Determination the concentration of amphotericin B in mouse plasma and tissues by liquid chromatography-mass spectrometry

CAI Liuyang; HE Haiyang; ZHANG Li; LIAO Wanqing; XUE Xiaochun; PAN Weihua

doi:10.3969/j.issn.1673-3827.2025.05.001

Abstract：Objective To establish a liquid chromatography-mass spectrometry(LC-MS/MS)method for the deter-mination of the concentration of the antifungal drug amphotericin B(AmB)in mouse plasma and tissues.Methods Using liver as representative tissue sample,the protein precipitation method was used for sample pretreatment of mouse plasma and liver.Chromatographic separation was performed using a shim-pack GIST-HP C18 AQ(2.10 mm×50.00 mm,3.00 μm)chromatographic column.Gradient elution was performed using water containing 2.00 mmol/L ammonium acetate and 0.10%formic acid as mobile phase A,and acetonitrile containing 0.10%formic acid as mobile phase B(sample vol-ume was 5.00 μL,mobile phase flow rate was 0.45 mL/min,column temperature was 40℃).The electrospray ion source was set up detection conducted in positive ion mode and multiple reaction monitoring mode.The quantitative ion pairs of AmB and the internal standard natamycin were m/z 924.50→906.50 and m/z 666.50→503.40,respectively.Re-sults The response of this LC-MS/MS method to interfering components was less than 10.00%of the response at the lower limit of quantification of the analyte,demonstrating good specificity.The lower limit of quantification for AmB and the internal standard detection in both plasma and tissue was 0.1 μg/mL,with good linearity in the range of 0.10 μg/mL-100.00 μg/mL,accuracies(recoveries)of intra-day and inter-day were all more than 95.00%,and the relative standard deviations(RSD)of precision,matrix effect,and dilution effect were all less than 10.00%.AmB could be stored stably under avoid light and low-temperature conditions.Conclusions The LC-MS/MS method for determining AmB concentra-tion in mouse plasma and tissues was successfully established,and all investigation results meet the methodological re-quirements.