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Toxicity has been emulated in tadpole species through chromatographic systems. The parameter studied to evaluate the non-specific toxicity of a compound is the narcosis concentration (Cnar), which is defined as the concentration needed for the immobilization of the organism. Because experimental investigation with animals is lengthy, costly, technically difficult, and ethically questionable, there is a great interest in developing surrogate physicochemical systems able to emulate biological systems to obtain the same information in a faster, more economic, and easier manner. In order to see which chromatographic systems would be able to emulate tadpole narcosis, both, tadpole narcosis data and data in several chromatographic and electrophoretic systems, were fitted to a linear solvation energy relationship (LSER) model. Thus, by comparison of the models it was possible to see which of the chromatographic systems were more similar to the biological one. The physicochemical systems that best emulate tadpole narcosis were an HPLC system based on an immobilized artificial membrane (IAM) column, and two micellar electrokinetic chromatography (MEKC) systems based on sodium taurocholate (STC) and a mixture of sodium dodecylsulphate (SDS) and Brij 35 as surfactants. A system based on a RP18 HPLC column also was selected for comparison because it is a common column in most analytical laboratories. To establish the models, a set of compounds with known Cnar values were analyzed in the chromatographic, and electrophoretic selected systems and, then, the retention factor (k) was correlated to the concentration of narcosis. Statistics showed that the system based on STC micelles was the best to emulate toxicity in tadpoles. The robustness and predictive ability of the developed models were validated.

The internal standard capillary electrophoresis method (IS-CE) has been implemented in a novel sequential injection-capillary electrophoresis instrument for the high-throughput determination of acidity constants (pK(a)) regardless of aqueous solubility, number of pK(a) values, or structure. This instrument comprises a buffer creation system that automatically mixes within a few seconds four reagents for in situ creation of the separation electrolyte with a pH range of 2-13, ionic strength of 10-100 mM and organic solvent content from 0% to 40%. Combined with 1.2 kV/cm and a short effective length (15 cm to the UV detector) fast 20 s electrophoretic separations can be obtained. The low standard deviation of the replicates and the low variation compared to reference values show that this system can accurately determine acidity constants of drugs by IS-CE. A single pK(a) can be determined in 2 min and a set of 20 measurements in half an hour, allowing rapid, simple, and flexible determination of pK(a) values of pharmaceutical targets.

Critical micelle concentration (CMC) of sodium dodecyl sulphate (SDS), lithium perfluorooctanesulfonate (LPFOS), hexadecyltrimethylammonium bromide (HTAB), tetradecyltrimethylammonium bromide (TTAB), and sodium cholate (SC), surfactants commonly used as pseudostationary phases in micellar electrokinetic chromatography (MEKC), have been determined by means of three different methods: MEKC, spectrophotometry, and conductometry. Determinations have been performed in water, and also in different concentrations of phosphate buffer at pH 7.0. CMC values ranging from 8.08 (water) to 1.99 (50 mM phosphate buffer) mM for SDS, from 7.16 (water) to 2,81 (30 mM phosphate buffer) mM for LPFOS, from 3.77 (water) to 1.93 (20 mM phosphate buffer) mM for TTAB, from 0.91 (water) to ∼0.34 (20 mM phosphate buffer) for HTAB, and around 13 mM (20 mM phosphate buffer) for SC, are obtained. The effect of the electrolyte concentration on the CMC, as well as the linear relationship between the electrolyte counter-ion concentration and the CMC are discussed. This linear relationship provides an easy way for users to estimate the CMC of a MEKC system, at a given electrolyte concentration. A comparison between experimental methods, as well as a discussion about the suitability of a given method for the determination of the CMC for a given surfactant system is also provided.

Kamlet-Taft solvatochromic scales have been used for the characterization of three different micellar electrokinetic chromatography (MEKC) systems, providing information about the polarity parameters of both the aqueous phase and the micellar phase of each system. The surfactants studied are sodium cholate (SC), which belongs to the family of bile salts, lithium perfluoro-n-octanesulfonate (LPFOS), a fluoro-surfactant, and n-hexadecyl-trimethylammonium bromide (HTAB), a cationic surfactant. Sodium n-dodecyl sulfate, the most commonly used surfactant in MEKC, was already studied in a previous work. The solvatochromic measurements allow the determination of the critical micellar concentration (cmc) of the surfactants and also the partition coefficients of the solvatochromic indicators in each system. Both cmc and partition coefficients have been also determined through MEKC experiments, and good agreement is observed between the two methods. The results of the solvatochromic study indicate that LPFOS is the surfactant that has properties more similar to those of the aqueous phase, whereas SC is the most hydrogen-bond basic and acidic surfactant. and HTAB has the lowest hydrogen-bond acidity.

