Studies on the analytical performance of a non-covalent coating with N,N-didodecyl-N,N-dimethylammonium bromide for separation of basic proteins by capillary electrophoresis in acidic buffers in 25- and 50-microm capillaries.

Capillaries (25- and 50-microm inner diameter) coated with a double-alkyl-chain cationic surfactant N,N-didodecyl-N,N-dimethylammonium bromide (DDAB) were used for the separation of four basic standard proteins in buffers of pH 4 at various ionic strengths. The choice of buffer is critical for the analytical performance. Ammonium ions must be avoided in the buffer used in the non-covalent coating procedure owing to competition with the surfactant. Phosphate buffer gave a better separation performance than some volatile buffers; the reason seems to be a complex formation between the proteins and dihydrogenphosphate ions, which decreases tendencies for adsorption to the capillary surface. The DDAB coating was easy to produce and stable enough to permit, without recoating, consecutive separations of the proteins for up to 100 min with good precision in migration times and peak areas. A strong electroosmotic flow gives rapid separations, which is of special importance when commercial instruments are used, since the choice of the length of the capillary is restricted.

Capillary electrochromatography of tricyclic antidepressants on strong cation exchangers with different pore sizes.

Four cation-exchange materials, possessing propanesulfonic acid ligands, for use in capillary electrochromatography were prepared from different commercially available 5-microm bare-silica particles ranging from 80 to 800 A in pore size. The performance of the materials was investigated at different compositions of the mobile phase (pH, ionic strength, and acetonitrile content) using tricyclic antidepressants and related quaternary ammonium analogues as test analytes. The wide-pore materials promoted pore flow, but this had no positive influence on the performance. The small-pore (highest surface area) particles gave, as could be expected, the best selectivity.

Capillary electrochromatography of hydrophobic amines on continuous beds.

The capillary electrochromatographic separation performance of hydrophobic amines and a related quaternary ammonium compound on continuous beds based on polymers of acrylamide has been studied. The chromatographic bed is polymerized in situ and the character of the polymers with regard to hydrophobicity and charge has been systematically changed by regulating its content of isopropyl and sulfonate ligands, respectively. The best performance was obtained for columns with a molar ratio of 1:80 for the sulfonate and isopropyl groups, and resulted in efficiencies up to 200000 plates per meter. The effects on retention, resolution and elution order by ionic strength, pH, and content of acetonitrile in the mobile phase have been investigated. The quaternary ammonium compound was always the least retained irrespective of pH. By increasing the pH, a reversal of the migration order between the tertiary and secondary amine was obtained. The results indicate a complex migration/retention mechanism where ion-exchange, adsorption and electrophoretic mobilities play a role. The concentration limit of detection could be lowered from 1.3 microg/mL to 50 pg/mL by using a high content of 2-propanol (96%) in the sample compared to dissolving the analytes in the mobile phase.

Effects of aliphatic amines on capillary electrochromatographic performance of tricyclic antidepressants on octadecylsilica.

Adding aliphatic amines to the mobile phase improves peak symmetry and efficiency in capillary electrochromatography of tricyclic antidepressants on octadecylsilica. The most hydrophobic aliphatic amine studied, dimethyloctylamine (DMOA), was the most efficient. Despite the fact that the amine additives substantially reduced the electroosmotic flow, the retention of the analytes decreased indicating a strong competitive effect of the additives. DMOA gave the largest retention decrease, and simultaneously reduced the resolution, indicating that silanophilic interaction is significant to the separation. Highest efficiencies were obtained at the lowest pH (2.8). Acetonitrile influenced both efficiency and peak symmetry, and best results were obtained at 60%.

Direct analysis of artemisinin in plasma and saliva using coupled-column high-performance liquid chromatography with a restricted-access material pre-column.

A previously established HPLC system with post-column derivatization for the analysis of artemisinin was coupled to an ADS (alkyl-diol silica) pre-column, allowing direct and repetitive injection of protein-rich fluids such as plasma. The limit of quantitation for 100 microl of plasma was 10 ng/ml (CV=10.5%) while concentrations down to 2 ng/ml could be quantified for 1.00 ml saliva samples (CV=11.1%). The system was linear in the tested range of 10-2000 ng/ml for plasma and 2-240 ng/ml for saliva samples, respectively. This paper introduces coupled column HPLC as a simplified method for the routine analysis of artemisinin in biological fluids.

Determination of the enantiomeric purity of S-ropivacaine by capillary electrophoresis with methyl-beta-cyclodextrin as chiral selector using conventional and complete filling techniques.

