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Papers by Clara Ràfols

Pharmaceutics, 2021

A new cocrystal of Norfloxacin, a poorly soluble fluoroquinolone antibiotic, has been synthetized... more A new cocrystal of Norfloxacin, a poorly soluble fluoroquinolone antibiotic, has been synthetized by a solvent-mediated transformation experiment in toluene, using resorcinol as a coformer. The new cocrystal exists in both anhydrous and monohydrate forms with the same (1:1) Norfloxacin/resorcinol stoichiometry. The solubility of Norfloxacin and the hydrated cocrystal were determined by the shake-flask method. While Norfloxacin has a solubility of 0.32 ± 0.02 mg/mL, the cocrystal has a solubility of 2.64 ± 0.39 mg/mL, approximately 10-fold higher. The dissolution rate was tested at four biorelevant pH levels of the gastrointestinal tract: 2.0, 4.0, 5.5, and 7.4. In a first set of comparative tests, the dissolution rate of Norfloxacin and the cocrystal was determined separately at each pH value. Both solid forms showed the highest dissolution rate at pH 2.0, where Norfloxacin is totally protonated. Then, the dissolution rate decreases as pH increases. In a second set of experiments, t...

ADMET and DMPK, 2018

Serum albumin is the main drug transporter of the bloodstream and contains two main binding sites... more Serum albumin is the main drug transporter of the bloodstream and contains two main binding sites: Sudlow I or acidic drug binding site, and Sudlow II or benzodiazepine binding site. Warfarin, a well-known anticoagulant drug commonly used in the prevention of thrombosis and thromboembolism, binds to Sudlow I site, whereas non-steroidal antiinflammatory drugs (NSAIDs) such as diflunisal bind preferentially to Sudlow II site. Albumin is a fluorophore that modifies its fluorescence (quenching or enhancement effect) when it is bound to a drug. The application of the double logarithm Stern-Volmer equation allows the calculation of the stoichiometry and the binding constant of the process. This procedure does not consider the possible interferences coming from the fluorescence of the drug though. Another strategy to evaluate the binding constants is to consider the whole spectrum, taking into account all the possible species in equilibrium; in this case we have used an extended version ...

ADMET and DMPK, 2018

The synthesis of cocrystals is presented as an alternative to improve the properties of active ph... more The synthesis of cocrystals is presented as an alternative to improve the properties of active pharmaceutical ingredients, especially those related to solubility and dissolution rate. In this work the dissolution rate of ciprofloxacin, a zwitterionic fluoroquinolone antibiotic, has been compared to its cocrystal with resorcinol. To this end, dissolution rate has been determined at several biorelevant pH values, and also in two simulated gastrointestinal fluids (FeSSIF and FaSSIF). Results show that both, ciprofloxacin and the cocrystal, dissolve more slowly as pH increases (from 2.0 to 7.4), as ionization degree of ciprofloxacin decreases. In addition, dissolution is not enhanced by the components of the gastrointestinal fluids.

ADMET and DMPK, 2017

In the present study the solubility-pH profiles of sibutramine free base and its hydrochloride sa... more In the present study the solubility-pH profiles of sibutramine free base and its hydrochloride salt were determined in the pH range between 2.0 and 9.5 by means of the recommended shake-flask method, and the solids collected were dried and studied by X-ray diffraction in order to elucidate their free base or salt structure. Above pH max (or Gibbs pK a) the solid collected was always identified as free base, whatever the sibutramine species (free base or hydrochloride salt) initially solved. However, in the pH range below pH max different solids were isolated depending on the buffers employed.

European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, Jan 23, 2018

The development of methods to increase the bioavailability of drugs is of great interest, especia... more 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 ...

