Determination of the enantiomeric composition of phenylalanine samples by chemometric analysis of the fluorescence spectra of cyclodextrin guest-host complexes

A novel strategy for determining the enantiomeric composition of phenylalanine samples that combines ordinary fluorescence spectroscopy, guest-host cyclodextrin chemistry, and multivariate regression modeling is investigated. Partial-least-squares regression (PLS-1) models were developed from fluorescence spectral data obtained with a series of samples containing cyclodextrin guest-host complexes of phenylalanine with different known enantiomeric compositions. The regression models were subsequently validated by determining the enantiomeric composition of a set of independently prepared phenylalanine samples. The ability of the models to correctly predict the enantiomeric compositions of future samples was evaluated in terms of the root-mean-square percent relative error (RMS%RE). The RMS%RE in the mol fraction of D-phenylalanine ranged from 1.3% to 3.0% when beta-cyclodextrin was used as the host molecule for different guest-host concentrations. The RMS%RE in the mol fraction of D-phenylalanine obtained in a similar validation study conducted with gamma-cyclodextrin ranged between 1.8% and 4.0% for different guest-host concentrations. Compared with previous studies done in absorption, fluorescence data were found to be more sensitive and the spectral differences observed as a function of enantiomeric composition were more uniformly spaced, making regression modeling more reliable. As a result, good regression models could be made at lower concentrations than were possible previously when absorption measurements were used.     

Determination of the enantiomeric composition of some molecules of pharmaceutical interest by chemometric analysis of the UV spectra of guest-host complexes formed with modified cyclodextrins

Multivariate regression modeling techniques (PLS-1 regression modeling) were applied to ordinary UV spectral absorption data obtained on solutions containing inclusion complexes formed between homochiral modified cyclodextrins (methyl-β-cyclodextrin, α-, β-, and γ-carboxymethyl cyclodextrins and α-, β-, and γ-hydroxypropyl cyclodextrins) and four guest molecules of pharmaceutical interest (ephedrine, norephedrine, norepinephrine-l-bitartrate, and tryptophan methyl ester). The PLS-1 regression models were developed by correlating the known enantiomeric composition of laboratory prepared samples with ordinary UV absorption spectral data. The regression models were subsequently validated with laboratory-prepared test sets. The rms percent relative error in the predicted mol fraction of (1S, 2R)-(+)-ephedrine, (1S, 2R)-(+)-norephedrine, (R)-(−)-norepinephrine-l-bitartrate, and d-tryptophan methyl ester obtained with the independently prepared test sets was heavily dependent on the host molecule used.     

Rapid enantiomeric separation of polychlorinated biphenyls by electrokinetic chromatography using mixtures of neutral and charged cyclodextrin derivatives

Electrokinetic chromatography with cyclodextrin derivatives (CD-EKC) was used to achieve the rapid enantiomeric separation of chiral polychlorinated biphenyls (PCBs). Thirteen of the 19 chiral PCBs stable at room temperature were individually separated into their two enantiomers by using 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.5) containing carboxymethylated gamma-cyclodextrin (CM-gamma-CD) as pseudostationary phase mixed with beta-cyclodextrin (beta-CD) or permethylated beta-cyclodextrin (PM-beta-CD). Urea was also added to increase the solubility of PCBs and cyclodextrins in the aqueous separation buffer. Several experimental parameters such as the nature, concentration, and pH of the buffer, nature and concentration of the cyclodextrin derivatives used, and the addition of different additives were studied in order to improve the enantiomeric separation. In addition, the effect of some instrumental parameters such as separation temperature and applied voltage was also investigated. PCBs were enantiomerically separated in less than 12 min by using a 50 mM MES buffer (pH 6.5) containing 20 mM CM-gamma-CD, 10 mM beta-CD or 20 mM PM-beta-CD, and 2 M urea at a temperature of 45 degrees C and an applied voltage of 20 kV.     

