A method for simultaneous determination of glycerol and mannitol in various human tissues was devised and for this we used high-performance liquid chromatography (HPLC). Specimens were homogenized in a mixture of chloroform and methanol, phosphate buffer (pH 7.0) and pentaerythritol (IS) solution. After centrifugation, an aliquot of the aqueous layer was evaporated to dryness and derivatized with p-nitrobenzoyl chloride at 50°C for 1h, then applied to HPLC with analytical conditions of: column, CAPCELL PAK C18 MG (250mm×3.0mm i.d., 5μm, Shiseido Co. Ltd., Tokyo, Japan); column temperature, 1-2°C; mobile phase, 75% acetonitrile-distilled water containing 0.05% trifluoroacetic acid, 0.05% heptafluoro-n-butyric acid and 0.1% triethylamine; flow rate, 0.5ml/min; wavelength, 260nm. Calibration curves for both substances were linear in concentration ranges from 1 to 500μg/0.1g and correlation coefficients exceeded 0.99. The relative standard deviation (R.S.D.) of the method was evaluated at concentrations of 10 and 100μg/0.1g, and ranged from 0.84 to 10.6%. Using this method, we determined the regional distribution levels of glycerol and mannitol in various tissues from an autopsied brain dead man.
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