Journal of pharmaceutical and biomedical analysis, Jan 20, 2018

Interaction thermodynamics between warfarin, a very popular anticoagulant, and Sudlow I binding s... more Interaction thermodynamics between warfarin, a very popular anticoagulant, and Sudlow I binding site of human (HSA) or bovine (BSA) serum albumin have been examined in strictly controlled experimental conditions (HEPES buffer 50 mM, pH 7.4 and 25 °C) by means of isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE). Each technique is based on measurements of a different property of the biochemical system, and then the results allow a critical discussion about the suitability of each approach to estimate the drug-protein binding parameters. The strongest interaction step is properly evaluated by the three assayed approaches being the derived binding constants strongly consistent: from 4 × 104 to 7 × 104 for HSA and from 0.8 × 105 to 1.2 × 105 for BSA. Binding enthalpy variations also show consistent results: -5.4 and -5.6 Kcal/mol for HSA and -4.3 and -3.7 Kcal/mol for BSA, as measured by ITC and FS, respectively...

Talanta, 2016

A study about the suitability of the chelation reaction of Ca 2+ with ethylendiamintetraacetic ac... more A study about the suitability of the chelation reaction of Ca 2+ with ethylendiamintetraacetic acid (EDTA) as a validation standard for Isothermal Titration Calorimeter measurements has been performed exploring the common experimental variables (buffer, pH, ionic strength and temperature). Results obtained in a variety of experimental conditions have been amended according to the side reactions involved in the main process and to the experimental ionic strength and, finally, validated by contrast with the potentiometric reference values. It is demonstrated that the chelation reaction performed in acetate buffer 0.1 M and 25ºC shows accurate and precise results and it is robust enough to be adopted as a standard calibration process.

Journal of molecular recognition : JMR, Jan 4, 2015

Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of... more Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target-drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A-site, with two well-known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin-dinucleotide and -diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin-diPNA conjugates as the best candidates for antimicrobial activity. Copyright © 2015 John Wiley & Sons, Ltd.

European Journal of Pharmaceutical Sciences, 2015

Several procedures based on the shake-flask method and designed to require a minimum amount of dr... more Several procedures based on the shake-flask method and designed to require a minimum amount of drug for octanol-water partition coefficient determination have been established and developed. The procedures have been validated by a 28 substance set with a lipophilicity range from-2.0 to 4.5 (log D7.4). The experimental partition is carried out using aqueous phases buffered with phosphate (pH 7.4) and n-octanol saturated with buffered water and the analysis is performed by liquid chromatography. In order to have accurate results, four procedures and eight different ratios between phase volumes are proposed. Each procedure has been designed and optimized (for partition ratios) for a specific range of drug lipophilicity (low, regular and high lipophilicity) and solubility (high and low aqueous solubility). The procedures have been developed to minimize the measurement in the octanolic phase. Experimental log D7.4 values obtained from different procedures and partition ratios show a standard deviation lower than 0.3 and there is a nice agreement when these values are compared with the reference literature ones.

Two different approaches were evaluated and used to estimate the aqueous pK a values of some flav... more Two different approaches were evaluated and used to estimate the aqueous pK a values of some flavonols sparingly soluble in water (morin, fisetin and quercetin) from their pK a values in methanol/water mixtures obtained by potentiometry. Both approaches lead to similar results, although one of them requires only one pK a value at one unique methanol/water mixture, whereas the other (the classical Yasuda-Shedlovsky plot) requires several pK a data at different methanol/water mixtures. Thus, the first approach is recommended because it is faster and simpler. The automated potentiometric method is strongly recommended for pK a determination of these types of compounds because of its simplicity and speed of operation.

ADMET & DMPK, 2014

Predictive software packages to estimate the lipophilicity of molecules have become key tools in ... more Predictive software packages to estimate the lipophilicity of molecules have become key tools in the new drug design. Six different well-known computational programs including the classical BioByte-clogP and the GALAS algorithm offered by ACDlabs were evaluated through a set of 103 drugs with different structures and functionalities. To evaluate the predictions accuracy, reliable experimental log P o/w values for the whole testing set were carefully selected. The best estimations are performed by GALAS/logP based on the fragmental method, corrected according to the similarity with compounds included in the software training set.

