Pressure-assisted capillary electrochromatography with electrospray ionization-mass spectrometry based on silica-based monolithic column for rapid analysis of narcotics

A pressure-assisted CEC (pCEC) with ESI-MS based on silica-based monolithic column was developed for rapid analysis of narcotics. Combining the extremely high permeability and separation efficiency of silica-based monolithic column with the high selectivity and sensitivity of pCEC-ESI-MS, the developed system exhibited its prominent advantages in separation and detection. A systematic investigation of the pCEC separation and ESI-MS detection parameters was performed. Experiment results showed that the optimized separation efficiency could be obtained at 8 bar assisted pressure with 25 kV separation voltage, using the solution containing 65% ACN v/v and 20 mmol/L ammonium acetate with pH 6.0 as running buffer. 3 microL/min of sheath liquid was considered as the optimized flow rate since it could provide the maximum signal intensity. Under the optimum conditions, the tested five narcotics could be completely separated within 10 min with the detection limit in the range of 2.0-80 nmol/L. The proposed method has been successfully used for detection of narcotics in real urine samples.     

Capillary electrochromatography‐mass spectrometry for the determination of 5‐nitroimidazole antibiotics in urine samples

The separation of eight antibiotics belonging to 5‐nitroimidazole family was carried out by means of CEC coupled with MS. Preliminary experiments were carried out with ultraviolet detection in order to select the proper stationary and mobile phase. Among the different stationary phases studied (namely Lichrospher C18, 5 μm particle size; Cogent^TM Bidentate C18, 4.2 μm; Pinnacle II? Phenyl, 3 μm; Pinnacle II? Cyano, 3 μm), Cogent? Bidentate C18 (4.2 μm) gave the best performance. For CEC‐MS coupling, a laboratory assembled liquid‐junction‐nano‐spray interface was used. In order to achieve a good sensitivity, special attention was paid to both optimization of the sheath liquid composition as well as selection of the injection mode. Under optimized CEC‐ESI‐MS conditions, the separation was accomplished within 22 min by using a column packed with a mixture of Bidentate C18:Lichrospher Silica‐60 (5 μm) 3:1 w/w, an inlet pressure of 11 bar, a voltage of 15 kV, and a mobile phase composed by 45:10:45 v/v/v ACN/MeOH/water containing ammonium acetate (5 mM pH 5). A combined hydrodynamic and electrokinetic injection of 8 bar, 15 kV, and 96 s was adopted. The method was validated in terms of repeatability and intermediate precision of retention times and peak areas, linearity, and LODs and LOQs. RSDs values were <2.9% for retention times and <16.1% for peak areas in both intraday and interday experiments. LOQ values were between 0.09 and 0.42 μg/mL for all compounds. Finally, the method was applied to the determination of three most employed 5‐nitroimidazole antibiotics (metronidazole, secnidazole, and ternidazole) in spiked urine samples, subjected to a SPE procedure. Recovery values in the 67–103% range were obtained. Furthermore, for the selected antibiotics, CEC‐MS² spectra were obtained providing the unambiguous confirmation of these drugs in urine samples.     

Analysis of phospholipids by NACE with on-line ESI-MS

A hyphenated method of nonaqueous capillary electrophoresis coupled to electrospray ionization mass spectrometry (NACE-ESI-MS) is described for the simultaneous analysis of phospholipids. The best results were obtained with a mixed solution of methanol/ACN (40:60 v/v) containing 20 mM ammonium acetate and 0.5% acetic acid, under the applied voltage of 30 kV and capillary temperature of 25 degrees C. ESI-MS measurements were performed in the negative mode with methanol/ACN (40:60 v/v) containing 50 mM ammonium acetate as sheath liquid at a flow rate of 2 microL/min. Different phospholipid classes have been successfully separated within 16 min, and the molecular species of every single class have been identified by using MS(2) or MS(3), which generates characteristic fragments through CID. The developed method has been applied to analyze the phospholipids extracted from rat peritoneal surface and the molecular species of phospholipid classes are presented.     