Nowadays, pharmaceutical research needs to deal with sparingly soluble compounds and requires high-throughput technology to rapidly and accurately determine the p K a values of drug candidates. In this chapter, on the one hand, the challenge of p K a determination in hydroorganic solvents and the most widely used approaches to obtain the aqueous extrapolated value are presented. On the other hand, high-throughput methods based on potentiometry, spectrophotometry, and capillary electrophoresis are discussed and compared.

This chapter reviews the most important features of the buffers commonly employed in capillary electrophoresis. Effect of the buffer properties such as pH, concentration, or ionic strength, on the electro-osmotic flow and on acid–base analytes migration is discussed. Other parameters that can affect buffer properties or analyte migration during CE separations, such as temperature, electrolysis time, or buffer-analyte interactions, are also reviewed.

This chapter reviews the most used classical methods, potentiometry and spectrophotometry, for acidity constant determination. For this purpose, the theoretical and operational definitions of pH and the equations to calculate acidity constants from potentiometric and spectrophotometric data are explained. In addition, capillary electrophoresis is presented as a very convenient technique for p K a determination, especially in the case of low-purity samples.

The solvation properties of a micellar electrokinetic chromatography (MEKC) system composed of an aqueous phosphate buffer at pH 7.0 and sodium n-dodecyl sulfate (SDS) have been determined with solvatochromic indicators and compared with those of pure unbuffered water−SDS micellar system. The solvatochromic measurements allow calculation of the critical micellar concentrations (CMC), partition coefficients for the test compounds, and solvation properties of the two micellar systems studied. The results obtained are in good agreement with data obtained here and in previous works from MEKC measurements for the phosphate−SDS system. The solvent properties and partition coefficients in phosphate−SDS buffer are very similar to those obtained in water−SDS system, but the CMC values are lower in the phosphate buffer. The obtained results indicate that SDS micelles are very polar (π* = 1.02±0.01), quite hydrogen-bond donor (HBD) acidic (α = 0.80±0.02), and moderately hydrogen-bond acceptor (HBA) basic (β = 0.62±0...

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.

Several micellar electrokinetic chromatography (MEKC) systems (sodium dodecyl sulfate, lithium dodecyl sulfate, lithium perfluorooctanesulfonate, sodium cholate, sodium deoxycholate, tetradecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide) have been characterized by means of the solvation parameter model. It has been observed that the coefficients of the correlation equations depend strongly on the particular set of compounds analyzed. Principal component analysis has been used to characterize the 2975 compounds with available solute descriptors and to select an appropriate subset of compounds to be analyzed by MEKC. With this set of compounds, the MEKC systems have been characterized. Principal component analysis has also been used to show the similarities and differences between the properties of the surfactants characterized by MEKC.

A novel high-throughput method for determining acidity constants (pKa) by capillary electrophoresis (CE) is developed. The method, based on the use of an internal standard (IS-CE), is implemented as a routine method for accurate experimental pKa determination of drugs undergoing physicochemical measurements in drug discovery laboratories. Just two electropherograms at 2 different pH values are needed to calculate an acidity constant. Several ISs can be used in the same buffer and run to enhance precision. With 3 ISs, for example, the pKa of the test compound (TC) can be obtained in triplicate in less than 3 min of electrophoresis. It has been demonstrated that the IS-CE method eliminates some systematic errors, maintaining, or even increasing the precision of the results compared with other methods. Furthermore, pH buffer instability during electrophoretic runs is not a problem in the IS-CE method. It is also proved that after 16 h of electroseparation using the same buffer vial, pH may change by around one unit; but the pKa calculated by the IS-CE method remains constant. Thus, IS-CE is a powerful high-throughput method for pKa determination in drug discovery and development.

