Modified Gaussian models applied to the description and deconvolution of peaks in chiral liquid chromatography.

The description of the profiles of chromatographic peaks has been studied extensively, with a large number of proposed mathematical functions. Among them, the accuracy achieved with modified Gaussian models that describe the deviation of an ideal Gaussian peak as a change in the peak variance or standard deviation over time, has been highlighted. These models are, in fact, a family of functions of different complexity with great flexibility to adjust chromatographic peaks over a wide range of asymmetries and shapes. However, an uncontrolled behaviour of the signal may occur outside the region being fitted, forcing the use of different strategies to overcome this problem. In this work, the performance of the LMG (Linear Modified Gaussian), PVMG (Parabolic Variance Modified Gaussian), and PLMG (Parabolic-Lorentzian Modified Gaussian) models is compared with variants obtained by combination of the modified Gaussian models with an equation that adds an exponential tail and with other functions that limit the growth of the independent variable. The behaviour of the approaches is checked through the simultaneous fitting of enantiomeric peaks showing a wide range of characteristics, obtained in the separation of drugs with chiral activity by liquid chromatography using enantioselective columns. The study is also carried out with the purpose of performing the deconvolution of the peaks of the enantiomers, when these are not completely resolved, in order to evaluate the enantiomeric fraction.

Prospective computational design and in vitro bio-analytical tests of new chemical entities as potential selective CYP17A1 lyase inhibitors.

The development and advancement of prostate cancer (PCa) into stage 4, where it metastasize, is a major problem mostly in elder males. The growth of PCa cells is stirred up by androgens and androgen receptor (AR). Therefore, therapeutic strategies such as blocking androgens synthesis and inhibiting AR binding have been explored in recent years. However, recently approved drugs (or in clinical phase) failed in improving the expected survival rates for this metastatic-castration resistant prostate cancer (mCRPC) patients. The selective CYP17A1 inhibition of 17,20-lyase route has emerged as a novel strategy. Such inhibition blocks the production of androgens everywhere they are found in the body. In this work, a three dimensional-quantitative structure activity relationship (3D-QSAR) pharmacophore model is developed on a diverse set of non-steroidal inhibitors of CYP17A1 enzyme. Highly active compounds are selected to define a six-point pharmacophore hypothesis with a unique geometrical arrangement fitting the following description: two hydrogen bond acceptors (A), two hydrogen bond donors (D) and two aromatic rings (R). The QSAR model showed adequate predictive statistics. The 3D-QSAR model is further used for database virtual screening of potential inhibitory hit structures. Density functional theory (DFT) optimization provides the electronic properties explaining the reactivity of the hits. Docking simulations discovers hydrogen bonding and hydrophobic interactions as responsible for the binding affinities of hits to the CYP17A1 Protein Data Bank structure. 13 hits from the database search (including five derivatives) are then synthesized in the laboratory as different scaffolds. Ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in vitro experiments reveals three new chemical entities (NCEs) with half maximal inhibitory concentration (IC50) values against the lyase route at mid-micromolar range with favorable selectivity to the lyase over the hydroxylase route (one of them with null hydroxylase inhibition). Thus, prospective computational design has enabled the design of potential lead lyase-selective inhibitors for further studies.

Fit-for-purpose chromatographic method for the determination of amikacin in human plasma for the dosage control of patients.

In this paper, a simple, rapid and sensitive method based on liquid chromatography with fluorimetric detection (HPLC-FLD) for the determination of amikacin (AMK) in human plasma is developed. Determination is performed by pre-column derivatization of AMK with ortho-phtalaldehyde (OPA) in presence of N-acetyl-L-cysteine (NAC) at pH 9.5 for 5 min at 80 °C. In our knowledge, this is the first time that NAC has been used in AMK derivatization. Derivatization conditions (pH, AMK/OPA/NAC molar ratios, temperature and reaction time) are optimized to obtain a single and stable, at room temperature, derivative. Separation of the derivative is achieved on a reversed phase LC column (Kromasil C18, 5 µm, 150 × 4.6 i.d. mm) with a mobile phase of 0.05 M phosphate buffer:acetonitrile (80:20, v/v) pumped at flow rate of 1.0 mL/min. Detection is performed using 337 and 439 nm for excitation and emission wavelengths, respectively. The method is fitted for the purpose of being a competitive alternative to the currently used method in many hospitals for AMK dosage control: fluorescence polarization immunoassay (FPIA). The method exhibits linearity in the 0.17-10 µg mL(-1) concentration range with a squared correlation coefficient higher than 0.995. Trueness and intermediate precision are estimated using spiked drug free plasma samples, which fulfill current UNE-EN ISO15189:2007 accreditation schemes. Finally, for the first time, statistical comparison against the FPIA method is demonstrated using plasma samples from 31 patients under treatment with AMK.