Capillary electrophoresis (CE) methods based on the conventional and complete filling techniques for determination of the enantiomeric purity of S-ropivacaine are described. The complete filling technique is a separation method which can be used instead of the partial filling technique in order to reduce the total analysis time, when the chiral selector solution does not absorb UV light. In the complete filling technique the total length of the capillary is filled with the chiral selector solution, prior to application of the analyte. During the run both ends of the capillary are connected to the background electrolyte, i.e. without chiral agent. An interlaboratory study was performed to validate the method. The limit of detection and quantification for R-ropivacaine were found to be about 0.6 and 1.6 microg/ml, respectively, corresponding to 0. 1 and 0.25% enantiomeric purity of S-ropivacaine. Good performances were demonstrated for the repeatability and linearity. The consumption of the chiral selector was about 160 times lower with the complete filling technique compared with the conventional CE technique.

Simultaneous analysis of endogenous neurotransmitters and neuropeptides in brain tissue using capillary electrophoresis--microelectrospray-tandem mass spectrometry.

Capillary electrophoresis was combined with highly sensitive microelectrospray-tandem mass spectrometry to simultaneously detect classical small molecule neurotransmitters as well as neuropeptides from discrete regions of the marmoset brain. A mixture of four classical neurotransmitters (glutamate, gamma-aminobutyric acid, acetylcholine, dopamine) and four neuropeptides (neurotensin, methionine-enkephalin, leucine-enkephalin and substance P 1-7) was studied to optimize the capillary electrophoresis conditions for separation, injection volume, and analysis time. Gamma-aminopropyltriethoxysilane-coated capillaries and acetic acid electrolytes were used to avoid interactions between the sample and the capillary surface and to obtain a high anodic electroosmotic flow, which resulted in a short analysis time. Detection was performed using tandem mass spectrometry in the selected reaction monitoring mode using a triple quadrupole mass spectrometer. Samples were dissolved in ammonium acetate to achieve a transient-isotachophoretic concentration step at the beginning of the separation and to make it possible to inject larger sample volumes, up to 140 nL. Small amounts of tissue from specific regions of the marmoset monkey brain were pretreated using solid-phase extraction as a clean-up and concentrating step. In the striatum we could detect endogenous glutamate, gamma-aminobutyric acid (GABA), acetylcholine and dopamine, as well as the neuropeptides methionine-enkephalin and substance P 1-7 in the same analysis, using only 58 mm3 of brain tissue.

Determination of association constants between enantiomers of orciprenaline and methyl-beta-cyclodextrin as chiral selector by capillary zone electrophoresis using a partial filling technique.

The principles for the determination of conditional association constants of enantiomers by capillary zone electrophoresis employing a partial filling technique (PFT) using methyl-beta-cyclodextrin as chiral selector is presented. Orciprenaline was used as a model compound. Partial filling is a separation technique, where different lengths of the chiral selector solution are introduced into the capillary to a final zone length shorter than the effective length of the capillary, prior to application of the solutes. Lengthening of the separation zone results in improving enantioresolution in addition to decreasing electrophoretic mobility of the enantiomers, because of longer interaction time between the solute and chiral selector. The degree of the reduction in electromobility depends on the affinity of the solute to the chiral selector, i.e. strength of the complex formed between the solute and cyclodextrin. The decrease in the electrophoretic mobility with increasing length of the separation zone is used for determination of the association constant. The association constants of the enantiomers of orciprenaline and the chiral selector were evaluated from the slope of the plot, observed electrophoretic mobility versus the ratio between the length of the separation zone and the effective length of the capillary. It was found that the association constants were independent of the chiral selector concentration. The mean values were 110 M(-1) and 160 M(-1) for respective enantiomer. Constants obtained by a conventional CE technique were in good agreement with those from the PFT experiments. The highest enantioselectivityy was obtained when about 50% of the solute was distributed to the selector phase.

Simultaneous separation and enantioresolution of racemic local anesthetic drugs by capillary zone electrophoresis with Tween 20 and methyl-beta-cyclodextrin as selectors, employing a double plug technique.

A new approach for simultaneous chiral and achiral separations by capillary zone electrophoresis is described. Two adjacent selector plugs, consisting of Tween 20 as an achiral and methyl-beta-cyclodextrin (CD) as a chiral selector, are employed and four related local anesthetics are used as model compounds. The principles of the partial filling technique, whereby the capillary is filled with the chiral selector solution followed by the micellar solution at different plug lengths and concentrations, prior to application of the solutes, was employed. During the run both capillary ends were dipped in a simple buffer, i.e., one without additives. The two separation media worked independently without any interaction. Separation of the solutes and their enantiomers was regulated by adjusting both the concentration and plug length (PL) of the micellar solution in the capillary, employing methyl beta-CD as chiral selector either at 38 or 76 mM. The solutes were separated on the basis of their affinity towards the micellar phase before they reached the methyl-beta-CD plug for enantioseparation. In the absence of the micellar plug, the enantiomers of prilocaine overlapped those of bupivacaine. The solutes and their enantiomers were completely separated by employing two adjacent plugs consisting of 100 mM Tween 20 solution (PL approximately 10 cm) and methyl-beta-CD solution at either 38 or 76 mM (PL approximately 30 cm).

