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 degrees 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 degrees 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.
University of North Dakota, School of Medicine and Health Sciences, Grand Forks, ND 58203, USA.
Ryan A
RA
Frieler
Dane J
DJ
Mitteness
Mikhail Y
MY
Golovko
Heidi M
HM
Gienger
Thad A
TA
Rosenberger
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005990:Glycerol / Q000097:blood* / Q000378:metabolism; D066298:In Vitro Techniques; D007294:Inositol / Q000097:blood* / Q000378:metabolism; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results
D007294:Inositol; D005990:Glycerol