Fast-multivariate optimization of chiral separations in capillary electrophoresis: anticipative strategies.

The design of experiments (DOE) is a good option for rationally limiting the number of experiments required to achieve the enantioresolution (Rs) of a chiral compound in capillary electrophoresis. In some cases, the modeled Rs after DOE analysis can be unsatisfactory, maybe because the range of the explored factors (DOE domain) was not the adequate. In these cases, anticipative strategies can be an alternative to the repetition of the process (e.g. a new DOE), to save time and money. In this work, multiple linear regression (MLR)-steepest ascent and a new anticipative strategy based on a multiple response-partial least squares model (called PLS2-prediction) are examined as post-DOE strategies to anticipate new experimental conditions providing satisfactory Rs values. The new anticipative strategy allows to include the analysis time (At) and uncertainty limits into the decision making process. To demonstrate their efficiency, the chiral separation of hexaconazole and penconazole, as model compounds, is studied using highly sulfated-ß-cyclodextrin (HS-ß-CD) in electrokinetic chromatography (EKC). Box-Behnken DOE for three factors (background electrolyte pH, separation temperature and HS-ß-CD concentration) and two responses (Rs and At) is used. Using commercially available software, the whole modeling and anticipative process is automatic, simple and requires minimal skills from the researcher. Both strategies studied have proven to successfully anticipate Rs values close to the experimental ones for EKC conditions outside the DOE domain for the two model compounds. The results in this work suggest that PLS2-prediction approach could be the strategy of choice to obtain secure anticipations in EKC.

Cyclodextrins in capillary electrophoresis: recent developments and new trends.

Despite the fact that extensive research in the field of separations by capillary electrophoresis (CE) has been carried out and many reviews have been published in the last years, a specific review on the use and future potential of cyclodextrins (CDs) in CE is not available. This review focuses the attention in the CD-CE topic over the January 2013-February 2014 period (not covered by previous more general CE-reviews). Recent contributions (reviews and research articles) including practical uses (e.g. solute-CD binding constant estimation and further potentials; 19% of publications), developments and applications (mainly chiral and achiral analysis; 38 and 24% of publications, respectively) are summarized in nine comprehensive tables and are commented. Statistics and predictions related to the CD-CE publications are highlighted in order to infer the current and expected research interests. Finally, trends and initiatives on CD-CE attending to real needs or practical criteria are outlined.

Modeling the chiral resolution ability of highly sulfated ß-cyclodextrin for basic compounds in electrokinetic chromatography.

Despite the fact that extensive research in the field of enantioseparations by capillary electrophoresis has been carried out, it is difficult to predict whether a concrete chiral selector would be useful for the separation of a racemic compound. Hence, several experimental effort is necessary to test the abilities of individual chiral selectors, usually by trial and error procedures. Thus, the enantioseparation of a new racemate becomes a time- and money-consuming task. In this work, the ability of highly sulfated ß-cyclodextrin (HS-ß-CD) as chiral selector in electrokinetic chromatography (EKC) is modeled for the first time, using exclusively directly-available structural data of forty compounds (structurally unrelated basic drugs and pesticides). A discriminant partial least squares (PLS)-based quantitative structure-property relationship (QSPR) approach is simplified, resulting in a consistent, predictive and descriptive model. It is converted into an explicit equation able to predict the enantioresolution level (Rs) of new compounds, from four structure properties available in an on-line open database: logarithm of octanol-water partition coefficient estimated at pH 7.4 (lgD), polar surface area (PSA), number of hydrogen bond donors (HBD) and acceptors (HBA). For the cases in which the model predicts good Rs only in concrete experimental conditions, a Box-Behnken experimental design is proposed for the fast PLS-based optimization of the most influential experimental variables: cyclodextrin concentration, temperature and pH.

In-line capillary electrophoretic evaluation of the enantioselective metabolism of verapamil by cytochrome P3A4.