Propolis is a complex hive product produced by honey bees, Apis mellifera. Its composition and biological activities depend on the vegetation where hives are placed. Propolis is often used as a food supplement. The aim of this research is to determine the antioxidant properties in vitro and the phenolic composition of six propolis collected from the region of Santiago of Chile. We obtained the ethanolic extracts dry and wax free (EEPs) and studied their antioxidant properties by FRAP, ORAC-FL, ORAC-PGR and DPPH radical methods. The total phenols were quantified by a spectrophotometric method and 30 phenolic compounds were identified by HPLC-ESI-MS/MS analysis. Curacavi EEP has the highest relative abundance of caffeic acid phenylethyl ester (CAPE) while Buin EEP has the highest relative abundance of caffeic acid benzyl ester and quercetin. Both have the highest antioxidant activity in vitro in all methods used. Our research shows, for the first time, a comparative analysis of the antioxidant activity and phenolic composition of six Chilean propolis. Pinobanksin is the only phenol present in the six samples of propolis so it may be a good candidate for the standardization of propolis ethanolic extracts in the region of Santiago.

Chamaecrista nictitans (L) extract possesses antivi- ral properties; it acts against the herpes simplex virus, and this may be attributed to its constituent phenolics. Here, high- resolution LC-ESI-MS/MS is used to identify the phenolic components of the most potent fraction of the extract. The fraction is a complex mixture rich in oligomeric proanthocyanidins with a high content of monohydroxyphenol moieties ((epi)fisetinidol, (epi)afzelechin and (epi)guibourtinidol) and A-type linkages, uncommon in other proanthocyanidin-rich phenolic extracts, such as those from grape seeds or pine bark. As monohydroxyphenolic structures and A-type linkages have been related to antiviral effects, par- ticularly through the inhibition of late transcription, we suggest that the fraction of C. nictitans extract exerts its action through a particularly effective combination of proanthocyanidins that include these two structural features.

The internal standard (IS) CE (IS-CE) method is an interesting alternative to other methods for the determination of acidity constants of compounds. Although some of the advantages of this method have been already reported, the method has not been tested yet as regards to temperature effects. This has been the aim of this work, where it is demonstrated that the method can be applied successfully for the determination of pK(a) s at different temperatures, if the acidity constant of the IS at the desired temperature is known. The fact of obtaining the acidity constants at different temperatures allows the calculation of some thermodynamic quantities, such as the molar standard enthalpy and the molar standard entropy in a fast way. It is also demonstrated that if the IS and the test compound have similar standard enthalpy increment, the IS compensates uncontrolled possible temperature fluctuations (e.g., due to Joule heat) inside the capillary obtaining reliable acidity constant values at the desired temperature.

Several chromatographic systems (three systems of high-performance liquid chromatography and two micellar electrokinetic chromatography systems) besides the reference octanol-water partition system are evaluated by a systematic procedure previously proposed in order to know their ability to model human skin permeation. The precision achieved when skin-water permeability coefficients are correlated against chromatographic retention factors is predicted within the framework of the solvation parameter model. It consists in estimating the contribution of error due to the biological and chromatographic data, as well as the error coming from the dissimilarity between the human skin permeation and the chromatographic systems. Both predictions and experimental tests show that all correlations are greatly affected by the considerable uncertainty of the skin permeability data and the error associated to the dissimilarity between the systems. Correlations with much better predictive abilities are achieved when the volume of the solute is used as additional variable, which illustrates the main roles of both lipophilicity and size of the solute to penetrate through the skin. In this way, the considered systems are able to give precise estimations of human skin permeability coefficients. In particular, the HPLC systems with common C18 columns provide the best performances in emulating the permeation of neutral compounds from aqueous solution through the human skin. As a result, a methodology based on easy, fast, and economical HPLC measurements in a common C18 column has been developed. After a validation based on training and test sets, the method has been applied with good results to the estimation of skin permeation of several hormones and pesticides.

A systematic approach for evaluating the goodness of chromatographic systems to model the sorption of neutral organic compounds by soil from water is presented in this work. It is based on the examination of the three sources of error that determine the overall variance obtained when soil-water partition coefficients are correlated against chromatographic retention factors: the variance of the soil-water sorption data, the variance of the chromatographic data, and the variance attributed to the dissimilarity between the two systems. These contributions of variance are easily predicted through the characterization of the systems by the solvation parameter model. According to this method, several chromatographic systems besides the reference octanol-water partition system have been selected to test their performance in the emulation of soil-water sorption. The results from the experimental correlations agree with the predicted variances. The high-performance liquid chromatography system based on an immobilized artificial membrane and the micellar electrokinetic chromatography systems of sodium dodecylsulfate and sodium taurocholate provide the most precise correlation models. They have shown to predict well soil-water sorption coefficients of several tested herbicides. Octanol-water partitions and high-performance liquid chromatography measurements using C18 columns are less suited for the estimation of soil-water partition coefficients.

