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Simultaneous Analysis of Antifungals in
Plasma by SPE with Agilent Bond Elut
Plexa and an Agilent 1260 Infinity
LC/MS/MS

Abstract
Multicompound analysis of six systemic antifungal drugs in human plasma is effectively achieved using an Agilent LC/MS/MS system composed of an Agilent Bond Elut Plexa polymeric sorbent, an Agilent Pursuit XRsUltra 2.8 Diphenyl column, and an Agilent 1260 Infinity LC Triple Quadrupole LC/MS. Agilent Bond Elut Plexa delivered extremely clean samples, which was demonstrated by detection down to pg levels. All compounds showed near 100% recovery and a single digit % RSD.
Superb linearity was achieved in the calibration curves with R2 ¡ 0.995 for allcompounds.
Introduction
Systemic antifungal medications are designed to treat fungal infections in thehuman body. Many single-compound analyses have been reported; however,multicompound analyses with low detection limits have limited availability and havenot yet been established [1-4]. The Agilent high quality, mono-disperse polymericsolid phase extraction (SPE) plate, Bond Elut Plexa, delivers clean samples toachieve extremely low-picogram (pg) detection limits of multi-antifungal drugs inhuman plasma. This application note presents good recovery, linearity, and precisiondata on antifungal drugs in human plasma.
Figure 1 shows examples of systemic antifungal drugs.
Figure 1. Structures of some systemic antifungal drugs.
Materials and Methods
LC/MS conditions
Column:
SPE reagents and solutions
Agilent Bond Elut Plexa 96-well plate (10 mg) Agilent 1260 Infinity Triple Quadrupole LC/MS 100 μL human plasma spiked with antifungals* SPE method
400 μL diluted sample from pretreatment ESI+ with Agilent Jet Stream Technology (JST) Evaporate under gentle air stream at room temperature and reconstitute in 100 μL 30% MeOH *Difenoconazole, internal standard (IS), was spiked at 50 ng/mL in all Calibration samples were prepared from 0.05 to 100 ng/mL in human plasma at 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 ng/mL.
For itraconazole, linezolid, and betertanol, a concentration range of 0.5 to 100 ng/mL was used due to detection limits.
MS/MS Collision
Antifungal
log P transition
Fragmentor
Results and Discussion
Figure 2 shows all compounds were baseline-separatedchromatographically with good retention using the AgilentPursuit XRsUltra 2.8 Diphenyl column. Excellent detection limitswere achieved for most compounds with the Agilent 1260Infinity Triple Quadrupole LC/MS, down to the 0.05 ng/mLlevel in human plasma.
Figure 2. MS chromatogram for antifungal drugs at 10 ng/mL spiked in human plasma (IS was spiked at 50 ng/mL).
All compounds showed great linearity with R2 ¡ 0.995 overthe entire calibration range (Figure 3).
Fluconazole - 8 Levels, 8 Levels Used, 8 Points, 8 Points Used, 0 QCs Linezolid - 6 Levels, 6 Levels Used, 6 Points, 6 Points Used, 0 QCs -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 Ketoconazole - 8 Levels, 8 Levels Used, 8 Points, 8 Points Used, 0 QCs Voriconazole - 8 Levels, 8 Levels Used, 8 Points, 8 Points Used, 0 QCs -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 Bitertanol - 6 Levels, 6 Levels Used, 6 Points, 6 Points Used, 0 QCs Itraconazole - 6 Levels, 6 Levels Used, 6 Points, 6 Points Used, 0 QCs -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 Figure 3. Calibration curves for antifungals in human plasma from 0.05 to 100 ng/mL (itraconazole, linezolid, and bitertanol use a0.5 to 100 ng/mL range due to LOD).
A recovery experiment was performed with n = 6 samples forfour different concentration levels: 1, 5, 50, and 100 ng/mLspiked in human plasma. Most of the compounds showedexcellent recovery and % RSD values for all concentrationlevels.
100 ng/mL
Correlation
(ng/mL) Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%) coefficient R2
Conclusion
For More Information
A reproducible and accurate analytical method was developed These data represent typical results. For more information on for several antifungal drugs in human plasma. Agilent Bond Elut our products and services, visit our Web site at Plexa delivered clean samples, which was demonstrated by the detection of pg levels of antifungals. All compounds showedclose to 100% recovery and a single digit % RSD with n = 6samples. Superb linearity was achieved in the calibration curveswith R2 ¡ 0.995 for all compounds.
References
K. Y. Beste, O. Burkhardt, and V. Kaever. (2011) “RapidHPLC-MS/MS method for simultaneous quantitation of fourroutinely administered triazole antifungals in humanplasma”. Clinica Chimica Acta, 413, 240 – 245.
R. J. Mitchell, R. Christian, H. Hughes, A. Miah, and D. K.
Walker. (2010) “The application of fully automated on-linesolid phase extraction in bioanalysis”. Journal ofPharmaceutical and Biomedical Analyisis, 52, 86 – 92.
M-C. Verdier, D. Bentué-Ferrer, O. Tribut, and E. Bellissant.
(2010) “Liquid chromatography-tandem mass spectrometry method for simultaneous quantification of four triazoleantifungal agents in human plasma”. Clinical Chemistry and Agilent shall not be liable for errors contained herein or for incidental or consequentialdamages in connection with the furnishing, performance, or use of this material.
Laboratory Medicine, 48 (10), 1515 – 1522.
Information, descriptions, and specifications in this publication are subject to change B. V. Araujo, D. J. Conrado, E. C. Palma, and T. Dalla Costa.
(2007) “Validation of rapid and simple LC-MS/MS methodfor determination of voriconazole in rat plasma”. Journal of Agilent Technologies, Inc., 2013Printed in the USA Pharmaceutical and Biomedical Analysis, 44, 985 – 990.

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