A rapid pretreatment method for the determination of pentosidine in human plasma by high performance liquid chromatography

抄録

Purpose: In HPLC-fluorescence measurement of pentosidine in blood, multiple-speciemen measurement has problems such as complexity of sample pretreatment, and measurement time. In this study, a sample pretreatment method using a spin column was verified for the purpose of simplifying sample processing. Methods: The blood sample (specimen) used was plasma provided by healthy men and women between the age of 20 to 100 years. For verification of the pretreatment method of the sample, plasma provided by 40 men and women in their 50s to 80s was mixed and used after homogenization. The sample was hydrolyzed under 6 N hydrochloric acid, added to a spin column (Monospin AG) having a mixed group consisting of a cation exchange group and a hydrophobic group as a carrier, followed by elution. Pentosidine was fluorescently detected by HPLC using a reverse phase column. Targeting 72 subjects aged between 20 and 80, a correlation analysis of plasma pentosidine was performed with subject age and age-related physical markers. This study was carried out under the deliberation and approval of the Ethics Review Committee of Doshisha University and the Society for Glycative Stress Research. Results: The recovery rate of pentosidine in the sample was 116% on average by this pretreatment method. The time required for pretreatment after hydrolysis was within 10 minutes. There was a positive correlation between the measured plasma pentosidine levels and age, and a weak positive correlation with HbA1c. Regarding the value of skin autofluorescence (AF) resulting from AGEs and the plasma pentosidine level, the value measured by AGE Reader mu was positively correlated, while by AGEs Sensor was weakly positively correlated. Conclusion: The pretreatment time of the blood sample was shortened from about 12 hours in the ion pair-HPLC method to approximately 10 minutes by using the spin column. Furthermore, this pretreatment could reduce contaminants, and the HPLC measurement time could be shortened to one-third that of the citric acid-HPLC method. It was verified that this method is useful as a multi-specimen assay for pentosidine in blood.