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Aliquots of compounds are mixed with either phosphate buffer (pH 7.4) or water for > 16 h at room temperature or 37 °C. Soluble and insoluble compounds are separated by filtration or double centrifugation. The preferred method is filtration accompanied with an assessment of filter binding. Following separation, the pH of the soluble fraction is determined and the soluble material diluted in mobile phase for analysis by HPLC or MS. Bulk form is assessed by microscopy.
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The stereoselectivity of the reduction of the 5′-ketone to the alcohol by ketone reductase was never determined. In addition, it was never identified whether reoxidation to the ketone occurred with the racemic alcohol or was specific to one of the enantiomers.
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