On-line micellar electrokinetic chromatography-mass spectrometry: feasibility of direct introduction of non-volatile buffer and surfactant into the electrospray interface

An on-line method for the coupling of micellar electrokinetic chromatography (MEKC) and mass spectrometry (MS) is presented which allows conventional MEKC conditions to be employed without further modification. The MEKC system is coupled directly to electrospray ionization (ESI) MS using a triaxial interface. A systematic study of the influence of the surfactant concentration, the nature and concentration of buffer salts and presence of organic modifier on the interface performance indicated the feasibility of the MEKC–MS approach. Effective interfacing of MEKC was achieved with both single quadrupole and ion-trap MS instruments. Using a background electrolyte containing 20 mM sodium dodecyl sulfate (SDS) and 10 mM sodium phosphate buffer, it is demonstrated that full MEKC runs of test mixtures of mebeverine and related compounds can be monitored by ESI-MS with satisfactory sensitivity. Sub-μg/ml levels of the analytes can still be detected in full scan mode, while detection limits are in the 10–50 ng/ml range when selected ion monitoring is applied. It is shown that such sensitivity would allow full-scan MS detection of 0.1% (w/w) levels of potential impurities in mebeverine. With the ion-trap instrument successful MEKC–MS/MS experiments were carried out providing information-rich MS spectra of the related compounds. Repeated MEKC–MS analyses proved that in the course of 1 day the migration time of mebeverine remained fairly constant while the MS-signal intensity only gradually decreased to approximately 65% of its original value. Once-a-day cleaning of the first part of the ion source, which takes only 5 min, suffices to preserve an optimal interface performance for a prolonged period of time.     

Multivariate approach for the enantioselective analysis in MEKC‐MS: II. Optimization of 1,1′‐binaphthyl‐2,2′‐diamine in positive ion mode

Enantiomeric separation and detection of 1,1′‐binaphthyl‐2,2′‐diamine (BNA) has been successfully optimized by MEKC‐ESI‐MS using a polymeric surfactant polysodium N‐undecenoxycarbonyl‐L‐leucinate (poly‐L‐SUCL) as a pseudostationary phase. In the first step, MEKC conditions were optimized by a five‐factor three‐level central composite design (CCD) of experiment. All five MEKC factors (buffer pH, percentage of ACN in the running buffer, concentration of surfactant, concentration of ammonium acetate (NH₄OAc), and voltage) were found significant to the responses (measured as the chiral resolution and analysis time). The interactions between MEKC factors were further evaluated using a quadratic model equation which allowed the generation of 3‐D response surface image to reach the optimum conditions. To obtain the best S/N, sheath liquid composition and spray chamber parameters were successfully optimized using the same strategy. Baseline enantiomeric resolution in less than 20 min and optimum MS signal of BNA enantiomers (S/N = 45 at 0.4 mg/mL) were ultimately achieved at the optimized conditions. The adequacy of the model was validated by experimental runs at the optimal predicted conditions. The predicted results were found to be in good agreement with the experimental data.     

Comparison of sheathless and sheath-flow electrospray interfaces for the capillary electrophoresis-electrospray ionization-mass spectrometry analysis of peptides

Capillary electrophoresis coupled to mass spectrometry via an electrospray interface provides a powerful system for separation and characterization of a high number of biomolecules. The present paper describes a home-made sheathless interface and compares it with a commercial sheath-flow interface, using a separation method based on a peptide hormone mixture of therapeutic interest. In a previous work, we optimized the parameters involved in a sheath-flow interface and obtained good results in sensitivity and reproducibility. The sheathless interface is performed with a graphite-coated electrospray ionisation (ESI) tip attached to the separation capillary. We demonstrate that electrolyte composition is the main parameter affecting signal sensitivity and separation resolution. The effect of the nature and concentration of the organic solvent added to the separation electrolyte is carefully studied. Furthermore, a general comparison of both interfaces is made in terms of separation, reproducibility, and sensitivity obtained under the optimized conditions described. Advantages and disadvantages of both coupling setups have been evaluated.     

