Quantitation of free and total amphotericin B in human biologic matrices by a liquid chromatography tandem mass spectrometric method

Amphotericin B remains the standard of care for the treatment of invasive a nd disseminated fungal infections. Various lipid-based formulations of amph otericin B have been developed to improve its therapeutic index by decreasi ng toxicity. Previous bioanalytic methods using microbial inhibition or hig h-pressure liquid chromatography quantified total amphotericin B (free, pla sma protein-bound, and lipidcomplexed). Sensitivity of this method with a l ow limit of quantitation of 0.05 mug/mL was inadequate to determine free (u nbound) amphotericin B. A sensitive LC/MS/MS method was developed to determ ine the total amphotericin B value in human plasma and other biologic matri ces and the free amphotericin B concentration in plasma. For determination of total plasma amphotericin B concentrations, the sample was diluted and i njected onto the LC/MS/MS. For total amphotericin B in other matrices and f ree amphptericin B in plasma, solid-phase extraction was used. Natamycin se rved as an internal standard. A PE Sciex API 3000 (Sciex; Concord, Ontario, Canada) was used to assess free amphotericin B in plasma ultrafiltrate det ermination and an API 3+ for the other matrices, with electrospray interfac ed to a C,, analytic column. The low limit of quantitation was 1 ng/mL for ultrafiltrate. For total amphotericin B, the low limits were 2 mug/mL for p lasma, 0.05 mug/mL for urine, and 0.4 mug/mL for fecal homogenate. The meth ods were validated to show the standard range linearity, sensitivity, selec tivity, accuracy, precision, and stability of amphotericin B in the matrice s tested.