Effects of lactate and acetate on the determination of serum ethyl glucuronide by CZE

The analysis of ethyl glucuronide (EtG), a marker of recent alcohol consumption, in serum with an optimized CZE assay is reported. The method uses a 0.1-mm id fused-silica capillary of 50 cm effective length that is coated with linear polyacrylamide, a pH 4.4 nicotinic acid/epsilon-aminocaproic acid (EACA) BGE, reversed polarity and indirect analyte detection. The assay is based on a 1:1 dilution of serum with deionized water and has LODs for EtG, lactate and acetate of 3.8 x 10(-7) M, 2.60 x 10(-6 )M and 2.18 x 10(-6 )M, respectively. Separation of EtG from endogenous macro- and microcomponents (anionic serum components of high and low concentration, respectively) and its quantification are shown to be possible for a wide range of lactate (stacker) and acetate (destacker) concentrations, macrocomponents that have an impact on the CZE behavior of EtG and that change after intake of ethanol. The assay has been successfully applied to the analysis of EtG, lactate and acetate in (i) sera of volunteers that ingested known amounts of alcohol and (ii) samples of patients that were classified (teetotalers and social drinkers vs. alcohol abusers) via analysis of carbohydrate-deficient transferrin.     

Analysis of ethyl glucuronide in human serum by capillary electrophoresis with sample self-stacking and indirect detection

Ethyl glucuronide (EtG), a metabolite of ethanol, is a marker of recent alcohol consumption. In the past few years, its analysis in body fluids has attracted considerable attention because it closes a gap between short time and long time alcohol markers such as ethanol and carbohydrate-deficient transferrin, respectively. The capillary zone electrophoresis (CZE) analysis of EtG in model mixtures and human serum is reported using uncoated and coated fused-silica capillaries together with acidic buffers in the pH range between 3.2 and 4.4 and indirect detection. In these approaches, separation of EtG from endogenous macro- and microcomponents (anionic serum components of high and low concentration, respectively) is based upon transient isotachophoretic stacking referred to as sample self-stacking. The selection of a favorable bufferco-ion and pH is shown to be crucial for optimized sensitivity. Abuffercomposed of 10 mM nicotinic acid and epsilon-aminocaproic acid (pH 4.3) is demonstrated to provide a detection limit for EtG in serum of 0.1 microg/ml, a value that is relevant for clinical and forensic purposes.     

Determination of adenosine deaminase activity in human erythrocytes by on-column capillary isotachophoresis-capillary zone electrophoresis in the presence of electroosmotic flow

Transient capillary isotachophoresis (CITP)-capillary zone electrophoresis (CZE) in presence of electroosmotic flow (EOF) was utilized for the measurement of adenosine deaminase activity in human erythrocytes. Phosphates, dominant anions of the sample matrix, were used as leading ions for transient isotachophoresis, and borates (0.3 M, pH 10) were used as terminating ions and background electrolyte for CZE. Final experimental conditions made it possible to inject 70% of the total capillary volume (1.45 microL) with the sample. Enzymatic conversion products (inosine and hypoxanthine), present in the sample in the low-micromolar range, were determined using optimized conditions. The limit of detection was 28 nM using UV detection at 202 nm. The presented data shows that CITP-CZE can be performed in uncoated capillaries in the presence of strong EOF.     

Confirmation analysis of ethyl glucuronide and ethyl sulfate in human serum and urine by CZE-ESI-MS(n) after intake of alcoholic beverages

CZE coupled to sheath liquid-based electrospray ionization (ESI) and multiple-stage ion trap mass spectrometry (MS(n) ) was used for the confirmation analysis of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in human serum and urine collected after intake of alcoholic beverages. Electrophoretic separations were performed in uncoated fused-silica capillaries using a pH 9.5 ammonium acetate background electrolyte and normal polarity. MS detection of EtG and EtS occurred after negative ionization using a spray liquid containing 0.5%?v/v ammonia in isopropanol/water (60:40%, v/v). CZE-MS and CZE-MS2 results obtained after injection of solid-phase extracts for EtG and EtS and of diluted urine confirmed the presence of EtG and EtS in samples whose levels were previously determined by CZE with indirect UV detection. Detection limits of each compound were estimated to be around 2.0 (injection of diluted urine) and 0.2?μg/mL (extracts).     

