Quantitative determination of free glycerol and myo-inositol from plasma and tissue by high-performance liquid chromatography

A high-performance liquid chromatographic method that accurately measures glycerol and myo-inositol from plasma and tissue is described. The method incorporates a pre-column derivatization reaction using aqueous extracts with benzoyl chloride as a modifying agent. The benzoylated derivatives are isolated by HPLC using reversed-phase gradient chromatography and quantified via absorbance detection at 231 nm. The benzoylated derivatives of glycerol and myo-inositol are well resolved from other known carbohydrates, internal standard and other contaminants encountered within samples and during incubation. The benzoylation of these analytes reach a maximum between 3.5 and 6 h of incubation and are stable for at least 24 days at 4 °C. The limit of quantization (LOQ) of glycerol was equal to 2.5 nmol/ml plasma and 6.4 nmol/g tissue and the LOQ of myo-inositol was 1.8 nmol/ml plasma and 3.6 nmol/g tissue. Incubation of known standards and samples with benzoyl chloride at 40 °C for 4 h showed fully benzoylated products as determined by mass spectral analysis. Calibration curves were linear between 2.7 and 174 nmol for glycerol and 1.4–89 nmol for myo-inositol. Comparison of tissue and plasma concentrations of glycerol and myo-inositol found using this method are in good agreement with other reported values using other techniques.