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.

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.

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.

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.

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.

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.

Estimation of the effect of the acidosis and alkalosis on the anesthetic potency of local anesthetics by biopartitioning micellar chromatography and micellar electrokinetic chromatography.

Local anesthetics are hydrophobic compounds and weak bases with protonation constants ranged between 7.5 and 8.8. These drugs block reversibly nerve conduction near their site of application or injection and thus produce temporary loss of feeling or sensation in a limited area of the body. The efficacy of anesthetic blockade of local anesthetics depends on the charged/uncharged form ratio and the hydrophobicity of the compounds. In addition their toxicological effects have been reported to be highly dependent on the physiological pH. Biopartitioning micellar chromatography (BMC) and micellar electrokinetic chromatography (MEKC), that use micellar solutions as mobile phases, have proven to be useful for describing the biological behavior of different kind of compounds. In this paper, relationships between the retention data in BMC and MEKC using Brij35 as surfactant (at pH 7.4) and some pharmacodynamic parameters of local anesthetics are obtained. These models are compared with those obtained using an immobilized artificial column (IAM). Finally, the effect of the corporal pH in situations of acidosis and alkalosis on the pharmacological and toxicological properties of local anesthetics is studied using the retention of compounds in BMC at different mobile phase pH values.

Evaluation of the pH effect of formulations on the skin permeability of drugs by biopartitioning micellar chromatography.

Dermal absorption of chemicals is an area of increasing interest for the pharmaceutical and cosmetic industries, as well as in dermal exposure and risk assessment processes. Biopartitioning micellar chromatography (BMC) is a mode of reversed phase micellar chromatography that has proved to be useful in the description and prediction of several pharmacological properties of xenobiotics including oral drug absorption, ocular and skin drug permeability. The present paper deals with the application of biopartitionig micellar chromatography to evaluate the pH effect on the skin permeability of twelve non-steroidal anti-inflammatory drugs and lidocaine. For this purpose the BMC retention of the whole set of compounds at several pHs between 3.5 and 8 was obtained. Using the BMC retention-permeability model previously reported, the permeability of the compounds at different pH values was estimated. The predicted permeability values at different pH values for ketoprofen, lidocaine, salicylic acid and ibuprofen agree with those experimental reported in literature for these compounds using excised human and rat skin.

Chiral separation of bupivacaine enantiomers by capillary electrophoresis partial-filling technique with human serum albumin as chiral selector.

Capillary electrophoresis (CE) is a powerful technique for enantiomer separations due to its intrinsic high separation efficiencies, speed of analysis, low reagent consumption and small sample requirements. However, some chiral selectors present strong background UV absorption providing high detection limits. The present paper deals with the application of the partial-filling technique to the separation of bupivacaine enantiomers by capillary electrophoresis using human serum albumin (HSA) as chiral selector. In this procedure the cationic surfactant cetyltrimethylammonium bromide (CTAB) was used as a dinamic capillary coating in order to reduce the electro-osmotic flow and detect both bupivacaine enantiomers out of the chiral selector plug. Several experimental conditions such as CTAB concentration, pH, HSA concentration and plug length, background electrolyte concentration, temperature and voltage were studied. Under the selected conditions it is possible to detect the separated enantiomers out of the HSA plug in less than 4 min using 50 mM Tris pH 8 as background electrolyte with 50 microM CTAB, at 30 degrees C and using a separation voltage of 25 kV. The proposed methodology was then validated for analytical purposes and applied to the analysis of pharmaceutical preparations commercially available. The results obtained with the proposed methodology were in good agreement with those declared by the manufacturers. The simplicity, sample throughput, accuracy, reproducibility and low cost of the proposed method make it suitable for the control of the enantiomeric composition of bupivacaine in pharmaceuticals.

Biopartitioning micellar chromatography to predict skin permeability.

Dermal absorption of chemicals is an area of increasing interest to the pharmaceutical and cosmetic industries, as well as in dermal exposure and risk assessment processes. In this paper the capability of biopartitioning micellar chromatography (BMC) as an in vitro technique to describe compound percutaneous absorption is evaluated. A multivariate study (principal component analysis, partial least squares) is performed in order to evaluate the importance of some physicochemical variables on the skin permeability constant values. From these results, a quantitative retention-activity relationship model for predicting the skin permeability constants that uses the BMC retention data and melting point as descriptor variables was obtained from a heterogeneous set of 43 compounds. The main advantage of the proposed methodology is that it allows the obtention of permeability constants of ionic compound and it can be very useful to predict the effect of pH of vehicle on the skin permeability of xenobiotics.

Rapid in vitro test to predict ocular tissue permeability based on biopartitioning micellar chromatography.

