Abstract
A highly sensitive method was developed to quantitate the antileishmanial agent paromomycin in human plasma, with a lower limit of quantification of 5 ng/mL. Separation was achieved using an isocratic ion-pair ultra-high performance liquid chromatographic (UPLC) method with a minimal concentration of heptafluorobutyric acid, which was coupled through an electrospray ionization interface to a triple quadrupole - linear ion trap mass spectrometer for detection. The method was validated over a linear calibration range of 5 to 1000 ng/mL (r2≥0.997) with inter-assay accuracies and precisions within the internationally accepted criteria. Volumes of 50 μL of human K2EDTA plasma were processed by using a simple protein precipitation method with 40 μL 20 % trichloroacetic acid. A good performance was shown in terms of recovery (100 %), matrix effect (C.V. ≤ 12.0 %) and carry-over (≤17.5 % of the lower limit of quantitation). Paromomycin spiked to human plasma samples was stable for at least 24 h at room temperature, 6 h at 35 °C, and 104 days at −20 °C. Paromomycin adsorbs to glass containers at low concentrations, and therefore acidic conditions were used throughout the assay, in combination with polypropylene tubes and autosampler vials. The assay was successfully applied in a pharmacokinetic study in visceral leishmaniasis patients from Eastern Africa.
Highlights
Paromomycin is an antimicrobial drug from the aminoglycoside branch
Hydrophilic interaction liquid chromatography (HILIC) [7,8], zwitterionic-HILIC (ZIC-HILIC) [9,10,11], derivatization of paromomycin [12] or ionpairing (IP) combined with an RP stationary phase chromatographic system [13,14,15,16] have been reported in the analysis of paromomycin
We evaluated the use of a zwitterion-HILIC column, but paromomycin did not elute well resulting in broad peaks and poor peak shapes and poor sensitivity
Summary
Paromomycin (aminosidine) is an antimicrobial drug from the aminoglycoside branch. It is the only aminoglycoside with a clinically important anti-leishmanial activity and was discovered in 1963 in the USSR [1]. Since 2006, intramuscular paromomycin has been licensed for the treatment of the neglected tropical disease visceral leishmaniasis in India. High efficacy rates in a series of clinical trials in India and Eastern Africa, in combination with sodium stibogluconate, combined with a good cost-effectiveness led to widespread implementation of this drug to treat visceral leishmaniasis [3,4,5]. Chemical characteristics of aminoglycosides include a high polarity, absence of hydrophobic side chains and poly-ionic charge in aqueous environments, which lack interaction with traditional reversed-phase (RP) liquid chromatography (LC) [6]. Internal standards used in these bioanalytical assays were synthesized permethylated aminoglycosides (spectinomycin, dihydrostreptomycin and kanamycin A) or chemical analogues (various other aminoglycosides)
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