Artificial neural networks for determination of enantiomeric composition of alpha-phenylglycine using UV spectra of cyclodextrin host-guest complexes: comparison of feed-forward and radial basis function networks

In this work feed-forward neural networks and radial basis function networks were used for the determination of enantiomeric composition of alpha-phenylglycine using UV spectra of cyclodextrin host-guest complexes and the data provided by two techniques were compared. Wavelet transformation (WT) and principal component analysis (PCA) were used for data compression prior to neural network construction and their efficiencies were compared. The structures of the wavelet transformation-radial basis function networks (WT-RBFNs) and wavelet transformation-feed-forward neural networks (WT-FFNNs), were simplified by using the corresponding wavelet coefficients of three mother wavelets (Mexican hat, daubechies and symlets). Dilation parameters, number of inputs, hidden nodes, learning rate, transfer functions, number of epochs and SPREAD values were optimized. Performances of the proposed methods were tested with regard to root mean square errors of prediction (RMSE%), using synthetic solutions containing a fixed concentration of beta-cyclodextrin (beta-CD) and fixed concentration of alpha-phenylglycine (alpha-Gly) with different enantiomeric compositions. Although satisfactory results with regard to some statistical parameters were obtained for all the investigated methods but the best results were achieved by WT-RBFNs.     

Enantiomeric separation of synthetic 2,3-dihydroxy-3-phenylpropionate compounds by beta-cyclodextrin-modified capillary electrophoresis

A new capillary electrophoretic method was developed for enantiomeric separation and optical impurity analysis of three synthetic 2,3-dihydroxy-3-phenylpropionate compounds using native beta-cyclodextrin (beta-CD) as chiral selector and borate as a background electrolyte. The separation was carried out in uncoated capillary (58.5 cm x 75 microm I.D., effective length 48.5 cm). The results showed that beta-CD as the chiral selector exhibited good enantioselectivity and the baseline separation was obtained at pH 9.8, 200 mM borate buffer containing 1.7% beta-CD at applied voltage 15 kV and capillary temperature 20 degrees C within 15 min. The precision of each tested compound was less than 1.0% at migration time and 5.0% in corrected peak area and the accuracy of the method was in the range of 98.7-105%. Furthermore, the developed method was successfully applied to the determination of the undesirable trace (2S,3R)-(+)-form impurity in the synthetic (2R,3S)-(-)-2,3-dihydroxy-3-phenylpropionate samples.     

Chiral CE separation of dopamine-derived neurotoxins.

An enantiomeric separation of dopamine-derived neurotoxins by capillary electrophoresis has been developed. Tetrahydroisoquinoline (TIQ), dopamine (DA), (R/S)-1-benzyl-TIQ (BTIQ), (R/S)-6,7-dihydroxy-1-methyl-TIQ (salsolinol, Sal), and (R/S)-6,7-dihydroxy-1, 2-dimethyl-TIQ (N-methyl-salsolinol, NMSal) were studied as model compounds. The CE running buffer (50 mM phosphate buffer at pH 3.0) contained 1.5 M urea and 12 mM beta-CD as a chiral selector. During separation, the (R)-enantiomers formed more stable inclusion complexes with beta-CD, and thus had a longer migration time than their optical antipodes. It was noticed that the recovery rates of these TIQ derivatives were very poor (< 15%) during protein precipitation, a procedure widely used for cleaning up biological samples. The recovery was significantly improved by pre-mixing the sample with a surfactant (e.g., sodium hexanesulfonate or Triton X-100) to reduce the co-precipitation. The present method in combination with electrospray ionization tandem mass spectrometry (ESI-MS/MS) was applied to study samples obtained from in vitro incubation of two catecholamines, dopamine and epinine, with aldehydes forming neurotoxins including (S)- and (R)-NMSal enantiomers. The later is known to induce Parkinsonism in rats.     

Enantioseparation of atropine by capillary electrophoresis using sulfated beta-cyclodextrin: application to a plant extract

