Ahmad Amini

Recent developments in chiral capillary electrophoresis and applications of this technique to pharmaceutical and biomedical analysis

This paper provides an overview of the current status of chiral capillary electrophoresis (CE). The emphasis is placed on the application of CE in chiral separation of various racemic compounds. During the last two years about 280 papers, several review articles, and two entire issues, edited by S. Fanali (Electrophoresis 1999, 20, 2577–2798, and H. Nishi and S. Terabe (J. Chromatogr. A 2000, 879, 1–471.) have been devoted to chiral CE. Enantiomeric separations of various compounds, e.g., pharmaceuticals, drug candidates, drugs and related metabolites in biological fluids, amino acids, di‐ and tri peptides, pesticides and fungicides, have been performed using different chiral selectors. Native and derivatized cyclodextrins continue to be the most widely used chiral selectors. Other chiral selectors such as natural and synthetic chiral micelles, crown ethers, chiral ligands, proteins, oligo‐ and polysaccharides, and macrocyclic antibiotics have also been applied to chiral CE separations.

Principle and applications of the partial filling technique in capillary electrophoresis

SummaryThe partial filling technique (PFT) in capillary electrophoresis (CE) is an efficient system where, only 50–800 nanolitres of a chiral selector solution needs to be added to each run. PFT is especially applicable when these additives to the background electrolyte (BGE) are expensive or absorb UV light. The selector dissolved in the BGE is applied to the capillary as a plug, shorter than the effective length of the capillary, prior to application of the analyte. During the run both ends of the capillary are connected to the BGE. The applied plug and the analyte may move in opposite directions or in the same direction at different velocities depending on their electrophoretic mobilities. Thus the final plug length is either longer or shorter than the original length. The technique has been successfully applied in a number of studies including enantiomeric separation with a variety of selectors, and for the determination of conditional association constants.

Chiral separation of local anaesthetics by a capillary electrophoresis/partial filling technique coupled on-line to micro-electrospray mass spectrometry

A novel procedure for analysing chiral compounds is described in which a capillary electrophoresis/partial filling technique was coupled on-line to microelectrospray mass spectrometry. Enantiomers of bupivacaine and ropivacaine were separated using methyl-β-cyclodetrin, a non-volatile buffer additive, as a chiral selector. To avoid introduction of the selector into the ion source, the capillary was coated with polyacrylamide to minimize the electroosmotic flow. Prior to sample injection, the capillary was partially filled with cyclodextrin dissolved in acetate buffer. When 30 kV was applied, the positively charged enantiomers migrated towards the mass spectrometer through the zone containing the neutral selector, which acted as a pseudo-stationary phase. A racemic mixture of bupivacaine and ropivacaine was successfully separated and an impurity of 0.25% of (R)-ropivacaine in (S)-ropivacaine could be detected.

Enantiomeric separation of local anaesthetic drugs by micellar electrokinetic capillary chromatography with taurodeoxycholate as chiral selector

Enantiomeric separation of local anaesthetic drugs by micellar electrokinetic capillary chromatography with taurodeoxycholate as chiral selector

Determination of association constants between enantiomers of orciprenaline and methyl-β-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‐β‐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.

Quantitative determination of salbutamol in tablets by multiple-injection capillary zone electrophoresis

A multiple-injection capillary zone electrophoresis (MICZE) method has been developed for the assay of salbutamol in Ventoline Depot tablets (GlaxoSmithKline). In the developed method, seven sample sets, each consisting of three samples, were sequentially injected into the capillary and analyzed within a single run. This enabled a total of twenty-one sequential injections, i.e., six standards and fifteen samples, containing salbutamol and the injection marker oxprenolol. The injected sample plugs were separated by plugs of background electrolyte, through application of a short-term voltage (30kV) over the capillary for different time periods, i.e., t(PE1) and t(PE2). The samples in each set were isolated from each other by partial electrophoresis for 2.35min (t(PE1)), while the sample sets were separated for 10.50min (t(PE2)). After the final injection, all the applied samples were subjected to electrophoresis for a time period corresponding to that in conventional single-injection CZE. The method was validated regarding linearity, accuracy, precision and robustness before it was applied to the determination of salbutamol in 15 tablets of Ventoline Depot with a labeled content of 8mg salbutamol. The average salbutamol content was determined to 7.8mg (+/-0.3mg) from simultaneous analyses of the 15 different tablets.

Determination of the enantiomeric purity of S-ropivacaine by capillary electrophoresis with methyl-β-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 separation and enantioresolution of racemic local anesthetic drugs by capillary zone electrophoresis with Tween 20 and methyl‐β‐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‐β‐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 β‐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‐β‐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 ≈ 10 cm) and methyl‐β‐CD solution at either 38 or 76 mM (PL ≈ 30 cm).

Principles for different modes of multiple-injection CZE

This paper introduces four different modes of multiple‐injection CZE (MICZE). The validity of these MICZE models was evaluated by the experimental data. Prior to the application of MICZE, the electrophoretic conditions are developed in the single‐injection mode by adjusting different experimental parameters such as pH, type and concentration of buffer additives and temperature. Based on the migration time difference (Δtmig) between the analyte and the internal standard or injection marker, one or more MICZE modes can be employed. The injection marker is added to the sample to compensate for injection‐volume fluctuations. The inter‐plug distance is regulated by applying an electrical field over the capillary for a short period of time between each injection. After the final injection, the separation is completed by electrophoresis for a time period corresponding to that in the single‐injection mode.

Dependence of chiral separations on the amount of cyclodextrins as selectors, employing the partial filling technique in capillary zone electrophoresis

SummaryThe enantioselectivity, resolution factor and migration times for some local anaesthetic drugs were evaluated at different selector concentrations and plug lengths with different cyclodextrins as chiral selectors using the partial filling technique. The resolution remained constant when equal amounts of the chiral selector at a concentration below the optimal concentration were introduced in the capillary. However, the selectivity factor increased with increasing concentration of the chiral selector.