Micellar electrokinetic chromatography-mass spectrometry

The combination of micellar electrokinetic chromatography (MEKC) with mass spectrometry (MS) is very attractive for the direct identification of analyte molecules, for the possibility of selectivity enhancement, and for the structure confirmation and analysis in a MS-MS mode. The direct coupling of MEKC with MS can be hazardous due to the effect of nonvolatile MEKC surfactants on MS performance, including the loss of analyte sensitivity and ion source contamination. The possibility of off-line coupling between MEKC and matrix-assisted laser desorption/ionization (MALDI)-MS remains to be investigated. Various approaches for on-line coupling MEKC with electrospray ionization (ESI)-MS, including the use of high-molecular-mass surfactant, an electrospray-chemical ionization (ES-CI) interface, a voltage switching and buffer renewal system, partial-filling micellar plug and anodically migrating micelles, are reviewed and evaluated. The use of an ES-CI interface is most promising for routine operation of on-line MEKC-MS under the influence of nonvolatile salts and surfactants. The use of a high-molecular-mass surfactants allows the formation of a micellar phase at very low surfactant concentrations and avoids the generation of a high level of background ions in the low m/z region. Alternatively, the application of a partial-filling micellar plug and anodically migrating micelles eliminate the introduction of MEKC micelles into the ESI-MS system. It is possible to directly transfer the conventional MEKC separations to partial-filling MEKC-ESI-MS and MEKC-ESI-MS using anodically migrating micelles without any instrument modifications.     

Sodium maleopimaric acid as pseudostationary phase for chiral separations of amino acid derivatives by capillary micellar electrokinetic chromatography

A new type of chiral surfactant, sodium maleopimaric acid (SMA), was synthesized, and employed for the enantioselective micellar electrokinetic chromatographic (MEKC) separation of amino acid enantiomers derivatized with naphthalene-2,3-dicarboxaldehyde (NDA-D/L-AAs). The effect of the surfactant concentration, type and concentration of the BGE, and buffer pH on the resolution was studied, and optimized conditions were used to evaluate the ability of this new surfactant to perform chiral separations toward NDA-D/L-AAs by MEKC. Enantiomeric separations of NDA-D/L-AAs were achieved with a running buffer consisting of 100 mM borate (pH 9.5) and 20 mM SMA in a 58.5 cm length x 50 microm id capillary. Under the conditions selected, two pairs of tested amino acid enantiomers including NDA-D/L-trptophan (Trp) and NDA-D/L-kynurenine (Kyn) were resolved.     

Study of the electrical connection mechanism of sheathless interface for capillary electrophoresis‐electrospray ionization‐mass spectrometry

With the combination of high separation ability of capillary electrophoresis (CE) and strong identification ability of mass spectrometry (MS), CE/MS is becoming a powerful tool for polar and ionic analytes analysis. Different interfaces have been developed to enhance the sensitivity and reliability since the first introduction of CE/MS in 1987. A sheathless porous interface based on a new ions transferring electric connection technique was reported to be with high sensitivity and reliability. In this work, a series of optical and electrochemical experiments were designed to study the electric connection process. The results indicated that closing CE electrical circuit and applying MS spray voltage were achieved by the small ions transferring through the interface porous wall. The new electric connection method significantly enhanced the sensitivity, resolution and stability of the CE/MS analysis. The interface was applied in CE/MS detection of morphine and 6‐monoacetylmorphine in urine sample and showed an equal sensitivity to LC/MS. With the significant improvement of sensitivity and stability, the CE/MS with the new interface showed strong potential for the determination of low abundance analytes. Copyright © 2012 John Wiley & Sons, Ltd.     

Micellar electrokinetic capillary chromatography for the determination of Viagra and its metabolite (UK-103,320) in human serum

Micellar electrokinetic capillary chromatography (MEKC) was investigated for the determination of Viagra (sildenafil citrate, SC) and its metabolite (UK-103,320) in human serum in a concentration range of clinical interest. For MEKC, human serum samples spiked with SC and UK were obtained directly after elution with methanol from a tC18 cartridge. The extract was evaporated and regenerated in a solution 1 mM of phosphate buffer (pH 12.3) which contained a methanol percentage of 20% that was analyzed using phosphate buffer (pH 12.3, 10 mM) containing 30 mM sodium dodecyl sulfate (SDS) as separation electrolyte and a fused-silica capillary. This method gave satisfactory interday precision with respect to migration times relative standard deviation (RSD < 1%) and linear responses for the concentration ranges investigated (0.50-3.50 mg L(-1) for the compound SC and 0.90-4.60 mg L(-1) for UK). An intraday RSD (n = 5 graphs) between the slopes of the calibration graphs was acceptable (6.40%) for SC and (3.37%) for UK. A satisfactory interday precision between slopes was also obtained (RSD 4.10% for SC and a RSD 2.72% for UK) which demonstrated the ruggedness of this method. Detection limits (S/N = 3) were about 200 ng/mL for both compounds in human serum. MEKC was shown as a good method with regards to simplicity, precision and sensitivity.     

