Development and validation of an HPLC-MS/MS method for the quantification of the anti-leishmanial drug miltefosine in human skin tissue.

Miltefosine is the only oral drug approved for the treatment of various clinical presentations of the neglected parasitic disease leishmaniasis. In cutaneous leishmaniasis and post-kala-azar dermal leishmaniasis, Leishmania parasites reside and multiply in the dermis of the skin. As miltefosine is orally administered and this drug is currently studied for the treatment of these skin-related types of leishmaniasis, there is an urgent need for an accurate assay to determine actual miltefosine levels in human skin tissue to further optimize treatment regimens through target-site pharmacokinetic studies. We describe here the development and validation of a sensitive method to quantify miltefosine in 4-mm human skin biopsies utilizing high-performance liquid chromatography coupled to tandem mass spectrometry. After the skin tissues were homogenized overnight by enzymatic digestion using collagenase A, the skin homogenates were further processed by protein precipitation and phenyl-bonded solid phase extraction. Final extracts were injected onto a Gemini C18 column using alkaline eluent for separation and elution. Detection was performed by positive ion electrospray ionization followed by a quadrupole - linear ion trap mass spectrometer, using deuterated miltefosine as an internal standard. The method was validated over a linear calibration range of 4-1000 ng/mL (r ≥ 0.9996) using miltefosine spiked digestion solution for calibration and quality control samples. Validation parameters were all within internationally accepted criteria, including intra- and inter-assay accuracies and precisions within± 15% and ≤ 15% (within± 20% and ≤ 20% at the lower limit of quantitation). There was no significant matrix effect of the human skin tissue matrix and the recovery for miltefosine, and internal standard were comparable. Miltefosine in human skin tissue homogenates was stable during the homogenization incubation (37 °C,± 16 h) and after a minimum of 10 days of storage at - 20 °C after the homogenization process. With our assay we could successfully detect miltefosine in skin biopsies from patients with post-kala azar dermal leishmaniasis who were treated with this drug in Bangladesh.