Separation of a range of cations by nonaqueous capillary electrophoresis using indirect and direct detection.

The use of nonaqueous media and indirect detection is reported for the separation and detection of a range of small cations. The novel applications involved separation of a range of metal ions, small nonchromophoric amines, cationic ion-pair reagents and cationic surfactants. Separations were achieved using acidified methanol containing imidazole as the UV co-ion for indirect detection. The methods produced different selectivity compared to aqueous methods using acidified aqueous imidazole solutions. Advantages of the methods include speed of analysis and prevention of sample micellerisation. The methods were shown to be quantitative and reproducible by their application to the determination of Tris content.

Response patterns with indirect UV detection in capillary zone electrophoresis.

Capillary zone electrophoresis with indirect UV-detection was used to separate mixtures containing both positively and negatively charged species. In order to understand the dependence of detector response patterns on the changes in compositions of the background electrolytes and the charge of marker ions (UV-absorbing ions), the separations were performed in two different systems. In a three-ion system (analyte ion, coion and counterion) a marker ion was the major ionic component of a buffer solution and in a two-coion or counterion system the marker ion was used as an additive. In the three-ion system the response profile of an analyte was in good agreement with the mathematical treatment based on the Kohlrausch regulation function. In the two-coion or counterion system the response patterns were more complicated; however, the experimental results agree well with data obtained from a computer simulation program. Peak directions of the analytes were not only determined by their relative charge to the marker ion, but were also associated with their relative mobilities to the buffer coion and the marker ion. The analytes with higher effective mobilities compared to the marker ion were detected as positive peaks and the ones with lower effective mobilities as negative peaks. Similarly to the three-ion system, the detector response of an analyte was stronger by applying a marker coion compared to a counterion. An interesting result was obtained in the separation of a mixture of quaternary ammonium ions and sugars by using a cationic marker ion. The highest and most symmetrical peak was not a cation, but raffinose anion, which appeared most closely to the system peak. The observation suggests that the electromigration dispersion in its zone was eliminated by migrating close to the electroosmosis. A system peak with the mobility corresponding to the electroosmotic flow was obtained in both systems, and an additional system peak with a mobility close to that the marker ion was present in the systems using marker ions as additives.

Detectability improvements in capillary zone electrophoresis by combining single capillary isotachophoretic preconcentration and frequency doubled argon ion laser-induced fluorescence detection.

Due to the small path length and low injection volume the concentration limit of detection is comparatively poor in capillary electrophoresis (CZE) with UV detection. This limitation can be overcome by means of preconcentration methods and/or improved detection techniques. This paper describes a strategy where isotachophoresis (ITP) is used to preconcentrate a new cholinesterase inhibitor (NXX-066) prior to a capillary zone electrophoresis analysis in the same single capillary. A hydrodynamic backpressure is used to prevent the analyte from migrating out of the capillary. Laser-induced fluorescence (LIF) is used to further increase the detectability. The total gain in detectability with ITP-CZE-LIF compared to CZE-UV was at least 5500-fold, and it is possible to determine NXX-066 at the 1 nM level. The ITP-CZE method was further evaluated for two beta-blockers; the mean coefficient of variation of the peak areas was 3.4% and the linearity of the calibration plots was satisfying.

Enantioseparation of anaesthetic drugs by capillary zone electrophoresis using cyclodextrin-containing background electrolytes.

