Nonaqueous capillary electrophoretic separation and thermo-optical absorbance detection of five tricyclic antidepressants and metabolism of amitriptyline by Cunninghamella elegans

We developed a technique based on nonaqueous capillary electrophoresis and laser-based thermo-optical absorbance detection to assay five antidepressants with similar structures and mass-to-charge ratios. A mixture of methanol and acetonitrile with ammonium acetate was essential to achieve baseline resolution of these compounds. We investigated the effects of ammonium acetate concentration, temperature, applied voltage, and capillary length on separation efficiency. The nonaqueous capillary electrophoresis and laser-based thermo-optical absorbance detection technique was used to study the metabolism of amitriptyline by Cunninghamella elegans. Sample preparation procedures were simplified for fast screening of the parent drug and its metabolites. Reproducible electropherograms were obtained from replicate cultures of C. elegans growing in the presence of amitriptyline.     

Unusual form of recurrent giant cell granuloma of the mandible and lower extremities in a patient with neurofibromatosis type 1

The high resolution of capillary zone electrophoresis/mass spectrometry (CZE/MS) offers a promising technique to characterize biomolecules in pharmaceutical and biotechnology industries. A novel capillary zone electrophoresis/electrospray ionization time-of-flight mass spectrometry (CZE/ESI-TOFMS) interface was designed in this study to successfully integrate ESI-TOFMS, nanospray, and CZE for biomolecular identification. The interface offers a novel way to take advantage of the high resolution separation achieved during CZE and the detection sensitivity of nanospray ESI-MS. The results showed mixtures of peptides were highly resolved within a few minutes (each CZE electropherogram of a peptide is 2-3 seconds). The novel CZE/ESI-TOFMS interface may simultaneously provide sensitivity, data acquisition speed, mass range, and mass resolution while maintaining resolution of the CZE separation.     

Analysis of peptides, proteins, protein digests, and whole human blood by capillary electrophoresis/electrospray ionization-mass spectrometry using an in-capillary electrode sheathless interface

An in-capillary electrode sheathless interface was applied to the capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) analysis of mixtures of small peptides, proteins, and tryptic digests of proteins. The effects of different experimental parameters on the performance of this CE/ESI-MS interface were studied. The distance of the in-capillary electrode from the CE outlet and the length of the electrode inside the capillary had no significant effects on the CE separation and ESI behavior under the experimental conditions used. However, significant enhancement of the sensitivity resulted from the use of narrower CE capillaries. Using a quadrupole mass spectrometer, an aminopropylsilane-coated capillary, and a wide scan mass-to-charge ratio range of 500-1400, detection limits of approximately 4, 1, and 0.6 fmol for cytochrome c and myoglobin were achieved for 75-, 50-, and 30-micron inner diameter capillaries, respectively. Approximately one order of magnitude lower detection limits were achieved under the multiple-ion monitoring mode. The application of the in-capillary electrode sheathless interface to real-world samples was demonstrated by CE/ESI-MS analysis of a human blood sample.     

Nonaqueous capillary electrophoresis--its applicability in the analysis of food, pharmaceuticals and biological fluids

The use of nonaqueous electrophoresis media for the application of capillary electrophoresis in the analysis of food, pharmaceuticals and biological fluids is reviewed. Some of the applications are discussed in detail and the benefits of using nonaqueous media in these cases are outlined. Three new applications within pharmaceutical analyses are presented. In these methods either a simple sample pretreatment by dilution with methanol (determination of chlorhexidine in a cream) or selective on-line capillary electrophoresis mass spectrometry (methods for identification of seizure drugs or opium alkaloids) are used. The choice of organic solvents and electrolytes for nonaqueous capillary electrophoresis are discussed. Furthermore, validation data obtained using capillary electrophoresis based on the nonaqueous principle are listed and discussed.     

