Improvement and validation of 125I-high-performance liquid chromatography method for determination of total human serum choline and ethanolamine plasmalogens

Abstract

Background Serum plasmalogens (Pls) have gained interest in several clinical symptoms such as metabolic syndrome/atherosclerosis or Alzheimer's disease possibly because of their antioxidant properties. We have developed a highly sensitive and simple method to determine plasmenylcholine (PlsCho; choline plasmalogen) and plasmenylethanolamine (PlsEtn; ethanolamine plasmalogen) separately, using a radioactive iodine and high-performance liquid chromatography (125I-HPLC method). The present study reports the improvement and validation of 125I-HPLC method by introducing a quantitative standard (QS) and online detection with a flow γ-counter. Methods 1-Alkenyl 2,3-cyclic glycerophosphate was prepared as QS from l- α-lyso plasmenylcholine by enzymatic treatment with phospholipase D. Online detection with a flow γ-counter was investigated to be available to quantify Pls. The method validation was carried out in terms of selectivity, sensitivity, linearity, precision, accuracy and recovery. Results Linearity was established over the concentration range 5–300 μmol/L for Pls and QS with regression coefficients >0.99. The accuracy and reliability were satisfactory. The method has been applied to the determination of human serum Pls from healthy subjects and the elderly with dementia or artery stenoses. Conclusions The improved 125I-HPLC method is useful as an autoanalytical system for a routine diagnostic test of human serum Pls.