In this paper a methodology for the in-line evaluation of enantioselective metabolism by capillary electrophoresis has been developed and applied to the study of verapamil metabolism by cytochrome P3A4. The developed methodology comprises an in-capillary reaction step carried out by electrophoretically mediated microanalysis and a separation step in which highly sulfated ß-cyclodextrin with partial filling technique has been employed as chiral selector for verapamil and norverapamil enantiomers resolution, joining the advantages of both methodologies in a unique assay. Kinetic parameters of the enzymatic reaction (Km and Vmax) have been evaluated for both verapamil enantiomers by non-linear fitting of experimental data obtained under intermediate precision conditions to Michaelis-Menten equation. Km and Vmax estimated values were 51±9 µM and 22±2 pmol min(-1) (pmol CYP)(-1) for S-VER and 47±9 µM and 21±2 pmol min(-1) (pmol CYP)(-1) for R-VER. Consequently, slight enantioselectivity was found for the CYP3A4 metabolism of verapamil. However, since confidence intervals of estimates overlap, we cannot assure a significant enantioselectivity. Intrinsic clearance values were also estimated from Km and Vmax for both enantiomers.

Experimental-like affinity constants and enantioselectivity estimates from flexible docking.

Experimental-like affinity constants and enantioselectivity estimates, not predicted so far computationally, were obtained using a novel flexible modeling/docking combined strategy. The S- and R-warfarin-human serum albumin (HSA, site I) complexes were used as an interaction model. The process for a verified estimation includes the following: (i) ionized open chain forming at physiological pH (a recent focus); (ii) conformational search (molecular mechanics and Monte Carlo methods); (iii) rigid protein-flexible ligand docking (GlideXP) generating low energy paired S- and R-poses; (iv) graphical comparison against the X-ray crystal structure (unsatisfactory verification step); (v) quantum polarized ligand docking (insufficient verification step); (vi) induced fit docking (one pose satisfying the verification criterion; selection step); (vii) converting docking scores to affinity and enantioselectivity estimates (log K(S) = 5.43, log K(R) = 5.34, ES = K(S)/K(R) = 1.23) and numerical comparison against equivalent literature data from bioanalytical techniques (validation step); (viii) intermolecular forces explaining ES (hydrogen bonding and π-π interactions).

On the zopiclone enantioselective binding to human albumin and plasma proteins. An electrokinetic chromatography approach.

In this work, a methodology for the chiral separation of zopiclone (ZPC) by electrokinetic chromatography (EKC) using carboxymethylated-ß-cyclodextrin as chiral selector has been developed and applied to the evaluation of the enantioselective binding of ZPC enantiomers to HSA and total plasma proteins. Two mathematical approaches were used to estimate protein binding (PB), affinity constants (K(1)) and enantioselectivity (ES) for both enantiomers of ZPC. Contradictory results in the literature, mainly related to plasma protein binding reported data, suggest that this is an unresolved matter and that more information is needed. Discrepancies and coincidences with previous data are highlighted.

Permeability and toxicological profile estimation of organochlorine compounds by biopartitioning micellar chromatography.

This paper points out the usefulness of biopartitioning micellar chromatography (BMC) as a high-throughput primary screening tool providing key information about the oral absorption, skin permeability (K(p)), brain-blood distribution coefficient (BB) and ecotoxicological parameters such as median lethal concentration (LC(50)) and bioconcentration factors of 15 organochloride compounds. The retention data of compounds in BMC conditions were interpolated in previously developed quantitative-retention activity relationships by our research group. Results show that the compounds studied readily cross the intestinal barrier (oral absorption > 90%) and the blood-brain barrier (log BB > 0.4). In addition, the organochlorines DDE, chlorobenzene, 1,3-dichlorobenzene and 1,2-dichlorobenzene are the compounds which can more quickly cross the skin barrier (log K(p ) > -0.74 cm/h). From a ecotoxicological point of view, it can be concluded that the most retained compounds, DDE, DDD, hexachlorobenzene and dicofol, are the most toxic and bioacumulative.

Biopartitioning micellar chromatography to predict blood to lung, blood to liver, blood to fat and blood to skin partition coefficients of drugs.

Biopartitioning micellar chromatography (BMC), a mode of micellar liquid chromatography that uses micellar mobile phases of Brij35 in adequate experimental conditions, has demonstrated to be useful in mimicking the drug partitioning process into biological systems. In this paper, the usefulness of BMC for predicting the partition coefficients from blood to lung, blood to liver, blood to fat and blood to skin is demonstrated. PLS2 and multiple linear regression (MLR) models based on BMC retention data are proposed and compared with other ones reported in bibliography. The proposed models present better or similar descriptive and predictive capability.

Chromatographic retention-activity relationships for prediction of the toxicity pH-dependence of phenols.

