Estimation of biological properties by means of chromatographic systems: evaluation of the factors that contribute to the variance of biological-chromatographic correlations.

The performance of chromatographic systems to emulate biological systems is evaluated in terms of the precision that can be achieved. The variance obtained when biological parameters are correlated against physicochemical ones can be decomposed in three terms: the variance of the biological data, the variance of the physicochemical data, and the variance caused by the dissimilarity between the two correlated systems (biological and physicochemical). The three terms contribute to the overall variance observed when measurements in chromatographic systems are correlated with experimental biological properties. The Abraham linear free energy relationships (LFERs) provide a very good approach to characterize biological and physicochemical systems and thus the variance of the analyzed data and the similarity/dissimilarity between them. The contribution of the three variances to the precision of the biological parameter estimated in this way is evaluated from the characterization of the biological and chromatographic systems by means of the Abraham model. The proposed method is able to estimate the goodness of chromatographic systems to predict particular biological properties. In particular, this method is illustrated by comparison of toxicity data (-log LC(50)) for the fish fathead minnow with retention data (log k) in several micellar electrokinetic chromatography (MEKC) systems and also by correlations between retention data (log k) in the sodium taurocholate (STC) MEKC system and data of several biological systems.

Separation of cytokinin isomers with a partial filling-micellar electrokinetic chromatography-mass spectrometry approach.

A new method based on partial filling-MEKC (PF-MEKC) directly coupled to ESI-MS was developed for the simultaneous separation and determination of 13 structurally similar cytokinins, including various geometric and positional isomers of cytokinins. On the basis of the resolution of the neighboring isomer peaks, different parameters (i.e., pH and concentration of buffer, surfactant concentrations, length of the injected micellar plug, organic modifier, and applied separation voltage) were optimized to achieve a satisfactory PF-MEKC separation. Under optimum conditions, the separation of 13 cytokinin standards was accomplished within 25 min. MS/MS with multiple reaction monitoring detection was carried out to obtain sufficient selectivity. PF-MEKC-MS/MS allowed for the direct identification and confirmation of the cytokinins present in banana (Musa spp.) pulp sample after extraction and purification. Finally, trans-zeatin riboside (ZR) and trans-zeatin (Z) were unambiguously identified in banana pulp. It is anticipated that the current PF-MEKC-MS method can be applied to analyze cytokinins in a wide range of biological samples.

Separation and determination of flavonoids using microemulsion EKC with electrochemical detection.

A selective and sensitive method of microemulsion EKC (MEEKC) with electrochemical detection (ED) was developed for separation and determination of 14 flavonoids. In order to obtain the better stability for the studied flavonoids, oil (ethyl acetate) with low interfacial surface tension was employed as organic solvent. A running buffer composed of 0.9% (w/v, 30 mM) SDS, 0.9% (w/v, 21 mM) sodium cholate (SC), 0.9% (w/v, 121 mM) butan-1-ol, 0.6% (w/v, 68 mM) ethyl acetate, and 98.2% v/v 10 mM Na(2)B(4)O(7)-20 mM H(3)BO(3) buffer (pH 7.5) was applied for the separation of flavonoids. Under the optimum conditions, the relationship between peak currents and analyte concentrations was linear over about 1.3 and 1.7 orders of magnitude with detection limits (defined as S/N = 3) ranging from 0.02 to 0.5 microg/mL for all analytes. This method was applied for the determination of flavonoids in real samples with simple extraction procedures, and the assay results were satisfactory.

Separation of very hydrophobic analytes by MEKC. II. Carbon number equivalents as analyte descriptors--influence of the composition of the separation electrolyte.