The performance of chromatographic systems to mimic aquatic toxicity to the fathead minnow fish is evaluated taking into account the factors that contribute to the variance of biological-chromatographic correlations. These factors are the precision to measure the fathead minnow toxicity, the precision of the surrogate chromatographic system, and the error from the dissimilarity between the fathead minnow and chromatographic systems. The precisions are estimated through the characterization of the systems by the solvation parameter model. Several chromatographic systems as well as the common reference octanol-water partition system have been selected to test their ability to model the nonspecific toxicity to the fathead minnow by means of the proposed approach. Predictions and experimental tests show that the micellar electrokinetic chromatography system of sodium taurocholate and chromatographic measurements using an immobilized artificial membrane column provide the most precise estimations of this biopartitioning property. The octanol-water partition system, the conventional C18 high-performance liquid chromatography systems, and the micellar electrokinetic chromatography system of sodium dodecylsulfate show worse performances.

El pdf del artículo es la versión post-print., D-Fagomine is an iminosugar first found in buckwheat (Fagopyrum esculentum Moench) which if used as a dietary supplement or functional food component may reduce the risks of developing insulin resistance, becoming overweight and suffering from an excess of potentially pathogenic bacteria. As D-fagomine may become increasingly important to the food industry, a reliable analytical method for its determination in natural plant sources and foodstuffs is desirable. We have devised a method to separate D-fagomine from its diastereomers 3-epi-fagomine and 3,4-di-epi-fagomine in a single run by cation exchange high-performance liquid chromatography (HPLC) with detection and quantification by mass spectrometry using electrospray ionisation and a simple quadrupole analyser (ESI-Q-MS). The method is validated and applied to the determination of D-fagomine in buckwheat groats (6.7-44 mg kg ?1), leaves, bran and flour.We show that buckwheat contains 3,4-di-epi-fagomine (1.0-43 mg kg ?1), which has not previously been reported in this source. The procedure is also applied to mulberry (Morus alba) leaves, which contain D-fagomine and 3-epi-fagomine as minor components. The new method provides a means for convenient and accurate determination of D-fagomine in plant samples and foodstuffs.© 2011 Springer-Verlag., This research was primarily funded by Bioglane SLNE. Financial support from the Spanish Ministry of Science and Innovation (CDTI-NEOTEC 20080474, AGL2009-12374-C03-03/ALI) and the Catalan regional government (Generalitat de Catalunya, 2009 SGR 00281) is also appreciated.

The inner bark of Ceylon cinnamon (Cinnamomum zeylanicum L.) is commonly used as a spice and has also been widely employed in the treatment and prevention of disease. The positive health effects associated with the consumption of cinnamon could in part be due to its phenolic composition; proanthocyanidins (PA) are the major polyphenolic component in commercial cinnamon. We present a thorough study of the PA profile of cinnamon obtained using matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. In addition to the advantages of MALDI-TOF as a sensitive technique for the analysis of high-molecular-weight compounds, the tandem arrangement allows the identification of the compounds through their fragmentation patterns from MS/MS experiments. This is the first time that this technique has been used to analyze polymeric PA. The results show that cinnamon PA are more complex than was previously thought. We show here for the first time that they contain (epi)gallocatechin and (epi)catechingallate units. As gallates (galloyl moieties) and the pyrogallol group in gallocatechins have been related to the biological activity of grape and tea polyphenols, the presence of these substructures may explain some of the properties of cinnamon extracts. MALDI-TOF/TOF reveals that cinnamon bark PA include combinations of (epi)catechin, (epi)catechingallate, (epi)gallocatechin, and (epi)afzelechin, which results in a highly heterogeneous mixture of procyanidins, prodelphinidins, and propelargonidins., This work was supported by the Spanish Ministry of Education and Science (research grants AGL2009-12374-C03-03/ALI). J.P-J thanks the Spanish Ministry of Science and Innovation for granting her a Sara Borrell postdoctoral contract (CD09/00068).

