Chromatographic resolution of closely related species in pharmaceutical chemistry: dehalogenation impurities and mixtures of halogen isomers.

In recent years, the use of halogen-containing molecules has proliferated in the pharmaceutical industry, where the incorporation of halogens, especially fluorine, has become vitally important for blocking metabolism and enhancing the biological activity of pharmaceuticals. The chromatographic separation of halogen-containing pharmaceuticals from associated isomers or dehalogenation impurities can sometimes be quite difficult. In an attempt to identify the best current tools available for addressing this important problem, a survey of the suitability of four chromatographic method development platforms (ultra high-performance liquid chromatography (UHPLC), core shell HPLC, achiral supercritical fluid chromatography (SFC) and chiral SFC) for separating closely related mixtures of halogen-containing pharmaceuticals and their dehalogenated isosteres is described. Of the 132 column and mobile phase combinations examined for each mixture, a small subset of conditions were found to afford the best overall performance, with a single UHPLC method (2.1 × 50 mm, 1.9 µm Hypersil Gold PFP, acetonitrile/methanol based aqueous eluents containing either phosphoric or perchloric acid with 150 mM sodium perchlorate) affording excellent separation for all samples. Similarly, a survey of several families of closely related halogen-containing small molecules representing the diversity of impurities that can sometimes be found in purchased starting materials for synthesis revealed chiral SFC (Chiralcel OJ-3 and Chiralpak IB, isopropanol or ethanol with 25 mM isobutylamine/carbon dioxide) as well as the UHPLC (2.1 × 50 mm, 1.8 µm ZORBAX RRHD Eclipse Plus C18 and the Gold PFP, acetonitrile/methanol based aqueous eluents containing phosphoric acid) as preferred methods.

Comparison of the influence of organic modifier on the secondary interactions of polar embedded and classical alkyl-silica reversed phase HPLC stationary phases.

Linear solvation energy relationships are used along with solute retention data to compare the influence of organic modifier on the secondary interactions of polar-embedded (EPG) and classical alkyl-silica (C18) stationary phases. System constants derived using the linear solvation energy relationship model measure the relative strength of solute interactions in the mobile phase and the solvated stationary phase. Differences in the e and s system constants, which, respectively, measure interactions of pi and lone pair electrons and dipole-type interactions, were larger between the two stationary phases when methanol was used versus ACN. A similar effect was not evident for the a system constant, which is a measure of hydrogen-bond acceptor ability. The b system constant, which measures hydrogen-bond donating ability, was consistently lower for the EPG stationary phases when water-methanol mobile phases were used, especially at lower methanol levels. Plots of the polar and specific selectivity for select solutes versus organic modifier level are used to demonstrate the differing influences of ACN and methanol on the selectivity obtained on representative EPG and C18 stationary phases. For the solutes studied, differences in both the polar and specific selectivity were increased between the two stationary phases when water-methanol mobile phases were used.

Challenges in the analytical method development and validation for an unstable active pharmaceutical ingredient.

A sensitive high-performance liquid chromatography (HPLC) impurity profile method for the antibiotic ertapenem is developed and subsequently validated. The method utilizes an Inertsil phenyl column at ambient temperature, gradient elution with aqueous sodium phosphate buffer at pH 8, and acetonitrile as the mobile phase. The linearity, method precision, method ruggedness, limit of quantitation, and limit of detection of the impurity profile HPLC method are found to be satisfactory. The method is determined to be specific, as judged by resolving ertapenem from in-process impurities in crude samples and degradation products that arise from solid state thermal and light stress, acid, base, and oxidative stressed solutions. In addition, evidence is obtained by photodiode array detection studies that no degradate or impurity having a different UV spectrum coeluted with the major component in stressed or unstressed samples. The challenges during the development and validation of the method are discussed. The difficulties of analyzing an unstable active pharmaceutical ingredient (API) are addressed. Several major impurities/degradates of the API have very different UV response factors from the API. These impurities/degradates are synthesized or prepared by controlled degradation and the relative response factors are determined.

Thermodynamic study of N-trifluoroacetyl-O-alkyl nipecotic acid ester enantiomers on diluted permethylated beta-cyclodextrin stationary phase.

Thermodynamic studies were performed on 12 pairs of N-trifluoroacetyl-O-alkyl nipecotic acid ester enantiomers on diluted permethylated beta-cyclodextrin stationary phase (CP Chirasil-Dex CB). The influence of ester alkyl group structure on interaction with permethylated beta-cyclodextrin (Me-CD) and enantioselectivity was studied. The types of alkyl groups studied included n-alkyl (C1-C5) and groups containing branching at differing locations relative to the chiral center of the molecule. The results show that for a given molecular weight, the n-alkyl esters have stronger interactions with Me-CD than esters containing branched alkyl groups. However, although having weaker interactions with Me-CD, esters containing alpha-branched alkyl groups exhibit higher enantioselectivity than the corresponding n-alkyl or beta-branched isobutyl esters. From the retention data, thermodynamic parameters were estimated using the retention increment method and enthalpy-entropy compensation plots (ln R' versus deltaH) were constructed. The results suggest that ester enantiomers with branching at the alpha-carbon of the ester alkyl group have additional and/or different types of enantioselective interactions with Me-CD than the C1-C5 n-alkyl esters or beta-branched isobutyl ester. In order to obtain a qualitative sense of the interaction with Me-CD, structures of the diastereomeric complexes formed between Me-CD and some of the ester enantiomers were modeled using simulated annealing molecular dynamics.