Highly sensitive UPLC-MS/MS method for the quantification of paromomycin in human plasma

J Pharm Biomed Anal. 2020 Jun 5;185:113245. doi: 10.1016/j.jpba.2020.113245. Epub 2020 Mar 9.


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.

Keywords: Assay; Bioanalytical validation; Human plasma; Ion-pair liquid chromatography; Paromomycin; Tandem mass spectrometry.

Publication types

  • Validation Study

MeSH terms

  • Adsorption
  • Africa, Eastern
  • Antiprotozoal Agents / administration & dosage
  • Antiprotozoal Agents / blood*
  • Antiprotozoal Agents / chemistry
  • Antiprotozoal Agents / pharmacokinetics
  • Calibration
  • Chromatography, High Pressure Liquid / methods
  • Chromatography, High Pressure Liquid / standards
  • Drug Monitoring / methods*
  • Drug Stability
  • Humans
  • Injections, Intramuscular
  • Leishmaniasis, Visceral / blood
  • Leishmaniasis, Visceral / drug therapy*
  • Limit of Detection
  • Paromomycin / administration & dosage
  • Paromomycin / blood*
  • Paromomycin / chemistry
  • Paromomycin / pharmacokinetics
  • Reference Standards
  • Reproducibility of Results
  • Tandem Mass Spectrometry / methods
  • Tandem Mass Spectrometry / standards
  • Trichloroacetic Acid / chemistry


  • Antiprotozoal Agents
  • Trichloroacetic Acid
  • Paromomycin