE) in borate electrolyte. Then, only a selected portion of the first-dimension separation was transferred into the second dimension of the separation by a specific voltage and pressure program. Finally, the zone of derivatized amino acids was separated by micellar electrokinetic chromatography in a borate-sodium dodecyl sulfate system. The whole 2D process can be performed in a conventional CE analyzer without any interface for connection of the two separation modes. Intraday repeatability of the total migration time was 2%. In general, the heart-cutting 2D-CE methodology in a single capillary can be adapted for any CE mode regardless of the direction and velocity of electroosmotic flow and position of the fraction of interest in the first dimension (i.e., first, last, or intermediate fraction).     

Condensation nucleation light scattering detection for capillary electrophoresis

We describe two means for interfacing condensation nucleation light scattering detection to capillary electrophoresis (CE). With the first method, a fused-silica capillary was used for the separation and the CE was grounded through a Nafion membrane that also connected the system to a microconcentric pneumatic nebulizer. Limits of detection (LODs) for underivatized amino acids were at the low microgram per milliliter level, and separation efficiencies were ～9 times lower than the optimum predicted for these species based on the injection plug width and axial dispersion by diffusion. LODs were limited by background nonvolatiles resulting from dissolution of fused silica at the high pHs used for the separations. An alternate system employed PEEK capillaries which acted as the separation capillary and also as the inner nebulizer capillary. In this case, the exit end of the capillary was coated with conductive paint which extended to the tip of the nebulizer, was in contact with the CE buffer, and was grounded to complete the CE circuit. Response was nonlinear and the separation efficiency of this system was somewhat lower than that for the Nafion membrane system. Response as peak heights for all of the amino acids and peptides studied was nearly identical on a mass basis. With this system, much lower background signals were obtained, and as a result, LODs for underivatized amino acids and peptides were below the 1 μg/mL level, corresponding to less than 10 pg or less than 100 fmol injected. Both systems were fairly simple, effective means to generate aerosols with the low flows of CE and should be applicable to interfacing of other aerosol-based detectors with CE.     

Enhanced neuropeptide profiling via capillary electrophoresis off-line coupled with MALDI FTMS

An off-line interface incorporating sheathless flow and counter-flow balance is developed to couple capillary electrophoresis (CE) to matrix-assisted laser desorption ionization Fourier transform mass spectrometry (MALDI FTMS) for neuropeptide analysis of complex tissue samples. The new interface provides excellent performance due to the integration of three aspects: (1) A porous polymer joint constructed near the capillary outlet for the electrical circuit completion has simplified the CE interface by eliminating a coaxial sheath liquid and enables independent optimization of separation and deposition. (2) The electroosmotic flow at reversed polarity (negative) mode CE is balanced and reversed by a pressure-initiated capillary siphoning (PICS) phenomenon, which offers improved CE resolution and simultaneously generates a low flow (<100 nL/min) for fraction collection. (3) The predeposited nanoliter volume 2,5-dihydroxybenzoic acid (DHB) spots on a Parafilm-coated MALDI sample plate offers an improved substrate for effective effluent enrichment. Compared with direct MALDI MS analysis, CE separation followed by MALDI MS detection consumes nearly 10-fold less sample (50 nL) while exhibiting 5-10-fold enhancement in S/N ratio that yields the limit of detection down to 1.5 nM, or 75 attomoles. This improvement in sensitivity allows 230 peaks detected in crude extracts from only a few pooled neuronal tissues and increases the number of identified peptides from 19 to 43 (Cancer borealis pericardial organs (n = 4)) in a single analysis. In addition, via the characteristic migration behaviors in CE, some specific structural and chemical information of the neuropeptides such as post-translational modifications and family variations has been visualized, making the off-line CE-MALDI MS a promising strategy for enhanced neuropeptidomic profiling.     

Analysis of underivatized amino acids and their D/L-enantiomers by sheathless capillary electrophoresis/electrospray ionization mass spectrometry

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of underivatized amino acids and the separation of their D/L-enantiomers. Under full-scan mode, all standard protein amino acids were separated and detected at low-femtomole levels using a 130-cm-long, 20-microm-i.d., 150-microm-o.d. underivatized fused-silica capillary with 1 M formic acid as the background electrolyte. The CE/ESI-MS technique was also applied to the separation of L-arginine from L-canavanine (a close analogue of arginine where the terminal methylene linked to the guanidine group of arginine is replaced by an oxygen atom) in a complex mixture containing all standard protein amino acids. The utility of CE/ESI-MS in the analysis of real-world samples was demonstrated by the identification of two metabolic diseases (PKU and tyrosinemia) through blood analysis with minimal sample preparation. In addition, the on-line separation of 11 underivatized L-amino acids from their D-enantiomers was achieved by using a 30 mM solution of (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as the background electrolyte.     