DNA fragment separations by on‐line combination of capillary isotachophoresis–capillary zone electrophoresis with UV detection

A high‐speed DNA fragment separation system based on an on‐line combination of capillary ITP with CZE (CITP‐CZE) and using UV detection at 260 nm was developed. A novel CITP‐CZE buffer system of pH 6.1 was designed for the separation of ten DNA fragments with sizes ranging from 100 to 1000 bp. An effect of underivatized α‐, β‐ and γ‐cyclodextrins on the resolution of DNA fragments in the CZE step of the CITP‐CZE combination was systematically investigated. Methylhydroxyethylcellulose present in the BGE was used to eliminate the EOF. DNA ladder fragments were separated within 10 min with LODs in the range of 1–5 ng/μL (S/N = 3). The RSDs of the migration time and peak area of individual DNA fragments were in the range of 1–3 and 3–9%, respectively. The developed CITP‐CZE system was further applied to the analysis of digest plasmid DNA samples.     

Determination of ethyl glucuronide in human serum by capillary zone electrophoresis and an immunoassay

Ethyl glucuronide (EtG) is a marker of recent alcohol consumption. For the optimization of the analysis of EtG by CZE with indirect absorbance detection, the use of capillaries with permanent and dynamic wall coatings, the composition of the BGE, and various sample preparation procedures, including dilution with water, ultrafiltration, protein precipitation, and SPE, were investigated. Two validated screening assays for the determination of EtG in human serum, a CZE‐based approach and an enzyme immunoassay (EIA), are described. The CZE assay uses a coated capillary, 2,4‐dimethylglutaric acid as an internal standard, and a pH 4.65 BGE comprising 9 mM nicotinic acid, ε‐aminocaproic acid and 10% v/v ACN. Proteins are removed via precipitation with ACN prior to analysis and the LOQ is 0.50 mg/L. The EIA is based upon commercial reagents which are promoted for the determination of urinary EtG. Krebs–Ringer solution containing 5% BSA is used as a calibration matrix. All samples are ultrafiltered prior to analysis of the ultrafiltrate on a Mira Plus analyzer. Assay calibration ranged between 0 and 2 mg/L and the upper reference limit was determined to be 0.05 mg/L. Both assays proved to be suitable for the analysis of samples from different individuals. For EtG levels above 0.50 mg/L, good agreement was observed for the comparison of the results of the two methods.     

Determination of Phenolic Acids in Herba Epilobi by ITP–CE in the Column-Coupling Configuration

The combination of capillary isotachophoresis (ITP) and capillary zone electrophoresis (CZE) in the column-coupling configuration has been optimized in a mode in which the background electrolyte employed in the CZE step was different from the leading and terminating electrolytes of the ITP step. The optimum composition of the electrolyte system was 0.01 M HCl, 0.02 M IMI, 0.2% HEC, pH 7.2 (leading electrolyte), 0.01 M HEPES, pH 8.2 (terminating electrolyte), and 25 mM MES, 50 mM TRIS, 30 mM boric acid, 0.2% HEC, pH 8.3 (background electrolyte). All solutions contained 20% methanol. The timing of the transfer of isotachophoretically stacked analyte zones into the CZE column was also optimized. An ITP–CZE method with UV detection at 270 nm was developed for separation of nine phenolic acids (protocatechuic, syringic, vanillic, cinnamic, ferulic, caffeic, ρ-coumaric, chlorogenic, and gentisic acids) in a model mixture and used for assay of some of these acids in a methanolic extract of herba epilobi. Application of ITP–CZE resulted in 100-fold better sensitivity than conventional CZE; limits of detection ranged between 10 and 60 ng mL⁻¹. When MES–TRIS–borate-based buffer, pH 8.3, was used in the CZE separation step the linearity of the ITP–CZE response was satisfactory (correlation coefficients were from 0.9937 to 0.9777). Repeatability was also satisfactory (RSD values ranged between 0.77% and 1.28% for migration times and between 1.65% and 13.69% for peak area). Revised: 23 March and 27 April 2006     

Analysis of water extracts from airborne dust samples by capillary isotachophoresis