A set of 25 monoprotic bases is proposed as internal standards for pK(a) determination by capillary electrophoresis. The pK(a) of the bases is determined and compared with available literature data. The capillary electrophoresis internal standard method offers numerous advantages over other typical methods for pK(a) determination, especially of analysis time and buffer preparation. However, it requires disposing of appropriate standards with reference pK(a) value. The set of bases established in this work together with the set of acids previously established provide a reference set of compounds with well-determined acidity constants that facilitate the process of selecting appropriate internal standards for fast pK(a) determination by capillary electrophoresis in high throughput screening of pharmaceutical drugs. In addition, the performance of the method when acidic internal standards are used for the determination of acidity constants of basic internal standards has also been tested. Although higher errors may be expected in this case, good agreement is observed between determined and literature values. These results indicate that in most cases structural similarity between the analyte and the internal standard might not be an essential requirement in the internal standard method.

The non-extractable fraction of many fruit and vegetables contains putatively bioactive polyphenolic compounds that, in most cases, have not been well characterised structurally. Non-extractable proanthocyanidins (NEPA) of a polymeric nature are part of the dietary fibre fraction of food. Using liquid chromatography coupled to a mass spectrometer equipped with an electrospray ionisation chamber and a triple quadrupole mass analyser for tandem analysis (HPLC–ESI–QqQ–MS/MS) techniques, we examine the phenolic metabolites present in urine and faeces from rats 24 h after ingestion of an NEPA-rich fraction. We show that NEPA are partially depolymerised during their transit along the intestinal tract, as evidenced by the presence of (epi)catechin (EC) monomers and dimers in faeces and phase II conjugates of EC in urine. Moreover, NEPA are further metabolised by the intestinal microbiota into smaller metabolites including phenolic acids that are present in urine as both free phenolics and conjugates with glucuronate or sulphate moieties. For the first time, we report evidence that NEPA behave in vivo as a source of phenolics that are released progressively and deliver phenolic species that come into contact with the intestinal walls and are bioavailable for at least 24 h after ingestion., The present work was supported by the Spanish Ministry of Education and Science (AGL2009-12374-C03-03/ALI).

Grape antioxidant dietary fiber (GADF) is a phenolic-rich dietary fiber matrix. The aim of this work was to determine which phenolic compounds come into contact with colonic epithelial tissue after the ingestion of GADF. By use of HPLC-ESI-MS/MS techniques phenolic metabolites were detected in feces, cecal content, and colonic tissue from rats. Free (epi)catechin (EC) was detected in all three sources, and more than 20 conjugated metabolites of EC were also detected in feces. Fourteen microbially derived phenolic metabolites were also identified in feces, cecal content, and/or colonic tissue. These results show that during transit along the digestive tract, proanthocyanidin oligomers and polymers are depolymerized into EC units. After ingestion of GADF, free EC and its conjugates, as well as free and conjugated microbially derived phenolic metabolites, come into contact with the intestine epithelium for more than 24 h and may be partly responsible for the positive influence of GADF on gut health., This work was supported by the Spanish Ministry of Education and Science (Research Grants AGL2004-07579-C04-02 and -03; AGL2009-12374-C03-03/ALI; and SAF2008-00164), the Instituto de Salud Carlos III and European Regional Development Fund (ISCIII-RTICC, RD06/0020/0046), the Generalitat de Catalunya (2009SGR1308), 2009CTP 00026 and Icrea Academia award 2010 granted to M. Cascante and the European Commission (Etherpaths Project KBBE-Grant Agreement 222639). J.P.-J. thanks the Spanish Ministry of Science and Innovation for granting her a Sara Borrell postdoctoral contract (CD09/00068).

Few regulators of phenylpropanoids have been identified in monocots having potential as biofuel crops. Here we demonstrate the role of the maize (Zea mays) R2R3-MYB factor ZmMYB31 in the control of the phenylpropanoid pathway. We determined its in vitro consensus DNA-binding sequence as ACC(T)/(A) ACC, and chromatin immunoprecipitation (ChIP) established that it interacts with two lignin gene promoters in vivo. To explore the potential of ZmMYB31 as a regulator of phenylpropanoids in other plants, its role in the regulation of the phenylpropanoid pathway was further investigated in Arabidopsis thaliana. ZmMYB31 downregulates several genes involved in the synthesis of monolignols and transgenic plants are dwarf and show a significantly reduced lignin content with unaltered polymer composition. We demonstrate that these changes increase cell wall degradability of the transgenic plants. In addition, ZmMYB31 represses the synthesis of sinapoylmalate, resulting in plants that are more sensitive to UV irradiation, and induces several stress-related proteins. Our results suggest that, as an indirect effect of repression of lignin biosynthesis, transgenic plants redirect carbon flux towards the biosynthesis of anthocyanins. Thus, ZmMYB31 can be considered a good candidate for the manipulation of lignin biosynthesis in biotechnological applications.