Application of capillary electrophoresis and related techniques to drug metabolism studies

The use of capillary electrophoresis (CE) for the separation of small organic molecules such as pharmaceutical agents and drug/xenobiotic metabolites has become increasingly popular. This has arisen, at least in part, from the complimentary mode of separation afforded by CE when compared to the more mature technique of HPLC. Other qualities of CE include relative ease of method of development, rapid analysis, and low solvent consumption. The recent introduction of a variety of detector systems (including UV diode array, laser-induced fluorescence, conductivity) and the demonstrated coupling of CE to MS have also aided acceptance of this technology. In the present report, we review the role of CE coupled to various detector systems including a mass spectrometer for the characterization of both in vitro and in vivo derived drug metabolite mixtures. Attributes of CE for this application are demonstrated by discussion of metabolism studies of the neuroleptic agent haloperidol. Various aspects of the development and use of CE and CE-MS for the characterization of haloperidol metabolites, including criteria for selection of parameters such as pH, ionic strength, extent of organic modification, and the use of nonaqueous capillary zone electrophoresis are discussed. We also consider potential limitations of CE and CE-MS for drug metabolism research and describe the introduction of membrane preconcentration-CE (mPC-CE) and mPC-CE-MS as a solution that overcomes the rather poor concentration limits of detection of CE methods without compromising the resolution of analytes or separation efficiency of this technique.     

Capillary liquid chromatography/electrospray mass spectrometry for the separation and detection of catechins in green tea and human plasma

The separation and detection of biologically active green tea catechins has been accomplished using capillary liquid chromatography/electrospray mass spectrometry (cLC/ESI-MS). Microscale determination (approximately 20 ng) of all six catechins in a green tea infusion, and the most extensively studied catechin, (-)epigallocatechin gallate (EGCG), in human plasma is demonstrated by cLC/ESI-MS with selected ion monitoring of protonated molecular ions. The overall quality of the analysis is shown to be dependent on the use of a capillary column with a deactivated, monomeric C18 stationary phase. The high chromatographic separation efficiency of this packed-capillary column, combined with the high sensitivity and selectivity afforded by the mass spectrometer as detector, provide a reliable approach to the analysis of picomolar quantities of these interesting compounds in complex matrices.     

Capillary electrophoresis, nuclear magnetic resonance and mass spectrometry studies of opposite chiral recognition of chlorpheniramine enantiomers with various cyclodextrins

Markedly different chiral separation abilities were observed for native beta-cyclodextrin (beta-CD), carboxymethyl-beta-CD (CM-beta-CD) and heptakis (2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD) towards the enantiomers of (+/-)-chlorpheniramine ((+/-)-CHL) in capillary electrophoresis (CE). Native beta-CD afforded almost baseline enantioseparation at a concentration of 18 mg/mL, whereas only 1 mg/mL solution of CM-beta-CD was required for adequate enantioseparation. TM-beta-CD allowed the nearly baseline enantioseparation only at a concentration as high as 80 mg/mL. Moreover, the migration order of (+/-)-CHL in the presence of TM-beta-CD was opposite to that with beta-CD and CM-beta-CD. 1H and 13C-NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) have been used in order to obtain preliminary information about the stoichiometry and the binding constants in the intermolecular diastereomeric complexes of (+/-)-CHL with these CDs.     

Protein identification by capillary zone electrophoresis/microelectrospray ionization-tandem mass spectrometry at the subfemtomole level

A method for the identification of proteins by their amino acid sequence at the low-femtomole to subfemtomole sensitivity level is described. It is based on an integrated system consisting of a capillary zone electrophoresis (CZE) instrument coupled to an electrospray ionization triple- quadrupole tandem mass spectrometer (ESI-MS/MS) via a microspray interface. The method consists of proteolytic fragmentation of a protein, peptide separation by CZE, analysis of separated peptides by ESI-MS/MS, and identification of the protein by correlation of the collision-induced dissociation (CID) patterns of selected peptides with the CID patterns predicted from all the isobaric peptides in a sequence database. Using standard peptides applied to a 20-microns-i.d. capillary, we demonstrate an ESI-MS limit of detection of less than 300 amol and CID spectra suitable for searching sequence databases obtained with 600 amol of sample applied to the capillary. Successful protein identification by the method was demonstrated by applying 50 and 38 fmol of a tryptic digest of the proteins beta-lactoglobulin and bovine serum albumin, respectively, to the system.     