Application of capillary isotachophoresis (CITP) for the analysis of water extracts of the dust samples collected in different periods in air-filtration devices in Prague car traffic tunnels and in Parisian metro station is presented. The extracts were analyzed in cationic mode with a leading electrolyte (LE) of 10 mM KOH, 25 mM acetic acid, pH 4.4, and a terminating electrolyte (TE) of 10 mM β-alanine, adjusted to pH 4.4 with acetic acid, and in anionic mode with LE 10 mM HCl, 20 mM histidine, pH 5.8 and TE 10 mM 2-(N-morpholino)ethanesulphonic acid, pH 3.7. Extracted amounts of UV-absorbing substances, including pollen allergens and organic pollutants, the number of the found components and concentrations of some inorganic ions (e.g. Cl⁻, K⁺, Na⁺, Ca²⁺) in the dust samples were determined. It was found that the extracted amounts of anionic components and their number were much higher than those of cationic components. Significant differences have been found in the analyses of the extracts of different origin. Much more material and more components were present in the extracts of samples from the pollen-rich period than from the pollen-free period, especially in anionic CITP mode.     

On-line coupling of capillary isotachophoresis and zone electrophoresis for the assay of phenolic compounds in plant extracts

The combination of capillary isotachophoresis (ITP) and capillary zone electrophoresis (CZE) in the column coupling configuration was optimized in a mode where the electrolyte for the CZE step is different from the leading and terminating ITP electrolytes. Two colored markers, picric acid and 1-nitroso-2-naphthol, were used for exact timing of the transfer of isotachophoretically stacked analyte zones into the CZE column and for the control of the residual amount of the leading and terminating ITP electrolytes entering the CZE capillary together with the analytes, thus controlling the duration of transient ITP migration in the CZE capillary and ensuring good separation of the analytes and reproducibility of the migration times (relative standard deviations 1%). ITP-CZE was applied to the simultaneous assay of several cinnamic acid derivatives and flavonoids in methanolic extracts of Sambucus flowers and Crataegus leaves and flowers. The preconcentrating and cleansing effect of the ITP step allowed injection of relatively large sample volumes (30 microL). The limits of detection were approximately 20-50 ng x mL(-1) and 100 ng x mL(-1) for the acids and flavonoids, respectively ( thick similar 200-times lower compared to conventional CE) with spectrophotometric detection at 254 nm. The ITP-CZE exhibited satisfactory linearity and precision when using CZE buffer of pseudo "pH" 9.0; 1-nitroso-2-naphthol was employed as the internal standard. The separation took approximately 35 min. The ITP-CZE results for rutin, hyperoside, and vitexin-2-O"-rhamnoside were in good accordance with those obtained previously by high-performance liquid chromatography.     

On-line coupling of capillary isotachophoresis and capillary zone electrophoresis for the determination of flavonoids in methanolic extracts of Hypericum perforatum leaves or flowers

Five flavonoids (hyperoside, isoquercitrin, quercitrin, quercetin and rutin) were separated and determined in extracts of Hypericum perforatum leaves or flowers by capillary zone electrophoresis (CZE) with isotachophoretic (ITP) sample pre-treatment using on-line column coupling configuration. The background electrolyte (BGE) used in the CZE step was different from the leading and terminating ITP electrolytes but all the electrolytes contained 20% (v/v) of methanol. The optimal leading electrolyte was 10 mM HCl of pH* approximately 7.2 (adjusted with Tris) and the terminating electrolyte was 50 mM H3BO3 of pH* approximately 8.2 (adjusted with barium hydroxide). This operational system allowed to concentrate and pre-separate selectively the flavonoid fraction from other plant constituents before the introduction of the flavonoids into the CZE capillary. The BGE for the CZE step was 50 mM Tris buffer of pH* approximately 8.75 containing 25 mM N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid as co-ion and 55 mM H3BO3 as complex-forming agent. The ITP-CZE method with spectrophotometric detection at 254 nm was suitable for the quantitation of the flavonoids in real natural samples; kaempferol was used as internal standard. The limit of detection for quercetin-3-O-glycosides was 100 ng ml(-1) and calibration curves were rectilinear in the range 1-10 microg ml (-1) for most of the analytes. The RSD values ranged between 0.9 and 2.7% (n=3) when determining approximately 0.07-1.2% of the individual flavonoids in dried medicinal plants.     

Analysis of orotic acid in human urine by on-line combination of capillary isotachophoresis and zone electrophoresis.

