Chiral x achiral multidimensional liquid chromatography. Application to the enantioseparation of dintitrophenyl amino acids in honey samples and their fingerprint classification.

Most HPLC enantiomer separations are performed with columns packed with a chiral stationary phase (CSP) operated with an achiral mobile phase. The intrinsically limited chemical selectivity of most CSPs to the simultaneous resolution of several pairs of enantiomers means that complex mixtures of diverse pairs of enantiomers cannot be resolved in a single run due to peak overlapping. Moreover, some drawbacks remain when the analyte is present in very complex samples containing other achiral compounds which can co-elute with the enantiomer peaks. Multidimensional chromatography becomes an option to increase peak capacity and resolve these samples. The aim of this work was to study an online fully comprehensive 2D-LC mode utilizing a combination of a chiral column in the first dimension and an achiral column in the second dimension. The 2D-LC system was built with an active flow splitter pump in order to easily adjust the volume of sample transferred into the second dimension and to independently optimize the flow rate in the first dimension. The present LCxLC method was optimized for the separation of amino acids present in honey samples, taking into account key parameters that influence the bidimensional peak capacity (orthogonality, sampling frequency, etc.). The amino acids have been preconcentrated on a cation-exchange column followed by derivatization. Several amino acids present in different honey samples have been identified and the data generated has been analyzed by principal component analysis.

High throughput method to characterize acid-base properties of insoluble drug candidates in water.

In drug design experimental characterization of acidic groups in candidate molecules is one of the more important steps prior to the in-vivo studies. Potentiometry combined with Yasuda-Shedlovsky extrapolation is one of the more important strategy to study drug candidates with low solubility in water, although, it requires a large number of sequences to determine pKa values at different solvent-mixture compositions to, finally, obtain the pKa in water (pwwKa) by extrapolation. We have recently proposed a method which requires only two sequences of additions to study the effect of organic solvent content in liquid chromatography mobile phases on the acidity of the buffer compounds usually dissolved in it along wide ranges of compositions. In this work we propose to apply this method to study thermodynamic pwwKa of drug candidates with low solubilities in pure water. Using methanol/water solvent mixtures we study six pharmaceutical drugs at 25 °C. Four of them: ibuprofen, salicylic acid, atenolol and labetalol, were chosen as members of carboxylic, amine and phenol families, respectively. Since these compounds have known pwwKa values, they were used to validate the procedure, the accuracy of Yasuda-Shedlovsky and other empirical models to fit the behaviors, and to obtain pwwKa by extrapolation. Finally, the method is applied to determine unknown thermodynamic pwwKa values of two pharmaceutical drugs: atorvastatin calcium and the two dissociation constants of ethambutol. The procedure proved to be simple, very fast and accurate in all of the studied cases.

Enantiomeric analysis of overlapped chromatographic profiles in the presence of interferences. Determination of ibuprofen in a pharmaceutical formulation containing homatropine.

In this work, we studied the combination of chemometric methods with chromatographic separations as a strategy applied to the analysis of enantiomers when complete enantioseparation is difficult or requires long analysis times and, in addition, the target signals have interference from the matrix. We present the determination of ibuprofen enantiomers in pharmaceutical formulations containing homatropine as interference by chiral HPLC-DAD detection in combination with partial least-squares algorithms. The method has been applied to samples containing enantiomeric ratios from 95:5 to 99.5:0.5 and coelution of interferents. The results were validated using univariate calibration and without homatropine. Relative error of the method was less than 4.0%, for both enantiomers. Limits of detection (LOD) and quantification (LOQ) for (S)-(+)-ibuprofen were 4.96×10-10 and 1.50×10-9mol, respectively. LOD and LOQ for the R-(-)-ibuprofen were LOD=1.60×10-11mol and LOQ=4.85×10-11mol, respectively. Finally, the chemometric method was applied to the determination of enantiomeric purity of commercial pharmaceuticals. The ultimate goal of this research was the development of rapid, reliable, and robust methods for assessing enantiomeric purity by conventional diode array detector assisted by chemometric tools.