Enantiomeric separation of amlodipine and its two chiral impurities by nano‐liquid chromatography and capillary electrochromatography using a chiral stationary phase based on cellulose tris(4‐chloro‐3‐methylphenylcarbamate)

In this work, a novel polysaccharide‐based chiral stationary phase, cellulose tris(4‐chloro‐3‐methylphenylcarbamate), also called Sepapak 4 has been evaluated for the chiral separation of amlodipine (AML) and its two impurities. AML is a powerful vasodilatator drug used for the treatment of hypertension. Capillary columns of 100 μm id packed with the chiral stationary phase were used for both nano‐LC and CEC experiments. The optimization of the mobile phase composed of ACN/water, (90:10, v/v) containing 15 mM ammonium borate pH 10.0 in nano‐LC allowed the chiral separation of AML and the two impurities, but not in a single run. With the purpose to obtain the separation of the three pairs of enantiomers simultaneously, CEC analyses were performed in the same conditions achieving better enantioresolution and higher separation efficiencies for each compound. To fully resolve the mixture of six enantiomers, parameters such as buffer pH and concentration sample injection have been then investigated. A mixture of ACN/water (90:10, v/v) containing 5 mM ammonium borate buffer pH 9.0 enabled the complete separation of the three couples of enantiomers in less than 30 min. The optimized CEC method was therefore validated and applied to the analysis of pharmaceutical formulation declared to contain only AML racemate.     

Simultaneous chiral separation and determination of ephedrine alkaloids by MEKC-ESI-MS using polymeric surfactant I: method development

In this work, simultaneous separation of eight stereoisomers of ephedrine and related compounds ((+/-)-ephedrine, (+/-)-pseudoephedrine, (+/-)-norephedrine and (+/-)-N-methylephedrine) was accomplished using a polymeric chiral surfactant, i.e. polysodium N-undecenoxycarbonyl-L-leucinate (poly-L-SUCL) by chiral (C)MEKC-ESI-MS. The conditions of CMEKC were first investigated. The baseline separation of all eight stereoisomers of ephedrine and related compounds was achieved under optimum CMEKC conditions (35 mM poly-L-SUCL, 15 mM NH(4)OAc, pH 6.0, 30% v/v ACN, 30 kV and 20 degrees C) in less than 30 min. Next, a central composite design for response surface modeling has been described to evaluate the electrospray chamber parameters and the sheath liquid conditions. Optimum mass abundance of stereoisomers of ephedrine and related compounds was observed using the spray chamber parameters, namely 250 degrees C drying gas temperature and 8 L/min drying gas flow rate at a nebulizer pressure of 4 psi. Furthermore, the experimental design indicates that the optimum mass abundance of the stereoisomers of ephedrine and related compounds can be obtained using a sheath liquid containing 80:20 v/v methanol-water, 5 mM NH(4)OAc at pH 8.5 delivered at 5 microL/min. Finally, compared to MEKC-UV, the use of poly-L-SUCL in MEKC-MS provided significantly higher sensitivity for stereoisomers of ephedrine and related compounds.     

Analysis of phenolic xenoestrogens by pressurized CEC with amperometric detection

A new method, pressurized CEC with end‐column amperometric detection using carbon paste electrode, has been developed for the separation and determination of five phenolic xenoestrogens in chicken eggs and milk powder samples. Efficient separation of five analytes was performed by pressurized CEC using a mobile phase consisting of 60% v/v ACN and 40% v/v Tris buffer (5 mmol/L, pH 8.0), +6 kV of applied voltage and 7.0 MPa of supplementary pressure. Detection limits of 50, 5, 2, 10 and 20 ng/mL for pentachlorophenol, bisphenol‐A, 2,4‐dichlorophenol, 4‐tert‐octylphenol and 4‐nonylphenol, respectively, were achieved using carbon paste electrode as working electrode and +0.8 V as detection potential. Matrix solid phase dispersion extraction method had been employed during sample preparation procedure, and mean recoveries ranged from 79.2 to 102.6% at different concentrations of phenolic xenoestrogens for spiked egg and milk powder samples were obtained.     