Journal of Pharmaceutical Sciences, 2003

The aqueous pK values (w w pK a) of several sparingly water-soluble drugs with amino groups have ... more The aqueous pK values (w w pK a) of several sparingly water-soluble drugs with amino groups have been calculated from pK values determined in several methanol/ water mixtures (s s pK a) by means of the Yasuda-Shedlovsky equation and by a linear equation that relates w w pK a of amino compounds with s s pK a obtained in any particular methanol/water mixture. Parameters of this last equation for amino compounds can be easily calculated from solely the methanol content of the solvent. Results from both approaches are consistent. However, Yasuda-Shedlovsky equation requires several s s pK a determinations in solutions with different methanol content and can be used only from measurements in solutions with a maximum methanol content of about 65% in weight. In contrast, the linear proposed equation allows a very good estimation of w w pK a from only one experimental s s pK a value, and it permits this estimation from s s pK a determined in a solution very rich in methanol. Therefore, it is suitable for very insoluble compounds. The examined amino compounds cover a wide range of w w pK a , from 6.7 to 10.6, which have been estimated from experimental s s pK a values in the whole composition range. In all instances consistent and satisfactory results have been achieved.

Journal of Pharmaceutical and Biomedical Analysis, 2009

The acidic dissociation enthalpies and constants of anilinium, protonated tris(hydroxymethyl)amin... more The acidic dissociation enthalpies and constants of anilinium, protonated tris(hydroxymethyl)aminomethane (HTris(+)), benzoic and acetic acids, have been determined at several temperatures in pure water and in methanol/water mixtures by potentiometry and by isothermal titration microcalorimetry (ITC). The pK(a) values determined by both techniques are in accordance when the dissociation process involves large amounts of heat. However, for the neutral acids the ITC technique gave slightly lower pK(a) values than those from potentiometry at the highest temperatures studied due to the small amounts of heat involved in the acidic dissociation. The dissociation enthalpies have been determined directly by calorimetry and the obtained values slightly decrease with the increase of temperature. Therefore, only a rough estimation of the dissociation enthalpies can be obtained from potentiometric pK(a) by means of the Van't Hoff approach.

Journal of Chromatography A, 2012

A systematic approach for evaluating the goodness of chromatographic systems to model the sorptio... more 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.

Journal of Chromatography A, 2003

The knowledge of the acid-base equilibria in water-solvent mixtures of both common buffers and an... more The knowledge of the acid-base equilibria in water-solvent mixtures of both common buffers and analytes is necessary in order to predict their retention as function of pH, solvent composition and temperature. This paper describes the effect of temperature on acid-base equilibria in methanol-water solvent mixtures commonly used as HPLC mobile phases. We measured the delta-correction parameter (delta = sw pH - ss pH = Ej - log sw(gamma)oh) between two pH scales: pH measured in the solvent concerned and referred to the same standard state, ss pH, and the pH measured in that solvent mixture but referred to water as standard state, sw pH, for several methanol compositions in the temperature range of 20-50 degrees C. These determinations suggest that the delta-term depends only on composition of the mixture and on temperature. In water-rich mixtures, for which methanol is below 40% (w/w), delta-term seems to be independent of temperature, within the experimental uncertainties, whereas for methanol content larger than 50% (w/w) the delta-correction decreases as temperature increases. We have attributed this decrease to a large increase in the medium effect when mixtures have more than 50% methanol. The pKa of five weak electrolytes of different chemical nature in 50% methanol-water at 20-50 degrees C are presented: the effect of temperature on pKa was large for amines, pyridine and phenol, but almost no dependence was found for benzoic acid. This indicates that buffers can play a critical role in affecting retention and selectivity in HPLC at temperatures far from 25 degrees C, particularlyfor co-eluted solutes.

Journal of Chromatography A, 2010

A set of 25 monoprotic bases is proposed as internal standards for pK a determination by capillar... more 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.