Structural characterization of mono- and dihydroxylated metabolites of paclitaxel in rat bile using liquid chromatography/ion spray tandem mass spectrometry

The capability of high performance liquid chromatography/ion spray mass spectrometry (HPLC/ISP-MS) and HPLC/ISP-tandem mass spectrometry (HPLC/ISP-MS/MS) were investigated to achieve mass separation as well as structural characterization of taxol metabolites directly in rat bile, without their previous isolation. HPLC/ISP-MS yielded information on the molecular weights of several hydroxylated derivatives while HPLC/ISP-MS/MS allowed the on-line structural characterization of all metabolites present in different ratios in rat bile. The approach led to the extraction of nine metabolites and their distinction from the other endogenous contaminants. These metabolites were recognized as three dihydroxytaxols, four monohydroxytaxols, one deacetyltaxol and one containing the taxane ring. Among the derivatives, we were able to identify four new metabolites of paclitaxel belonging to the dihydroxy and monohydroxy series, never previously detected. HPLC/ISP-MS/MS enabled the classification of all di- and monohydroxy isomers. These results demonstrate that the high sensitivity of this method, based on the combined use of tandem mass spectrometry with chromatographic separation, can be considered as offering a valid approach to the detection of new taxol derivatives directly in biological fluids.     

Capillary electrophoresis/electrospray ionization high mass accuracy time-of-flight mass spectrometry for protein identification using peptide mapping

Capillary electrophoresis/electrospray ionization (CE/ESI) high mass accuracy time-of-flight mass spectrometry was used for the first time to characterize small proteins using peptide mapping. To identify small proteins, the intact proteins were first analyzed to obtain their average molecular weights with errors less than 1 Da. On-line capillary electrophoresis mass spectrometry of the tryptic digests of these small proteins was then performed to obtain the accurate molecular weights of the peptides with accuracies of approximately 10 ppm. Next, this information was used for the identification of the proteins using a protein database. It was found that high mass accuracy is an effective tool in reducing the list of most-likely proteins generated by the database. In addition, on-line collision-induced dissociation of the completely or partially resolved capillary electrophoresis peaks of the protein digests was used to unambiguously identify the sequences of these peptides. Each CE/ESI-MS analysis used only 5 nL of sample containing approximately 120 fmol of each peptide in protein digests. The results indicate that the combination of capillary electrophoresis and high resolution, high mass accuracy time-of-flight mass spectrometry is a viable option for the identification of small proteins using peptide mapping.     

Nonaqueous capillary zone electrophoresis for separation of free fatty acids with indirect fluorescence detection

The feasibility of combining nonaqueous capillary zone electrophoresis with indirect fluorescence detection was studied for the efficient separation and sensitive detection of ionizable hydrophobic substances which do not possess practically suitable detection properties. Medium- and long-chain free fatty acids, C6-C24, were selected as test compounds. The results showed that such a wide range of medium- and long-chain free fatty acids could be separated between any two consecutive homologs in one run and be detected at a level of about 0.01-0.02 mM in highly basic methanol/acetonitrile media containing fluorescein as coion of background electrolyte for indirect fluorescence detection.     

Capillary electrophoretic separation of tricyclic antidepressants using charged carboxymethyl-beta-cyclodextrin as a buffer additive

Charged carboxymethyl-beta-cyclodextrin was successful in the capillary electrophoretic separation of a series of tricyclic antidepressants. The cyclodextrin alone was successful in the separation of carbamazepine, protriptyline, desipramine, clomipramine, and opipramol using a 3-(trimethoxysilyl)propyl methacrylate capillary coating to reduce the electroosmotic flow. The ideal buffer pH was found to be in the range of 6-7 and the ideal cyclodextrin concentration to be 10 mM. All nine antidepressants were resolved using the charged cyclodextrin in the micellar electrokinetic chromatography (MEKC) mode with sodium dodecyl sulfate as the surfactant. Neither the cyclodextrin nor the surfactant alone were successful in resolving the whole series of compounds under investigation but a combination of both produced the separation. Separations were performed on a linear polyacrylamide coated capillary. The ideal pH of the buffer was in the range of 5-7.     