Proanthocyanidins (PAs) or condensed tannins, a major group of dietary polyphenols, are oligomers and polymers of flavan-3-ol and flavan-3, 4-diols widely distributed in plant foods. Most literature data on PAs' metabolic fate deal with PAs that can be extracted from the food matrix by aqueous-organic solvents ( extractable proanthocyanidins). However, there are no data on colonic fermentation of non-extractable proanthocyanidins (NEPAs), which arrive almost intact to the colon, mostly associated to dietary fibre (DF). The aim of the present work was to examine colonic fermentation of NEPAs associated with DF, using a model of in vitro small intestine digestion and colonic fermentation. Two NEPA-rich materials obtained from carob pod (Ceratonia siliqua L. proanthocyanidin) and red grapes (grape antioxidant dietary fibre) were used as test samples. The colonic fermentation of these two products released hydroxyphenylacetic acid, hydroxyphenylvaleric acid and two isomers of hydroxyphenylpropionic acid, detected by HPLC-ESI-MS/MS. Differences between the two products indicate that DF may enhance the yield of metabolites. In addition, the main NEPA metabolite in human plasma was 3,4-dihydroxyphenyl acetic acid. The presence in human plasma of the same metabolites as were detected after in vitro colonic fermentation of NEPAs suggests that dietary NEPAs would undergo colonic fermentation releasing absorbable metabolites with potential healthy effects., The present research was performed under the financial support of the Spanish Ministry of Science and Innovation, National Program of I+D (AGL-2004-0759 and AGL-2008-01633).

In this article, we describe a recently developed capillary-electrophoresis method for the determination of acidity constants and compare it with other existing methods. The new method is based on the use of an internal standard (compound similar in nature and pKa value to the analyte), and offers several benefits, since it has all the advantages of capillary electrophoresis. In addition, it is very fast, because the exact measure of the pH of the separation electrolytes is not needed, and only a few electrophoretic runs are required to perform a pKa determination. The acidity constants of some monoprotic weak acids and bases were determined by this fast method, yielding a very good agreement with literature values.

Micellar electrokinetic chromatography (MEKC) systems with mixed pseudostationary phases of the bile surfactants sodium cholate (SC) and sodium deoxycholate (SDC) have been characterized by means of the solvation parameter model. The importance of characterizing systems with an appropriate set of solutes that embrace a wide range of descriptor values has been proven as they can significantly influence the value of the system constants. The fit of the solvation parameter model to the experimental log k data has been compared for each SC–SDC system when the Abraham descriptors and the Poole optimized descriptors, recently proposed, are used. In both cases, the variation in MEKC surfactant composition results in similar changes in the coefficients of the correlation equations, which in turn leads to similar information on solute–solvent and solute–micelle interactions. It is demonstrated that SDC is more hydrogen-bond acidic and hydrophobic but slightly less polarizable than SC. Systems with intermediate selectivity are obtained through mixtures of both surfactants.

A new and fast method to determine acidity constants of monoprotic weak acids and bases by capillary zone electrophoresis based on the use of an internal standard (compound of similar nature and acidity constant as the analyte) has been developed. This method requires only two electrophoretic runs for the determination of an acidity constant: a first one at a pH where both analyte and internal standard are totally ionized, and a second one at another pH where both are partially ionized. Furthermore, the method is not pH dependent, so an accurate measure of the pH of the buffer solutions is not needed. The acidity constants of several phenols and amines have been measured using internal standards of known pK(a), obtaining a mean deviation of 0.05 pH units compared to the literature values.

