Quantifying Isavuconazole in Dried Blood Spots Using HPLC

Researchers from Otto-von-Guericke University in Magdeburg, Germany explored a new means of quantifying isavuconazole, an antimycotic agent that can linger in blood, during dried blood spot (DBS) sampling (1). The new study, which was published in the Journal of Chromatography B, looked at DBS spots that were extracted in methanol:water 10:1 and analyzed by an established high-performance liquid chromatography (HPLC) method using fluorescence detection.

The study investigated isavuconazole, a newer antimycotic agent used to treat systematic fungal infections, especially invasive aspergillosis or mucormycosis. It is typically applied when patients are intolerant of alternative antifungal agents (voriconazole for aspergillosis or amphotericin B for mucormycosis) (2). Commonly named Cresemba, the drug is typically administered orally or intravenously. Renal extraction of isavuconazole is minimal, while it is eliminated mainly by metabolic conversion in the liver by the enzymes CYP 3A4 and CYP 3A5. As such, isavuconazole is prone to drug-drug interactions that can lead to reduced and potentially sub-therapeutic concentration levels. However, impaired liver function can prolong the half-life of isavuconazole in circulation, potentially leading to increased drug exposure in such patients.

There are various methods of quantifying isavuconazole in human plasma, typically alongside other antifungal drugs. One increasingly popular method is dried blood spot (DBS) sampling. This microsampling method involves removing blood from an individual, then immediately applying it to pre-prepared DBS filter paper for trying (3). It has been used for the qualitative or semi-quantitative analysis of various molecules, including nucleic acids, elements, proteins, and antibodies. According to the scientists, DBS’s main advantages include less invasive sampling, no biohazard while shipping samples to the laboratory, easy and better-tolerated sampling in infants and convenient bio banking of samples. However, the approach can also involve low sampling volume necessitating sensitive analytic methods and the potential influence of factors like variable values of hematocrit or viscosity of whole blood on the quantitative results of the analytical procedure.

The researchers compared DBS quantification results with corresponding plasma values. DBS were created by spotting 15 µL of ethylenediaminetetraacetic acid (EDTA)-blood onto DBS cards. The isavuconazole proved stable in DBS over three weeks, whether kept at room temperature or refrigerated at 6 °C. Intra-day precision and accuracies of the quantification from DBS were better than 5 %, while inter-day results were better than 12%.

Fourteen plasma samples, which were taken from intensive care patients that showed isavuconazole concentrations of <0.1 μg/mL to 3.52 μg/mL (median 1.42 μg/mL), were compared to the results obtained from corresponding DBS samples. Each concentration value was covered by the analytical method’s calibration range (0.1–20 μg/mL). Differences between the plasma and DBS results were less than 0.2 µg/mL. with underestimations of less than 6% in the DBS values. However, these were not therapeutically relevant.

Overall, the scientists found that in patients treated with Cresemba, DBS could be a viable matrix for TDM of isavuconazole. The plasma concentrations calculated from the DBS measurements closely corresponded with the directly measured plasma concentrations, potentially informing decision making in TDM. One precondition for this favorable result was that exact volumes of whole blood were spotted on the DVS cards, with complete blood spots being extracted in laboratories. However, this made minimally invasive blood sampling by finger pricking unsuitable. In the future, potential improvements can involve application of disposable metering devices able to spot exact volumes of whole blood onto the DBS cards, making DBS a preferable sampling method.

References

(1) Martens-Lobenhoffer, J.; Angermair, S.; Bode-Böger, S. M. Quantification of Isavuconazole from Dried Blood Spots: Applicability in Therapeutic Drug Monitoring. J. Chromatogr. B 2025, 1258, 124590. DOI: 10.1016/j.jchromb.2025.124590

(2) Isavuconazole (Cresemba). University of Nebraska Medical Center 2025. https://www.unmc.edu/intmed/divisions/id/asp/protected-antimicrobials/isavuconazole.html (accessed 2025-5-20)

(3) Dried Blood Spot Testing – Part 1. Capitainer 2023. https://capitainer.com/dried-blood-spot-testing-part-1/ (accessed 2025-5-20)