Yoshihide Tanaka

Estimation of binding constants by capillary electrophoresis

Capillary electrophoresis (CE) has become a useful technique for measuring binding constants. This review is focused on recent trends in the estimation of binding constants by affinity CE. First, we introduce several mathematical equations in which it is assumed that the stoichiometry of the binding between drug and protein is 1:1 as a simple model. In order to calculate accurate binding constants by affinity CE, several experimental considerations are described in this review. In addition, some recent methodologies, such as partial filling technique and multiple-step ligand injection method, are introduced. Among research publications within 3 years, recent applications for determining binding constants are reviewed.

Partial separation zone technique for the separation of enantiomers by affinity electrokinetic chromatography with proteins as chiral pseudo-stationary phases

Abstract A previously reported technique which employed a discontinuous separation zone built in a part of a capillary was applied to the separation of enantiomers by affinity electrokinetic chromatography using proteins as chiral pseudo-stationary phases to avoid a detection problem caused by the presence of UV-absorbing materials in the separation solution. The technique was named “partial separation zone technique”, where the capillary was partially filled with a solution containing a protein and the protein was not in the detector cell when analytes reached that cell. It was shown that the technique was successfully performed automatically using a commercial capillary electrophoresis instrument. The migration velocity of the protein was practically reduced to zero by using a coated capillary. The protein concentration could be increased in this technique to enhance resolution without the deterioration of the detector sensitivity. The detection sensitivity was remarkably improved using short-wavelength light in comparison with the conventional technique where the protein was added to the whole separation solution. The reproducibilities of the migration time and the peak area were evaluated, giving results comparable to those with the conventional method. Successful separations of basic racemates were achieved by the technique using bovine serum albumin, α1-acid glycoprotein, ovomucoid and conalbumin.

Enantiomer separation of acidic racemates by capillary electrophoresis using cationic and amphoretic β-cyclodextrins as chiral selectors

Enantiomer separations of various acidic racemates were performed by capillary electrophoresis (CE) using a commercial cationic beta-cyclodextrin (beta-CD), quaternary ammonium beta-CD (QA-beta-CD), and a commercial amphoteric beta-CD (AM-beta-CD) as chiral selectors. Eleven acidic racemates were successfully separated using QA-beta-CD by changing the CD concentration and the buffer pH. These enantiomer separations were compared with the results using five neutral CD derivatives. Although most racemates were separated with some of the neutral CDs, relatively high CD concentrations were required to obtain baseline separations. In contrast, when QA-beta-CD was employed, the enantiomer separations were successful at low concentrations below 5 mM. Enantiomers of five acidic racemates and ten dansylated amino acids (Dns-amino acids) were separated using AM-beta-CD. Although the baseline separation of racemic 4-chloromandelic acid was not achieved with either QA-beta-CD or five neutral CDs, AM-beta-CD showed complete resolution. Furthermore, the simultaneous enantiomer separation of eight Dns-amino acids was also achieved with AM-beta-CD. Both QA-beta-CD and AM-beta-CD were analyzed by CE and mass spectrometry (MS) in order to identify their compositions because they consisted of a mixture having different degrees of substitution. QA-beta-CD consisted of six components having from one to six quaternary ammonium groups. The composition of AM-beta-CD, however, was very complicated and could not be identified.

Separation of acidic enantiomers by capillary electrophoresis-mass spectrometry employing a partial filling technique

Abstract Enantiomer separations were performed by capillary electrophoresis–mass spectrometry (CE–MS). Because high concentrations of nonvolatile chiral selectors were not compatible for the CE–MS interface and/or the MS instrument, a partial filling technique was employed to avoid the problem. In this method, a separation solution containing a chiral selector was filled in only a part of the capillary. Since the electroosmotic flow (EOF) was almost completely suppressed in a polyacrylamide-coated capillary, neither the uncharged chiral selector nor the positively charged chiral selector migrated towards the MS detector under the negative polarity mode. Using either cyclodextrins (CDs) or avidin as a chiral selector, racemic mixtures of camphorsulfonic acid, tropic acid, arylpropionic acid and warfarin were successfully separated and detected by MS. This method was also applied to the optical purity test of commercial camphorsulfonic acid enantiomers.

Separation of the enantiomers of basic drugs by affinity capillary electrophoresis using a partial filling technique and α1-acid glycoprotein as chiral selectorglycoprotein as chiral selector

SummarySeparation of the enantiomers of a variety of basic drugs by affinity capillary electrophoresis has been investigated using α1-acid glycoprotein (α1-AGP) as chiral selector. In order to use a high concentration of α1-AGP without causing low detection sensitivity, the partial filling technique was employed. Enantiomer separations were performed under conditions (a running buffer at pH 5.0 or 6.0) causing the protein to migrate toward the injection end. Twenty nine basic racemates were successfully separated by optimizing the protein concentration, buffer pH and organic modifier. α1-AGP obtained from three different suppliers was used to investigate differences among the proteins from different sources. Although most of the racemates were similarly separated with any of the three types of α1-AGP, some racemates, e.g. acebutolol behaved differently with the three types. The reasons for the different enantioselectivities of the three types of α1-AGP has not yet been clarified. The method was used to test the optical purity of commercial sulpiride enantiomers and it was found that the method was suitable and applicable for the purpose.

Recent advances in enantiomer separations by affinity capillary electrophoresis using proteins and peptides.