Journal of Chromatography A, 2006

A fast RP-HPLC method for the simultaneous separation of eleven main flavonoid aglycones in food ... more A fast RP-HPLC method for the simultaneous separation of eleven main flavonoid aglycones in food samples was developed. Flavonoids were divided in three groups according to their hydrophobicity and were resolved by a ternary gradient elution and diode array detection within analysis times less than 15 min. The detection limits of the flavonoids studied were in the range between 0.04 and 0.31 microg/mL, with repeatabilities values between 0.4 and 5%. The method was successfully applied to the analysis of these compounds in complex natural samples, like propolis and Ginkgo biloba.

Journal of Chromatography A, 2004

Residual silanol acidity and activity of one immobilized artificial membrane (IAM) column have be... more Residual silanol acidity and activity of one immobilized artificial membrane (IAM) column have been measured from the retention of LiNO 3 in the column with a methanol/buffer (1 mM in Na +) (60:40, v/v) mobile phase buffered to different pH values. Just one type of silanol with s s pK a = 7.61 (near the pH limit recommended by the manufacturer) was found, although these silanols show large activity. The results obtained have been compared with those obtained previously for Resolve C18, Resolve Silica, Symmetry C18, Symmetry Silica, XTerra MS C18, underivatized XTerra, Lichrospher 100 RP-18, Purospher RP-18e, Luna C18 (2) and Chromolith Perfomance RP-18e, showing that the IAM column is similar to Luna C18 and Symmetry C18 in terms of silica quality, as measured by Li + retention. A warning about the use of IAM columns for emulation of biological systems at physiological pH 7 is given because the ionized silanols may contribute to the retention of some drugs at this pH.

Journal of Chromatography A, 2005

The retentive behavior of weak acids and bases in reversed-phase liquid chromatography (RPLC) upo... more The retentive behavior of weak acids and bases in reversed-phase liquid chromatography (RPLC) upon changes in column temperature has been theoretically and experimentally studied. The study focuses on examining the temperature dependence of the retention of various solutes at eluent pH close to their corresponding pK a values, and on the indirect role exerted by the buffer ionization equilibria on retention and selectivity. Retention factors of several ionizable compounds in a typical octadecylsilica column and using buffer solutions dissolved in 30% (v/v) acetonitrile as eluent at five temperatures in the range from 25 to 50 • C were carefully measured. Six buffer solutions were prepared from judiciously chosen conjugated pairs of different chemical nature. Their pK a values in this acetonitrile-water composition and within the range of 15-50 • C were determined potentiometrically. These compounds exhibit very different standard ionization enthalpies within this temperature range. Thus, whenever they are used to control mobile phase pH, the column temperature determines their final pH. Predictive equations of retention that take into account the temperature effect on both the transfer and the ionization processes are evaluated. This study demonstrates the significant role that the selected buffer would have on retention and selectivity in RPLC at temperatures higher than 25 • C, particularly for solutes that coelute.

Journal of Chromatography A, 2002

The polarity parameter model previously developed: log k = (log k) 0 + p(P N m − P N s) has been ... more The polarity parameter model previously developed: log k = (log k) 0 + p(P N m − P N s) has been successfully applied to study several chromatographic systems involving new generation RPLC columns (Luna C18, Resolve C18, XTerra MSC18, and XTerra RP18). In this model the retention of the solutes (log k) is related to a solute parameter (p), a mobile phase parameter (P N m) and two chromatographic system parameters [P N s and (log k) 0 ]. The studied systems have been characterized with different acetonitrile-water and methanol-water mobile phases, using a set of 12 neutral solutes of different chemical nature. The polarity parameter model allows prediction of retention of any solute in any mobile phase composition just using the retention data obtained in one percentage of organic modifier and the polarity parameters established in the characterization of the chromatographic systems. This model also allows the solute polarity data transference between RPLC characterized systems, so it is possible to predict the retention in various RPLC systems working experimentally with just one of them. Moreover, the global solvation parameter model has also been applied to the same chromatographic systems using a wide set of solutes in order to compare its predictive ability with the one of the polarity parameter model. The results clearly show that both models predict retention with very similar accuracy but the polarity parameter model requires much less preliminary experimental measurements to achieve equivalent results than the global solvation approach.