The use of the parameter carbon number equivalent, N(c)(*), as analyte descriptor for neutral hydrophobic solutes in EKC is investigated. The carbon number equivalent of an analyte is defined analogously to the retention index, RI, however, it is directly calculated from the regression line plotting for the members of a homologous series the logarithm of the retention factor against the number of methylene units N(c) in the alkyl chain. This plot can be obtained by using simultaneously an iteration procedure for the calculation of t(MC). For separation electrolytes containing SDS as micellar phase employing ACN, urea, and CaCl(2) as additives within a wide span of concentrations the parameter N(c)(*) is quasi-independent of the composition of the separation electrolyte, being of importance concerning identification of analytes and improving repeatability and reproducibility. With known octanol-water partition coefficients for the members of the homologous series taken as reference it is possible to transform the conventional time scale of an electropherogram into a scale of N(c)(*) or a scale of log P(OW). Due to the quasi-independence of N(c)(*) from the composition of the separation electrolyte these transformed electropherograms can be used as normalized electropherograms for identification purposes. It is also shown that the linear dependence of log P(OW) on N(c) can be the basis of the determination of octanol-water partition coefficients of solutes within a very large range (-4 < or = log P(OW) < or = 9).

Separation and determination of epinephrine and dopamine in traditional Chinese medicines by micellar electrokinetic capillary chromatography with laser induced fluorescence detection.

A micellar electrokinetic capillary chromatography method with laser-induced fluorescence detection was developed for the analysis of epinephrine and dopamine after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The optimum derivatization conditions were: 30 mM sodium borate (pH adjusted to 8.0 with 1.0 M HCl), reaction time 30 min at 60 degrees C. Baseline separation was achieved within 14 min with a running buffer composed of 10 mM sodium borate + 25 mM sodium dodecyl sulfate (pH adjusted to 9.5 with 0.1 M NaOH) and an applied voltage of 15 kV. Good linearity relationships (correlation coefficients: 0.9991 for epinephrine and 0.9985 for dopamine) between peak areas and concentrations of the analytes were obtained. The detection limits and quantification limits for epinephrine and dopamine were 0.0038 mg/L and 0.013 mg/L, and 0.065 mg/L and 0.020 mg/L, respectively. The method was applied to the analysis of the two compounds in two Chinese medicines with recoveries in the range of 92.6-108.7%.

Method for enantiomeric purity of a quinuclidine candidate drug by capillary electrophoresis.

A chiral procedure based on EKC was developed and validated for determination of the enantiomeric purity of PHA-543613, a drug candidate that was under development for treatment of the cognitive deficits of Alzheimer's disease and schizophrenia. Separation of enantiomers is accomplished via differential, enantiospecific complexation with a single-isomer, precisely sulfated beta-CD and heptakis-6-sulfato-beta-CD (HpS-beta-CD). Both neutral and sulfated CDs were screened before selecting HpS-beta-CD as the chiral selector. The separation is conducted in a 61 cm x 50 microm uncoated fused silica capillary with 25 mM HpS-beta-CD in pH 2.50, 25 mM lithium phosphate as the separation buffer with detection at 220 nm. Application of reverse polarity at -30 kV results in an elution time of about 12 min for PHA-543613 and 13 min for the undesired S-enantiomer. Quantification is versus an authentic reference S-enantiomer as an external standard in combination with an internal standard. The procedure was validated over the range 0.1-2.0% w/w. The detection limit is 0.01-0.02%. The amount of distomer intrinsic to the drug substance is about 0.1% or less. The developed method was used to generate stability data on multiple lots: in one case for up to 3 years.

Comparison of two capillary electrophoresis online stacking modes by analysis of polycyclic aromatic hydrocarbons in airborne particulates.

Naphthalene, fluorene, pyrene, anthracene, phenanthrene, and chrysene were successfully separated by CD-modified MEKC (CD-MEKC) using 20 mM borate (pH 9.0) containing 90 mM SDS and 75 mM beta-CD. Two online stacking methods, i.e., sweeping and field-enhanced sample injection (FESI), were explored to enhance the detection sensitivity. The influences of some crucial parameters in sweeping and FESI procedures were investigated. For FESI method, a plug of water and low-conductivity sample matrix was used to increase the stacking efficiency. Compared with the sweeping method, FESI can increase the sensitivity in the range of 10-20-fold. The proposed method was used for the analysis of polycyclic aromatic hydrocarbons in airborne particulates.