The enantiomers of five racemic anaesthetic drugs were resolved with cyclodextrins using capillary zone electrophoresis. Parameters which affected the chiral resolution, such as type and concentration of cyclodextrin, temperature, and addition of organic modifier were investigated. The results show that the enantiomeric discrimination of the solutes is influenced by the structural shape of the solute molecules, separation temperature, and type of cyclodextrin. It was found that alpha-cyclodextrin was the best enantioselector for resolution of prilocaine and ketamine, while the enantiomers of mepivacaine, ropivacaine, and bupivacaine were resolved with beta-cyclodextrin and/or modified beta-cyclodextrins, i.e., methyl- and 2-hydroxypropyl-beta-cyclodextrin, as chiral selectors. The length of the alkyl chain on the amino group of the drug molecule had a strong effect on the enantioresolution of mepivacaine, ropivacaine, and bupivacaine. Baseline separation of racemic ketamine was achieved with alpha- and methyl-beta-cyclodextrin at 15 degrees C. Addition of 5 M urea to the running buffer containing beta-cyclodextrin at high concentrations resulted in the enantioseparation of prilocaine, mepivacaine, and ketamine. Enantioresolution was improved upon the addition of 10% methanol to the buffer containing urea and beta-cyclodextrin. Generally, the complex formed between the S-enantiomers and modified beta-cyclodextrins was stronger than the corresponding R-forms. An exception was prilocaine where the R-form gave a more stable complex both with alpha- and beta-cyclodextrin.

Evaluation of association constants between drug enantiomers and human alpha 1-acid glycoprotein by applying a partial-filling technique in affinity capillary electrophoresis.

The principles for evaluation of conditional association constants between drug enantiomers and proteins, exemplified here by alpha 1-acid glycoprotein (AGP), using capillary zone electrophoresis employing a partial filling technique, is presented. In the partial filling technique only the first part of the capillary is filled with the selector, and this selector zone (plug) length can be varied by introducing the selector solution at different times at constant pressure. An important feature of the technique is the low consumption of selector solution in this study only 40-290 nL is used per run, of special importance when the availability of the selector is limited, and also in case it is expensive. Conditions are chosen so that the protein has a net negative charge and migrates toward the anode, while the analytes migrate toward the detector at the cathodic side. The resolution is linearly related to the effective plug length, as shown in separations of the enantiomers of disopyramide and remoxipride. The effective plug length can be calculated, which forms the basis to apply this technique for determinations of association constants. The association between the enantiomers of the solutes and AGP varied with increasing temperature, as shown by determined association constants. It was found that the association between the enantiomers and AGP was strongest at 25 degrees C and decreased at both lower and higher temperatures. This unexpected finding may indicate conformational changes of the protein with temperature variations.

Enantioresolution of disopyramide by capillary affinity electrokinetic chromatography with human alpha1-acid glycoprotein (AGP) as chiral selector applying a partial filling technique.

A method using alpha1-acid glycoprotein (AGP) as chiral selector for disopyramide by means of affinity electrokinetic chromatography has been developed. In order to avoid UV absorbance interferences, less than the effective length of the capillary was filled with the chiral selector. The electrophoretic conditions were chosen to give opposite migration directions for the chiral selector and the analyte; AGP migrated away from the detector. Enantiomers of disopyramide were separated on a methylcellulose-coated capillary with 20 cm length to the detector. The enantioresolution of the solute was affected by the concentration of the chiral selector, the plug length of the selector in the capillary, and the applied voltage. Resolution factors and migration times decreased with reduction of the plug length, while the efficiency of the separation system and peak performance were improved by decreasing the separation zone. A special feature of the technique is an enhanced selectivity due to increasing separation of the enantiomers when the fastest has migrated from the selector zone, while the second one still is retained. Equations relating selectivity and resolution with the difference in effective plug lengths between the two enantiomers are developed. Optimized conditions yielding complete resolution, requiring an 0.75 mM AGP plug of only 4.5 cm effective length, also gave high efficiencies (about 400,000 plates/m) for both enantiomer peaks.

Determination of methotrexate and its metabolite 7-hydroxymethotrexate by direct injection of human plasma into a column-switching liquid chromatographic system using post-column photochemical reaction with fluorimetric detection.

A simple, sensitive and fully automated column-switching system by direct injection of plasma samples for determination of methotrexate and its metabolite 7-hydroxymethotrexate was developed. The system utilized a C8 alkyl-diol silica precolumn coupled with a LiChrospher RP-18 analytical column, followed by a photoreactor and fluorimetric detection. The photo-oxidative irradiation was accomplished at UV 254 nm in the presence of 0.1% hydrogen peroxide in the eluent. Studies showed that the fluorimetric response was influenced by the reaction time, the degree of the reactor's transparency and the choice of the working wavelengths. By optimizing the content of acetonitrile in the eluent, methotrexate can be separated from 7-hydroxymethotrexate completely. The method validation revealed quantitative recoveries (> or = 94%) with coefficients of variation < or = 4.4%. The limits of detection and quantitation for determination of methotrexate were 0.20 and 0.36 ng, respectively, corresponding to 2.0 and 3.6 ng/ml for an injection volume of 100 microliters. It was possible to enhance the sensitivity further by injecting larger plasma volumes, up to 500 microliters.