Simultaneous analysis of cationic, anionic, and neutral compounds using monolithic CEC columns

A new capillary electrochromatography (CEC) column for the simultaneous analysis of cationic, neutral, and anionic compounds using CEC-ESI-MS is described. Three different silica monolith columns were prepared by changing the poly(ethylene glycol) (PEG) contents for comparison of the separation property of these columns. Different separation programs were used for the simultaneous separation of different charged compounds under the same conditions. The column prepared with 80 mg of PEG separated typical compounds within 15 min using 1 M formic acid as the electrolyte. The analytes migrated in the order of cationic, neutral, and anionic compounds, which means that the migration order was mainly determined by the electrophoresis. The hydrodynamic flow by pressure from the inlet side was significant for a stable analysis to be achieved. The effect of the composition of the sheath liquid was also examined. All analytes (14 amino acids, thiourea, urea, citric acid, and ATP) were detectable when 1% acetic acid in 50% (v/v) methanol was used as the sheath liquid.     

Analysis of phenolic compounds in extra virgin olive oil by using reversed-phase capillary electrochromatography

In this work, the simultaneous separation of ten phenolic compounds (protocatechuic, p-coumaric, o-coumaric, vanillic, ferulic, caffeic, syringic acids, hydroxytyrosol, tyrosol and oleuropein) in extra virgin olive oils (EVOOs) by isocratic RP CEC is proposed. A CEC method was optimized in order to completely resolve all the analyzed compounds by studying several experimental parameters. The influence of the stationary phase type (C(18) and C(8) modified silica gel), buffer concentration and pH as well as the organic modifier content of the mobile phase on retention factors, selectivity and efficiency were evaluated in details. A capillary column packed with Cogent bidentate C(18) particles for 23 cm and a mobile phase composed by 100 mM ammonium formate buffer pH 3/H(2)O/ACN (5:65:30 v/v/v) allowed the baseline resolution of the compounds under study in less than 35 min setting the applied voltage and temperature at 22 kV and 20 degrees C, respectively. A study, evaluating the intra- and interday precision as well as LOD and LOQ and method linearity was developed in accordance with the analytical procedures for method validation. LODs were in the range of 0.015-2.5 microg/mL, while calibration curves showed a good linearity (r(2) >0.997). The CEC method was applied to the separation and determination of these compounds in EVOO samples after a suitable liquid-liquid extraction procedure. The mean recovery values of the studied compounds ranged between 87 and 99%.     

Rapid separation and determination of carbamate insecticides using isocratic elution pressurized capillary electrochromatography

An isocratic elution pressurized CEC (pCEC) system was used to separate and determine ten carbamate insecticides. It was found that introduction of the electrical field, supplementary pressure, and SDS in the proposed method greatly improved the speed, column efficiency, selectivity, and repeatability for separation and determination of carbamates. On a capillary column of 75 microm ID packed with 3 microm octadecyl silica, baseline separation and detection of ten analytes was performed by using a mobile phase consisting of 30% v/v ACN and 70% v/v of 5 mmol/L ammonium acetate (pH 6.5) containing 1 mmol/L SDS and 0.01% triethylamine (TEA). Under the optimum conditions ten carbamate insecticides could be completely separated within 20 min. For the real vegetable samples, an SPE procedure for the cleanup of matrices was carried out prior to pCEC analysis. The detection limits of 0.05-1.6 mg/kg for ten carbamates and mean recoveries of 51.3-109.2% for eight kinds of vegetable samples at different concentrations of carbamates with RSD less than 11.4% were obtained, respectively. The proposed method has been proved to be effective in the rapid analysis of carbamate residues in vegetables.     

