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.In the fluticasone study, it was shown that HPLCNMR spectroscopy could detect and characterise impurities below the 0.1 % peaklevel, which is the relevant limit for submissions to regulatory authorities [27].However, NMR spectra on the peaks which were at a level of 0.2 % or less ofthe parent drug required considerable and time-consuming data acquisition inorder to achieve acceptable signal-to-noise ratios.Although this appears toinvolve a high cost in NMR analysis time it would be justified in cases such asthat found with fluticasone propionate where there are few alternative analyticalapproaches.It may, therefore, be beneficial to concentrate the impurities, ifstable, before the HPLC NMR analysis by the application of solid-phase extrac-tion chromatography, column switching or concentration/enrichment of theimpurities by preparative HPLC.Such techniques can be feasible because ofthe availability of large amounts of sample from drug production batches.The fluticasone study focused on the evaluation of HPLC NMR spectro-scopy for characterising impurities in raw bulk drug substances.However, thetechnique could also be applicable to the investigation of formulated drugsubstances where impurities often appear as a result of the drug substance re-acting with the formulation compound or on samples from degradation studies.These types of adduct can sometimes be unstable during sample extraction andpurification.This area is of considerable practical importance in the pharma-ceutical industry and is worthy of further study.Directly coupled HPLC NMR spectroscopy has been used in a number ofother studies of chemical impurities.An impurity in a bulk drug sample of theglycinamide ribonucleotide transformylase inhibitor AG2034, shown below:has been characterised by using both 1D and 2D TOCSY analysis [32].Thestudies were actually performed on an impurity-enriched sample of the finaldrug synthetic precursor and this demonstrated that the impurity was a dimericmolecule of the desired parent structure.The technique has also been used to characterise the synthetic route forfluticasone in the preparation of [S-fluoromethyl-18F] fluticasone propionate 62 LC NMR and Related Techniquesfor use as a radio-tracer substance for lung deposition studies employingpositron emission tomography [33].HPLC 1H NMR spectroscopy using an isocratic separation with acetoni-trile and D2O in the ratio 7:13 has also been used to study photo-isomerisationof the natural material azadirachtin extracted from the seeds of the neem treeand which is a powerful insect anti-feedant [34].This has a complex structurewith an (E)-2-methylbutyl-2-enoate fragment but which after exposure to UViradiation is converted to the Z -isomer:OOOORREZAnother different area of application where there is a need to characteriselow-level impurities occurs in environmental studies.A recent study has usedHPLC NMR spectroscopy and HPLC MS in the MSn mode to identifyorganic components in the industrial waste-water effluent of a textile company.Generally in the past, GC MS and HPLC MS have been used for such ana-lyses where the identity of many of the pollutants are not known beforehand.However, it has recently been shown that HPLC NMR can provide usefulinformation and if this is used with complementary information from HPLCMS then this is the most efficient approach.This study evaluated the impurity profile of untreated water from a textileplant in Portugal [35].The organic material was concentrated by extraction from1 l of water into dichloromethane and HPLC NMR and HPLC MS experi-ments were carried out using a reverse-phase separation with an acetonitrile/1D2O gradient elution with H NMR spectroscopic observation at 600 MHz.Forthe HPLC NMR studies, the samples were further fractionated into two poolsaccording to their HPLC retention times.The HPLC NMR studies were carriedout in the stop-flow mode and the combination of NMR and MS results yieldedthe identification or tentative identification of 14 compounds, comprisingmainly surfactants, anthraquinone dyes and nonylphenol-related molecules.3.5 APPLICATION TO CHIRAL SEPARATIONS OFPHARMACEUTICAL MIXTURESMany pharmaceutical products are chiral molecules, either as single isomers ormore commonly as racemic mixtures
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