Enantiomeric separation of baclofen by capillary electrophoresis tandem mass spectrometry with sulfobutylether-beta-cyclodextrin as chiral selector in partial filling mode

Capillary electrophoresis (CE) coupled to tandem mass spectrometry was applied to the chiral separation of baclofen using sulfobutylether-beta-cyclodextrin chiral selector in partial filling counter current mode. On-line UV detection was simultaneously used. Method optimization was performed by studying the effect of cyclodextrin and BGE concentration as well as sheath liquid composition on analyte migration time and enantiomeric resolution. The cyclodextrin showed stereoselective complexation towards baclofen enantiomers, allowing chiral resolution at low concentration. The CE capillary protrusion from the ESI needle relevantly affected the chiral resolution and the analyte migration time. Complete enantiomeric separation was obtained by using 0.25 M formic acid BGE containing 1.75 mM of chiral selector and water/methanol (30:70, v/v) 3% formic acid as sheath liquid. The method exhibited a LOD of 0.1 microg/mL (racemic concentration) in MS3 product ion scan mode of detection and was applied to the analysis of racemic baclofen in pharmaceutical formulations.     

Capillary electrophoresis-mass spectrometry as a characterization tool for therapeutic proteins

With the increasing use of capillary electrophoresis (CE) in the biotechnology industry, there is a demand for analytical tools and methodology that can be used to characterize CE profiles. This article describes the implementation and optimization of a robust online CE-mass spectrometry (CE-MS) system used for the characterization of several CE assays developed at Genentech Inc. These assays include CE as a complement to reverse-phase peptide mapping for the identification of small peptides eluting in the void volume, profiling N-linked glycopeptide heterogeneity, and determining O-linked site occupancy. In addition, CE-MS was used to confirm major 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans released from recombinant antibodies that are routinely profiled by CE-laser-induced fluorescence (CE-LIF). For each study, CE-MS was able to successfully identify components seen in UV or LIF electropherograms, thereby expanding the capability of CE and CE-MS for profiling biomolecules.     

Stability Enhancement for Peptide Analysis by Electrospray Using the Triple Quadrupole Mass Spectrometer

Electrospray ionization sources, used with triple quadrupole mass spectrometers from PE/Sciex (API III+), Micromass (Quattro II), and Finnigan (TSQ 7000), were modified with a 35-gauge stainless steel needle. The dimensions of the needle were 63 μm i.d. by 145 μm o.d. with variable length, depending on the specific instrument. This modification led to enhanced signal stability, improved signal/noise ratios, and lowered sample consumption for a wide range of peptides. Stable baselines were observed with flow rates in the range of 50 nL/min to 5 μL/min. An alternative design, based on a metal wire housed within a fused silica capillary, led to the most stable signals of all during infusion, but caused excessive peak broadening with capillary chromatography. The Finnigan interface was further modified with an external postcolumn addition tee, used in conjunction with capillary liquid chromatography columns of 30 and 50 μm internal diameter. The best results with the modified Finnigan interface were acquired using the 50-μm column at a flow rate of 150 to 200 nL/min.     

Electrophoretic and mass spectrometric strategies for profiling bacterial lipopolysaccharides

Capillary electrophoresis (CE) is a high-resolution separation technique that has been widely used for trace analysis in biological samples. On-line capillary electrophoresis-electrospray mass spectrometry (CE-MS) was developed for the analysis of lipopolysaccharide (LPS) glycoforms from the gram-negative bacteria, Haemophilus influenzae. In this paper, we report on the application of CE-MS to characterize structural differences in O-deacylated LPS samples from H. influenzae strains Rd 11.7 and 375.1. The resolution capability of on-line CE-MS was first demonstrated by analysis of a complex LPS mixture from H. influenzae strain Rd 11.7. This strain contains a mixture of isomeric glycoforms differing in the number and positions of hexose moieties. Sialic acid containing glycoforms were also determined. Structural features of LPS from a lic1 mutant of H. influenzae strain 375 (375.1) were studied using on-line CE-MS/MS. With the separation provided by CE, two isomeric glycoforms differing in the location of phosphoethanolamine substituents were characterized by tandem mass spectrometry.     