Automatized sspKa measurements of dihydrogen phosphate and Tris(hydroxymethyl) aminomethane in acetonitrile/water mixtures from 20 to 60°C.

We measured pKa values of Tris(hydroxymethyl)aminomethane and dihydrogen phosphate; both are commonly used to prepare buffers for reverse-phase liquid chromatography (RPLC), in acetonitrile/water mixtures from 0% to 70% (v/v) (64.6% (w/w)) acetonitrile and at 20, 30, 40, 50, and 60°C. The procedure is based on potentiometric measurements of pH of buffer solutions of variable solvent compositions using a glass electrode and a novel automated system. The method consists in the controlled additions of small volumes of a thermostated solution from an automatic buret into another isothermal solution containing exactly the same buffer-component concentrations, but a different solvent composition. The continuous changes in the solvent composition induce changes in the potentials. Thus, only two sequences of additions are needed: increasing the amount of acetonitrile from pure water and decreasing the content of acetonitrile from 70% (v/v) (64.6% (w/w)). In the procedure with homemade apparatus, times for additions, stirring, homogenization, and data acquisition are entirely controlled by software programmed for this specific routine. This rapid, fully automated method was applied to acquire more than 40 potential data covering the whole composition range (at each temperature) in about two hours and allowed a systematic study of the effect of temperature and acetonitrile composition on acid-base equilibria of two widely used substances to control pH close to 7. The experimental pKa results were fitted to empirical functions between pKa and temperature and acetonitrile composition. These equations allowed predictions of pKa to estimate the pH of mixtures at any composition and temperature, which would be very useful, for instance, during chromatographic method development.

Fast enantioseparations of basic analytes by high-performance liquid chromatography using cellulose tris(3,5-dimethylphenylcarbamate)-coated zirconia stationary phases.

In this work, we study the influence of the mobile phase and column temperature on the enantioresolution of basic compounds on microparticulate porous zirconia coated with cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC). The chiral analytes are amino compounds, including a number of beta-blockers. Analytes are eluted with hexane-alcohol mobile phases. We investigated the effect of alcohol (type and concentration), basic eluent additives, and column temperature on the parameters that control resolution (column efficiency, retention and selectivity). Conditions for achieving an adequate separation in the least time have been determined for numerous racemic mixtures. For most solutes, baseline resolution of the enantiomeric pair was achieved in less than 1 min; 12 of 13 pairs were separated in less than 2 min.

Cellulose tris(3,5-dimethylphenylcarbamate)-coated zirconia as a chiral stationary phase for HPLC.

In this work we explore the use of microparticulate porous zirconia coated with cellulose tris(3,5-dimethylphenyl-carbamate) (CDMPC) as a support for separation of chiral compounds by HPLC. The surface of zirconia, previously sintered but not rehydroxylated, provides a stable surface for depositing the chiral polymer. Zirconia's surface prior to coating was investigated by diffuse reflectance FT-IR. The spectra indicate the presence of residual hydroxyl groups even after treatment at 750 degrees C for 5 h. The amount of chiral polymer deposited was systematically varied, and the pore structure of the resulting particles was assessed by nitrogen sorptometry. Dynamic studies of columns packed with these stationary phases were also conducted. We found that columns packed with about 3-4% (w/w) CDMPC coated on 2.5-micron zirconia particles provide an excellent compromise between loading need to impart good chiral recognition ability to the stationary phase and column's chromatographic efficiency. Preliminary results show resolutions higher than 1 for 9 out of 16 racemic mixtures in packed 5-cm columns. The use of shorter columns combined with reduced particle size to provide sufficient resolution has the advantage of decreasing the analysis times and reducing eluent volumes. CDMPC-coated zirconia columns exhibit high stability under normal-phase conditions at relatively high linear velocities.