Pharmaceutics, 2021

A new cocrystal of Norfloxacin, a poorly soluble fluoroquinolone antibiotic, has been synthetized... more A new cocrystal of Norfloxacin, a poorly soluble fluoroquinolone antibiotic, has been synthetized by a solvent-mediated transformation experiment in toluene, using resorcinol as a coformer. The new cocrystal exists in both anhydrous and monohydrate forms with the same (1:1) Norfloxacin/resorcinol stoichiometry. The solubility of Norfloxacin and the hydrated cocrystal were determined by the shake-flask method. While Norfloxacin has a solubility of 0.32 ± 0.02 mg/mL, the cocrystal has a solubility of 2.64 ± 0.39 mg/mL, approximately 10-fold higher. The dissolution rate was tested at four biorelevant pH levels of the gastrointestinal tract: 2.0, 4.0, 5.5, and 7.4. In a first set of comparative tests, the dissolution rate of Norfloxacin and the cocrystal was determined separately at each pH value. Both solid forms showed the highest dissolution rate at pH 2.0, where Norfloxacin is totally protonated. Then, the dissolution rate decreases as pH increases. In a second set of experiments, t...

ADMET and DMPK, 2018

Serum albumin is the main drug transporter of the bloodstream and contains two main binding sites... more Serum albumin is the main drug transporter of the bloodstream and contains two main binding sites: Sudlow I or acidic drug binding site, and Sudlow II or benzodiazepine binding site. Warfarin, a well-known anticoagulant drug commonly used in the prevention of thrombosis and thromboembolism, binds to Sudlow I site, whereas non-steroidal antiinflammatory drugs (NSAIDs) such as diflunisal bind preferentially to Sudlow II site. Albumin is a fluorophore that modifies its fluorescence (quenching or enhancement effect) when it is bound to a drug. The application of the double logarithm Stern-Volmer equation allows the calculation of the stoichiometry and the binding constant of the process. This procedure does not consider the possible interferences coming from the fluorescence of the drug though. Another strategy to evaluate the binding constants is to consider the whole spectrum, taking into account all the possible species in equilibrium; in this case we have used an extended version ...

ADMET and DMPK, 2018

The synthesis of cocrystals is presented as an alternative to improve the properties of active ph... more The synthesis of cocrystals is presented as an alternative to improve the properties of active pharmaceutical ingredients, especially those related to solubility and dissolution rate. In this work the dissolution rate of ciprofloxacin, a zwitterionic fluoroquinolone antibiotic, has been compared to its cocrystal with resorcinol. To this end, dissolution rate has been determined at several biorelevant pH values, and also in two simulated gastrointestinal fluids (FeSSIF and FaSSIF). Results show that both, ciprofloxacin and the cocrystal, dissolve more slowly as pH increases (from 2.0 to 7.4), as ionization degree of ciprofloxacin decreases. In addition, dissolution is not enhanced by the components of the gastrointestinal fluids.

ADMET and DMPK, 2017

In the present study the solubility-pH profiles of sibutramine free base and its hydrochloride sa... more In the present study the solubility-pH profiles of sibutramine free base and its hydrochloride salt were determined in the pH range between 2.0 and 9.5 by means of the recommended shake-flask method, and the solids collected were dried and studied by X-ray diffraction in order to elucidate their free base or salt structure. Above pH max (or Gibbs pK a) the solid collected was always identified as free base, whatever the sibutramine species (free base or hydrochloride salt) initially solved. However, in the pH range below pH max different solids were isolated depending on the buffers employed.

European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, Jan 23, 2018

The development of methods to increase the bioavailability of drugs is of great interest, especia... more 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 ...