毛细管电泳-质谱联用技术研究进展

毛细管电泳-质谱（CE-MS）联用技术兼具毛细管电泳高效分离能力与质谱高灵敏检测、高真度定性的优势,已成为物质分离分析研究的一种非常重要的工具。本文对近几年来 CE-MS 联用的关键技术及 CE-MS 在中药分析、环境检测等领域的一些应用进展进行综述,对其发展进行了展望。     

Separation methods for pharmacologically active xanthones

Xanthones, as a kind of polyphenolic natural products with many strong bioactivities, are attractive for separation scientists due to the similarity and diversity of their structures resulting in difficult separation by chromatographic methods. High performance liquid chromatography (HPLC) and thin layer chromatography (TLC) are traditional methods to separate xanthones. Recently, capillary electrophoresis (CE), as a micro-column technique driven by electroosmotic flow (EOF), with its high efficiency and high-speed separation, has been employed to separate xanthones and determine their physicochemical properties such as binding constants with cyclodextrin (CD) and ionization constants. Since xanthones have been used in clinic treatment, the development of chromatographic and CE methods for the separation and determination of xanthones plays an essential role in the quality control of some herbal medicines containing xanthones. This article reviewed the separation of xanthones by HPLC, TLC and CE, citing 72 literatures. This review focused on the CE separation for xanthones due to its unique advantages compared to chromatographic methods. The comparison of separation selectivity of different CE modes including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic capillary chromatography (MEEKC) and capillary electrochromatography (CEC) was discussed. Compared with traditional chromatographic methods such as HPLC and TLC, CE has higher separation efficiency, faster separation, lower cost and more flexible modes. However, because of low sensitivity of UV detector and low contents of xanthones in herbal medicines, CE methods have seldom been applied to the analysis of real samples although CE showed great potential for xanthone separation. The determination of xanthones in herbal medicines has been often achieved by HPLC. Hence, how to enhance CE detection sensitivity for real sample analysis, e.g. by on-line preconcentration and CE-MS, would be a key to achieve the quantitation of xanthones.     

Dynamic coating for fast and reproducible determination of basic drugs by capillary electrophoresis with diode-array detection and mass spectrometry

The double coating principle of CEofix buffers was evaluated for the analysis of some basic drugs by capillary electrophoresis-diode-array detection (CE-DAD) and capillary electrophoresis-mass spectrometry (CE-MS). The involatile phosphate present in original low pH CEofix, was replaced with formic acid for hyphenation of CE with MS. The double coating produces a substantial and highly reproducible electroosmotic flow (EOF), even at low pH. The rinsing procedure and electrolyte composition were optimized for both CE-DAD and CE-MS. The system was evaluated with the analysis of a mixture of basic drugs and a spiked urine sample enriched by solid-phase extraction (SPE). The R.S.D. values on the migration time and peak area measured for 28 analyses with CE-DAD were below 0.25 and 2.40%, respectively. For CE-MS, the R.S.D. on the migration time was 0.85% or less and the area precision ranged from 5.65 to 14.33% (for seven injections). The LOD with the developed CE-MS method was below 50 ppb for all five drug standards tested.     

Site-specific sampling of taurine from rat brain followed by on-line sample pre-concentration, throughout in-capillary derivatization and capillary electrophoresis

A method of pinpoint-sampling followed by on-line pre-concentration of the sample, throughout in-capillary derivatization and capillary electrophoretic separation was evaluated by demonstrating the detection of taurine, 2-aminoethanesulfonic acid at a specific location of a rat brain. The direct sampling of taurine from the rat brain was accomplished by using voltage injection associated with two kinds of driving forces, electrophoretic flow and electroosmotic flow (EOF). The capillary tube (75 microm of inner diameter x 375 microm of outer diameter) of the capillary electrophoresis (CE) apparatus was already filled with a CE run buffer, viz., 40 mM phosphate-borate buffer (pH 10) containing 2mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) as the derivatization reagent. One end of a platinum wire (0.5mm o.d.), used as the anode, and the inlet end of capillary tube (from which a 1.0 cm long polyimide coating was removed), were pricked down onto the surface of either the cerebrum or cerebellum of a rat brain at a location of very small dimension. When a low voltage (5 kV, 30s) was applied, taurine began to move from the rat brain into the capillary tube, and, simultaneously, electric focusing of taurine occurred by the action of "the pH-junction effect" at the inlet end of the capillary tube. After completing the injection, both the platinum wire and capillary tube were detached from the brain and dipped into the run buffer in an anode reservoir filed with the same solution as that in the capillary tube for the CE apparatus. Then, by applying a high voltage (20 kV) between the ends of the capillary tube, taurine was automatically derivatized to yield the fluorescent derivative, separated and detected with fluorescence (E(x)=340 nm, E(m)=455 nm) during migration throughout the capillary tube. The migration profiles obtained from cerebrum and cerebellum appeared to be different, but the peak corresponding to taurine was identified on both electropherograms. The efficacy of the present method including sample on-line pre-concentration prior to throughout in-capillary derivatization CE was first verified with several preliminary experiments by using samples of taurine in water, saline and a piece of 1.5% agar-gel block, as an alternate standard for the rat brain used in this study.     