A capillary zone electrophoresis (CZE) method, with sulfated beta-CD as chiral selector, was optimized by means of an experimental design for the enantioseparation of atropine. In this study, a central composite design was used and the following factors were varied simultaneously: buffer concentration, buffer pH and sulfated beta-CD concentration. The resolutions between littorine and its positional isomer ((-)-hyoscyamine) and between atropine enantiomers, as well as the separation time and generated current were established as responses. A model was obtained for each response by linear multiple regression of a second-degree mathematical expression. The most favorable conditions were determined by maximizing the resolution between atropine enantiomers and by setting the other responses at threshold values. Successful results were obtained with a 55 mM phosphate buffer at pH 7 in the presence of 2.9 mM sulfated-beta-CD at 20 degrees C and 20 kV. Under these optimized conditions, a baseline separation of littorine and atropine enantiomers was achieved in less than 5 min. Finally, the method allowed the enantiomeric separation of atropine in a pharmaceutical formulation and was also found to be suitable for the enantiomeric purity evaluation of (-)-hyoscyamine in plant extracts, in relation with the extraction procedure. It was demonstrated that supercritical fluid extraction induced less racemization than classical liquid-solid extraction procedures.     

Determination of enantiomeric composition of samples by multivariate regression modeling of spectral data obtained with cyclodextrin guest-host complexes-Effect of an achiral surfactant and use of mixed cyclodextrins

The determination of the enantiomeric composition of samples by chemometric modeling of spectral data was investigated for samples of N,N′-bis-(-methylbenzyl) sulfamide and tryptophan methyl ester hydrochloride. Multivariate regression models (PLS-1) were developed from spectral data obtained on solutions containing N,N′-bis-(-methylbenzyl)sulfamide or tryptophan methyl ester hydrochloride in the presence of sodium dodecyl sulfate and mixed cyclodextrin host molecules. The regression models were subsequently used to predict the enantiomeric composition of laboratory-prepared test samples of N,N′-bis(-methylbenzyl)sulfamide or tryptophan methyl ester hydrochloride. The capability of the models to accurately predict the enantiomeric composition was evaluated in terms of the root-mean-square percent relative error (RMS %R.E.) as calculated from the results obtained with independently prepared validation sets of samples. It was found that the presence of SDS in most cases either had little effect on the predictive ability of the model or it actually reduced the predictive ability of the model. Moreover, it was found that the use of mixed CDs, either in the presence or absence of SDS, reduced the predictive ability of the regression model when compared with results obtained with individual CDs.     

Fast enantiomeric separation of basis drugs by electrokinetic chromatography. Application to the quantitation of terbutaline in a pharmaceutical preparation

Electrokinetic chromatography (EKC) using micelles of bile salts alone or mixed with sodium dodecyl sulfate (SDS) and neutral, anionic, or cationic cyclodextrins (CDs) in the separation buffer has been employed in order to achieve fast enantiomeric separation of basic drugs. A study of the enantiomeric separation ability of these chiral selectors concerning four basic drugs (epinephrine, terbutaline, clenbuterol, and salbutamol) has been carried out under different experimental conditions. The best chiral selectors to perform the enantiomeric separation of these drugs were neutral beta-CD derivatives, specifically permethylated beta-CD PM-beta-CD. The effect of the PM-beta-CD concentration, temperature, and applied voltage on the enantiomeric resolution of the basic drugs was investigated. The use of a 25 mM ammonium acetate buffer (pH 5.0), 30 mM in PM-beta-CD together with an applied voltage of 20 kV and a temperature of 15 degrees C enabled the individual and fast enantiomeric separation of epinephrine, norepinephrine, terbutaline, clenbuterol, and salbutamol each one into its two enantiomers in less than 3 min. The EKC method was validated (precision and accuracy) to quantitate terbutaline in a pharmaceutical preparation, obtaining a limit of detection of 4 microg/mL.     

Cyclodextrin-mediated micellar electrokinetic chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the enantiomer separation of racemorphan in human urine.

Micellar electrokinetic chromatography (MEKC) was successfully and conveniently applied to the chiral separation with the addition of cyclodextrins (CDs) as chiral selector to the running buffer. Chiral separation depended on the type of CD; in particular, beta-CD was effective for the chiral separation of racemorphan. We investigated the optimal conditions of type and concentration of CD as chiral selector for the routine enantiomeric separation of racemorphan with good reproducibility. The effects of other parameters such as buffer pH and detection wavelength were also investigated to obtain the optimum conditions for the enantiomeric separation of racemorphan. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for confirmation of racemorphan. The optimal conditions for enantiomeric separation of the racemorphan were as follows: 50 mM borate buffer at pH 9.4 with 50 mM SDS, 10 mM beta-CD and 20% 1-propanol, 57 cm x 50 microns fused-silica capillary column, and UV detection at 192 nm. Based on the developed method, racemorphan in human urine was also separated and determined using solid-phase extraction and MEKC.     

