Adam McShane (Presenter)
Cleveland Clinic
Bio: Dr. Adam J. McShane’s commitment to patient health commenced early in his career when he obtained emergency medical technician certification as a senior in high school, and 3 years later achieved paramedic certification. In 2009, he obtained a BS in chemistry from the University of Pittsburgh. Adam continued his studies by enrolling in the graduate chemistry program at the University of Connecticut, where he obtained a PhD in biological chemistry. His doctoral research focused on novel chemical tools and methods for quantitative proteomics. Adam is currently the clinical biochemistry fellow at the Cleveland Clinic, where his service partially involves the development and validation of clinical mass spectrometry methods.
Authorship: Adam J. McShane and Sihe Wang
Department of Laboratory Medicine, Cleveland Clinic, Cleveland, OH
| Short Abstract Invasive fungal infections are deadly and prevalent in certain high-risk patient populations: patients with hematological malignancies, the immunosuppressed, and the critically ill. Therapeutic drug monitoring of azole antifungal medications may potentially decrease morbidity and mortality in patients undergoing azole treatment. Therefore, a liquid chromatography tandem mass spectrometry method was developed and validated for 6 analytes: fluconazole, voriconazole, posaconazole, isavuconazole, itraconazole, and its active metabolite hydroxyitraconazole. Analytically, simple sample preparation is required, and the injection-to-injection window is less than 2 minutes. The implementation of this method will be a great asset to our infectious disease service. |
Long Abstract
Introduction:
Invasive fungal infections (IFI) are deadly and prevalent in certain high-risk patient populations: patients with hematological malignancies, the immunosuppressed, and the critically ill (1-3). Successful azole antifungal medication therapy, prophylactically and empirically, can be life-saving for these patients. Therapeutic drug monitoring (TDM) of the azole medications may potentially decrease morbidity and mortality in patients undergoing azole treatment (4-8). However, for TDM-based programs to be successful, result turn-around times must be short enough to meet the desired dosing interval. The aim of this work was to develop a fast and accurate assay for the quantification of selected azoles that are routinely administered at our hospital. There are 3 major analytical methodologies for azole quantification: bioassays, liquid chromatography (LC), and LC coupled to mass spectrometry (MS) (9). Due to its speed, analyte specificity and sensitivity, analytical measuring range (AMR), and ability to multiplex, LC-MS has emerged as the current platform of choice (10-15). Therefore, an LC-MS method was developed and validated for 6 analytes: fluconazole, voriconazole, posaconazole, isavuconazole, itraconazole, and its active metabolite hydroxyitraconazole. This represents the first known, clinical-use method to include all 6 of these analytes into a single LC-MS assay. Analytically, only simple preparation is required, and the injection-to-injection window is less than 2 minutes.
Methods:
Materials
Burdick and Jackson LC-MS grade acetonitrile and methanol was purchased from Fisher. Ultrapure water was prepared with a Milli-Q water purification system. Formic acid was purchased from Fluka and hydrochloric acid from Fisher. The charcoal-stripped serum was purchased from Seracare. Voriconazole, voriconazole-D3, fluconazole, fluconazole-13C3, posaconazole, posaconazole-D4, itraconazole, itraconazole-D4, and hydroxyitraconazole-D4 were purchased from Cerilliant. Isavuconazole and hydroxyitraconazole were purchased from Toronto Research Chemicals. Isavuconazole-D4 was purchased from Medical Isotopes.
Sample preparation
During preparation, 50 µL of sample (serum or lithium heparin plasma) was precipitated with an acidified acetonitrile solution [0.1% (v/v) 1M HCl] containing the internal standards for all 6 analytes (1 µg/mL). After 60 seconds of vortexing, the sample was centrifuged at 13,000 g for 10 minutes. A 50 µL aliquot was then diluted with 200 µL 0.1% (v/v) formic acid in ultrapure water. Calibrators were prepared in charcoal-stripped serum at the following concentrations: 0.2, 0.3, 0.6, 1.3, 2.5, 5.0, and 10.0 µg/mL for voriconazole, posaconazole, itraconazole, hydroxyitraconazole, and isavuconazole. The fluconazole calibrators were prepared at 0.5, 0.9, 1.9, 3.8, 7.5, 15.0, and 30.0 µg/mL in charcoal-stripped serum.