Rugged gap reactor device for postcolumn fluorescence detection in capillary electrophoresis

In this paper, the construction and performance of a rugged device for postcolumn derivatization in capillary electrophoresis (CE) are described. The device was based on a gap design, and a gap with a very small distance (<3 μm, estimated under microscope) could be easily constructed without micromanipulation. Addition of derivatizing reagents into the reaction capillary was attributable to gravity flow. The concentration of derivatizing reagents can be controlled through manipulating the electroosmotic flow in the reaction capillary and the height of the liquid levels from the derivatizing reagents to the buffer reservoirs. The device has been applied in fluorescence detection of amino acids using a mixture of o-phthaldialdehyde/2-mercaptoethanol as derivatizing reagent. Theoretical plate numbers for 11 amino acids separated in a pH 9.5 borate buffer were obtained in the order of 40?000-250?000. The detection limit for glycine (S/N = 2) was found to be 6.7 × 10(-)(7) mol/L using a commercial HPLC fluorescence detector modified for CE. Free amino acids in a wine sample were also determined. Because the device is quite stable, we believe that it can be used routinely in analytical laboratories.     

Electrogenerated chemiluminescence detection of amino acids based on precolumn derivatization coupled with capillary electrophoresis separation

A novel method for highly sensitive detection of primary and secondary amino acids with selective derivatization using acetaldehyde as a new derivatization reagent was proposed by capillary electrophoresis (CE) coupled with electrogenerated chemiluminescence (ECL) of tris(2,2'-bipyridine)ruthenium(II). The precolumn derivatization of these amino acids with acetaldehyde was performed in aqueous solution at room temperature for 1 h. Upon optimized derivatization, the ECL intensities and detection sensitivities of the amino acids were significantly enhanced by 20-70 times. Using four amino acids, arginine, proline, valine, and leucine, as model compounds, their derivatives could be completely separated by CE and sensitively detected by ECL within 22 min. The linear ranges were 0.5-100 microM for arginine and proline and 5-1000 microM for valine and leucine with the detection limits of 1 x 10(-7) (0.5 fmol, arginine), 8 x 10(-8) (0.4 fmol, proline), 1 x 10(-6) (5 fmol, valine), and 1.6 x 10(-6) M (8 fmol, leucine) at a signal-to-noise ratio of 3. The derivatization reactions and ECL process of amino acids were also proposed based on in situ Fourier transform infrared and ultraviolet spectrometric analyses.     

Coupling capillary electrophoresis and high-field asymmetric waveform ion mobility spectrometry mass spectrometry for the analysis of complex lipopolysaccharides

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is a new technology for atmospheric pressure, room temperature separation of gas-phase ions. The FAIMS system acts as an ion filter that can continuously transmit one type of ion, independent of mass-to-charge ratio (m/z). Capillary electrophoresis-electrospray mass spectrometry (CE-MS) has been extensively used for the analysis of complex bacterial lipopolysaccharides (LPS). The coupling of FAIMS to CE-MS provides a sensitive technique for the characterization of these complex glycolipids, permitting the separation of trace-level LPS oligosaccharide glycoforms for subsequent structural characterization using tandem mass spectrometry. This was demonstrated for LPS from nontypeable Haemophilus influenzae strain 375 following O-deacylation with anhydrous hydrazine. This strain of H. influenzae can express a triheptosyl-containing glycoform to which four hexose residues are linked forming the outer-core region of the molecule. This has been referred to as the Hex4 glycoform. Glycoforms have been identified which differ in the number of phosphoethanolamine substituents in the inner-core. With the use of CE-FAIMS, isomeric Hex4 glycoforms containing two PEtn groups were separated and characterized by MS/MS. FAIMS provided a significant reduction in mass spectral noise, leading to improved detection limits ( approximately 70 amol of the major glycoform). The extracted mass spectrum showed that the apparent noise was virtually eliminated. In addition to the reduction of chemical background, the ion current was increased by as much as 7.5 times as a result of the atmospheric pressure ion-focusing effect provided by the FAIMS system. The linearity of response of the CE-FAIMS-MS system was also studied. The calibration curve is linear for approximately 3 orders of magnitude, over a range of 40 pg/microL to 10 ng/microL.     