The techniques of the on-line combination of capillary isotachophoresis with zone electrophoresis in two coupled capillaries (ITP-CZE) and a single capillary zone electrophoresis (CZE) were used for the sensitive determination of orotic acid (OA) in human urine. The simple CZE system was successfully applied for fast and reliable analyses of urine of healthy adult volunteers (the detection limit 1.7.10(-6) M OA, the total time of analysis 6 min). However, this method failed in analyses of OA in urine of ill children due to more complex matrix of the samples. Here, the ITP preconcentration and preseparation step coupled on-line with CZE proved to serve well with an electrolyte system developed and optimized for this purpose. The maximum selectivity and resolution of OA from other sample constituents in ITP-CZE was achieved by use of an electrolyte system of very low pH 2.15 both for ITP and CZE stage. The sensitivity of detection and simplicity of OA identification were enhanced by use of an external UV scanning detector. High sensitivity of ITP-CZE combination (limit of detection 3.10(-7) M OA), low sample consumption (1 microliter), good reproducibility of migration times (inter-day RSD < 1.86%) and acceptable reproducibility of the determination of OA in urine samples (average RSD = 7.27%) make this technique suitable for routine determination of trace concentration of OA especially in urine of ill children under various pathological conditions and medication.     

Determination of effective charges and ionic mobilities of polycationic antimicrobial peptides by capillary isotachophoresis and capillary zone electrophoresis

Capillary ITP (CITP) and CZE were applied to the determination of effective charges and ionic mobilities of polycationic antimicrobial peptides (AMPs). Twelve AMPs (deca‐ to hexadecapeptides) containing three to seven basic amino acid residues (His, Lys, Arg) at variable positions of peptide chain were investigated. Effective charges of the AMPs were determined from the lengths of their ITP zones, ionic mobilities, and molar concentrations, and from the same parameters of the reference compounds. Lengths of the ITP zones of AMPs and reference compounds were obtained from their CITP analyses in cationic mode using leading electrolyte (LE) composed of 10 mM NH₄OH, 40 mM AcOH (acetic acid), pH 4.1, and terminating electrolyte (TE) containing 40 mM AcOH, pH 3.2. Ionic mobilities of AMPs and singly charged reference compounds (ammediol or arginine) were determined by their CZE analyses in the BGE of the same composition as the LE. The effective charges numbers of AMPs were found to be in the range 1.65–5.04, i.e. significantly reduced as compared to the theoretical charge numbers (2.86–6.99) calculated from the acidity constants of the analyzed AMPs. This reduction of effective charge due to tightly bound acetate counterions (counterion condensation) was in the range 17–47% depending on the number and type of the basic amino acid residues in the AMPs molecules. Ionic mobilities of AMPs achieved values (26.5‐38.6) × 10⁻⁹ m²V⁻¹s⁻¹ and in most cases were in a good agreement with the ratio of their effective charges and relative molecular masses.     

Analysis of 5-methyltetrahydrofolate in human blood, serum and urine by on-line coupling of capillary isotachophoresis and zone electrophoresis

The predominant circulating folate coenzyme in plasma/serum, 5‐methyltetrahydrofolate (5‐MTHF) was determined in human blood, serum and urine using a method based on the hyphenation of capillary ITP and zone electrophoresis. Measurements were done with a commercially available instrument for capillary isotachophoresis equipped with a column‐switching system. The choice of electrolytes was limited by the instability of 5‐MTHF and volatility of electrolytes for the potential coupling of the instrumentation with MS detector. To get an insight into the separability of individual sample components in an isotachophoretic analysis, we constructed zone existence diagrams for isotachophoretic electrolyte systems having a leading electrolyte composed of acetate and ammonium of pH 4.5 and 7.0, hydrocarbonate and ammonium, pH 7.8, chloride and ammonium, pH 5.6, and chloride and creatinine, pH 5.0, with hydroxide ion as the terminator. For isotachophoretic preseparation, the non‐volatile leading electrolyte with good buffering capacity composed of 1×10⁻² M HCl and 2.5×10⁻² M creatinine, pH 5.0, and terminating electrolyte composed of 1×10⁻² M MES was selected as the most suitable. The optimum BGE for CZE analysis from the standpoint of analyte stability, separability and volatility for MS coupling was 1×10⁻² M acetate with 3.5×10⁻² M ammonium, pH 4.5. Using this combination of electrolytes, LODs reached with optical detection at 220 nm were 1.6×10⁻⁷ M in human blood, 1.1×10⁻⁷ M in human serum and 4.7×10⁻⁶ M in human urine. Estimated content of 5‐MTHF in blood and serum samples of women following oral daily administration of 0.8 mg of folic acid was 1.2×10⁻⁵ and 5.8×10⁻⁶ M, respectively.     