Enantiomer separations by capillary electrophoresis (CE), using proteins as chiral selectors--affinity capillary electrophoresis (ACE) with free solutions and capillary electrochromatography (CEC)--with protein immobilized capillaries, are reviewed. The separation principle, recent advances in this field and some interesting topics are presented. In ACE, various enantiomer separations have been already reported using either plasma proteins or egg white ones. Miscellaneous proteins were also explored in the last few years. On the contrary, only a limited number of enantiomer separations have been successfully achieved in CEC. CEC is not yet mature enough to date, and further investigations, such as efficiency, durability and reproducibility of capillaries, will be necessary for the use of routine analyses. The study of enantioselective drug-protein binding is important in pharmaceutical developments. Some applications including high-performance CE/frontal analysis (HPCE/FA) are introduced in this paper.

Separation of enantiomers by capillary electrophoresis–mass spectrometry employing a partial filling technique with a chiral crown ether

Enantiomer separations were performed by capillary electrophoresis-mass spectrometry (CE-MS) with (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H4) as a chiral selector. In order to prevent the introduction of the nonvolatile chiral, selector, 18C6H4, into the nozzle of the CE-MS interface and/or the orifice plate, a partial filling technique was employed in this study. By the partial filling technique, the contamination caused by the nonvolatile chiral selector was avoided not only during the analysis but also during the washing of capillary with the separation solution prior to the run. Several racemic compounds having a primary amino group were successfully separated. Racemic 3-aminopyrrolidine and racemic alpha-amino-epsilon-caprolactam have no strong UV absorption, but such compounds were detected with a high sensitivity by MS detection. In this paper, the effects of the length of separation zone and those of the 18C6H4 concentration were described. As the length of the separation zone was longer or as the concentration of 18C6H4 was higher, the enantiomer resolution was enhanced more and more. However, the optimization of 18C6H4 concentration was practically enough to obtain the baseline separation.

Evaluation of an atmospheric pressure chemical ionization interface for capillary electrophoresis–mass spectrometry

A simple and inexpensive approach to modify a commercial atmospheric pressure chemical ionization (APCI) interface was proposed for capillary electrophoresis-mass spectrometry (CE-MS). In order to accommodate lower flow rates in the range 1-10 microl/min, both sheath liquid and nebulizing gas were coaxially supplied to the nebulizer as an arrangement of pneumatically assisted electrospray interface. Since this paper focused on the primary study of the modified APCI interface, the performance of the interface was first evaluated by the direct infusion of a reserpine solution. Optimization of several APCI parameters, such as temperature of APCI vaporizer, nebulizing gas flow and APCI corona discharge current, were accomplished. The orifice dimension for the nebulizing gas flow was largely independent of the MS sensitivity when the nebulizing gas flow rate was ca. 0.4 l/min. A successful CE-APCI-MS separation is obtained using the modified APCI interface.

Strategy for selecting separation solutions in capillary electrophoresis–mass spectrometry

Abstract A simultaneous determination of cationic, neutral and anionic analytes was performed by capillary zone electrophoresis–mass spectrometry (CZE–MS). An ammonium acetate buffer (pH 6.0) was used as a weakly acidic running solution, an ammonium phosphate buffer (pH 7.0) was used as a neutral one, and an ammonium carbonate buffer (pH 8.5) was used as an alkaline one. The best CZE separation was achieved using the alkaline running solution because several analytes having acidic hydroxyl groups were deprotonated near their p K a values. The ammonium phosphate buffer could also be used as the running solution in CZE–MS without a deterioration of the detection sensitivity. As for a sheath liquid, a mixture of the alkaline buffer (pH 8.5)–methanol (1:1) was suitable because a stable negative electrospray was obtained with an alkaline solution for the detection of anionic analytes by negative ion mode. Electrokinetic chromatography–MS (EKC–MS) was performed for the separation of neutral analytes. Not only sodium dodecyl sulfate (SDS) but also sulfobutyl ether β-cyclodextrin [β-CD-SBE(IV)] and 4-sulfonated calix[6]arene (Cx-SO 3 ) were employed as pseudo-stationary phases. The interference of the MS detection by SDS was investigated using the alkaline running solution (pH 8.5), but the deterioration of the detection sensitivity of analytes was not observed. A significantly different selectivity was achieved when β-CD-SBE(IV) and Cx-SO 3 were used as pseudo-stationary phases in comparison with SDS micelles. Furthermore, no background signal on the MS detection caused by the elution of the pseudo-stationary phases was observed. The charged CD and the charged calixarene were also useful for the separation of neutral analytes by EKC–MS.

Studies on enantioselectivities of avidin, avidin-biotin complex and streptavidin by affinity capillary electrophoresis

SummaryVarious enantiomer separations were performed by affinity capillary electrophoresis with a partial filling technique (PFACE) using egg white avidin (AVI) and two avidin analogues: succinylated avidin (Suc-AVI) and streptavidin (STAV). Basic AVI was useful for enantiomer separation of acidic racemates, while the chemical modified acidic Suc-AVI was useful for that of basic racemates. Such a chemical modification of protein was effective in extending the applicability of the chiral selector in PFACE. STAV, which was a neutral nonglycosylated protein, was useful for both acidic and basic enantiomer separations. Although AVI and AVI analogues show similar biotin-binding activities, enantioselectivity was different among the proteins in this study. Additionally, we investigated whether the enantioselectivity was due to interaction of enantiomers with biotin-binding sites or not. Not only enantioselevtivity but also interaction with enantiomers was entirely lost by the formation of AVI-biotin complex.