LC-MS/MS method
Utilizing a Transcend LC system (Thermo Scientific), 20 µL of the sample was injected onto a reversed-phase column (Accucore RP-MS, 50 x 2.1 mm, 2.6 µm). Solvent A was comprised of 0.1% (v/v) formic acid in ultrapure water and solvent B was 0.1% (v/v) formic acid in acetonitrile. At a flow rate of 0.8 mL/min, the gradient was ramped from 40% B to 60% B over 20 seconds, held at 60% B for 30 seconds, and finally equilibrated back to 40% B for 60 seconds. A Thermo Scientific TSQ Vantage triple quadrupole mass spectrometer, operating in multiple reaction monitoring (MRM) mode, was utilized. The source was positive heated electrospray ionization (HESI) with a spray voltage of 4000 V, a vaporizing temperature of 350 oC, a sheath gas of 50, an auxiliary gas of 10, and a capillary temperature of 200 oC. Two mass-to-charge (m/z) transitions were monitored for each analyte (quantifier and qualifier): fluconazole 307.2->238.1 and 220.1, voriconazole 350.2->127.0 and 281.1, posaconazole 701.5->683.4 and 614.3, hydroxyitraconazole 721.4->408.2 and 430.2, itraconazole 705.4->392.2 and 432.3, and isavuconazole 438.3->224.0 and 127.0. One transition was monitored for each corresponding internal standard: fluconazole-13C3 310.2->223.1, voriconazole-D3 353.1->127.0, posaconazole-D4 705.6->687.5, hydroxyitraconazole-D4 725.5->412.2, itraconazole-D4 709.5->396.2, and isavuconazole-D4 442.2->224.0.
Method validation
To validate the method, the following experiments were performed: ion suppression, mixing study, interference, AMR, carryover, stability, precision, and sample comparisons. The use of leftover patient samples was approved by the Cleveland Clinic Institutional Review Board.
Results:
Ion suppression
All 6 analytes, with their corresponding internal standards, were evaluated for matrix effects. The analytes were infused post-column, while extracted serum samples (3 male and 3 female) were injected onto the column. Only fluconazole exhibited signal suppression versus a blank sample.
Mixing study
A mixing study was performed to verify that the analytes behaved similarly to their internal standards (i.e. fluconazole and its internal standard did not exhibit differential ion suppression). Pooled-patient serum and charcoal-stripped serum were first mixed to evaluate charcoal-stripped serum’s suitability as a calibrator matrix. The mean percent difference for the analytes in these 2 matrices ranged from -5.2% to 4.3%. A mixing study was also performed between pooled-patient serum and pooled-patient lithium heparin plasma, to expand the acceptable sample type. The mean percent difference ranged from -7.5% to 5.5%.
Interferences
The method was found to be free (percent difference <20%) from endogenous (hemolysis, icterus, lipemia, and uremia) and exogenous (>100 therapeutic drugs and common analytes) interferences.
AMR
The AMR for voriconazole, posaconazole, itraconazole, hydroxyitraconazole, and isavuconazole was established by triplicate analysis at 7 levels in spiked, pooled-patient serum: 0.2, 0.3, 0.6, 1.3, 2.5, 5.0, and 10.0 µg/mL. Fluconazole’s AMR was evaluated for higher levels to be consistent with therapeutic target ranges: 0.5, 0.9, 1.9, 3.8, 7.5, 15.0, and 30.0 µg/mL. The analytical recovery for all of the analytes ranged from 91.6% to 121.3%. The triplicate coefficient of variation (CV) was less than 14.3% for all analytes at all levels.
Carryover
Pooled, patient specimen spiked at 3 times the upper AMR level was injected prior to a low level sample. No carryover was found (percent difference <1.8%) for all analytes.