New carrier electrolytes for the separation of chlorophenols by capillary electrophoresis

Optimum conditions for the separation of positional isomers of chlorophenols by capillary zone electrophoresis (CZE) were established. The behavior of five volatile electrolytes (L-cysteic acid, 3-amino-1-propanesulfonic acid, aminomethanesulfonic acid, diethylmalonic acid, and ammonium acetate) was compared. The best performance based on low electrophoretic current and high separation efficiency was obtained for diethylmalonic acid as working electrolyte. The influence of pH on the separation, using both uncoated fused-silica capillaries and modified capillaries (NaAMPS from EKT) with anionic coating, was discussed. Moreover, the effect of electrolyte concentration and applied voltage using fused-silica capillaries was studied. The optimum CZE conditions that allowed the separation of 16 chlorophenols were 20 kV, 30 mM diethylmaIonic acid, pH 7.25, and uncoated fused-silica capillary. Figures of merit such as run-to-run and day-to-day precision, linearity, and limits of detection were calculated.     

Determination of quinine in beverages by online coupling capillary isotachophoresis to capillary zone electrophoresis with UV spectrophotometric detection

The present study illustrates the possibilities of capillary isotachophoresis (CITP) online coupled with capillary zone electrophoresis (CZE) and hyphenated with fiber-based spectrophotometric diode array detection (DAD) for the direct, highly reliable, and ultrasensitive determination of quinine (QUI) in real multicomponent ionic matrices (beverages). Here, the CITP provided an effective online sample pretreatment (preseparation and preconcentration) prior to the CZE separation. Due to the CITP sample preconcentration, a simple UV-visible absorbance spectrophotometric detection was sufficient for obtaining very low concentration limits of detection (~2.3 ng/mL). Enhanced separation selectivity due to the combination of different separation mechanisms (CITP vs. CZE) enabled to obtain a pure analyte zone, suitable for its detection and quantitation in the directly injected real samples. The spectrophotometric DAD, unlike single wavelength UV detection, enabled to characterize the purity (i.e. spectral homogeneity) of the analyte zone and preliminary data indicate structurally related compounds via characteristic spectra recorded in the interval of 200-600 nm. The proposed CITP-CZE-DAD method was characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, and selectivity) and successfully applied to the control of QUI and potential QUI impurities in commercial beverages. This method is proposed as a routine automatized method for the highly reliable quality food control.     

Determination of ethyl sulfate in human serum and urine by capillary zone electrophoresis

The use of capillary zone electrophoresis (CZE) with indirect absorbance detection for the analysis of ethyl sulfate (EtS) in serum and urine was investigated. EtS is a direct metabolite of ethanol employed as marker for recent alcohol consumption. Fused-silica capillaries of 60 cm total length were either coated with cetyltrimethylammonium bromide (CTAB, 50 microm I.D. capillary) or poly(diallyldimethylammonium chloride) (PDADMAC, 100 microm I.D. capillary) to allow CZE analyses to be performed with reversed polarity. At pH 2.2 with a maleic acid/phthalic acid background electrolyte, both approaches provided reliable EtS serum levels down to 0.2 mg L(-1) (1.6 microM) for the analysis of solid-phase extracts that were prepared after chloride precipitation. Analysis of urines diluted to a conductivity of 5 S m(-1) and analyzed in the two capillary formats resulted in limits of quantification (LOQs) of 2 and 1 mg L(-1), respectively. With urines adjusted to 10 S m(-1) via dilution or condensation, an LOQ of 0.6 mg L(-1) (4.8 microM) was obtained in the CTAB coated capillary whereas in the PDADMAC-coated capillary of equal length not all matrix components were resolved from EtS. The developed assays are robust and suitable to monitor EtS in samples of individuals who consumed as little as one standard drink of an alcoholic beverage containing about 14 g of ethanol.     