The performance of the most common and also some other less common CE buffers has been tested for the pK a determination of several types of compounds (pyridine, amines, and phenols). The selected buffers cover a pH ranging from 3.7 to 11.8. Whereas some buffers, like acetic acid/acetate, BisTrisH + /BisTris, TrisH + /Tris, CHES/CHES - , and CAPS/CAPS - can be used with all type of analytes, others like ammonium/ammonia, butylammonium/ butylammonia, ethylammonium/ethylammonia, diethylammonium/diethylammonia, and hydrogenphosphate/phosphate are not recommended because they interact with a wide range of compounds. The rest of the tested buffers (dihydrogenphosphate/hydrogenphosphate, MES/MES - , HEPES/HEPES - , and boric acid/borate) can show specific interactions depending on the nature of the analytes, and their use in some applications should be restricted.

A model that relates the chromatographic hydrophobicity index (CHI) to the pH of the mobile phase has been tested in two of the most common high-performance liquid chromatography (HPLC) solvents: methanol and acetonitrile. A set of eight monoprotic acids and nine monoprotic bases of different chemical nature (phenols, benzoic acids, anilines, and pyridine) have been selected for the validation. The variation of CHI values with the pH of the mobile phase shows a good fit to the model for almost all compounds, regardless of their nature, and similar CHI values of the neutral form of the substances are obtained in both organic modifiers. On the contrary, higher differences are observed for the ionic form of the test solutes. The values of the other parameters obtained with the model (s, pKa) are discussed according to the nature of the compounds, and the variation of solvent pH and compounds pKa along the gradient elution.

Encapsulates having shells of cross-linked mixtures of proteins and polysaccharides are widely used in the food and pharmaceutical industry for controlled release of actives and flavour compounds. In order to be able to predict the behaviour and the release characteristics of the microcapsules, a better understanding of the nature and extent of the cross-linking reaction is needed. Several analytical techniques were applied for the characterisation of glutardialdehyde (GDA) cross-linked encapsulates made of gelatine and gum arabic. To allow the use of sensitive, high-resolution methods such as chromatography and mass spectrometry, the sample first had to be hydrolysed. In this way, a mixture of amino acids, small peptides and the cross-link moieties was obtained. High-resolution liquid chromatography coupled to high-resolution mass spectrometry (HPLC-MS) was applied to detect possible cross-link markers through a comparison of HPLC-MS mass-chromatograms obtained for cross-linked and non-cross-linked coacervates. HPLC-MS/MS was used to identify the species responsible for the differences. Cross-linking occurred between GDA molecules and lysine and hydroxylysine epsilon-amino groups, and up to eight cross-link products of different nature could be identified. They included pyridinium ions and Schiff bases, and also unreacted GDA condensation products. Next, based on the insight gained in the possible chemical structures present in the cross-link markers, methods for selective labelling of these functionalities were employed to allow easier detection of related reaction products. Both liquid chromatography (LC) and gas chromatography (GC) were used in these experiments. Unfortunately, these approaches failed to detect new cross-link markers, most likely as a result of the low levels at which these are present.

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.

A suite of small unilamellar vesicles (SUVs) composed of mixtures of phospholipids and cholesterol (CH) or the synthetic surfactant dihexadecyl phosphate (DHP) and cholesterol was investigated using a homologous series of solvatochromic pi* indicators coupled with size exclusion methods and photon correlation spectroscopy (PCS). The solvatochromic method, which is based on the measurement of solvent-dependent shifts in lambda(max) from UV-Vis spectra of solubilized indicators, was used to quantify the dipolarity and polarizability (pi*) of probe solvation environments. The partitioning of the series of individual di-n-alkyl-p-nitroaniline (DNAP) pi* indicators in PG(24)PC(46)Chol(30) and DHP(70)Chol(30) SUVs was examined as a function of the head group structure as well as the method of dye-vesicle preparation. Solubilization of the larger more hydrophobic probes in the bilayer portion of PG(24)PC(46)Chol(30) SUVs was aided through physical entrapment. Such physical methods were not needed for the smaller indicators or for the range of indicators in the DHP(70)Chol(30) dispersions. Extrusion and size-exclusion chromatographic methodologies for the preparation of physically entrapped dopants in SUVs of fixed size range demonstrated that the larger (longer alkyl chain) dopants in the series resided in PG(24)PC(46)Chol(30) liposomes with a wider range of sizes, while the smaller more polar solutes tended to be entrapped in smaller vesicles with a narrower size range.

