title: Quantitation of free and total amphotericin B in human biologic matrices by a liquid chromatography tandem mass spectrometric method.
abstract: Amphotericin B remains the standard of care for the treatment of invasive and disseminated fungal infections. Various lipid-based formulations of amphotericin B have been developed to improve its therapeutic index by decreasing toxicity. Previous bioanalytic methods using microbial inhibition or high-pressure liquid chromatography quantified total amphotericin B (free, plasma protein-bound, and lipid-complexed). Sensitivity of this method with a low limit of quantitation of 0.05 microg/mL was inadequate to determine free (unbound) amphotericin B. A sensitive LC/MS/MS method was developed to determine the total amphotericin B value in human plasma and other biologic matrices and the free amphotericin B concentration in plasma. For determination of total plasma amphotericin B concentrations, the sample was diluted and injected onto the LC/MS/MS. For total amphotericin B in other matrices and free amphotericin B in plasma, solid-phase extraction was used. Natamycin served as an internal standard. A PE Sciex API 3000 (Sciex; Concord, Ontario, Canada) was used to assess free amphotericin B in plasma ultrafiltrate determination and an API 3+ for the other matrices, with electrospray interfaced to a C18 analytic column. The low limit of quantitation was 1 ng/mL for ultrafiltrate. For total amphotericin B, the low limits were 2 microg/mL for plasma, 0.05 microg/mL for urine, and 0.4 microg/mL for fecal homogenate. The methods were validated to show the standard range linearity, sensitivity, selectivity, accuracy, precision, and stability of amphotericin B in the matrices tested.
authors.affiliation: MDS Pharma Services, Inc., Lincoln, Nebraska, USA. jean.lee@mdsps.com
authors.forename: J W
authors.initials: JW
authors.lastname: Lee
authors.forename: M E
authors.initials: ME
authors.lastname: Petersen
authors.forename: P
authors.initials: P
authors.lastname: Lin
authors.forename: D
authors.initials: D
authors.lastname: Dressler
authors.forename: I
authors.initials: I
authors.lastname: Bekersky
mesh_terms: D000666:Amphotericin B / Q000032:analysis* / Q000302:isolation & purification; D000935:Antifungal Agents / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D006801:Humans; D013058:Mass Spectrometry
chemical_list: D000935:Antifungal Agents; D000666:Amphotericin B