Application of polymeric surfactants in micellar electrokinetic chromatography-electrospray ionization mass spectrometry of benzodiazepines and benzoxazocine chiral drugs

Chiral micellar EKC (CMEKC) coupled to ESI-MS using polymeric surfactants as pseudostationary phases is investigated for simultaneous enantioseparation of two benzodiazepines, (+/-)-oxazepam ((+/-)-OXA) and (+/-)-lorazepam ((+/-)-LOR), and one benzoxazocine, (+/-)-nefopam ((+/-)-NEF). First, enantioselectivity and electrospray sensitivity of six chiral polymeric surfactants for all three chiral compounds are compared. Second, using poly(sodium N-undecenoyl-L-leucinate) as pseudostationary phase, the organic modifiers (methanol (MeOH), isopropanol, and ACN) are added into the running buffer to further improve chiral resolution (RS). Next, a CMEKC-ESI-MS method for the simultaneous enantioseparation of two benzodiazepines is further developed by using a dipeptide polymeric surfactant, poly(sodium N-undecenoxy carbonyl-L,L-leucyl-valinate) (poly-L,L-SUCLV). The CMEKC conditions including nebulizer pressure, capillary length, ammonium acetate concentration, pH, poly-L,L-SUCLV concentration, and capillary temperature were optimized to achieve maximum chiral RS and highest sensitivity of MS detection. The spray chamber parameters (drying gas temperature and drying gas flow rate) as well as sheath liquid conditions (MeOH content, pH, flow rate, and ionic strength) were found to significantly influence MS S/N of both (+/-)-OXA and (+/-)-LOR. Finally, a comparative study between simultaneous UV and MS detection showed high plate numbers, better chiral RS, and enhanced detectability with CMEKC-MS. However, speed of analysis was faster using CMEKC-UV.     

Capillary electrochromatography of biologically relevant flavonoids

Flavonoids were separated utilizing CEC technique. Baseline separation of biologically relevant flavonoids was obtained using a 100 microm ID fused-silica capillary filled with 3 microm Silica-C18 material and an optimized mobile phase comprising of 20 mM Tris-HCl (pH 6.5), ACN and water at a ratio of 10/40/50 v/v/v. Separations were carried out at 25 kV and a column temperature of 25 degrees C. The influence of relevant parameters for the CEC separation, such as buffer concentration, pH, separation voltage, and ACN concentration, was investigated and optimized. Dependencies of the electroendoosmotic flow (EOF) on these parameters and effects on the resolution of the analytes were studied. During analyses the solvents used for dissolving the samples turned out to have significant effects on the separation of flavonoids. The optimized system was then successfully used for the separation of the flavonoids epicatechin, myricetin, quercetin, naringenin, and hesperetin. CEC turned out to be a useful complementary tool for the economic analysis of flavonoids in addition to common HPLC, muHPLC, and CE methodologies. This method can be used for real applications in phytomics.     

Chromatographic behaviour of peptides on a mixed-mode stationary phase with an embedded charged group by capillary electrochromatography and high-performance liquid chromatography

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.     

Simultaneous enantioseparation and sensitive detection of eight beta-blockers using capillary electrochromatography-electrospray ionization-mass spectrometry

The feasibility of using vancomycin chiral stationary phase (CSP) and polar organic eluent is investigated for simultaneous enantioseparation of eight beta-blockers using CEC coupled to ESI mass spectrometric detection (ESI-MS). The internally tapered capillaries were utilized to pack CEC-MS columns. As compared to externally tapered columns, the use of internally tapered columns demonstrated enhanced stability, durability, and reproducibility. A mixture containing methanol/ACN/acetic acid/triethylamine at 70:30:1.6:0.2 v/v/v/v was considered as optimum mobile phase since it provided a good compromise between resolution and analysis time. As expected, sheath liquid and ESI-MS parameters mainly influenced the detection sensitivity. Interestingly, structural information of beta-blockers was available by varying the MS fragmentor voltage using in-house CID in the scan mode. In order to maximize the chiral/achiral resolution, various column-coupling approaches using teicoplanin as complementary CSP to vancomycin were tested. Several changes in the elution order of beta-blockers were observed using multimodal CSPs with some improvement in chiral or achiral resolution. The quantitative aspects of the CEC-MS method were demonstrated using R- and S-talinolol as internal standards. The calibration curves of beta-blockers showed good linearity in the range of 3-600 microM. The enantiomer of beta-blockers at a concentration of 30 nM was detectable. Furthermore, both 0.1 and 1% of the S-enantiomer could be precisely quantified in the presence of 99.9 and 99% of the R-isomer of beta-blocker.     