Analysis of human blood serum using the off-line coupling of capillary isoelectric focusing to matrix-assisted laser desorption/ionization time of flight mass spectrometry

Off-line coupling of capillary IEF (CIEF) with matrix-assisted laser desorption/ionization mass spectrometry was utilized for the analysis of human blood serum. Serum proteins were initially separated by CIEF, and fractions of the isoelectric separation were eluted sequentially to a MALDI-TOF MS sample target. During pressure elution of the CIEF sample, voltage was maintained across the capillary system utilizing a sheath flow arrangement to minimize band broadening induced by the laminar flow field. Both pI and mass information were obtained from the complex biological sample, similar to traditional 2-DE techniques, and the platform was faster (hours versus days), more automatable, and simpler than 2-DE. The volume of raw sample present in the actual analysis was approximately 100 nL, making this technique well suited for very rare specimens. Additionally, the speed and simplicity of the technology make it an attractive technique for performing initial comparative analyses of complex samples.     

Determination of dopamine and methoxycatecholamines in patient urine by liquid chromatography with electrochemical detection and by capillary electrophoresis coupled with spectrophotometry and mass spectrometry

The applicability of capillary electrophoresis (CE) with UV and mass spectrometric (MS) detection for the determination of dopamine and methoxycatecholamines in urine was evaluated in comparison with the liquid chromatography-electrochemical detection (LC-EC) method widely used in catecholamine analysis. The catecholamines in urine were deconjugated with acid or enzyme hydrolysis, purified by cation exchange (CEX) or solid-phase extraction (SPE) with a copolymer of N-divinylpyrrolidone and divinylbenzene and analyzed by LC-EC, CE-UV, and CE-MS. Acid hydrolysis was more effective in the deconjugation than enzymatic hydrolysis with Helix pomatia. However, the recoveries of HMBA, DA and NMN from spiked samples were less than 30% after acid hydrolysis and SPE purification. The CEX purification was more efficient than SPE in removing matrix compounds from the urine samples. The limits of detection were lower in LC-EC analysis than in CE-UV or CE-MS. Many factors in the analytical procedure caused deviations in the concentrations measured for urinary dopamine and methoxycatecholamines. The recovery of HMBA, which was used as the internal standard, was poor after acid hydrolysis and SPE purification. The purification methods were validated in conjunction with the analytical methods and therefore cross analysis was unsuccessful. The LC-EC method was the most sensitive, but CE-UV and CE-MS were sensitive enough for the determination of dopamine and methoxycatecholamines even in healthy patient urine. The EC and MS detections were superior to the UV detection in specificity since, after acid hydrolysis, some matrix compounds were migrating close to I.S., DA and 3MT.     

Quantitative metabolome analysis using capillary electrophoresis mass spectrometry

A new approach for the comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS) is proposed. Metabolites are first separated by CE based on charge and size and then selectively detected using MS by monitoring over a large range of m/z values. This method enabled the determination of 352 metabolic standards and its utility was demonstrated in the analysis of 1692 metabolites from Bacillus subtilis extracts, revealing significant changes in metabolites during B. subtilis sporulation.     