A novel buffer system for separation of metal cations by capillary electrophoresis with indirect UV detection

Generally, the buffers used for metal ion separations in capillary electrophoresis (CE) consist of a UV-active substance, pH-adjuster, and weak complexing reagent. This paper describes the successful separation of metal ions with a new buffer that contains no complexing reagent. Of several weakly basic compounds tested, 2-aminopyridine was selected as the most useful UV-active substance. It was used at a concentration of 15 mM with pH adjusted to 5.0 +/- 0.1 by acetic acid. The degree of protonation of the UV-active substance played an important role in detection. The stacking phenomenon was a significant contributor to efficiency in this buffer system, and water-diluted samples gave especially high efficiencies. When a 75-micron-i.d. fused-silica capillary was used, a separation efficiency of 1.8 x 10(5) was observed. Quantitative determinations of Ca2+, Mn2+, Zn2+, and Cd2+ were achieved with good linear calibration curves over the range of concentration from a few milligrams per liter to 100 mg/L. The detection limits were 0.2 mg/L for Ca2+, 0.4 mg/L for Mn2+ and Zn2+, and 0.6 mg/L for Cd2+, based on three times the baseline noise.     

Protein identification by solid phase microextraction-capillary zone electrophoresis-microelectrospray-tandem mass spectrometry

We describe an analytical system for the rapid identification of proteins by correlation of tandem mass spectra with protein sequence databases. The system consists of an integrated solid phase microextraction/capillary zone electrophoresis peptide separation device that is connected through a microelectrospray ion source to a tandem mass spectrometer. The limits of detection are 660 amol of sample at a concentration limit of < 33 amol/microliters for peptide mass measurement, and < 10 fmol of sample, at a concentration limit of < 300 amol/microliters for peptide analysis by collision-induced dissociation. Using this system, we have identified low nanogram amounts of yeast proteins separated by high-resolution two-dimensional gel electrophoresis.     

Development of a three-dimensional topographic map display for capillary electrophoresis/mass spectrometry with an ion trap/reflectron time-of-flight mass spectrometer detector: applications to tryptic digests of isoforms of myelin basic protein

A three-dimensional (3-D) contour map format has been developed to display the large amount of data continuously collected throughout an on-line capillary separation using an ion trap storage/reflectron time-of-flight detector (IT/reTOF). The resulting data are displayed on a single computer screen with a mass-to-charge ratio value-elution time-intensity representation. The intensity of various components is represented by 16 different colors so that the mass-to-charge ratio value, the elution time, and the intensity can be conveniently determined for each component. In addition, the mass spectrum and total ion chromatogram or total ion electropherogram (TIE) are shown on the same screen as the 3-D map that enables the correlation of a single spot in the 3-D map to the peaks in the TIE and the corresponding mass spectrum. The 3-D map has been used to identify various posttranslational modification sites of bovine myelin basic protein charge isomers, where the datafiles of tryptic digests of proteins analyzed by capillary electrophoresis/mass spectrometry were processed by this software and a comparison could be performed among the isoforms. The feature of in-screen integration over both the separation domain and the mass domain makes the acquisition of the selected ion chromatogram very convenient and greatly improves the ability to detect modified components present in low amounts.     

Direct evidence of methylglucose lipopolysaccharides/palmitoyl-CoA noncovalent complexes by capillary zone electrophoresis-electrospray/mass spectrometry

Mycobacterial methylglucose lipopolysaccharides (MGLPs) play an important regulatory role in the biosynthesis of long-chain fatty acids by forming complexes with neosynthesized acyl-CoA fatty acid derivatives. The MGLPs from Mycobacterium smegmatis were purified by high-performance anion-exchange chromatography and characterized by LSIMS and CE/ESI-MS. We investigated their interaction with palmitoyl-CoA using capillary zone electrophoresis with both direct and indirect UV detection. In the latter mode, the signal of the UV-transparent MGLPs decreased upon addition of increasing amounts of palmitoyl-CoA; while using direct UV detection, the addition of palmitoyl-CoA to the MGLPs revealed characteristic profiles. The major peak was assigned to the noncovalent MGLP/palmitoyl-CoA complex on the basis of its electrophoretic mobility. The abundance of the complex was found to increase until the MGLP/palmitoyl-CoA molar ratio reached a 1/1 stoichiometry. The existence of and the stoichiometry of this complex were assessed by CE/ESI mass spectrum analysis, showing pseudomolecular ions of the MGLP/palmitoyl-CoA complex. These results confirm that CE/ESI-MS is a powerful tool to characterize noncovalent molecular association.     