Phanquinone as a suitable derivatization reagent in micellar electrokinetic chromatography and HPLC analysis of amino acids

Phanquinone (chemically: 4,7-phenanthroline-5,6-dione) was applied as an original precolumn derivatization reagent for amino acids followed by separation using MEKC with UV detection (240 nm). The derivatization reaction was carried out at 68 degrees C in the presence of aqueous phosphate buffer (pH 8.0) and it was found to be complete after 30 min. Twelve derivatized standard amino acids were separated in about 22 min under MEKC conditions using sodium cholate (250 mM) as the surfactant in phosphate buffer (20 mM, pH 9.0). The developed method was validated for the analysis of D,L-phosphoserine (D,L-p-Ser) and L-glutamine (L-Gln); good linearity (r > 0.999) was achieved in the calibration range of 0.25-2.5 micromol/mL. The sensitivity of the MEKC method (LOD 0.1 micromol/mL; LOQ 0.25 micromol/mL, RSD% <5.0%, n = 3) was found to be adequate for quantitation of amino acids in pharmaceuticals. Quantitative applications of the validated MEKC method were carried out by the analysis of commercially available oral polyaminoacid formulations (tablets and extemporaneous solutions) containing L-Gln and D,L-p-Ser; the obtained results were found to be in agreement with those from a validated reference RP-HPLC method.     

Towards a general approach for the impurity profiling of drugs by micellar electrokinetic chromatography

A general micellar electrokinetic chromatographic (MEKC) strategy for the impurity profiling of drugs was developed involving a sodium dodecyl sulfate (SDS) and a cetyltrimethylammonium bromide (CTAB) MEKC system. With this combination, in principle, each sample component passes the detector in at least one of the two MEKC systems provided that separation buffers of the same pH are used in both systems. In order to select the proper MEKC systems, the electroosmotic flow (EOF) and micelle migration time (t(mc)) were determined for separation buffers of several pH values, containing various amounts of surfactant and organic modifier. The selectivity of the MEKC systems was studied using a mixture of compounds with a wide range of physico-chemical properties. The final selection of two adequate MEKC systems for this approach was based on the requirements that the t(mc) (i.e., analysis time) of both systems was below 20 min and that the t(mc)/t(eof) ratio was above 3 or 2 for the SDS and CTAB system, respectively. Furthermore, the systems should provide high efficiency, exhibit differences in selectivity and use moderate concentrations of modifier and surfactant, so that, if needed, further optimization is possible. The selected MEKC systems contained 60 mM SDS or 10 mM CTAB, respectively, in a phosphate buffer (pH 7.5) with 10% acetonitrile. Some test compounds with extreme mobilities were used to demonstrate the suitability of the MEKC approach to detect each component of a sample. The potential of the proposed MEKC combination for impurity profiling was demonstrated by the analysis of fluvoxamine with several impurities at the 0.1% level.     

A new sheathless electrospray interface for coupling of capillary electrophoresis to ion-trap mass spectrometry

A simple laboratory-made sheathless electrospray interface for coupling of capillary electrophoresis to ion-trap mass spectrometry (CE/MS) was developed. The interface was machined in-house and it was designed to be freely interchangeable with the commercially available ionization sources for the mass spectrometer. Sharpened fused-silica capillaries were coated with nickel by a simple electrodeless plating procedure and were used as all-in-one columns/emitters. The electrodeless plating produced a 2-5- micro m thick smooth nickel layer that lasted for more than 8 h of continuous electrospraying. The performance of the CE/MS interface was examined by using four cationic imipramine derivatives as test substances. Relative detection limits were calculated on the basis of the extracted ion electrophorograms and were in the range 6-130 nmol/L, corresponding to absolute detection limits in the range of 20-400 amol. The system was applied for analysis of impurities in an impure imipramine N-oxide preparation, and two of the impurities could be identified on the basis of online-MS(MS) spectra recorded in scan-dependent mode.     

