Abstract: Objective To establish a method for detecting rifampicin in human cerebrospinal fluid (CSF) with restricted access
media coupled with high-performance liquid chromatography that allows online direct sample injection and enrichment.
Methods We used the column of restricted access media as the pre-treatment column and a C18 column as the analytical
column. The mobile phase of pre-treatment column was water-methanol (95∶5, V/V) and the flow rate was 1 mL/min; the
mobile phase of the analytical column was methanol-acetonitrile-10 mmol/L ammonuium acetate (volume ratio of 60∶5∶35).
The detection wavelength was 254 nm and the column temperature was set at 25 ℃．Results For an injection volume of 100
μL, the peak area of rifampicin was 5.33 times that for an injection volume of 20 μL, and the limit of detection was effectively
improved. The calibration curve showed an excellent linear relationship (r=0.9997) between rifampicin concentrations and
peak areas within the concentration range of 0.25 to 8 μg/mL in CSF. The limits of detection and quantification was 0.07 μg/mL
and 0.25 μg/mL, respecetively, with intra-day and inter-day assay precisions and relative standard deviation (RSD%) all below
5%. The recoveries of rifampicin at 3 blank spiked levels (low, medium, and high) ranged from 87.69% to 102.11%. In patients
taking oral rifampicin at the dose of 10 mg/kg, the average rifampicin concentration was 0.29 in the CSF at 2 h after medication.
Conclusion The method we established is simple and fast for detecting rifampicin in CSF and allows direct online injection
and enrichment with good detection precisions and accuracies.