Nonaqueous capillary electrophoresis-mass spectrometry of synthetic polymers

In this work, the separation and characterization of ionizable organic polymers nonsoluble in water is carried out using nonaqueous capillary electrophoresis-ion trap mass spectrometry (NACE-MS). The polymers studied are poly(N(epsilon)-trifluoroacetyl-l-lysine) (poly(TFA-Lys)) obtained by ring-opening polymerization of the corresponding N-carboxyanhydride. Different parameters (i.e., liquid sheath nature and flow rate, electrospray temperature, and separation buffer composition) are optimized in order to obtain both an adequate CE separation and a high MS signal of the samples under study. The optimum NACE-MS separation conditions allow the molecular mass characterization of poly(TFA-Lys) up to a degree of polymerization of 38. NACE-MS provides interesting information on the chemical structure of (i). the polymer end groups and (ii). other final byproducts. The MS spectra obtained by using this CE-MS protocol confirm that the polymerization was initiated by the reaction of n-hexylamine (initiator) on the monomer. CE-MS-MS and CE-MS-MS-MS results demonstrate that two different termination reactions occurred during the polymerization process leading to the transformation of the reactive amine end group into a carboxylic or a formyl groups. Byproducts such as 3-hydantoinacetic acid or diketopiperazine were also detected. To our knowledge, this is the first work in which the great possibilities of NACE-MS and NACE-MS(n) for characterizing synthetic polymers are demonstrated.     

Mass transport in a micro flow-through vial of a junction-at-the-tip capillary electrophoresis-mass spectrometry interface

When coupling capillary electrophoresis with postcolumn detection methods, such as mass spectrometry, the presence of postcolumn band broadening must be considered. The band broadening effects introduced by junction-at-the-tip CE-MS interfaces using a postcolumn micro flow-through vial are investigated by studying the hydrodynamic flow patterns and mass transport process inside the micro vial at the end of the CE separation capillary. Simulation results obtained by solving the Navier-Stokes and mass balance equations provide insights into the velocity field and concentration distribution of the analytes in the micro vial and demonstrate that, with a low flow rate of chemical modifier solution, the laminar flow streams confine the analyte molecules to the central part of the micro vial and thus maintain major features of the peak shapes. Peaks detected by UV and MS under similar experimental conditions were compared to verify the numerical prediction that the main features of the UV peak can be retained in the MS peak. Experiments also show that band broadening can be minimized when an appropriate chemical modifier flow rate is selected.     

Metabolic differentiation of neuronal phenotypes by single-cell capillary electrophoresis-electrospray ionization-mass spectrometry

Single-cell mass spectrometry (MS) is a rapidly emerging field in metabolic investigations. The inherent chemical complexity of most biological samples poses analytical challenges when using MS platforms to measure sample content without prior chemical separation. Here, a single-cell capillary electrophoresis (CE) system was coupled with electrospray ionization (ESI) MS to enable the simultaneous measurement of a vast array of endogenous compounds in over 50 identified and isolated large neurons from the Aplysia californica central nervous system. More than 300 distinct ion signals (m/z values) were detected from a single neuron in the positive ion mode, 140 of which were selected for chemometric data analysis. Metabolic features were evaluated among six different neuron types (B1, B2, left pleural 1 (LPl1), metacerebral cell (MCC), R2, and R15) chosen for their various physiological functions. The results indicated chemical similarities among some neuron types (B1 to B2 and LPl1 to R2) and distinctive features for others (MCC and R15 cells). The quantitative nature of the MS platform allowed the comparison of metabolite levels for specific neurons. The CE-ESI-MS approach for examination of individual nanoliter-volume cells as described herein is readily adaptable to other volume-limited samples.     