Optimization of an HPLC peroxyoxalate chemiluminescence detection system for some dansyl amino acids

Bis(2,4,6-trichlorophenyl) oxalate (TCPO)-hydrogen-peroxide-generated chemiluminescence (CL) of four dansyl amino acids has been used as a model system for the optimization of a detection system in reversed-phase high-performance liquid chromatography. Dansylated alanine, glutamic acid, methionine, and norleucine were subjected to peroxyoxalate induced CL in a static system and in a flow system under various conditions with respect to TCPO (ethyl acetate) and hydrogen peroxide (acetone) concentrations, solvent composition and flow, using a two-pump or a one-pump post-column reagent system. From the CL-decay curve, the influence on the emission signal from the total flow rate in the detector was investigated. Special attention was focused on the mixing of the LC eluate and the reagent in order to combine an efficient collection of the emitted light using a 74μI flow cell (originally 10μI in the fluorescence detector) with minimal extra column band broadening. Therefore, a capillary fused-silica tubing of about 100μm i.d. was inserted against the end-frit of the column and brought through a mixing tee, in which the solutions of TCPO and hydrogen peroxide were added. The column end tubing ended in the flow cell and the LC eluate and the reagents were mixed when entering the flow-cell. Average detection limits (S/N=2) of 200fmol injected dansylated amino acid could be reached. A comparison is made between the use of TCPO and DNPO (bis (2, 4-dinitrophenyl) oxalate).     

Chromatographic and capillary electrophoretic methods for the analysis of nicotinic acid and its metabolites

Methods for the assay of nicotinic acid (NiAc) and its metabolites in biological fluids using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are reviewed. Most of the references cited in this review concern HPLC methods. A few CE methods that have been recently reported are also included. As these compounds are relatively polar and have a wide range of physico-chemical properties, the sample pre-treatment or clean-up process prior to analysis is included. Most HPLC methods using an isocratic elution system allow determination of a single or few metabolites, but gradient HPLC methods enable simultaneous determination of five to eight compounds. Simultaneous determination of NiAc including many metabolites in a single run can be achieved by CE. We also discuss the pharmacokinetics of NiAc and some of its metabolites.     

Capillary electrophoresis and capillary electrophoresis-ion trap multiple-stage mass spectrometry for the differentiation and identification of oxycodone and its major metabolites in human urine

Oxycodone (OCOD) and its metabolites, including oxymorphone (OMOR), noroxycodone (NOCOD) and noroxymorphone (NOMOR), are opioids that carry an OH group at position 14. Using capillary electrophoresis (CE) with a binary phosphate buffer containing 60% ethylene glycol (pH 7.9), the migration order of OCOD and OMOR with respect to their N-demethylated analogs was found to be reversed compared to that observed for codeine, dihydrocodeine, morphine and dihydromorphine, compounds that do not have an OH group at position 14. OCOD and structurally related compounds can also be distinguished from these opioids by their absorbance spectra at low wavelengths and via a characteristic neutral H2O loss at the MS2 level. Using the binary phosphate buffer, CE with UV detection is shown to be capable of monitoring OCOD, NOCOD, OMOR (after hydrolysis only) and NOMOR (after hydrolysis and in patient urine only) in alkaline liquid-liquid extracts of urines that were collected after ingestion of 10 mg OCOD hydrochloride and in a patient urine collected at steady state (80 mg OCOD hydrochloride daily). Using an aqueous pH 9 ammonium acetate buffer, these results were confirmed by CE-MS3. Based on CE-MS, MS2 and MS3 data, the absorbance spectra measured across the CE peaks and the relative position within the electropherogram, two peaks monitored in the UV absorbance electropherograms could be assigned to the two keto-reduced metabolites 6oxycodol (60COL) and nor6oxycodol, for which no standards were available. Comparison of data obtained with urines pretreated with two different enzyme products (beta-glucuronidase and beta-glucuronidase/arylsulfatase) suggest that OCOD, NOCOD and 6OCOL are mainly glucuronidated, whereas OMOR mainly forms other conjugates. Furthermore, in a first attempt to directly measure conjugates of the compounds of interest, solid-phase extracts were analyzed by CE-MS4, which revealed the presence of the acyl glucuronides of 6OCOL and OMOR and an unidentified OMOR conjugate. The quantitation of free OCOD and NOCOD by CE-MS using deuterated internal standards is also discussed briefly.     