Determination of inorganic anions in saliva by capillary isotachophoresis

Nitrite, nitrate, iodide and thiocyanate have been quantified in non-smoker and smoker saliva by capillary isotachophoresis (CITP). Hydrochloric acid (10 mmol l⁻¹) adjusted with histidine to pH 6.0 plus 6% poly(vinylpyrrolidone) was used as the leading electrolyte (LE) and 5 mmol l⁻¹ acetic acid as the terminating electrolyte (TE). Linearity was observed from 0.005 to 0.500 mmol l⁻¹ with a coefficient of determination (r²) of 0.999. The separation of anions was achieved in less than 19 min. The minimal sample pretreatment and relatively low running cost make isotachophoresis good alternative to existing methods.     

Flow Injection Coupled Capillary Isotachophoresis to ICP-AES and On-line Determination of Metal Species

A new technique for coupling capillary isotachophoresis (CITP) to Inductive coupled plasmas atomic emission spectrometry (ICP-AES) using flow injection (FI) is developed. Great attention to CITP have been paid owing to its broad application prospect in biochemistry, clinic, pharmacy, food,environment detection and conventional ionic analysis. Its zone-boundary sharpening, large size capillary and self-concentrating effects are different from those of other electrophoretic techniques. Recently, ICP-AES/MS, as a special metal detector, has been combined with the capillary zone electrophoresis (CZE). However, in CZE system, typical sample volume and flow rate are only in the range of 0.02 - 1 μl/min and 0.2-2μl, which results in the difficult for the coupling of CZE and ICP with conventional sampling system and the obtaining enough sensitivity.     

Application of capillary isotachophoresis-based multidimensional separations coupled with electrospray ionization-tandem mass spectrometry for characterization of mouse brain mitochondrial proteome

By employing a capillary ITP (CITP)/CZE-based proteomic technology, a total of 1795 distinct mouse Swiss-Prot protein entries (or 1705 nonredundant proteins) are identified from synaptic mitochondria isolated from mouse brain. The ultrahigh resolving power of CITP/CZE is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. The degree of peptide overlapping among CITP fractions is even lower than that achieved using combined CIEF/nano-RP LC separations for the analysis of the same mitochondrial sample. When evaluating the protein sequence coverage by the number of distinct peptides mapping to each mitochondrial protein identification, CITP/CZE similarly achieves superior performance with 1041 proteins (58%) having 3 or more distinct peptides, 233 (13%) having 2 distinct peptides, and 521 (29%) having a single distinct peptide. The reproducibility of protein identifications is found to be around 86% by comparing proteins identified from repeated runs of the same mitochondrial sample. The analysis of the mouse mitochondrial proteome by two CITP/CZE runs results in the detection of 2095 distinct mouse Swiss-Prot protein entries (or 1992 nonredundant proteins), corresponding to 59% coverage of the updated Maestro mitochondrial reference set. The collective analysis from combined CITP/CZE and CIEF-based proteomic studies yields the identification of 2191 distinct mitochondrial protein entries (or 2082 nonredundant proteins), corresponding to 76% coverage of the MitoP2-database reference set.     

Capillary zone electrophoresis of metal-binding proteins in formic acid with UV- and mass spectrometric detection using cationic transient capillary isotachophoresis for preconcentration

A capillary zone electrophoresis (CZE) method with preceding cationic transient capillary isotachophoresis (tCITP-CZE) was developed for uncoated fused-silica capillaries to analyze metal-binding proteins (MBPs) of clinical relevance. UV detection was followed by mass spectrometry (MS). Optimization was done with model proteins of properties similar to relevant human MBPs. Using 1.0 mol x L(-1) formic acid (pH 1.78) as electrolyte resulted in up to 165000 plates m(-1) in CZE and 230000 plates m(-1) in combination with tCITP and analysis time was less than 5 min in uncoupled mode. Cationic tCITP with 125 mmol x L(-1) ammonium formate, buffered to pH 4.00, as leading electrolyte improved sample loadability considerably in comparison with sample stacking without impairing resolution. Following systematic optimization of the electrospray ionization process (ESI) the coupled system ((tCITP)-CZE-UV-ESI-MS) was tested with protein model mixtures and human MBPs. Repeatability of migration times was < 0.64% in pure CZE mode and in tCITP-CZE mode and < 0.83% in CZE-ESI-MS coupled mode. Mass accuracy was < 0.015%. Limits of detection were found to be in the range 50-160 fmol.