Rapid separation and sensitive detection method for beta-blockers by pressure-assisted capillary electrochromatography-electrospray ionization mass spectrometry

A new method for rapid separation and sensitive detection of beta-blockers by pressure-assisted capillary electrochromatography (pCEC) with electrospray ionization mass spectrometry (ESI-MS) using silica-based monolithic column was studied in this paper. The proposed method has been confirmed to be very powerful since the fast mass transfer property and good permeability of silica monolithic column was used in this pCEC-ESI-MS system. In this work, a silica monolithic column was prepared with sol-gel method for simultaneous fast separation of beta-blockers. Furthermore, in order to obtain the highly selective and sensitive result of pCEC-ESI-MS, both the CEC separation and MS detection parameters were optimized in detail. Under the optimized conditions, namely 80% acetonitrile and 20% 20 mmol/L ammonium acetate (pH 6.0) as the mobile phase, 20 kV and 8 bar as the separation voltage and the assisted pressure, isopropanol/water (1:1, v/v) containing 7.5 mmol/L acetic acid as the sheath liquid, and 3 microL/min as the flow rate of sheath liquid, seven beta-blockers were well separated within 11 min with detection limits in the range of 0.15-0.80 ng/mL (defined as S/N=3). The recoveries of spiked urine samples of these beta-blockers were between 86.3 and 103% with the RSDs lower than 8.0%. The real samples from some male volunteers were successfully analyzed and confirmed with the proposed method. Comparing with GC-MS or LC-MS, the new method has some superiority (such as fast analysis capacity and simple pretreatment) in clinical practice and doping control.     

Use of a polar-embedded stationary phase for the separation of tocopherols by CEC

A polar-embedded stationary phase (ULTIMA C18) has been investigated for the separation of alpha-, beta-, gamma- and delta-tocopherols by CEC in comparison with commercially available C(18) and C(30) n-alkyl RPs. The behavior of this stationary phase was tested for different mobile phases based on methanol, ACN, or mixtures thereof and different separation parameters such as retention factors and resolution were evaluated. The main feature of this stationary phase is the improved selectivity for the separation of beta- and gamma-tocopherols (positional isomers) when compared with the pure n-alkyl C(18) material, which was unable to resolve these compounds. Additionally, it is possible to observe a reversal in the elution order of the beta- and gamma-tocopherol isomers with respect to that obtained on the C(30) column. The resulting data indicate that the enhanced selectivity obtained with the polar-embedded stationary phase, with respect to the conventional C(18) material, is due to the participation of both hydrophobic and polar interactions: these latter are of the hydrogen bridge type with the amide group of the polar-embedded stationary phase, which increases the retention of the tocopherols and facilitates the discrimination between the beta- and gamma-isomers. Adequate separation of the four tocopherols was obtained by CEC using the polar-embedded stationary phase and 95:5 v/v methanol/water (5 mM Tris, final concentration) as the mobile phase.     

In-line coupling of low-pressure short capillary electrochromatography columns to electrospray ionisation mass spectrometry

A new in-house designed and constructed injection valve for capillary electrochromatography (CEC) based on a rotating injection part with compartments for the eluent as well as for the sample has been coupled to a mass spectrometer via a sheath flow electrospray ionisation (ESI) interface, using short capillary columns of 15 cm length. The CEC columns were packed with 3 microm C(18) bonded silica particles, and a mixture of peptides was analysed using an ammonium acetate/acetonitrile eluent. A significant increase in the signal-to-noise ratio was obtained when the peptides were dissolved in water with the same content of organic modifier as in the eluent with an addition of 0.5% (v/v) acetic acid. When the CEC analysis was performed without any additional pressure, the separation current sometimes dropped tremendously due to bubble formation, caused by different permeability in the first and packed part of the column causing an extremely low electroosmotic flow. The separation current was restored to its original value by applying only 7 bar at the inlet of the CEC column, and the separation performance for the test peptides was recovered. A comparison of the CEC performance of peptides in pure CEC mode and in low-pressure CEC mode is reported.     