An investigation of the use of the chromatographic retention (log k) as an in vitro approach for modeling the pH-dependence of the toxicity to Guppy of phenols is developed. A data set of 19 phenols with available experimental toxicity-pH data was used. The importance of the mechanism of toxic action (MOA) of phenols was studied. log k data at three pH values were used for the phenols classification and two groups or 'MODEs' were identified. For one 'MODE' a quantitative retention-activity relationship (QRAR) model was calculated. Finally, the model was used to assess the toxicity to Guppy of phenols at different pH values. The results of this investigation suggest that chromatographic retention data allows fish toxicity modeling, in the 5.5-8 pH range of interest.

Enantiomeric quality control of antihistamines in pharmaceuticals by affinity electrokinetic chromatography with human serum albumin as chiral selector.

The present paper deals with the enantiomeric separation of six antihistaminic enantiomers by affinity electrokinetic chromatography (AEKC)-partial filling technique using human serum albumin (HSA) as chiral selector. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and HSA plug length (SPL) was carried out since there are interactions between variables that could not be considered in an univariate optimization. The estimated and experimental resolution values obtained for antihistaminic enantiomers varied from 1.13 (for orphenadrine) to 2.15 (for brompheniramine). The optimum experimental conditions for enantioresolution of each compound were: brompheniramine, pH 8.5, [HSA] 180 microM, SPL 180 s; chlorcyclizine, pH 6.5, [HSA] 180 microM, SPL 150 s; chlorpheniramine, pH 8.25, [HSA] 160 microM, SPL 150 s; hydroxyzine, pH 7.0, [HSA] 180 microM, SPL 150 s; and orphenadrine, pH 7.8, [HSA] 160 microM, SPL 150 s. pH and the quadratic term of pH seem to be the most critical factors that determine enantioresolution of antihistamines. The validity of the developed methodologies to enantiomeric quality control of antihistamines in pharmaceutical formulations is demonstrated analyzing the content of brompheniramine, chlorpheniramine and hyroxyzine enantiomers in commercially available pharmaceutical formulations containing racemic mixtures of compounds. Resolution, accuracy, reproducibility, cost and sample throughput of the proposed methodologies make them suitable for quality control of the enantiomeric composition of antihistamines in pharmaceutical preparations.

Biopartitioning micellar chromatography to predict mutagenicity of aromatic amines.

Mutagenicity is a toxicity endpoint associated with the chronic exposure to chemicals. Aromatic amines have considerable industrial and environmental importance due to their widespread use in industry and their mutagenic capacity. Biopartitioning micellar chromatography (BMC), a mode of micellar liquid chromatography that uses micellar mobile phases of Brij35 in adequate experimental conditions, has demonstrated to be useful in mimicking the drug partitioning process into biological systems. In this paper, the usefulness of BMC for predicting mutagenicity of aromatic amines is demonstrated. A multiple linear regression (MLR) model based on BMC retention data is proposed and compared with other ones reported in bibliography. The proposed model present better or similar descriptive and predictive capability.

Biopartitioning micellar chromatography: an alternative high-throughput method for assessing the ecotoxicity of anilines and phenols.

An investigation of the use of the chromatographic retention (log k) as an in vitro approach for modelling the toxicity to Fathead Minnows of anilines and phenols is developed. A data set of 65 compounds with available experimental toxicity data was used. Log k data at three pH values were used for the compounds classification and two groups or 'MODEs' were identified. For one 'MODE' a quantitative retention-activity relationship (QRAR) model was calculated. Finally, it was used to estimate the toxicity to Fathead minnows of anilines and phenols for which experimental data are not available. These estimations were compared to those obtained from another toxicity (to Tetrahymena pyriformis) data set and those estimated from a U.S. EPA QSAR approach (ECOSAR software) to decide on the toxicity level according to the Directive 3/21/EEC.

Harmonized internal quality aspects of a multi-residue method for determination of forty-six semivolatile compounds in water by stir-bar-sorptive extraction-thermal desorption gas chromatography-mass spectrometry.

Three main aspects of internal quality-internal method validation, internal quality control (IQC), and sample result uncertainty-have been established for a multi-residue method for determination of 46 organic micropollutants (pesticides and polycyclic aromatic hydrocarbons) in water by stir-bar-sorptive extraction (SBSE) and thermal desorption (TD) coupled to capillary gas chromatography-mass spectrometry (GC-MS). From data obtained with increasing time, the process mean and standard deviation were used to harmonize the internal quality statistics. The relationship between these statistics and the hydrophobicity of the compounds was evaluated.

