Determination of acid sphingomyelinase activity in biological samples with ultra-performance liquid chromatographic assay

摘要

A simple, precise and accurate ultra-performance liquid chromatographic (UPLC) method was developed for the quantitative determination of the acid sphingomyelinase (ASM) activity in L-02 hepatocytes. The biological samples of cell homogenate were collected from L-02 hepatocytes and incubated with the fluorescent substrate BODIPY C-12-Spm for 2hr. The samples were determined using ACQUITY UPLC (R) BEH Amide chromatographic column with mobile phase containing acetonitrile and 13mM sodium acetate (97:3, v/v) at a flow rate of 0.8mL/min. The injection volume was 5L. The eluent was monitored using a fluorescence detector set to excitation and emission wavelengths of 435nm and 525nm, respectively. Fluorescent product B-12-Cer in chromatography analytical can be effectively separated in 2min, showing good linearity in the range of 6.25-300ng/mL with the correlation coefficient of 0.9926. The mean ASM activity in L-02 hepatocytes with different sample pretreatment methods, lysis buffer, ultrasonic disruption, and freeze-thawing with liquid nitrogen was 3225.76 +/- 323.63, 3330.98 +/- 277.99 and 3355.05 +/- 267.77ng/mgprot/h, respectively. Thus we think the current method can be used to detect ASM activity in traces of biological samples.