Capillary electrochromatography of biomolecules with on-line electrospray ionization and time-of-flight mass spectrometry

Capillary electrochromatography (CEC) is considered a hybrid of liquid chromatography and capillary electrophoresis. It is expected to combine the high peak efficiency of capillary zone electrophoresis with the versatility and loading capacity of HPLC to bring about another high-performance MS-compatible chromatographic system. This paper explores the potential of CEC coupled with the electrospray ionization and time-of-flight mass spectrometry in biochemical analysis. The packed columns used in this study were tapered at the outlet to retain the packing material, thereby obviating the need for an outlet frit. Electrosmotically driven solvent gradients were employed for the separation of phenylthiohydantoin (PTH)-amino acids by reversed-phase chromatography, and a time-of-flight (TOF) mass spectrometer was employed as the detector for the CEC column effluent. The effect of CEC operating parameters, such as gradient shape, column length, and electric field, on the analytical results from the separation and MS detection of a standard mixture of PTH-amino acids was investigated. Particular attention was paid to the effect of sheath flow-rate, sheath composition and mass spectra acquisition rate on the performance of the electrospray TOF-MS.     

Determination of illicit and/or abused drugs and compounds of forensic interest in biosamples by capillary electrophoretic/electrokinetic methods

The application of capillary electrophoresis (CE) methods in forensic toxicology for the determination of illicit and/or misused drugs in biological samples is reviewed in the present paper. Sample pretreatments and direct injection modes used in CE for analysis of drugs in biological fluids are briefly described. Besides, applications of separation methods based on capillary zone electrophoresis or micellar electrokinetic chromatography with UV absorbance detection to (i) analysis of drugs of abuse, (ii) analysis of other drugs and toxicants of potential forensic interest and (iii) for metabolism studies are reviewed. Also, alternative CE methods are briefly discussed, including capillary isotachophoresis and separation on mixed polymer networks. High sensitivity detection methods used for forensic drug analysis in biological samples are then presented, particularly those based on laser induced fluorescence. A glimpse of the first examples of application of CE-mass spectrometry in forensic toxicology is finally given.     

Capillary electrophoresis interfaced to inductively coupled plasma mass spectrometry for element selective detection in arsenic speciation

A method is presented to separate and detect six arsenic species by capillary electrophoresis (CE) interfaced to inductively coupled plasma mass spectrometry (ICP-MS). CE was used as a highly resolving separation system, whereas ICP-MS served as an element selective detector providing low detection limits. The special mode of operation included sample stacking and a differentiation of separation and detection. This provided separation and detection of six As species, uncharged and anionic, to be monitored within a single run. Detection limits were calculated according to IUPAC recommendation at 15 microg As/L for As (III), dimethyl arsinic acid (DA), monomethyl arsonic acid (MA) and As (V), or 65 microg As/L for arsenobetaine (AsB) and arsenocholine (AsC). Investigations were focused on possibly occurring interferences, e.g., ArCl+ interference at the monoisotope 75As. Finally, real samples from biomedical field (urine) and environmental field (sewage sludge) were analyzed.     

Habit-specific locus of control scales for drinking, smoking, and eating

A modified version of capillary isoelectric focusing (cIEF) was developed to separate hemoglobin variants contained within single human erythrocytes. Laser-induced native fluorescence with 275 nm excitation was used to detect the separated hemoglobins. In this method, baseline fluctuations were minimized and detection sensitivity was improved by using dilute solutions of anolyte, catholyte, and carrier ampholytes (with methylcellulose). Since electroosmotic flow was used for mobilization of the focused bands, separation and detection were integrated into a single step. The capillary was first filled with only ampholyte solution, and the cell (or standard) was injected as in capillary zone electrophoresis. The approximately 90 fl injection volume for individual cells is 7 x 10(4) times lower than those previously reported. Adult (normal and elevated A1), sickle (heterozygous), and fetal erythrocytes were analyzed with the amounts of hemoglobins AO, A1c, S and F determined. The pH gradient for cIEF was linear (r = 0.9984), which allowed tentative identification of Hb Fac. Variants differing by as little as 0.025 pl units were resolved.