Evaluation of liquid chromatographic behavior of restricted-access media precolumns in the course of direct injection of large volumes of plasma samples in column-switching systems.

The chromatographic behavior of an alkyl-diol silica (ADS, 25 x 4 mm I.D.) and a semipermeable surface (SPS, 10 x 10 mm I.D.) supports two types of restricted-access media (RAM), which served as precolumns in column-switching systems for direct injection of large volumes of plasma samples (500 microl), was studied with regard to peak performance, retention and column back pressure. The adsorption of matrix proteins both on sealings (porous frits and sieves) and packings was also examined. Columns of ADS and SPS were unchanged after the injection of 10-20 ml human plasma under normal working conditions. Even when changes occurred on the precolumns (>50 ml of plasma in total), it was still possible to regenerate the column performance by replacing the column sieves, or by washing and removing columns from the system for a period, since the changes were more related to the blockage of sealings and/or the adsorption of proteins on the hydrophilic surfaces. Proteins could eventually be unspecifically adsorbed on the hydrophobic ligand of the support. It was found on one ADS column that the retention decreased by 20% and the pressure increased 30 bar after an intensive loading of 75 ml plasma (injection volume, 500 microl) without reconditioning procedure. Studies showed that the column sealings played the most important role for the lifetime of RAM columns. For ADS columns, using sieves without polytetrafluoroethylene (PTFE) nets were the best. No significant difference in column life span between SPS and ADS was found.

Ion-pair chromatography of methotrexate in a column-switching system using an alkyl-diol silica precolumn for direct injection of plasma.

The retention behaviour of methotrexate as an ion-pair with tetrabutylammonium in a column-switching system, based on an alkyl-diol silica C8 precolumn, combined with an analytical column, LiChrospher RP 18, was studied. Methotrexate is mainly present as a divalent anion at pH 7.4, however, the retention data was consistent with the formation of a 1 + 1 ion pair with the counter ion. The concentration of the tetrabutylammonium and the acetonitrile in the mobile phase could be used to regulate the retention in the system. Relevant chromatographic parameters to estimate the enrichment effect in the column-switching system are also identified and discussed. The column-switching system was applied to direct injection of plasma (100 microliters) giving a limit of detection of 10 ng/ml for methotrexate using UV detection at 307 nm.

Direct injection of large volumes of plasma in a column-switching system for the analysis of local anaesthetics. I. Optimization of semi-permeable surface precolumns in the system and characterization of some interference peaks.

Possibilities for accomplishing direct injection of large volumes (500 microliters) of plasma samples into a column-switching HPLC system were investigated. A new format of precolumn containing a semi-permeable surface (SPS) support (1 cm x 1 cm) was used for the sample clean-up and trace enrichment and was combined with a Kromasil C18 column for the final separation. A stable chromatographic system with respect to the separation selectivity and separation time was constructed and evaluated. The main parameters were the hydrophobicity of the SPS column, pH of the eluents, concentration of the organic modifier in the eluents and the detection wavelength. Two main interference peaks that were eluted in front of the ropivacaine peak were systematically characterized by varying the loading conditions for the SPS precolumn. The SPS column could tolerate large volumes (< or = 500 microliters) of plasma injections with a total volume of more than 50 ml. The developed system is stable, which permits the detection of 30 ng/ml ropivacaine in human plasma.

Direct injection of large volumes of plasma in a column-switching system for the analysis of local anaesthetics. II. Determination of bupivacaine in human plasma with an alkyldiol silica precolumn.

A column-switching high-performance liquid chromatographic system was applied for the determination of bupivacaine in plasma. A 500-microliter plasma sample was directly introduced onto a C18-alkyl-diol silica (ADS) precolumn separating analytes from proteins and polar endogenous compounds. The fraction containing bupivacaine and ropivacaine (internal standard) was back-flushed and transferred to a conventional reversed-phase column (Kromasil C18) for final separation. A single ADS precolumn could withstand more than 50 ml of plasma injections without changing analytical performance. Quantitative studies showed a broad range of linearity (0.033-3.31 micrograms/ml) and high recovery (95-99.9%) with coefficients of variation less than 3.1%. The advantages of the ADS material are its high capability of sample clean-up, due to rapid elution of plasma proteins and endogenous compounds to waste, and its ability to elicit a stable baseline. As a result, UV detection could be performed at 210 nm and clean chromatograms with baseline separation for desired peaks were obtained within 15 min. The detection limit of this system was 10 ng/ml defined by a signal-to-noise ratio of 3:1. The concentration of bupivacaine in patients determined by this method agreed well with the values obtained from an alternative method, making the technique applicable for pharmacokinetic studies in humans.