Development and validation of ivermectin quantification method in volumetric absorptive microsampling using liquid chromatography-tandem mass spectrometry

Ivermectin is a broad-spectrum anthelmintic used to control onchocerciasis from nematode parasites. As an anthelmintic, ivermectin is designed to have high levels in the gastrointestinal tract, so that the systemic intake is relatively low. Due to the very small concentration of ivermectin, a selective and sensitive approach is needed for the analysis of ivermectin in blood. Several methods have been developed using plasma and Dried Blood Spots, but there are still shortcomings due to hematocrit effects. Therefore, this study was conducted to establish a validated ivermectin analysis method with doramectin as the internal standard in using Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Mass spectrometry equipped with triple quadrupole and positive electrospray ionization mode was used to conduct the analysis. For the biological matrix, whole blood was used by Volumetric Absorptive Microsampling and extracted using a protein precipitation technique with a combination of acetonitrile and methanol (1:1). VAMS has some advantages such as not being affected by hematocrit, requires a small and fixed volume of sample, also a more efficient sampling process.

The optimum conditions were achieved with an Acquity® UPLC BEH C18 column (1,7 μm; 2.1 × 100 mm); extracted-flow rate was 0,2 mL/min; mobile phase was 5 mM ammonium formate pH 3.00 and acetonitrile (10:90) with isocratic elution. Multiple Reaction Monitoring (MRM) detection by m/z values was 892.41 > 569.5 for ivermectin and 916,41 > 331,35 for doramectin.

The method has been appropriately validated in compliance with the 2018 guidelines laid out by the US Food and Drug Administration. Resulting the minimum detection (LLOQ) was 1 ng/mL with a linear concentration range spanning from 1 to 150 ng/mL.