Semipermanent capillary coatings in mixed organic-water solvents for CE

This report describes the creation of semipermanent capillary coatings that are compatible with organic-water solvent systems in CE. The coatings are created by simply rinsing the fused-silica capillary with long double-chain cationic surfactants, such as dimethyl-ditetradecyl ammonium bromide (2C(14)DAB), dihexadecyldimethyl ammonium bromide (2C(16)DAB), and dimethyldioctadecyl ammonium bromide (2C(18)DAB). These surfactants generate semipermanent bilayer coatings on the capillary surface, which display a high degree of stability in buffers containing up to 60% v/v of organic solvents, such as methanol and ACN. The coating stability increases with increasing hydrophobicity of the surfactant, i.e., with increasing chain length. For instance, the EOF changes by only 1.2% in a 2C(18)DAB-coated capillary after 130 capillary volumes of rinsing with 60% v/v methanol containing buffer. The bilayer coatings allow separations to be performed without the need to regenerate the coating between runs or to maintain the EOF modifier in the run buffer. Rapid separations (<2 min) of anions and basic drugs with migration time reproducibility of less than 0.5% RSD and efficiencies of 0.4-0.6 million plates/m are obtained. In addition, selectivity changes for small anions and cationic drugs are also observed when the organic solvent content is adjusted.     

Pressurized liquid extraction-capillary electrophoresis-mass spectrometry for the analysis of polar antioxidants in rosemary extracts

A method based on capillary electrophoresis-electrospray-mass spectrometry (CE-ESI-MS) was developed to qualitatively characterize natural antioxidants from rosemary (Rosmarinus officinalis L.) in different fractions obtained by pressurized liquid extraction (PLE) using subcritical water. The parameters of CE-ESI-MS were adjusted allowing the separation and characterization of different compounds from rosemary in the PLE fractions. These parameters for CE are kind, pH and concentration of the separation buffer, parameters for ESI-MS are dry gas temperature and flow, nebulizing gas pressure, and make-up flow. The following analytical conditions were found most favorable: aqueous CE buffer (40 mM ammonium acetate/ammonium hydroxide, pH 9); sheath liquid containing 2-propanol-water (60:40, v/v) and 0.1% (v/v) triethylamine at a flow rate of 0.24 mL/h; drying gas flow rate equal to 7 L/min at 350 degrees C, nebulizing gas pressure of 13.8 kPa (2 psi), using a compound stability of 50%. Different antioxidant compounds (e.g., rosmarinic acid and carnosic acid) could be detected in the rosemary extracts by CE-ESI-MS without any additional treatment, enabling the determination of variations in the extract composition caused by the different PLE conditions (i.e., 60 and 100 degrees C). The results provide complementary information to HPLC analysis.     

Separation of catechins and methylxanthines in tea samples by capillary electrochromatography

In this paper, the simultaneous separation of several polyphenols such as (+)‐catechin, (–)‐epicatechin, (–)‐epigallocatechin, theophylline, caffeine in green and black teas by capillary electrochromatography (CEC) was developed. Several experimental parameters such as stationary phase type, mobile phase composition, buffer and pH, inner diameter of the columns, sample injection, were evaluated to obtain the complete separation of the analysed compounds. Baseline resolution of the studied polyphenols was achieved within 30 min by using a capillary column (id 100 μm) packed with bidentate C₁₈ particles for 24.5 cm and a mobile phase composed of 5 mM ammonium acetate buffer pH 4 with H₂O/ACN (80:20, v/v). The applied voltage and the temperature were set at 30 kV and 20°C. Precision, detection and quantification limits, linearity, and accuracy were investigated. A good linearity (R² > 0.9992) was achieved over a concentration working range of 2–100 μg/mL for all the analytes. LOD and LOQ were 1 and 2 μg/mL, respectively, for all studied compounds. The CEC method was applied to the analysis of those polyphenols in green and black tea samples after an extraction procedure. Good recovery data from accuracy studies ranged between 90% and 112% for all analytes.     

Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides

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