Journal of pharmaceutical and biomedical analysis, Jan 20, 2018

Interaction thermodynamics between warfarin, a very popular anticoagulant, and Sudlow I binding s... more Interaction thermodynamics between warfarin, a very popular anticoagulant, and Sudlow I binding site of human (HSA) or bovine (BSA) serum albumin have been examined in strictly controlled experimental conditions (HEPES buffer 50 mM, pH 7.4 and 25 °C) by means of isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE). Each technique is based on measurements of a different property of the biochemical system, and then the results allow a critical discussion about the suitability of each approach to estimate the drug-protein binding parameters. The strongest interaction step is properly evaluated by the three assayed approaches being the derived binding constants strongly consistent: from 4 × 104 to 7 × 104 for HSA and from 0.8 × 105 to 1.2 × 105 for BSA. Binding enthalpy variations also show consistent results: -5.4 and -5.6 Kcal/mol for HSA and -4.3 and -3.7 Kcal/mol for BSA, as measured by ITC and FS, respectively...

Talanta, 2016

A study about the suitability of the chelation reaction of Ca 2+ with ethylendiamintetraacetic ac... more A study about the suitability of the chelation reaction of Ca 2+ with ethylendiamintetraacetic acid (EDTA) as a validation standard for Isothermal Titration Calorimeter measurements has been performed exploring the common experimental variables (buffer, pH, ionic strength and temperature). Results obtained in a variety of experimental conditions have been amended according to the side reactions involved in the main process and to the experimental ionic strength and, finally, validated by contrast with the potentiometric reference values. It is demonstrated that the chelation reaction performed in acetate buffer 0.1 M and 25ºC shows accurate and precise results and it is robust enough to be adopted as a standard calibration process.

Journal of molecular recognition : JMR, Jan 4, 2015

Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of... more Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target-drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A-site, with two well-known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin-dinucleotide and -diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin-diPNA conjugates as the best candidates for antimicrobial activity. Copyright © 2015 John Wiley & Sons, Ltd.

European Journal of Pharmaceutical Sciences, 2015

Several procedures based on the shake-flask method and designed to require a minimum amount of dr... more Several procedures based on the shake-flask method and designed to require a minimum amount of drug for octanol-water partition coefficient determination have been established and developed. The procedures have been validated by a 28 substance set with a lipophilicity range from-2.0 to 4.5 (log D7.4). The experimental partition is carried out using aqueous phases buffered with phosphate (pH 7.4) and n-octanol saturated with buffered water and the analysis is performed by liquid chromatography. In order to have accurate results, four procedures and eight different ratios between phase volumes are proposed. Each procedure has been designed and optimized (for partition ratios) for a specific range of drug lipophilicity (low, regular and high lipophilicity) and solubility (high and low aqueous solubility). The procedures have been developed to minimize the measurement in the octanolic phase. Experimental log D7.4 values obtained from different procedures and partition ratios show a standard deviation lower than 0.3 and there is a nice agreement when these values are compared with the reference literature ones.

Two different approaches were evaluated and used to estimate the aqueous pK a values of some flav... more Two different approaches were evaluated and used to estimate the aqueous pK a values of some flavonols sparingly soluble in water (morin, fisetin and quercetin) from their pK a values in methanol/water mixtures obtained by potentiometry. Both approaches lead to similar results, although one of them requires only one pK a value at one unique methanol/water mixture, whereas the other (the classical Yasuda-Shedlovsky plot) requires several pK a data at different methanol/water mixtures. Thus, the first approach is recommended because it is faster and simpler. The automated potentiometric method is strongly recommended for pK a determination of these types of compounds because of its simplicity and speed of operation.

ADMET & DMPK, 2014

Predictive software packages to estimate the lipophilicity of molecules have become key tools in ... more Predictive software packages to estimate the lipophilicity of molecules have become key tools in the new drug design. Six different well-known computational programs including the classical BioByte-clogP and the GALAS algorithm offered by ACDlabs were evaluated through a set of 103 drugs with different structures and functionalities. To evaluate the predictions accuracy, reliable experimental log P o/w values for the whole testing set were carefully selected. The best estimations are performed by GALAS/logP based on the fragmental method, corrected according to the similarity with compounds included in the software training set.

