Development and validation of a high-performance liquid chromatography tandem mass spectrometry method for the quantification of the antiparasitic and antifungal drug amphotericin B in human skin tissue

Amphotericin B is an antifungal and antiparasitic drug used in first-line treatment of the parasitic neglected tropical disease leishmaniasis. Liposomal amphotericin B is currently studied for the treatment of cutaneous and post-kala-azar dermal leishmaniasis, where the dermis of the skin is infected with Leishmania parasites. For the optimization of known treatment regimens, accurate target-site concentrations of the drug are required. To date, no assay was available to assess human skin concentrations of amphotericin B. We here present a bioanalytical assay for the quantification of amphotericin B in 4-mm human skin biopsies. Human skin biopsies were ho-mogenized by overnight digestion using collagenase A and were processed afterwards by simple protein pre-cipitation using methanol. Separation and detection were achieved using a Gemini C18 column with slightly acidic chromatographic conditions and a quadrupole - linear ion trap mass spectrometer, respectively. The method was validated in digestion solution over a range of 10-2,000 ng/mL using natamycin as internal stan-dard, with a correlation coefficient (r2) of at least 0.9974. The assay performance, accuracy and precision, were acceptable over the validated range, using international (EMA and FDA) acceptance criteria. In the skin tissue extracts, amphotericin B ion enhancement was observed, however, the internal standard (IS) corrected for this effect hence calibration standards in digestion solvent could be used as a surrogate matrix for the quantification in skin tissue. Sample preparation recoveries were low (around 27%) because of degradation of amphotericin B during digestion and sample preparation processes, albeit highly reproducible, without compromising the ac-curacy and precision of the method. Using this assay, amphotericin B could be detected and quantified in skin biopsies originating from treated Indian post-kala-azar dermal leishmaniasis patients.

Automated summary

This study presents a bioanalytical assay for the quantification of amphotericin B in human skin biopsies, which can optimize known treatment regimens by accurately measuring drug concentrations in the skin. The assay achieved acceptable accuracy and precision, and demonstrated the presence of amphotericin B in skin biopsies from treated patients.