The drug permeability prediction across the ocular tissues is important in the development of new drugs and drug delivery strategies. Physicochemical characteristics of drugs, mainly acid-base character, hydrophobicity and the molecular size determine both their transport across the eye tissue barriers and their retention in biopartitioning micellar chromatography (BMC). An in vitro model able to describe and predict the whole cornea drug permeability is proposed. The model uses the retention of drugs in BMC and molecular weight (MW) as predictive variables. The relationships between drug retention data in BMC and their bibliographic permeability values in stroma, epithelium-plus-stroma and endothelium-plus-stroma are also studied. The results show that BMC can be a useful tool to select drug candidates according to their whole cornea permeability at the early stage of the drug discovery process.

QRAR models for central nervous system drugs using biopartitioning micellar chromatography.

The capability of biopartitioning Micellar Chromatography, BMC, to describe and estimate pharmacokinetic and pharmacodynamic parameters of central nervous system drugs is reviewed in this article. BMC is a mode of micellar liquid chromatography, MLC, that uses micellar mobile phases of Brij35 (polyoxyethilene(23) lauryl ether) prepared in physiological conditions (pH, ionic strength). The retention of a drug in this system depends on its hydrophobic, electronic and steric properties, which also determine its biological activity. The results of BMC studies suggest that this in vitro approach is an attractive useful tool to be implemented into the lead optimization step of drug development scheme.

Enantiomeric separation of chiral phenoxy acid herbicides by electrokinetic chromatography. Application to the determination of analyte-selector apparent binding constants for enantiomers.

The enantiomeric resolution of chiral phenoxy acid herbicides was performed by electrokinetic chromatography using a cyclodextrin as chiral pseudophase (CD-EKC). A systematic evaluation of several neutral and charged cyclodextrins was made. Among the cyclodextrins tested, (2-hydroxy)propyl beta-cyclodextrin (HP-beta-CD) was found to be the most appropriate for the enantioseparation of phenoxy acids. The influence of some experimental conditions, such as nature and pH of the background electrolyte, chiral selector concentration, and temperature, on the enantiomeric separation of phenoxy acids was also studied. The use of a 50 mM electrolyte solution in ammonium formate at pH 5 and a temperature of 40 degrees C enabled the enantiomeric resolution of four of the six phenoxy acids investigated (2-phenoxypropionic acid, 2(3-chlorophenoxy)propionic acid, 2-(4-chlorophenoxy)propionic acid, and 2-(2,4-dichlorophenoxy)propionic acid) obtaining migration times ranging from 9 to 15 min. Mixtures of the two phenoxy acids not enantiomerically resolved (2-(4-chlorophenoxy)-2-methylpropionic acid and 2-(2,4,5-trichlorophenoxy)propionic acid) and up to three of the phenoxy acids enantiomerically resolved were separated in about 15 min. Finally, the apparent binding constants for each enantiomer-HP-beta-CD pair were calculated at two temperature values (20 and 40 degrees C).

Development of predictive retention-activity models of butyrophenones by biopartitioning micellar chromatography.

The predictive and interpretative capability of quantitative chromatographic retention-biological activity models is supported by the fact that in adequate experimental conditions the solute partitioning into the chromatographic system can emulate the solute partitioning into lipid bilayers of biological membranes, which is the basis of drug and metabolite uptake, passive transport across membranes and bioaccumulation. The use of retention data obtained in biopartitioning micellar chromatography (BMC) has been demonstrated to be helpful in describing the biological behaviour of different kinds of drugs. In this chromatographic system, polioxyethylene 23 lauryl ether Brij35 micellar mobile phases and C(18) reversed stationary phase in adequate experimental conditions are used. The RP-HPLC capacity factors of butyrophenones were determined using different Brij35 concentrations as micellar mobile phases. Relationships between seven biological activities of butyrophenones reported in bibliography and retention data were established and their predictive and interpretative ability evaluated. These relationships were significant between preclinical pharmacology and therapeutic efficacy parameters and the retention factors of butyrophenones (0.89 < R(2) < 0.98). The results indicate that the retention of compounds in BMC is capable of describing and predicting in vitro the biological activities of butyrophenones. This approach can be very useful in the development of new neuroleptic drugs, avoiding the use of experimental animals.

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

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

Multivariate data analysis of quality parameters in drinking water.

The quality of water destined for human consumption has been treated as a multivariate property. Since most of the quality parameters are obtained by applying analytical methods, the routine analytical laboratory (responsible for the accuracy of analytical data) has been treated as a process system for water quality estimation. Multivariate tools, based on principal component analysis (PCA) and partial least squares (PLS) regression, are used in the present paper to: (i) study the main factors of the latent data structure and (ii) characterize the water samples and the analytical methods in terms of multivariate quality control (MQC). Such tools could warn of both possible health risks related to anomalous sample composition and failures in the analytical methods.