Determining Ivermectin in Plasma and Whole Blood Using LC-MS/MS

A recent study conducted at Mahidol University (Bangkok, Thailand) developed and validated a high-throughput and sensitive method to quantify ivermectin in plasma and whole blood samples, using automated sample extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A paper based on this research was recently published in Wellcome Open Research (1).

Ivermectin, a drug commonly used to treat parasitic infections and shows potential for fighting malaria because it can kill mosquitoes (1), is widely recognized as a crucial drug for the treatment of helminthiasis (a medical condition characterized by the infestation of parasitic worms in the human body [2]) and is particularly valued for its effectiveness against filarial (stemming from a type of parasitic found in tropical regions [3]) infections (4).

Method validation was performed in accordance with the Guidance for Industry from the U.S. Food and Drug Administration (FDA) (5) and the European Medicines Agency (EMA) (6). The calibration curves were analyzed as duplicate samples of each concentration (0, 0.970, 3.39, 11.9, 41.6, 146, and 384 ng/ml). Non-weighted and weighted (1/x and 1/x2) linear regression models were evaluated for the best performing calibration curve model. The selected model was chosen based on the accuracy of back-calculated concentrations of the calibration curves and quality control (QC) samples from four independent validation runs (7). Intra-assay and inter-assay accuracy (mean relative error, %) and precision (%CV) were determined by analyzing five replicates of the lower limit of quantification sample, upper limit of quantification sample, QC samples, and over curve sample. A single factor analysis of variance was used for %CV calculation (1).

The chromatographic methods used by the researchers used a low sample volume of 100 µl and demonstrated a robust quantification of ivermectin over a range of clinically relevant concentrations related to therapy. The use of extraction plates (phospholipid removal) allowed the team to perform a simple sample extraction technique with a high recovery, effectively adsorbing blood components such as hemoglobin from whole blood samples without the loss of ivermectin. This technique was also easy to automate by using a liquid handler for high-throughput analysis of clinical whole blood and plasma samples. The validated high-throughput method demonstrated to be reliable and reproducible when implemented in the research team’s bioanalytical clinical laboratory, enabling them to elucidate the pharmacokinetic and pharmacodynamic relationships of ivermectin when used for its many indications. The authors of the paper report that no part of the developed method was particularly susceptible to stability issues, and the method should be able to straightforwardly be implemented into a routine bioanalytical high-throughput laboratory (1).

Red blood cells travel in an artery. © Digilife - stock.adobe.com

References

1. Kaewkhao, N.; Hanpithakpong, W.; Tarning, J.; Blessborn, D. Determination of Ivermectin in Plasma and Whole Blood Using LC-MS/MS. Wellcome Open Res. 2024, 5 (9), 231. DOI: 10.12688/wellcomeopenres.20613.2

2. Helminthiasis. Yale Medicine website. https://www.yalemedicine.org/clinical-keywords/helminthiasis#:~:text=Helminthiasis%20is%20a%20medical%20condition,%2C%20or%20trematodes%20(flukes) (accessed 2024-10-03)

3. Filarial worms. U. S. Centers for Disease Control and Prevention website.https://www.cdc.gov/filarial-worms/about/index.html (accessed 2024-10-03)

4. WHO: Web Annex A. World Health Organization model list of essential Medicines – 23rd list, 2023. In: The Selection and Use of Essential Medicines 2023: Executive Summary of the Report of the 24th WHO Expert Committee on the Selection and Use of Essential Medicines, 24 – 28 April 2023. Geneva: World Health Organization. https://iris.who.int/bitstream/handle/10665/371090/WHO-MHP-HPS-EML-2023.02-eng.pdf?sequence=1 (accessed 2024-07-01)

5. Food and Drug Administration (FDA): Bioanalytical Method Validation Guidance for Industry. US Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research and Center for Veterinary Medicine  2018.  https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf (accessed 2024-07-01)

6. European Medicines Agency (EMA): Guideline on Bioanalytical Method Validation 2012. https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdf (accessed 2024-07-01)

7. Singtoroj, T.; Tarning, J.; Annerberg, A. et al. A New Approach to Evaluate Regression Models During Validation of Bioanalytical Assays. J. Pharm. Biomed. Anal. 2006, 41 (1), 219–227. DOI: 10.1016/j.jpba.2005.11.006