Abstract: Objective To establish a method based on restricted access media-high performance liquid chromatography for
direct online sample injection and detection of plasma and urine furosemide in rats. Methods The column of restricted access
media was used as the pre-treatment column and a C18 column as the analytical column. The mobile phase of the pretreatment
column was water-methanol (95∶5, V/V) with a volume percentage of formic acid of 0.1%. The mobile phase of the
analytical column was methanol-water (65∶35, V/V) for plasma and methanol-water (55∶45, V/V) for urine samples, all
containing a volume percentage of formic acid of 0.1% with a flow rate of 1 ml/min. The detection wavelength was 274 nm and
the column temperature was maintained at 25 ℃. Results The calibration curve showed an excellent linear relationship in rat
plasma furosemide concentration range of 0.1-3.2 μg/ml (r=0.9995) and in urine concentration range of 0.5-32 μg/ml (r=0.9991).
The average recoveries of furosemide at 3 spiked levels ranged from 101.82% to 113.36% for plasma and from 98.75% to
112.27% for urine samples. The detection showed good intra- and inter-day assay precisions and accuracies with the relative
standard deviations all below 5%. The pharmacokinetic parameters AUC0→24 was 6.265 g/(ml·h) with a t1/2 of 2.447 h and a CMax
of 1.414 g/ml. The mean cumulative excretory rate of furosemide in the urine of rats over 24 h was 32.50%-39.08%. Conclusion
Detection of furosemide in plasma and urine samples using restricted access media-high performance liquid chromatography
is simple and efficient and allows direct online injection of the samples.