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The method of classifying LC chiral stationary phases (CSPs) introduced by Irving Wainer in 1987 became widely adopted largely because it was based on the mechanisms by which the different CSP Types (I-V) achieved chiral recognition and was an integral part of a 'how to do chiral LC' package. The classification became less used perhaps because it was thought that it was not clear to which Type some of the newer CSPs should be assigned. Its only modern day significance seemed to be in single enantiomer drug patent litigation cases in defining the 'state-of-the-art' of chiral LC in the late 1980s. However, on closer inspection, it is clear that even with the introduction of many new commercially available CSPs, the Wainer classification, perhaps with minor modifications, remains a useful suitable vehicle for distinguishing between different groups (Types) of CSPs as an aid to chiral LC method development., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Screening approaches adopted in pharmaceutical companies for chiral LC method development may be quite complicated and sophisticated in order to guarantee a high success rate. However in other environments it may be of more value to assess how simple a screen might be used to still have a good chance of achieving success. The genuine need to develop chiral separations for the former 'legal-high' drug mephedrone and related cathinones of topical interest presented a good opportunity to develop this theme. In initial work on mephedrone itself, no chiral separation was observed on Chirobiotic V, Cyclobond I 2000 DNP, Whelk-O1 and AmyCoat using reversed phase mobile phases. However, using normal phase solvents, chiral separation was observed on all the chiral stationary phases (CSP) used except Chiralcel OJ-H. Of the chiral separations observed on RegisPack, RegisCell and Whelk-O1, some optimisation work was carried out on the latter two which had showed the greatest enantioselectivity. Following optimisation, the best enantioselectivity (1.59) and enantioresolution (5.90) was found with a 250 mm × 4.6 mm I.D. Whelk-O1 column using a propan-2-ol (IPA)-hexane-trifluoroacetic acid (TFA)-triethylamine (TEA) (10:90:0.05:0.05, v/v/v/v) mobile phase. Subsequent screening on other cathinones was restricted to RegisPack, RegisCell and Whelk-O1 or equivalent phases with two mobile phases and this gave a very good success rate. Indeed it was possible to separate all six cathinones on one column, RegisCell, with one mobile phase, propan-2-ol-hexane-TFA (15:85:0.1, v/v/v) but obviously it had been necessary to go through the 3-column screen to arrive at this finding. While Whelk-O1 was not so successful, ease of optimisation on this phase was again a feature. To illustrate the applicability of these separations, it was shown that, as a basis for semi-preparative work, the optimsed mephedrone separation on Whelk-O1 could be scaled-up to a 2000μl injection of a 1.0 mg ml(-1) solution in mobile phase (2.0mg on-column) while still using the 250 mm × 4.6 mm I.D. analytical column., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Chiral stationary phases (CSP) for LC had a major impact on pharmaceutical R&D when they first became commercially available in the 1980s. Even although the use of CSP in pharmaceutical R&D is now very much a mature area, there is still scope for using CSP more effectively to bring about efficiencies. One such instance is the possibility of combining the chiral LC test for the level of a trace enantiomeric impurity in a chiral drug substance and the LC test for related substances into one test. It was envisaged that this could be achieved by carrying out reversed-phase LC on an ODS silica/CSP coupled column system. In evaluating Chiralpak QD-AX, Cyclobond I 2000 DNP and Whelk-O1 CSP using a polar organic - aqueous mobile phase it was found that the Whelk-O1 CSP had good achiral selectivity, the required match of retentivity with the ODS silica material, ACE 5 C18 and also exhibited an encouraging degree of enantioselectivity in the reversed-phase mode. Following consideration of the selectivity of the ACE 5 C18 and Whelk-O1 phases it became apparent that it might be possible to achieve the desired goal of achieving both the enantiomeric impurity and related substances separations in one system by using the Whelk-O1 CSP on its own. This was subsequently demonstrated to be the case using S-naproxen, laevokalim and S-flurbiprofen as illustrative examples., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The cleaning verification of pharmaceutical manufacturing equipment prior to further use is a cGMP requirement. Typically, relevant data are generated by HPLC with UV detection using methods individually developed and validated for each product. This work describes the use of HPLC with mass spectrometry to analyse cleaning verification samples, a novel means of utilising this analytical technology. The initial aim was to produce a single, generic method capable of quantifying a broad range of pharmaceuticals. Ultimately, however, a more effective strategy, in terms of efficiency and reliability, proved to be application of a well-defined approach to the rapid generation of compound specific methods. Results of studies to optimise the sample preparation for a basic compound in drug development (compound 1), together with experimental results for two further compounds are presented. These demonstrated that the combination of a well defined approach to chromatographic method development and mass spectrometric detection provided methodology with advantages in terms of sensitivity. Additionally, and by virtue of its potential for general applicability, the approach proposed has the potential to improve the overall efficiency with which methods for cleaning verification samples can be developed and applied.