Characterization of antihistamine-human serum protein interactions by capillary electrophoresis.

An important topic in the drug discovery and development process is the role of drug binding to plasma proteins. In this paper the characterization of the interaction between antihistamines (cationic drugs) towards human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP) under physiological conditions by capillary electrophoresis-frontal analysis is presented. Furthermore, the binding of these drugs to all plasma proteins is evaluated by using ultrafiltration and capillary electrophoresis. Antihistamines present a wide-ranging behaviour with respect to their affinities towards plasma proteins. Orphenadrine, phenindamine, tripelenamine and tripolidine principally bind to HSA; carbinoxamine, dimetindene and etintidine principally bind to AGP; brompheniramine, chlorpheniramine and ranitidine present an important binding to lipoproteins and/or globulins and finally, chlorcyclizine, cinarizine, cyclizine, doxylamine, hydroxyzine, perphenazine and terfenadine do not bind to lipoproteins and/or globulins but bind to HSA and AGP in different extension. The interaction of antihistamines with HSA is determined by the hydrophobicity (direct relationship) and the polar surface area (indirect relationship) of the compounds. The steric parameters and hydrogen bonding character of compounds seems to be related with the binding of antihistamines to AGP. The antihistamine-HSA affinity constants were evaluated and the K(1) values ranged from 7 x 10(2)M(-1) (for doxylamine) to 4 x 10(4)M(-1) (for phenindamine).

A diagnostic tool for determining the quality of accuracy validation. Assessing the method for determination of nitrate in drinking water.

Realistic internal validation of a method implies the performance validation experiments under intermediate precision conditions. The validation results can be organized in an X (NrxNs) (replicates x runs) data matrix, analysis of which enables assessment of the accuracy of the method. By means of Monte Carlo simulation, uncertainty in the estimates of bias and precision can be assessed. A bivariate plot is presented for assessing whether the uncertainty intervals for the bias (E +/- U(E)) and intermediate precision (RSDi +/- U(RSDi) are included in prefixed limits (requirements for the method). As a case study, a method for determining the concentration of nitrate in drinking water at the official level set by 98/83/EC Directive is assessed by use of the proposed plot.

Comparison between micellar liquid chromatography and capillary zone electrophoresis for the determination of hydrophobic basic drugs in pharmaceutical preparations.

The determination of highly hydrophobic basic compounds by means of conventional reversed-phase liquid chromatographic methods has several drawbacks. Owing to the characteristics of micellar liquid chromatography (MLC) and capillary electrophoresis (CE), these techniques could be advantageous alternatives to reversed-phase chromatographic methods for the determination of these kinds of compounds. The objective of this study was to develop and compare MLC and CE methods for the determination of antipsychotic basic drugs (amitryptiline, haloperidol, perphenazine and thioridazine) in pharmaceutical preparations. The chromatographic determination of the analytes was performed on a Kromasil C(18) analytical column; the mobile phase was 0.04 m cetyltrimethylammonium bromide (CTAB), at pH 3, containing 5% 1-butanol, at a flow rate of 1 mL/min. The CE separation was performed in a fused-silica capillary with a 50 mm tris-(hydroxymethyl)-aminomethane buffer, pH 7, at an applied voltage of 20 kV, using barbital as internal stardard. The proposed methods are suitable for a reliable quantitation of these compounds in the commercial tablets and drops in terms of accuracy and precision and require a very simple pre-treatment of the samples. By comparing the performance characteristics and experimental details of the MLC and CE methods we conclude that CE seems to be slightly better than MLC in the determination of highly hydrophobic compounds in pharmaceuticals in terms of resolution and economy, taking into account that the limits of detection are not a handicap in pharmaceutical samples.

Uncertainty-based internal quality control. Harmonization considerations.

Three main quality aspects for analytical laboratories are internal method validation, internal quality control (IQC), and sample result uncertainty. Unfortunately, in the past they have been used in a nonharmonized way. The most universal IQC tool is the mean chart, but some criteria used to fix their control limits do not fit the real nature of analytical results. A new approach for fixing these limits is proposed (the u-approach). The key is the combined uncertainty, u, obtained from the method validation information, also used for estimating the sample result uncertainty. A comparative study on "in-control" simulated, bibliographic, and real laboratory data suggests that the u-approach is more reliable than other well-established criteria. In addition, the u-approach mean chart emerges as an IQC tool, consistent with chemical assays, which harmonizes the validation-control-uncertainty process.