Abstract
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 C12-Spm for 2 hr. The samples were determined using ACQUITY UPLC® BEH Amide chromatographic column with mobile phase containing acetonitrile and 13 mM sodium acetate (97:3, v/v) at a flow rate of 0.8 mL/min. The injection volume was 5 μL. The eluent was monitored using a fluorescence detector set to excitation and emission wavelengths of 435 nm and 525 nm, respectively. Fluorescent product B12-Cer in chromatography analytical can be effectively separated in 2 min, showing good linearity in the range of 6.25–300 ng/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 ± 267.77 ng/mgprot/h, respectively. Thus we think the current method can be used to detect ASM activity in traces of biological samples.
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