Glycolamide esters (compounds 1-17) of 2-(3-trifluoromethyl-phenylamino)nicotinic acid (niflumic acid, CAS 4394-00-7) have been synthesized and evaluated as possible prodrugs. In-vitro hydrolysis studies were conducted at selected pH values (1.2, 3.5, 4.8, 7.4 and 7.8) and in human plasma at 37 +/- 0.5 degree C using HPLC with UV detection. The aqueous (pH 7.4 and 7.8) and enzymatic rates of hydrolysis were substantially affected by the nature of promoieties in this series. The compounds showed good chemical stability in the buffers of low pH values (1.2, 3.5 and 4.8) and appreciable hydrolysis under alkaline conditions and in human plasma. They exhibited long hydrolytic half-lives of 7-46 h in aqueous buffer solutions (pH 7.4 and 7.8) and 14-21 min in human plasma, respectively. It was observed that N,N-disubstituted and cyclic glycolamide derivatives showed 2 fold more hydrolysis in the alkaline pH than monosubstituted derivatives, whereas the piperidino and thiomorpholino derivatives did not undergo chemical hydrolysis. The compounds contain two possible sites for hydrolysis with an increased hydrolytic susceptibility at the terminal aliphatic carbonyl site in aqueous buffers and human plasma solutions. They were found to be cleaved at two hydrolytic carbonyls, namely the nicotinyl (2-5 % in enzymatic hydrolysis) and the aliphatic site (7-55 % and 70-85 % in buffer and plasma hydrolysis, respectively) as revealed by HPLC analysis. The glycolamide ester prodrugs of niflumic acid underwent chemical and enzymatic hydrolysis to release mainly the metabolite 2-(3-trifluoromethyl-phenylamino) nicotinic acid carboxymethyl ester (III) and not the parent drug 2-(3-trifluoromethyl-phenylamino)nicotinic acid. The structure of the metabolite was confirmed by liquid chromatography-mass spectroscopy (LCMS).

Gossypol 1, gossypolone 2, and a series of bis 3 and half Schiff's bases 4 of gossypol were synthesised and tested for anti-proliferative and anti-oxidant activity. (-)-Gossypol (-)-1 was the most potent inhibitor of the proliferation of the HPV-16 keratinocyte cell line (using an MTT viability assay) with a GI50 of 4.8 microM. The bis Schiff's base of (-)-gossypol with L-tyrosine ethyl ester (-)-3b was the most potent inhibitor of iron/ascorbate dependent lipid peroxidation (using the thiobarbituric acid test), with an IC50 of 11.7 microM, with (-)-gossypol being the next most potent of the series, with an IC50 of 13.1 microM. The results from these initial assays suggest that gossypol, as either a racemic mixture rac-1, or the individual atropisomers (-)-1 or (+)-1, has potential for the treatment of psoriasis.

Assessing the short-term stability of drug development candidates in one or more formulations to be used during their preclinical evaluation is a routine, but important, task. Typically, this is based on data generated by HPLC with ultraviolet detection of the species of interest and using methodology specifically developed and validated for the purpose. This work describes a feasibility study conducted into the use of HPLC with mass spectroscopic detection for work of this type. Experimental details and the results of trials with three different drugs, each in a different vehicle, are given. It was concluded that, by using mass spectroscopic detection, a well-defined strategy could be used to generate stability data, which offered advantages in terms of specificity, speed and sensitivity over that typically used.

HPLC analysis of the urine of autistic subjects indicated the presence of an unidentified component in greatly increased concentrations. We have reported the isolation of this component by HPLC and its identification. Mass spectrometry, NMR and UV spectroscopy identified the peak as corresponding to indolyl-3-acryloylglycine (IAG, 3), and this has been confirmed by an independent synthesis.