Influence of (hydroxy)alkylamino substituents on enantioseparation ability of single-isomer amino-beta-cyclodextrin derivatives in chiral capillary electrophoresis

A family of single-isomer amino-beta-cyclodextrin (amino-beta-CD) derivatives containing an amino or (hydroxy)alkylamino group in one of the primary positions has been synthesized. The steric effect and hydrogen bond forming ability of the different substituents on enantioseparation of acidic enantiomers has been studied by capillary electrophoresis (CE). Three enantiomeric model compounds (mandelic acid, cis-permethrinic acid, and cis-deltamethrinic acid) having significantly different apparent complex stability constants with beta-CD were applied in the experiments. Dependence of separation selectivity, resolution as well as mobility difference on chiral selector concentration (0.1-20 mM, pH 6.0) was investigated. Each amino-beta-CD showed higher enantioselectivity than the native beta-CD. One hydroxyalkyl group attached to the primary amino N-atom significantly increased both the enantioselectivity and the resolution compared to the primary amino-beta-CD, while two hydroxyalkyl moieties decreased them due to the predominance of steric hindrance. The value of the apparent complex stability constants obtained suited well the mobility difference model (by Wren). On the other hand, the optimum selector concentrations calculated according to the model were slightly lower than the experienced concentrations giving the maximum enantioresolution of enantiomers.     

Multicomponent analyses of chiral samples by use of regression analysis of UV–visible spectra of cyclodextrin guest–host complexes

We report the first combined use of analytical spectroscopy, guest–host chemistry, and multivariate regression analysis for determination of enantiometric composition of multicomponent samples of chiral analytes. Sample solutions containing multicomponent analytes of ephedrine, tryptophan, propranolol, and proline of varying enantiomeric composition with beta-cyclodextrin (BCD) or methyl-beta-cyclodextrin (Me-BCD) as chiral host molecules were investigated using ultraviolet (UV)–visible spectroscopy. The interactions of enantiomers of chiral analytes with chiral hosts resulted in the formation of transient diastereomeric inclusion complexes with varying spectral properties. Multivariate analysis using partial-least-square (PLS) regression was used to correlate subtle changes in the UV–visible spectra of the guest–host complexes with the enantiomeric composition of the calibration samples. These PLS regressions were carefully optimized and then used to predict the enantiomeric composition of multicomponent chiral analytes of validation samples. The results of these validation studies demonstrate the predictive ability of the regression models for determination of future enantiomeric composition of samples. The accuracy of the models to correctly predict the enantiomeric composition of samples, evaluated by use of the root mean square percent relative error (RMS%RE) was analyte and chiral host dependent. In general, better prediction of enantiomeric composition of samples and low RMS%RE values were obtained when Me-BCD was used as the chiral host. The analyses procedure reported here is simple, rapid, and inexpensive. In addition, this approach does not require prior separation of chiral analytes, thus reducing analysis time and eliminating the need for expensive chiral columns.     

Liquid chromatography determination of citalopram enantiomers using beta-cyclodextrin as a chiral mobile phase additive

A reliable and specific method for the determination of citalopram enantiomers was developed and validated. Chromatographic resolution of citalopram enantiomers was made on a Shim-pack (5 microm particle size) cyanopropyl column with beta-cyclodextrin (beta-CD) as an effective chiral mobile phase additive. The composition of the mobile phase was (90 + 10, v/v) aqueous 0.1% triethylammonium acetate buffer, pH 4.0 (adjusted with acetic acid), and acetonitrile, containing 12 mM beta-CD. The flow rate was 0.8 mL/min with ultraviolet detection at 240 nm. The effects of the mobile phase composition, concentration of beta-CD, and pH of the triethylammonium acetate buffer on peak shape and resolution of the enantiomers were investigated. The calibration graphs were linear (r = 0.9999, n = 8) in the range of 1-40 microg/mL for S(+) citalopram and R-(-) citalopram. The limit of detection values were 5.51 x 10(-3) and 4.35 x 10(-3) pg/mL, while the limit of quantification values were found to be 1.84 x 10(-2) and 1.45 x 10(-2) microg/mL for S-(+) citalopram and R-(-) citalopram, respectively.     