Studies of histidine as a suitable isoelectric buffer for tryptic digestion and isoelectric trapping fractionation followed by capillary electrophoresis-mass spectrometry for proteomic analysis

The use of histidine as a protein digestion buffer followed by isoelectric trapping separations using "membrane separated wells for isoelectric focusing and trapping" (MSWIFT) and mass spectrometry (MS) analysis is described. Tryptic digestion of bovine serum albumin (BSA) performed in histidine buffered solutions yields similar amino acid sequence coverage values to those obtained using ammonium bicarbonate buffer. Time course studies suggest that histidine buffers provide faster migration of peptides from the loading compartment compared to digestions prepared in ammonium bicarbonate due to differences in conductivities of the two buffers. In addition, this sample preparation method and MSWIFT separations have been coupled with capillary electrophoresis (CE) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) as an alternative separation approach for proteomic studies. Tryptic peptides of ribosomal proteins in histidine are fractionated using MSWIFT followed by CE-MALDI-MS, which further illustrates the ability to couple fractions from a pI based separation device to CE-MS. Specifically, two-dimensional CE-MS plots provide a direct correlation between the numbers of basic residues within the peptide sequence displayed in charge-state trend lines. Combining MSWIFT and CE-MS provides added information regarding peptide sequence, specifically pI and in-solution charge state. Post-translational modifications can also be identified using this method.     

Mechanism of signal suppression by anionic surfactants in capillary electrophoresis-electrospray ionization mass spectrometry

Micellar-mediated capillary electrophoresis (CE) is used for a wide variety of applications, including the separation of pharmaceuticals, environmental contaminants, illicit drugs, DNA fragments, and many other biological samples. The electrospray ionization interface is one of the most common CE-MS interfaces. Coupling micellar-mediated CE separations with MS detection combines two very powerful, widely applicable analytical techniques. Some types of surfactants strongly interfere with electrospray ionization mass spectrometric (ESI-MS) detection of analytes, and in many cases the ESI-MS analyte signals are completely quenched. Only a few reports have appeared that describe the ESI-MS detection of analytes in the presence of surfactants; however, the exact mechanism of ionization suppression has not yet been addressed. In this work, a modified aerosol ionic redistribution (AIR) model is presented that qualitatively explains the results of previous studies, including those using "polymeric surfactants". Analyte ionization suppression by surfactants appears to be caused by Coulombic interaction between oppositely charged solute and surfactant ions in the ESI-produced offspring droplets. It appears that the ability of surfactants to quench electrospray ionization is directly related to the surface activity and the charge of the surfactant. Also, highly surface active components tend to be enriched in ESI-produced offspring droplets. Analyte ion signals can be detected under conditions that lower the surface concentration of oppositely charged surfactant ions in aerosol droplets. The mechanistic information outlined here may be used to design micellar-mediated CE separations that allow detection of analyte ions by ESI-MS.     

Chip-based quantitative capillary electrophoresis/mass spectrometry determination of drugs in human plasma

A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system is described for the on-chip separation and coupled electrospray detection of selected small drug molecule compounds. These studies include the quantitative determination of carnitine and acetylcarnitine in analytical standard solutions as well as imipramine and desipramine in fortified human plasma samples. A clinical human plasma sample was also analyzed following the normal administration of desipramine to a volunteer, and the parent drug was determined using the described chipbased CE/MS technique. In each instance, stable isotope-incorporated internal standards were used. The chip-based CE system was microfabricated from glass and coupled to a micro ion spray device constructed in-house. The atmospheric pressure ionization system employed in this work was a PE Sciex API III tandem triple quadrupole system operated in the selected ion monitoring (SIM) mode. The results from the work reported here demonstrate the feasibility for carrying out rapid (30 s) chipbased quantitative CE/MS determinations of samples containing small-molecule compounds. Using SIM CE/ MS techniques, the described API III quadrupole system provided acceptable ion current electropherograms from subpicomole levels of the targeted compounds loaded onto the chip. The corresponding electropherograms for the standard solution of carnitines at the 1-500 microg/mL level were obtained via SIM CE/MS techniques (R2 > 0.99). In addition, analyses of fortified samples of imipramine desipramine were measured relative to their corresponding d3 internal standards to obtain calibration curves ranging from 5 to 500 microg/mL in human plasma (R2 > 0.99). The intra-assay precision ranged from 4.1 to 7.3% RSD. The intra-assay accuracy ranged from 94.0 to 104%. These results demonstrate the feasibility for on-chip CE separation and electrospray mass spectrometric determination in applications for bioanalytical measurements for these important compounds in synthetic mixtures and human plasma extracts.