Micellar electrokinetic chromatography of polychlorinated biphenyl congeners using a polymeric surfactant as the pseudostationary phase

Micellar electrokinetic chromatography (MEKC) of highly hydrophobic compounds is generally difficult using sodium dodecyl sulfate micellar solutions. The polymeric surfactant, polysodium undecyl sulfate (poly-SUS) has been used to separate moderately to highly hydrophobic polychlorinated biphenyl (PCB) congeners by MEKC in the absence of cyclodextrins. Parameters such as concentration of acetonitrile (ACN), polymeric surfactant concentration, and the effect of pH were examined. Optimum MEKC conditions to get baseline resolution of nine PCBs was 7.5 mM borate in 40% (v/v) ACN fraction buffered at pH 9.2 using 0.5% (w/v) poly-SUS. The applied voltage was 30 kV and the temperature was maintained at 25 degrees C. Elution order for each PCB congener was found to be dependent on the degree of chlorination and hydrophobic character.     

Use of high‐conductivity sample solution with sweeping‐micellar electrokinetic capillary chromatography for trace‐level quantification of paliperidone in human plasma

Paliperidone is a new antipsychotic drug with a relatively low therapeutic concentration of 20–60 ng/mL. We established an accurate and sensitive CE method for the determination of paliperidone concentrations in human plasma in this study. To minimize matrix effect caused by quantification errors, paliperidone was extracted from human plasma using Oasis HLB SPE cartridges with three‐step washing procedure. To achieve sensitive quantification of paliperidone in human plasma, a high‐conductivity sample solution with sweeping‐MEKC method was applied for analysis. The separation is performed in a BGE composed of 75 mM phosphoric acid, 100 mM SDS, 12% acetonitrile, and 15% tetrahydrofuran. Sample solution consisted of 10% methanol in 250 mM phosphoric acid and the conductivity ratio between sample matrix and BGE was 2.0 (γ, sample/BGE). The results showed it able to detect paliperidone in plasma samples at concentration as low as 10 ng/mL (S/N = 3) with a linear range between 20 and 200 ng/mL. Compared to the conventional MEKC method, the sensitivity enhancement factor of the developed sweeping‐MEKC method was 100. Intra‐ and interday precision of peak area ratios were less than 6.03%; the method accuracy was between 93.4 and 97.9%. This method was successfully applied to the analysis of plasma samples of patients undergoing paliperidone treatment.     

Analysis of ecdysteroids by micellar electrokinetic chromatography with on-line preconcentration

In order to enhance the UV detection sensitivity, an application study of an on-line preconcentration technique for micellar electrokinetic chromatographic (MEKC) was carried out. The simultaneous determination of four test ecdysteroids, 20-hydroxyecdysone, ajugasterone C, polypodine B and ponasterone A has been investigated by using the normal stacking mode in MEKC with UV detection. The effects of anionic surfactant composition and concentration, the applied voltage, the pH buffer, the kind and the amount of organic solvent and the injection time on the analyte resolution were evaluated. The optimised conditions for the separation involved the use of a 50 mM borate as the running buffer containing 50 mM of a mixture of sodium dodecyl sulphate (SDS) and sodium cholate (SC) in the ratio of 1:1 together with a concentration of 10% (v/v) of 2-PrOH at pH 9.0. Hydrodynamic injection of 12 s at 50 mbar and separation voltage of 20 kV at temperature of 20 degrees C were employed. These conditions allowed a repeatability separation within 21 min. Concentration detection limit for the neutral analytes studied improve about an order of magnitude. The method was also applied to the determination of ecdysteroids in a real sample.     