Stability
Pooled-patient samples spiked at 3 concentrations were evaluated for analyte stability. The unextracted samples were found to be stable (percent difference <8%) for 15 days at room temperature and 4 oC. At -20 oC, all analytes were stable for 60 days (percent difference <8%). The extracted stability at 4 oC (percent difference <8%) was 7 days.
Precision
Utilizing the Clinical & Laboratory Standards Institute (CLSI) EP10-A3 guideline each analyte's precision was determined at 3 levels in spiked, pooled patient serum. For fluconazole, the levels were 1.9, 8.8, and 15.2 µg/mL. For the other analytes, the levels were approximately 0.7, 3.0, and 5.0 µg/mL. The intraday CVs ranged from 1.5% to 3.4%. The total CVs ranged from 1.8% to 3.6%.
Method comparison
A reference laboratory, utilizing similar methodology, was used as a comparator for itraconazole, hydroxyitraconazole, voriconazole, fluconazole, and posaconazole. For isavuconazole, the University of Texas Health Science Center at San Antonio was the comparator. Approximately 40 patient samples (spiked and endogenous) were compared for each analyte. The results were then compared via Deming regression. The correlation coefficients (R) ranged from 0.9658 to 0.9981. The range for the slopes and intercepts were 0.947 to 1.105 and -0.296 to 0.127, respectively.
Conclusion:
An LC-MS/MS method was developed and validated to support TDM of patients undergoing azole antifungal medication treatment. The method only requires simple sample preparation and a sub 2 minute analysis time. During the validation, no interferences from potential endogenous and exogenous sources were found. All analytes displayed a correlation coefficient above 0.96 with comparator laboratories. The highest imprecision found displayed a CV of 3.6%. No carryover was found at 3 times the upper limit of quantitation. The unextracted stability was 15 days at room temperature and 4 oC. This laboratory-friendly assay will be a great asset to our infectious disease service.
References & Acknowledgements:
References:
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2. Eggimann P, Garbino J, Pittet D. Epidemiology of candida species infections in critically ill non-immunosuppressed patients. The Lancet Infectious diseases 2003;3:685-702.
3. Ruping MJ, Vehreschild JJ, Cornely OA. Patients at high risk of invasive fungal infections: When and how to treat. Drugs 2008;68:1941-62.
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9. Laverdiere M, Bow EJ, Rotstein C, Autmizguine J, Broady R, Garber G, et al. Therapeutic drug monitoring for triazoles: A needs assessment review and recommendations from a canadian perspective. The Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale 2014;25:327-43.
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11. Farowski F, Cornely OA, Vehreschild JJ, Hartmann P, Bauer T, Steinbach A, et al. Quantitation of azoles and echinocandins in compartments of peripheral blood by liquid chromatography-tandem mass spectrometry. Antimicrobial agents and chemotherapy 2010;54:1815-9.
12. Jourdil JF, Tonini J, Stanke-Labesque F. Simultaneous quantitation of azole antifungals, antibiotics, imatinib, and raltegravir in human plasma by two-dimensional high-performance liquid chromatography-tandem mass spectrometry. Journal of chromatography B, Analytical technologies in the biomedical and life sciences 2013;919-920:1-9.
13. Mak J, Sujishi KK, French D. Development and validation of a liquid chromatography-tandem mass spectrometry (lc-ms/ms) assay to quantify serum voriconazole. Journal of chromatography B, Analytical technologies in the biomedical and life sciences 2015;986-987:94-9.
14. Reddy TM, Tama CI, Hayes RN. A dried blood spots technique based lc-ms/ms method for the analysis of posaconazole in human whole blood samples. Journal of chromatography B, Analytical technologies in the biomedical and life sciences 2011;879:3626-38.
15. Xiong X, Zhai S, Duan J. Validation of a fast and reliable liquid chromatography-tandem mass spectrometry (lc-ms/ms) with atmospheric pressure chemical ionization method for simultaneous quantitation of voriconazole, itraconazole and its active metabolite hydroxyitraconazole in human plasma. Clinical chemistry and laboratory medicine 2013;51:339-46.
Acknowledgements:
We are grateful to Dr. Nathan Wiederhold at the University of Texas Health Science Center at San Antonio for participating in the sample exchange.
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