Spectroscopic study of orange G-beta-cyclodextrin complex and its analytical application

The mechanism of the inclusion of orange G and beta-cyclodextrin (beta-CD) has been studied by using both spectrophotometry and infrared spectroscopy. Effects of the pH, concentrations of beta-CD, and ionic strength on the inclusion complex of beta-CD and orange G were examined. The result showed that orange G reacts with beta-CD to form a 1:1 host-guest complex with an apparent formation constant of 3.03 x 10(3)mol(-1)l. The thermodynamic parameters of inclusion complex, DeltaG(0), DeltaH(0), and DeltaS(0) were obtained. Based on the enhancement of the absorbance of orange G produced through complex formation, a ratiometric method spectrophotometrically for the determination of orange G in bulk aqueous solution in the presence of beta-CD was developed, which overcome the effect of condition change on the determination of orange G. The linear relationship between the absorbance and orange G concentration was obtained in the range of 1.0 x 10(-5) to 4.0 x 10(-5)mol l(-1), with a correlation coefficient of 0.9998. The detection limit was 3.6 x 10(-6)mol l(-1). The principal advantage of the proposed method is high accuracy because ratiometry was used in measurement.     

Sucrose as chiral selector for determining enantiomeric composition of phenylalanine by UV-vis spectroscopy and chemometrics

The determination of enantiomeric composition by partial least squares(PLS) modeling of UV-vis spectral data was investigated for samples of phenylalanine(phe) using sucrose as a chiral auxiliary.And a new data preprocess method,reference band normalization,was introduced to eliminate the spectral variations due to the changes of total concentration of phe.The determination coefficient(R~2) and the standard error of calibration set(SEC) of 13 standard samples are 0.9987 and 0.0128 respectively.The standard error of validation set(SECV) of 7 validation samples is 0.0049.The standard error of predict(SEP) of 6 blind samples for evaluating the robustness of the model is 0.0366.The regression model is robust to determine enantiomeric composition when total concentration varied.It is demonstrated that the reference band normalization is a convenient method of compensating for variations in total concentrations without knowing that in advance.     

Kinetic study for the inclusion complex of carboxylic acids with cyclodextrin by the ultrasonic relaxation method

Ultrasonic absorption coefficients in the frequency range of 0.8-95 MHz were measured in aqueous solutions containing both beta-cyclodextrin (beta-CD) (host) and butanoic acid (in its dissociated form and undissociated one) (guest). A single relaxational phenomenon was observed only when the solutes were coexisting, although no relaxation was found in the beta-CD solution or in the acid solutions. The absorption was also measured in a solution of pentanoic acid (dissociated form) with beta-CD, and single relaxation was detected. The ultrasonic relaxation observed in these solutions was due to a perturbation of a chemical equilibrium related to a reaction of an inclusion complex formed by the host and guest. The equilibrium constant was obtained from the dependence of the maximum absorption per wavelength on the guest concentration. The rate constant for the inclusion process of the guest into a cavity of beta-CD and that for the leaving process from the cavity were determined from the obtained relaxation frequency and the equilibrium constant. The standard volume change of the reaction was also computed from the maximum absorption per wavelength. These results were compared with those in solutions containing both beta-CD and different guest molecules. It was found that the hydrophobicity of guest molecules played an important role in the formation of the inclusion complex and also that the charge on the carboxylic group had a considerable effect on the kinetic characteristics of the complexation reaction.     