Journal of Pharmaceutical Sciences, 2003

The aqueous pK values (w w pK a) of several sparingly water-soluble drugs with amino groups have ... more The aqueous pK values (w w pK a) of several sparingly water-soluble drugs with amino groups have been calculated from pK values determined in several methanol/ water mixtures (s s pK a) by means of the Yasuda-Shedlovsky equation and by a linear equation that relates w w pK a of amino compounds with s s pK a obtained in any particular methanol/water mixture. Parameters of this last equation for amino compounds can be easily calculated from solely the methanol content of the solvent. Results from both approaches are consistent. However, Yasuda-Shedlovsky equation requires several s s pK a determinations in solutions with different methanol content and can be used only from measurements in solutions with a maximum methanol content of about 65% in weight. In contrast, the linear proposed equation allows a very good estimation of w w pK a from only one experimental s s pK a value, and it permits this estimation from s s pK a determined in a solution very rich in methanol. Therefore, it is suitable for very insoluble compounds. The examined amino compounds cover a wide range of w w pK a , from 6.7 to 10.6, which have been estimated from experimental s s pK a values in the whole composition range. In all instances consistent and satisfactory results have been achieved.

Journal of Pharmaceutical and Biomedical Analysis, 2009

The acidic dissociation enthalpies and constants of anilinium, protonated tris(hydroxymethyl)amin... more The acidic dissociation enthalpies and constants of anilinium, protonated tris(hydroxymethyl)aminomethane (HTris(+)), benzoic and acetic acids, have been determined at several temperatures in pure water and in methanol/water mixtures by potentiometry and by isothermal titration microcalorimetry (ITC). The pK(a) values determined by both techniques are in accordance when the dissociation process involves large amounts of heat. However, for the neutral acids the ITC technique gave slightly lower pK(a) values than those from potentiometry at the highest temperatures studied due to the small amounts of heat involved in the acidic dissociation. The dissociation enthalpies have been determined directly by calorimetry and the obtained values slightly decrease with the increase of temperature. Therefore, only a rough estimation of the dissociation enthalpies can be obtained from potentiometric pK(a) by means of the Van't Hoff approach.

Journal of Chromatography A, 2012

A systematic approach for evaluating the goodness of chromatographic systems to model the sorptio... more 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.

Journal of Chromatography A, 2003

The knowledge of the acid-base equilibria in water-solvent mixtures of both common buffers and an... more The knowledge of the acid-base equilibria in water-solvent mixtures of both common buffers and analytes is necessary in order to predict their retention as function of pH, solvent composition and temperature. This paper describes the effect of temperature on acid-base equilibria in methanol-water solvent mixtures commonly used as HPLC mobile phases. We measured the delta-correction parameter (delta = sw pH - ss pH = Ej - log sw(gamma)oh) between two pH scales: pH measured in the solvent concerned and referred to the same standard state, ss pH, and the pH measured in that solvent mixture but referred to water as standard state, sw pH, for several methanol compositions in the temperature range of 20-50 degrees C. These determinations suggest that the delta-term depends only on composition of the mixture and on temperature. In water-rich mixtures, for which methanol is below 40% (w/w), delta-term seems to be independent of temperature, within the experimental uncertainties, whereas for methanol content larger than 50% (w/w) the delta-correction decreases as temperature increases. We have attributed this decrease to a large increase in the medium effect when mixtures have more than 50% methanol. The pKa of five weak electrolytes of different chemical nature in 50% methanol-water at 20-50 degrees C are presented: the effect of temperature on pKa was large for amines, pyridine and phenol, but almost no dependence was found for benzoic acid. This indicates that buffers can play a critical role in affecting retention and selectivity in HPLC at temperatures far from 25 degrees C, particularlyfor co-eluted solutes.

Journal of Chromatography A, 2010

A set of 25 monoprotic bases is proposed as internal standards for pK a determination by capillar... more 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.

