Paper spray high-resolution accurate mass spectrometry for quantitation of voriconazole in equine tears
Paper spray high-resolution accurate mass spectrometry is a fast and versatile analysis method. This ambient ionization technique enables the quantitation of xenobiotics in complex biological matrices without chromatography or conventional sample extraction. The simplicity, rapidity, and affordability of the paper spray mass spectrometry (PS-MS) method make the technique especially attractive for clinical investigations where fast and affordable sample analysis is crucial. A new PS-MS method for the quantitation of voriconazole in equine tears was developed and validated. For a concentration range of 10 to 1000 ng/mL, good linearity (R2 > 0.99), inter- and intra-run precision (coefficient of variation (CV) max. 11.9%), accuracy (bias of the nominal concentration ± 13.9%), and selectivity (signal areas of the double blanks represent 0.13 ± 0.05% of the lower limit of quantitation (LLOQ) signal in equine tears) were observed. The quantitation of voriconazole was based on three product ions and calculated relative to the isotope-labeled internal standard, voriconazole-d3, which had a final concentration of 250 ng/mL in the standards and samples. The matrix effect of the method showed an ionization suppression by reduction of the voriconazole response to 63.6%, 70.2%, and 81.9% for 30 ng/mL, 450 ng/mL, and 900 ng/mL in equine tears compared with voriconazole in solvent (methanol:water, 50:50, v:v). The method was used to analyze 126 study samples collected for a pharmacokinetic study investigating a novel approach for treatment of fungal keratitis in horses. Therefore, the integrity of the sample dilution (n = 6, CV 6.90%, and bias of nominal concentration + 8.40%) and the carryover effect (increase from 0.33 ± 0.21% to 1.33 ± 0.89% of the signal of the LLOQ) was further investigated. To our knowledge, this method is the first application of PS-MS for quantitation of drug concentrations in tears from any species.
KeywordsPaper spray high-resolution accurate mass spectrometry Equine tears Antifungal drug Voriconazole Quantitation
The authors are grateful to Prosolia, Inc., especially Donna Hollinshead, for providing the Velox 360 paper spray system, the Velox Sample Cartridges, as well as technical support. Lastly, thanks to Eva Eckstein for help with the illustration.
This project was financed with the Faculty Start-up Funds of Prof. Dr. Borts provided by the Department of Veterinary Diagnostic and Production Animal Medicine at Iowa State University (ISU), the ISU College of Veterinary Medicine, and the ISU Office of the Vice President for Research.
Compliance with ethical standards
Ethical conduct of research
The use of horses and all procedures in this study were approved by the Institutional Animal Care and Use Committee at Iowa State University (protocol no. 12-16-8403-E). Horses were obtained by client donation due to severe and/or chronic lameness and following completion of our study were humanely euthanized for reasons unrelated to ophthalmic research. Horses were housed individually in stalls and provided free choice hay and water. Ophthalmic examinations were performed, and all the eyes of all horses were deemed healthy with no confounding ocular conditions.
All equine horse owners donated their horses to the Iowa State University College of Veterinary Medicine Lloyd Veterinary Medical Center. They did this by signing a form that officially transferred ownership of the horse and provided informed consent for the horses to be used for approved teaching and research procedures, both before and after euthanasia.
Conflict of interest
The authors declare that they have no conflict of interest.
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