Separation of hydrophobic solutes by organic-solvent-based micellar electrokinetic chromatography using cation surfactants

In this study, the separation of 13 homologous stick-like hydrophobic solutes, i.e., biphenyl nitrile derivatives, by organic-solvent-based micellar electrokinetic chromatography (MEKC) was investigated in terms of separation medium composition, species and concentration of surfactant, other additives, separation voltage and temperature. The results showed that the 13 strong hydrophobic compounds were baseline separated in 25 min with a repeatability of less than 1.3% (RSD) for migration time. The separation medium was a mixture of methanol, 2-propanol and water (58.5:10:31.5), containing 150 mM cetyltrimethylammonium bromide (CTAB) and 20 mM sodium borate. Variety of solvent composition, temperature and applied voltage all showed remarkable effect on the separation. The organic-solvent-based MEKC method proved to be superior to the aqueous MEKC and microemulsion electrokinetic chromatography (MEEKC) methods for the separation of strongly hydrophobic compounds.     

Dodecyl thioglycopyranoside sulfates: novel sugar-based surfactants for enantiomeric separations by micellar electrokinetic capillary chromatography

Four novel chiral anionic surfactants having carbohydrate hydrophilic heads, sodium n-dodecyl 1-thio-beta-D-glucopyranoside 6-hydrogen sulfate (6-betaGlcD), sodium n-dodecyl 1-thio-beta-L-glucopyranoside 6-hydrogen sulfate (6-betaGlcL), sodium n-dodecyl 1-thio-beta-L-fucopyranoside 3-hydrogen sulfate (3-betaFucL), and sodium n-dodecyl 1-thio-alpha-L-rhamnopyranoside 3-hydrogen sulfate (3-alphaRhaL), were synthesized by selective sulfation of the corresponding thioglycosides. Their CMC determined by fluorescence using pyrene as a probe in water was 1.3-2.7 mM. These surfactants found to be useful as chiral selectors for enantiomeric separation by MEKC. The enantiomeric separation was optimized with respect to pH, buffer concentration, and surfactant concentration. Under the optimized conditions (50 mM phosphate buffer at pH 6.5, 30 mM surfactant, 20 kV), the enantiomeric separations of five dansylated amino acids (Dns-AAs) were achieved within approximately 20 min with the migration order of Val相似文献   

Direct coupling of micellar electrokinetic chromatography to mass spectrometry using a volatile buffer system based on perfluorooctanoic acid and ammonia

Direct coupling of micellar electrokinetic chromatography (MEKC) to mass spectrometry (MS) without employing partial filling is considered to be a challenge. One way of solving the problem would be the use of an MS-compatible surfactant. In the present study, the applicability of a series of surfactants (sodium dodecyl sulfate (SDS), lauric acid, cholic acid and perfluorated carboxylic acids) have been investigated both in terms of separation performance and MS compatibility. It was found that a MEKC system based on perfluorooctanoic acid (PFOA) and ammonia gave excellent results. The separation performance of the suggested system is comparable to the one obtained with standard systems based on SDS and sodium borate buffer although the selectivity is different. The electrospray ionization MS signal of the analytes is not seriously suppressed even at a PFOA concentration of 100 mM. Clusters are formed but their intensities are relatively low and comparable to those obtained with acetic acid. PFOA is volatile enough to allow long-term use, 30 h of continuous use has been recorded without any signs of decreasing performance. After use residual PFOA is easily removed from the ion-source (no memory effects). Furthermore, quantitation of trace impurities is possible at 25 ppb level when employing selected ion monitoring.     

A robust approach for the analysis of peptides in the low femtomole range by capillary electrophoresis-tandem mass spectrometry

A capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) approach has been developed for routine application in proteomic studies. Robustness of the coupling is achieved by using a standard coaxial sheath-flow sprayer. Thereby, greater stability than nanoelectrospray ionization-mass spectrometry coupling of sheathless capillary electrophoresis or nanoliquid chromatography (nano-LC) is achieved, resulting in stable operation for several weeks and unattended overnight sequences. The applied sheath flow is reduced to 1-2 microL/min in order to increase sensitivity. Standard peptides and those of digests of standard proteins and gel-separated proteins can be detected in the low femtomole range (full scan and MS/MS). Detection limits are found to be as low as 500 amol. Low femtomole amounts are required for unequivocal identification by MS/MS experiments in the ion trap and subsequent database search. By applying a simple pH-mediated stacking the concentration sensitivity can be lowered to some tens of fmol/microL (nM), depending on capillary size. This sensitivity is close to published values for sheathless CE-MS and nano-LC-MS, respectively (a comparison to reference values is presented). Moreover, with capillaries of about 50 cm in length separations in less than 10 min are possible resulting in a throughput of up to four analyses per hour. This is a factor of 4-12 times faster than nano-LC separation, being the state-of-the-art techniques for proteomic studies.     