Spectrophotometric study of the inclusion complex between beta-cyclodextrin and dibenzoyl peroxide and its analytical application

The noncovalent interaction of dibenzoyl peroxide and beta-cyclodextin (beta-CD) has been studied by spectrophotometry. The mechanism of the inclusion was studied. The results showed that beta-CD reacts with dibenzoyl peroxide to form a 2:1 host-guest complex with an apparent formation constant of 2.5 x 10(4)mol(-2)L(2). The beta-CD reacts with benzoic acid to form a 1:1 host-guest complex with an apparent formation constant of 6.9 x 10(2)mol(-1)L after the dibenzoyl peroxide was reduced by hydroxyl ammonium. Based on the enhancement of the absorbance of dibenzoyl peroxide produced through complex formation, a spectrophotometric method for determination of dibenzoyl peroxide in bulk aqueous solution in the presence of beta-CD was developed. A linear relationship between the absorbance and dibenzoyl peroxide concentration was obtained in the range of 0.300-50.0 microg mL(-1). Linear regression equation of the calibration graph C=0.02926+53.25 A, with a correlation coefficient of 0.9984 and a relative standard deviation (R.S.D.) of 3.4%. The detection limit was 0.200 microg mL(-1), and the recovery was from 98.00 to 105.0%. The proposed method was used to determine the dibenzoyl peroxide in the flour with satisfactory results. The principal advantage of the proposed method is its excellent selectivity based on molecule recognition of beta-CD.     

Enantioseparation of cetirizine by sulfated-beta-cyclodextrin-mediated capillary electrophoresis

A sulfated beta-cyclodextrin (sulfated beta-CD)-mediated capillary electrophoresis method is described for the enantioseparation of cetirizine using achiral cefazolin as an internal standard. The enantioseparation of the drug was performed in a borate buffer (5 mM, pH 8.7) with 1% sulfated beta-CD (w/v) as chiral selector at 10 kV. Several parameters affecting the separation were studied, including the pH and the concentration of borate buffer and chiral selector. Under optimized conditions, a baseline separation of two enantiomers was achieved in less than 7 min. Using cefazolin as an internal standard (IS), the linear range of the method for the determination of levocetirizine was over 1.0 to 50.0 microg/mL; the detection limit (signal-to-noise ratio = 3) of levocetirizine was 0.5 microg/mL. The method allowed the enantioseparation of cetirizine in bulk samples and enantiomeric purity evaluation of levocetirizine (R-enantiomer) in pharmaceutical tablets (Xyzal), and it was also found to be suitable for enantioseparation in human plasma.     

毛细管区带电泳法研究肾上腺素类药物的手性分离

使用β－环糊精（β－ＣＤ）及β－ＣＤ－羧甲基（ＣＭ－β－ＣＤ）作为手性选择剂,采用毛细管区带电泳法（ＣＺＥ）对去甲肾上腺素、肾上腺素和异丙肾上腺素的手性分离进行了研究。对影响这类药物手性分离的主要因素〔手性选择剂、背景电解质（ＢＧＥ）、分离体系的酸度和温度〕进行了讨论,并对手性识别机理进行了探讨。     

Chiral separation of amino acids derivatized with fluoresceine-5isothiocyanate by capillary electrophoresis and laser-induced fluorescence detection using mixed selectors of beta-cyclodextrin and sodium taurocholate

Chiral separation of 20 pairs of amino acids derivatized with fluoresceine-5-isothiocyanate (FITC) by capillary electrophoresis and laser-induced fluorescence detection was studied using the mixture of beta-cyclodextrin (beta-CD) and sodium taurocholate (STC) as selector. Resolution was considerably superior to that obtained by using either beta-CD or STC alone. The molar ratio of beta-CD to STC of about 2:3 was found to be critical to achieve maximum separation. At this beta-CD-to-STC ratio, chiral separation occurred at really low total concentration of beta-CD and STC (<0.1 mM). Other impacting factors were investigated including the total concentration of beta-CD and STC, pH, and capillary conditioning procedure between two successive runs. Using a running buffer of 80 mM borate containing 20 mM beta-CD and 30 mM STC at pH 9.3, all of the 20 pairs of FITC-amino acid enantiomers were baseline resolved. The resolutions of the most pairs of the amino acid enantiomers (17 of 20) were higher than 3.0, only three pairs gave a resolution lower than 3.0 but higher than 1.90 (beta-phenylserine, pSer). The highest resolution reached 14.58 (Glu). Two derivatives of beta-CD, 2-hydroxypropyl-beta-CD (HP-beta-CD) and heptakis(2,6-di-O-methyl)-beta-CD (DM-beta-CD) were also explored. HP-beta-CD showed similar cooperative effect with STC, while DM-beta-CD together with STC led to poorer chiral separation.