Journal of Chromatography A, 2006

A fast RP-HPLC method for the simultaneous separation of eleven main flavonoid aglycones in food ... more A fast RP-HPLC method for the simultaneous separation of eleven main flavonoid aglycones in food samples was developed. Flavonoids were divided in three groups according to their hydrophobicity and were resolved by a ternary gradient elution and diode array detection within analysis times less than 15 min. The detection limits of the flavonoids studied were in the range between 0.04 and 0.31 microg/mL, with repeatabilities values between 0.4 and 5%. The method was successfully applied to the analysis of these compounds in complex natural samples, like propolis and Ginkgo biloba.

Journal of Chromatography A, 2004

Residual silanol acidity and activity of one immobilized artificial membrane (IAM) column have be... more Residual silanol acidity and activity of one immobilized artificial membrane (IAM) column have been measured from the retention of LiNO 3 in the column with a methanol/buffer (1 mM in Na +) (60:40, v/v) mobile phase buffered to different pH values. Just one type of silanol with s s pK a = 7.61 (near the pH limit recommended by the manufacturer) was found, although these silanols show large activity. The results obtained have been compared with those obtained previously for Resolve C18, Resolve Silica, Symmetry C18, Symmetry Silica, XTerra MS C18, underivatized XTerra, Lichrospher 100 RP-18, Purospher RP-18e, Luna C18 (2) and Chromolith Perfomance RP-18e, showing that the IAM column is similar to Luna C18 and Symmetry C18 in terms of silica quality, as measured by Li + retention. A warning about the use of IAM columns for emulation of biological systems at physiological pH 7 is given because the ionized silanols may contribute to the retention of some drugs at this pH.

Journal of Chromatography A, 2005

The retentive behavior of weak acids and bases in reversed-phase liquid chromatography (RPLC) upo... more The retentive behavior of weak acids and bases in reversed-phase liquid chromatography (RPLC) upon changes in column temperature has been theoretically and experimentally studied. The study focuses on examining the temperature dependence of the retention of various solutes at eluent pH close to their corresponding pK a values, and on the indirect role exerted by the buffer ionization equilibria on retention and selectivity. Retention factors of several ionizable compounds in a typical octadecylsilica column and using buffer solutions dissolved in 30% (v/v) acetonitrile as eluent at five temperatures in the range from 25 to 50 • C were carefully measured. Six buffer solutions were prepared from judiciously chosen conjugated pairs of different chemical nature. Their pK a values in this acetonitrile-water composition and within the range of 15-50 • C were determined potentiometrically. These compounds exhibit very different standard ionization enthalpies within this temperature range. Thus, whenever they are used to control mobile phase pH, the column temperature determines their final pH. Predictive equations of retention that take into account the temperature effect on both the transfer and the ionization processes are evaluated. This study demonstrates the significant role that the selected buffer would have on retention and selectivity in RPLC at temperatures higher than 25 • C, particularly for solutes that coelute.

Journal of Chromatography A, 2002

The polarity parameter model previously developed: log k = (log k) 0 + p(P N m − P N s) has been ... more The polarity parameter model previously developed: log k = (log k) 0 + p(P N m − P N s) has been successfully applied to study several chromatographic systems involving new generation RPLC columns (Luna C18, Resolve C18, XTerra MSC18, and XTerra RP18). In this model the retention of the solutes (log k) is related to a solute parameter (p), a mobile phase parameter (P N m) and two chromatographic system parameters [P N s and (log k) 0 ]. The studied systems have been characterized with different acetonitrile-water and methanol-water mobile phases, using a set of 12 neutral solutes of different chemical nature. The polarity parameter model allows prediction of retention of any solute in any mobile phase composition just using the retention data obtained in one percentage of organic modifier and the polarity parameters established in the characterization of the chromatographic systems. This model also allows the solute polarity data transference between RPLC characterized systems, so it is possible to predict the retention in various RPLC systems working experimentally with just one of them. Moreover, the global solvation parameter model has also been applied to the same chromatographic systems using a wide set of solutes in order to compare its predictive ability with the one of the polarity parameter model. The results clearly show that both models predict retention with very similar accuracy but the polarity parameter model requires much less preliminary experimental measurements to achieve equivalent results than the global solvation approach.