Concentration of amphotericin B in blood based on two-dimensional high performance liquid chromatography

Objective To establish a method to determine concentration of amphotericin B in blood based on two-dimensional high performance liquid chromatography method.

Methods Aston SX1 (3.5 mm×25.0 mm, 5.0 μm) was used as the first-dimensional chromatographic column, mobile phase V_methanol∶V_acetonitrile∶V_water (25.0 mmoL/L ammonium phosphate salt solution)=1∶3∶3, flow rate was 0.7 mL/min. The second-dimensional column was used Aston SCB (4.6 mm×125.0 mm, 5.0 μm), mobile phase V_water (25.0 mmol/L ammonium phosphate solution)∶V_water (1.0 mmol/L diammonium hydrogen phosphate solution)∶V_methanol∶V_Acetonitrile=10∶35∶10∶45, flow rate was 1.0 mL/min. Aston SCB (4.6 mm×10.0 mm, 3.5 μm) was used in the middle column, and the auxiliary mobile phase was pure water. The detection wave length was 380 nm, the column temperature was 40 ℃, and the injection volume was 300 μL.

Results Amphotericin B had a good linear relationship with peak area in the range of 0.059 to 12.31 μg/mL (r=0.999 9), the lowest limit of quantification was 0.04 μg/mL, the detection limit was 0.01 μg/mL, and the recovery rate was 98.79% to 99.39%. The extraction recovery rate was 98.50% to 99.00%, and relative standard deviation (RSD) of the intra-day and inter-day precision was less than 3.00%.

Conclusion The plasma concentration of amphotericin B based on two-dimensional high performance liquid chromatography has the characteristics of high sensitivity, good reproducibility, high degree of automation and simple sample processing. It meets the requirements of serum sample detection and can be used for clinical monitoring of amphotericin B plasma concentration.