The optimization of the composition of mixed surfactants used as micellar electrokinetic chromatography (MEKC) pseudostationary phases is proposed as an effective method for the separation of complex mixtures of analytes. The solvation parameter model is used to select two surfactants (lithium dodecyl sulfate, LDS, and lithium perfluorooctanesulfonate, LPFOS) with contrasting solvation properties. Combination of these two surfactants allows variations of the solvation properties of MEKC pseudostationary phase along a wide range. Thus, the convenient variation of the proportion of both surfactants allows an effective control of the selectivity in such systems. An algorithm that predicts the overall resolution of a given mixture of compounds is described and applied to optimize the composition of the mixed surfactant for the separation of the mixture. The algorithm is based on the calculation of peak purities on simulated chromatograms as a function of the composition of the mixed LDS/LPFOS micellar buffer from data at several micellar buffer compositions. Successful separations were achieved for mixtures containing up to 20 compounds, in less than 12 min.

Several micellar electrokinetic chromatography (MEKC) systems (sodium dodecyl sulfate, lithium dodecyl sulfate, lithium perfluorooctanesulfonate, sodium cholate, sodium deoxycholate, tetradecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide) have been characterized by means of the solvation parameter model. It has been observed that the coefficients of the correlation equations depend strongly on the particular set of compounds analyzed. Principal component analysis has been used to characterize the 2975 compounds with available solute descriptors and to select an appropriate subset of compounds to be analyzed by MEKC. With this set of compounds, the MEKC systems have been characterized. Principal component analysis has also been used to show the similarities and differences between the properties of the surfactants characterized by MEKC.

A wide study of the compounds and procedures mostly used to determine the electroosmotic flow (EOF) and micelle elution times has been done in seven different micellar electrokinetic chromatography (MEKC) systems. These systems are formed from mixtures of an aqueous buffer with the surfactants sodium dodecyl sulfate, lithium dodecyl sulfate, lithium perfluorooctane sulfonate, sodium cholate, sodium deoxycholate, tetradecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide. The solvation parameter model has been used to evaluate the usefulness of the compounds studied as EOF or micellar markers in each of the seven MEKC systems. It is demonstrated that methanol, acetonitrile and formamide are the best EOF markers, and that dodecanophenone is the best micellar marker.

Thermodynamic solubility is a decisive physicochemical property in drug development. The Chasing Equilibrium method offers an alternative to the classical procedures to measure the solubility of compounds with acid-base properties. The method is fast and yields accurate results. In this work, the solubility of several compounds including acids and bases was determined through the Chasing Equilibrium approach. A study of experimental conditions in terms of sample weight was performed to measure solubilities. The study shows that only a limited range of weights, depending on the nature and solubility of the compounds, is adequate to obtain reliable results.

In 1997 Valko et al. developed a generic fast gradient HPLC method, based on the calculation of the chromatographic hydrophobicity index (CHI) from the gradient retention times, in order to measure lipophilicity. We have enhanced the correlations between CHI and log P o/w and adapted the rapid gradient HPLC method to UHPLC obtaining excellent resolution and repeatability in a short analysis time (

The development of methods to increase the bioavailability of drugs is of great interest, especially for those which are poorly soluble or permeable. One of the strategies to enhance the solubility (which in turn has the potential of increase bioavailability) of drugs is the use of additives in the formulation process, so that the drug can stay supersaturated in biological fluids for a period of time long enough to allow absorption. The use of polymers as pharmaceutical excipients in order to stabilize the supersaturation of drugs is common practice. In this work, the ability of different polymers of vinylpyrrolidone (K-12, K-17, K-25, K-29/32, K-90) and a copolymer of vinylpyrrolidone and vinylacetate (S-630) have been tested for their impact on the supersaturation of drugs. Sixteen drugs of different chemical nature have been selected, and analyzed using the Cheqsol method. The results of the drug alone, and of physical mixtures with the different polymers at several polymer:drug ratios have been compared in terms of supersaturation extent and duration. It has been observed that acidic compounds displayed enhanced solubility in different ways: sometimes the supersaturated state of the drug is maintained for a long time, due to the precipitation of an amorphous solid, as determined by X-ray diffraction studies; on other occasions supersaturation increases but only for a short time, compared to the drug alone, and then the drug precipitates to a crystalline form. Only a few basic drugs displayed enhanced solubility in the presence of PVP polymers, in contrast to acidic compounds.