Selectivity of single, mixed, and modified pseudostationary phases in electrokinetic chromatography.

The selectivity of a compilation of single, mixed, and modified EKC pseudostationary phases, described in the literature and characterized through the solvation parameter model, is analyzed. Not only have micellar systems of different nature been included but also microemulsions, polymeric, and liposomial phases. In order to compare the systems, a principal component analysis of the coefficients of the solvation equation is performed. From this analysis, direct information of the system properties, differences in selectivity, as well as evidence of lack of accuracy in some system characterizations are obtained. These results become a very useful tool to perform separations with mixtures of surfactants, since it is possible to know which mixtures will provide a greater selectivity variation by changing only the composition of the pseudostationary phases. Furthermore, the variation of the selectivity of some mixtures, as well as the effect of the addition of organic solvents on selectivity, is also discussed.

Estimation of partition coefficients by MEKC part 2: anthocyanins.

The octanol-water partition coefficients of seven anthocyanins (in neutral form, pH = 7.0) were measured by means of MEKC. A new iterative method for the estimation of migration time of electroosmotic flow (EOF) and micelles in MEKC is presented. This calculation is based on Gauss-Newton linearization of the dependence between migration indices and migration times of a set of suitable standards and the application of an advanced statistical evaluation procedure. The values of partition coefficients obtained with the aid of an iterative process are compared with values obtained on use of EOF and micelle markers.

Verification of statistical-overlap theory in micellar electrokinetic chromatography.

The limited peak capacity of neutral compounds in micellar electrokinetic chromatography (MEKC) causes peak overlap in a simple 38-compound sample that is predicted by statistical-overlap theory (SOT). The low-concentration sample was prepared in-house from several compound classes to span the entire migration-time range and was resolved partially in a pH=7 phosphate buffer containing 50 mM sodium dodecyl sulfate. Peaks, singlets, doublets, and other multiplets were identified on the basis of known migration times and were counted at 13 voltages spanning 4 - 26 kV. These numbers agreed well with predictions of a simple SOT based on the assumption of an inhomogeneous Poisson distribution of migration times. Because the dispersion theory of MEKC is simple, the standard deviations of single-component peaks were modeled theoretically. As part of a new way to implement SOT, probability distributions of the numbers of peaks, singlets, and so on, were computed by Monte Carlo simulation. These distributions contain all theoretical information on peak multiplicity predictable by SOT and were used to evaluate the agreement between experiment and theory. The peak capacity of MEKC was calculated numerically and substituted into the simplest equations in SOT, affirming that peak overlap arises from limited peak capacity.

Determination of the chiral and achiral related substances of methotrexate by cyclodextrin-modified micellar electrokinetic chromatography.

A cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method for the determination of the most important potential impurities of methotrexate (MTX): 2,4-diamino-6-(hydroxymethyl)pteridine, aminopterine hydrate, 4-[N-(2-amino-4-hydroxy-6-pteridinylmethyl)-N-methylamino] benzoic acid, 4-[N-(2,4-diamino-6-pteridinylmethyl)-N-methylamino] benzoic acid, and the distomer D-MTX is presented. The MEKC separation of these compounds was optimized by applying a step-by-step approach. The addition of beta-CD to a conventional MEKC system, based on sodium dodecyl sulfate (SDS) as surfactant, showed to be essential for the enantioresolution of racemic MTX as well as for the separation of the achiral impurities. To achieve high-resolution factor between the peaks adjacent to the main component (L-MTX), as required in the analysis of related impurities, the separation conditions were stressed; in particular, the addition of methanol to the CD-MEKC system resulted in a very effective choice. Under the optimized final conditions (100 mM SDS and 45 mM beta-CD in a mixture of 50 mM borate buffer, pH 9.30-methanol (75:25 v/v)), the method was validated showing a general adequate accuracy (93-106% recovery) in the determination of L-MTX related substances at the impurity level of 0.12% w/w with a relative standard deviation (RSD)% lower than 8% (n = 4). The method was successfully applied to the analysis of pharmaceuticals (tablets and injections) which showed to contain the distomer D-MTX as major impurity and aminopterine hydrate as a further related substance in the commercial tablets.