Bottom‐up proteome analysis of E. coli using capillary zone electrophoresis‐tandem mass spectrometry with an electrokinetic sheath‐flow electrospray interface

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE‐ESI‐MS/MS. The separation was performed in a 60‐cm‐long linear polyacrylamide‐coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath‐flow electrospray interface was used to couple the separation capillary with an Orbitrap‐Velos operating in higher‐energy collisional dissociation mode. Each CZE‐ESI‐MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom‐up analysis of prokaryote proteomes.     

Simultaneous determination of the main metabolites in rice leaves using capillary electrophoresis mass spectrometry and capillary electrophoresis diode array detection

The study of the metabolomics of primary metabolites using conventional chemical analyses requires a high-throughput method. Chemical derivatizations are a prerequisite for gas-chromatographic separation, and a large sample quantity is needed for liquid-chromatographic separation and nuclear magnetic resonance detection systems. Recently, we have developed a capillary electrophoresis-mass spectrometry (CE-MS) technology that can simultaneously quantify a large number of primary metabolites, using only a small quantity of samples, and without any chemical derivatizations. Parallel use of a capillary electrophoresis-diode array detector (CE-DAD) system further enables almost all water-soluble intracellular metabolites to be analyzed. We demonstrate, with rice leaves, a simple and rapid method of sample preparation for CE analysis; using this method, we have successfully measured the levels of 88 main metabolites involved in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, photorespiration, and amino acid biosynthesis.     

Analysis of recombinant human growth hormone by capillary electrophoresis with bilayer-coated capillaries using UV and MS detection

The characterization of recombinant human growth hormone (rhGH; somatropin) by capillary electrophoresis (CE) with UV-absorbance and mass spectrometric (MS) detection using capillaries noncovalently coated with polybrene (PB) and poly(vinyl sulfonic acid) (PVS) is demonstrated. Compared with bare fused-silica capillaries, PB-PVS coated capillaries yielded more favorable migration-time reproducibilities and higher separation efficiencies. Optimal separation conditions for the bilayer-coated capillaries comprised a background electrolyte (BGE) of 400 mM Tris phosphate (pH 8.5) yielding migration-time R.S.D.s of less than 1.0% and plate numbers above 300,000 for intact rhGH. The protein was also analyzed using the CE method described in the European Pharmacopoeia (Ph. Eur.) monograph. The pharmacopoeial method gave much longer analysis times (22 min versus 8 min), lower resolution and plate numbers, and consecutive shifts in migration time for rhGH, indicating possible interactions between the protein and the inner capillary wall. Due to stable migration times obtained with the coated capillaries, reliable profiling and quantification of rhGH and its byproducts in time was possible. Analysis of thermally degraded rhGH revealed the formation of two main degradation products. CE-mass spectrometry (MS) of this sample, using a PB-PVS coated capillary and a BGE of 75 mM ammonium formate (pH 8.5), suggests that these products are desamido forms of rhGH. Analyses of expired rhGH preparations with CE-UV and CE-MS indicated the presence of both deamidation and oxidation products.     

Analytical characterisation of crude extracts from an African Ancistrocladus species using high-performance liquid chromatography and capillary electrophoresis coupled to ion trap mass spectrometry

The analysis by HPLC, CE and CE-MS/MS of root bark extracts of a, so far undescribed, Central-African Ancistrocladus species (family Ancistrocladaceae) is described. Owing to the complexity of the extract, the application of reversed-phase HPLC resulted in a partially incomplete separation of the naphthylisoquinoline alkaloids, whilst CE using a non-aqueous buffer proved to be a very valuable complementary method for a first characterisation of the crude extract. By performing additional CE-MS/MS experiments, in combination with parallel isolation studies and structural elucidation using conventional methods, six alkaloidal substances present in the plant could be identified.     

Clinical applications of capillary electrophoresis

Capillary electrophoresis (CE) is an extremely sensitive technique, which has been used in the clinical laboratory for almost 10 yr. The components of CE instrumentation are described, as are injection modes, buffers, and effects of electroosmotic flow. The modes of separation used in CE, namely, capillary zone electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, and micellar electrokinetic capillary chromatography, are explained. References for 26 different clinical applications of CE are included, among them assays that are used routinely as well as niche assays for specialized applications of CE. Verification of CE assays, current instrumentation, and future development of CE in the clinical laboratory are addressed.