Analytical potential of fluorescein analogues for ultrasensitive determinations of phosphorus-containing amino acid herbicides by micellar electrokinetic chromatography with laser-induced fluorescence detection

The analytical potential of three fluorescein analogues, fluorescein isothiocyanate isomer I (FITC), 5-(4,6-dichlorotriazinylamino) fluorescein (DTAF) and 5(6)-carboxyfluorescein N-succinimidyl ester (CFSE), as labelling reagents for the ultrasensitive determination of phosphorus-containing amino acid herbicides (glufosinate and glyphosate) and aminomethylphosphonic acid (the major metabolite of glyphosate) by nonionic surfactant micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection was investigated. Practical aspects related to label chemistry and MEKC separation showed that DTAF is the best choice for the determination of these herbicides; in addition, the most important features of these reagents for the derivatization of amino compounds are discussed. The optimum procedure includes a derivatization step of the herbicides at 40 degrees C with DTAF for 1 h and a 2-fold dilution prior to MEKC analysis, which is conducted within about 10 min using Brij-35 in the running buffer. This nonionic surfactant improves the selectivity and therefore the sensitivity of the method at low analyte concentrations by shifting the interfering peaks of the DTAF excess. The lowest detectable analyte concentration ranged from 0.06 to 0.16 microg/L with a precision of 2.1-3.2%. These results indicate that nonionic surfactant MEKC-LIF is useful as a selective, rapid and sensitive tool for the determination of these herbicides showing a great potential for their analysis in environmental samples without previous enrichment steps. The proposed method surpasses other chromatographic alternatives in terms of limit of detection and sample requirements for the analysis.     

Determination of nonylphenol and nonylphenol ethoxylates in wastewater using MEKC

Nonylphenol ethoxylates (NPEO_x) are surfactants which are used worldwide and can be transformed in the environment by microorganisms to form nonylphenol (NP). Analysis of these compounds was carried out with micellar electrokinetic capillary chromatography (MEKC). Different parameters such as background electrolyte (BGE) solution, pH, type of surfactant, and sample stacking were optimized. The use of CHES (20 mM, pH 9.1) in combination with 50 mM sodium cholate as a surfactant as BGE solution, together with sample stacking using 50 mM NaCl in the sample and an injection time of 20 s, provided the best separation of the compounds studied. The method was applied to the determination of target analytes in two types of sludge water coming from two steps of a wastewater treatment plant. Liquid–liquid extraction was carried out using toluene as solvent, resulting in recoveries around 100% for all studied analytes. The presence of NPEO_x was observed in the first step of the sludge water treatment, based on migration time and UV spectra. Identification was confirmed using tandem MS. LOQs of the studied compounds were in the range of 12.7 to 30.8 ng/mL, which is satisfactory for the analysis of real wastewater samples.     

Separation of basic compounds of pharmaceutical interest by using nano-liquid chromatography coupled with mass spectrometry

Nano-liquid chromatography-mass spectrometry (nano-LC-MS) was evaluated for the separation of basic compounds of pharmaceutical interest. The separation of selected beta-blockers, namely nadolol, oxprenolol, alprenolol and propranolol in the presence of terbutaline was performed using two 75 microm I.D. capillaries packed with two different RP18 stationary phases (SP). The best results concerning resolution and efficiency were achieved using the SP where free silanol groups were not present. As expected, this latter SP proved to be very efficient and symmetry factors were observed mainly in the case of the more retained analytes. Baseline resolution of all studied basic compounds was achieved with the Cogent bidentate C18 silica phase (CBC18) eluting analytes at 800 nL/min with a mobile phase containing 500 mM ammonium acetate pH 4.5-water-methanol (1:8:91, v/v/v). The separated basic compounds were revealed using on-column UV detector at 205 nm and electrospray-ion-trap mass spectrometer (ESI-MS). The packed capillary was connected to the MS through a commercial sheath liquid interface or a sheathless nano-spray interface and in both cases the sensitivity was studied and the results compared. Limit of detection (LOD) as low as 0.1 ng/mL was measured for nadolol using the sheathless nano-spray interface and the capillary column packed with the CBC18 stationary phase.