Application of stepwise discriminant analysis to classify commercial orange juices using chiral micellar electrokinetic chromatography-laser induced fluorescence data of amino acids.

The use of chiral amino acids content and stepwise discriminant analysis to classify three types of commercial orange juices (i.e., nectars, orange juices reconstituted from concentrates, and pasteurized orange juices not from concentrates) is presented. Micellar electrokinetic chromatography with laser-induced fluorescence (MEKC-LIF) and beta-cyclodextrins are used to determine L- and D-amino acids previously derivatized with fluorescein isothiocyanate (FITC). This chiral MEKC-LIF procedure is easy to implement and provides information about the main amino acids content in orange juices (i.e., L-proline; L-aspartic acid, D-Asp, L-serine, L-asparagine, L-glutamic acid, D-Glu, L-alanine, L-.arginine, D-Arg, and the non-chiral gamma-amino-n-butyric acid (GABA), i.e., gamma-aminobutyric acid). From these results, it is clearly demonstrated that some D-amino acids occur naturally in orange juices. Application of stepwise discriminant analysis to 26 standard samples showed that the amino acids L-Arg, L-Asp and GABA were the most important variables to differentiate the three groups of samples. With these three selected amino acids a 100% correct classification of the samples was obtained either by standard or by leave-one-out cross-validation procedures. These classification functions based on the content in L-Arg, L-Asp and GABA were also applied to nine test samples and provided an adequate classification and/or interesting information on these samples. It is concluded that chiral MEKC-LIF analysis of amino acids and stepwise discriminant analysis can be used as a consistent procedure to classify commercial orange juices providing useful information about their quality and processing. To our knowledge, this is the first report about the combined use of chiral capillary electrophoresis and discriminant techniques to classify foods.

Solute-solvent interactions in micellar electrokinetic chromatography: V. Factors that produce peak splitting.

The experimental conditions that produce analyte peak splitting in micellar electrokinetic capillary chromatography (MEKC) have been systematically investigated. The system studied was a neutral phosphate buffer and sodium dodecyl sulfate (SDS) micelles as pseudostationary phase. A number of analytes showing a wide variety of hydrophobicity values and several organic solvents as sample diluents have been tested. Peak splitting phenomena are mainly due to the presence of organic solvent in the sample solution. They increase with the hydrophobicity of the analyte and decrease with the increase of the surfactant concentration. When hydrophobic compounds are analyzed the suggested ways to avoid split peaks are: (i) the use of 1-propanol or 1-butanol as sample diluent instead of methanol or acetonitrile or (ii) the use of high concentration of surfactant in the separating solution when the analyte must be dissolved in pure methanol or acetonitrile.

Development and optimization of an analytical method for the determination of UV filters in suntan lotions based on microemulsion electrokinetic chromatography.

Microemulsion electrokinetic chromatography (MEEKC) has been applied to the separation of some UV filters (Eusolex 4360, Eusolex 6300, Eusolex OCR, Eusolex 2292, Eusolex 6007, Eusolex 9020, Eusolex HMS, Eusolex OS, and Eusolex 232) commonly found in suntan lotions. The composition of the microemulsion employed was optimized with respect to the best possible separation of the selected analytes using artificial neural networks (ANNs). Two parameters namely the composition of the mixed surfactant system comprising the anionic sodium dodecyl sulfate (SDS) and neutral Brij 35 and the amount of organic modifier (2-propanol) present in the aqueous phase of the microemulsion were modeled. Using an optimized MEEKC buffer consisting of 2.25 g SDS, 0.75 g Brij 35, 6.6 g 1-butanol, 0.8 g n-octane, 17.5 g 2-propanol, and 72.1 g of 10 mM borate buffer (pH 9.2), eight target analytes could be separated in under 25 min employing a diode-array detector to segregate the overlapping signals obtained for Eusolex 9020 and Eusolex HMS. Detection limits from 0.8 to 6.0 nug/mL were obtained and the calibration plots were linear over at least one order of magnitude. The optimized method could be applied to the determination of Eusolex 6300 and Eusolex 9020 in a commercial suntan lotion.

Simultaneous analysis of sulfur-containing excitatory amino acids using micellar electrokinetic chromatography with diode array and laser-induced fluorescence detection.

The suitability of micellar electrokinetic chromatography (MEKC) coupled with diode array or laser-induced fluorescence (LIF) detection to analyze the four sulfur-containing excitatory amino acids (SEAA), homocysteine sulfinic acid (HCSA), homocysteic acid (HCA), cysteine sulfinic acid (CSA), and cysteic acid (CA) was investigated. 5-Carboxy-fluorescein succinimidyl ester was chosen as fluorescent reagent to derivatize HCSA, HCA, CSA, and CA. During method development, the yield of reaction dependent on pH and incubation time as well as the stability of the products were analyzed. The maximum yield was obtained after 30 min using a 0.1 M borate buffer (pH 8.9) as derivatization buffer. Each labeled amino acid exhibited high stability at room temperature over a period of 5 days. Baseline separation of labeled HCSA, HCA, CSA, and CA was obtained using a buffer consisting of 0.1 M borate, 50 mM sodium dodecyl sulfate (SDS), and 5% v/v methanol (pH 9.0). By applying LIF detection, limits of detection ranged from 0.9 x 10(-10) M for HCSA to 6.0 x 10(-10) M for CA, respectively. Slightly modified separation conditions enabled the analysis of SEAA in cerebrospinal fluid in the presence of the neurotransmitters glutamate and aspartate. In conclusion, MEKC coupled with LIF detection is a suitable technique for the simultaneous and sensitive analysis of SEAA. Further work will focus on the validation of the method with cerebrospinal fluid as sample matrix.

Determination of dye/protein ratios in a labeling reaction between a cyanine dye and bovine serum albumin by micellar electrokinetic chromatography using a diode laser-induced fluorescence detection.

The degree of labeling, i.e., dye/protein ratio (D/P) is important for characterizing properties of dye labeling with proteins. A method for the determination of this ratio between a fluorescent cyanine dye and bovine serum albumin (BSA), based on the separation of the labeling mixture using micellar electrokinetic chromatography with diode laser-induced fluorescence detection, is described. Two methods for the determination of D/P were examined in this study. In these methods, a hydrolysis product and impurities, which are usually unfavorable compounds that are best excluded for protein analysis, were utilized to determine the amounts of dye bound to BSA. One is a direct method in which a ratio of the peak area of BSA to the total peak area of all the products produced in the labeling reaction was used for determining the average number of dye molecules bound to a single BSA molecule. The other is an indirect determination, which is based on diminution of all peak areas related to the products except for the labeled BSA. These methods were directly compared by means of a spectrophotometric method. The experimental results show that the indirect method is both reliable and sensitive. Therefore, D/P values can be determined at trace levels using the indirect method.

Chiral separation of pemoline enantiomers by cyclodextrin-modified micellar capillary chromatography.

Micellar electrokinetic chromatography (MEKC) was successfully applied to the chiral separation with the addition of cyclodextrins (CDs) as chiral selector to running buffer. Chiral separation depended on the type of CDs. Mono-3-O-phenylcarbamoyl-beta-CD was effective for the chiral separation of pemoline. We investigated the type and concentration of CD and other parameters such as buffer pH, the concentration of SDS and the effect of organic modifier. The conditions for enantiomeric separation of pemoline were as follows: 40 mmol/l borate buffer at pH 9.0 with 40 mmol/l SDS, 20 mmol/l mono-3-O-phenylcarbamoyl-beta-CD and 10% 2-propanol. Baseline separation (Rs=2.21) of pemoline can be achieved.