Yi Xiao (Presenter)
Children's Hospital Los Angeles
Bio: I am working at the Biomedical Genetics and Special Chemistry laboratory at Children's Hospital Los Angeles to develop and validate mass spectrometry based approaches for daily clinical pathological and diagnostic tests. I also work with research faculties to provide mass spec expertise for their research projects. I obtained my Ph.D. in biochemistry from Vanderbilt University. My research project was focused on functional studies of cytochrome P450 enzymes by LC-MS based metabolomics approaches. During my Ph.D. study, I spent 6 months in an internship at the Drug Metabolism and PharmacoKinetics (DMPK) department at Sanofi and investigated the metabolism pathway of several drug candidates.
Authorship: Yi Xiao (1), Nemesio Duran (1), Yan-Kang Xu (1), Paul Pattengale (1, 2), Xiaowei Fu (1, 2)
(1) Department of Pathology and Laboratory Medicine, Children’s Hospital of Los Angeles (2) Department of Pathology, Keck School of Medicine, University of Southern California
| Short Abstract A simple and fast sample extraction and HPLC-MS/MS method was developed for simultaneous quantitation of voriconazole, voriconazole-N-oxide, posaconazole, fluconazole, itraconazole, and hydroxyitraconazole in human serum for therapeutic drug monitoring of antifungal treatments. Sample preparation involves only protein precipitation and dilution; LC-MS/MS method takes only three minutes. The inter-day CVs ranged from 4% to 6%, and the intra-day CVs ranged from 2% to 4%. The recoveries were between 95% and 103% for three concentrations tested. This method is accurate and robust, and offers a cost-effective tool for dosing adjustment for single-drug antifungal regimens and combination salvage therapies on a daily basis. |
Long Abstract
Introduction:
Antifungal agents are commonly used treatments for life-threatening fungal infections (e.g. Aspergillus and Candida). Their narrow therapeutic indexes require therapeutic drug monitoring to achieve efficacy and safety at the same time. We have developed a simple sample extraction and fast high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to simultaneously quantify four antifungal agents (voriconazole, posaconazole, fluconazole, and itraconazole) and two metabolites (voriconazole-N-oxide and hydroxyitraconazole) in human serum. The sample preparation method involves only protein precipitation and dilution, and the isocratic HPLC method takes only three minutes. The method is accurate, simple, fast, and cost-effective, and can be either implemented at regular clinical laboratory settings at hospitals or performed with high throughput at major reference laboratories.
Method:
Sample preparation:
100 µl of serum sample was spiked with 200 µl of mobile phase B (see below) and a 100 µl of a acetonitrile stock of six isotope-labeled internal standards. Protein was removed by centrifugation, and supernatant was diluted four times with a stock of methanol:mobile A (see below) = 50:50 before HPLC-MS/MS analysis.
HPLC-MS/MS:
HPLC-MS/MS analysis was performed on a Prominence Shimadzu HPLC system connected to a 4000 Qtrap (AB SCIEX) mass spectrometer with electrospray and positive ionization mode. Separation of the antifungal agents/metabolites was achieved on a Luna C8(2), 3u, 50 x 2mm HPLC column (Phenomenex) with a three-minute isocratic HPLC method (mobile phase A:B = 50:50; mobile phase A consisted of 0.1% formic acid and 10 mM ammonium formate in water; mobile phase B consisted of 0.1% formic acid in acetonitrile; flow rate was 0.7 ml min-1). 10 µl of sample was introduced onto the column by a CTC HTC PAL auto-sampler (Leap Technologies). Quantitation was performed with multiple reaction monitoring (MRM) mode with optimized MS/MS parameters to achieve ultimate sensitivity for each analyte. The collision energy for fluconazole was manually adjusted to decrease sensitivity so that higher concentration of fluconazole can be covered. Calibration curves were constructed with the corresponding deuterated internal standards with a weighting factor of 1/X.
Method validation:
Pooled human serum free of these antifungal agents/metabolites was used for method validation. Pooled human serum samples spiked with different concentrations of antifungal agents/metabolites (n = 3 per concentration) were processed as described above and injected in a random order to evaluate linearity (by EP evaluator) and carryover effect. Calibration curves were designed to cover reference values for each antifungal agents. Recovery was evaluated by comparing antifungal agents/metabolites-containing serum with neat standard stocks at three different concentrations (n = 3 per concentration). Precision was evaluated with two quality control samples from UTAK. Accuracy was evaluated with the quality control samples from UTAK, as well as by comparing results from reference laboratories.
Results:
The assay is linear from 0.01 – 10 mcg/ml for voriconazole and voriconazole-N-oxide; 0.1 – 20 mcg/ml for posaconazole; 0.2 – 200 mcg/ml for fluconazole; 0.1 – 20 mcg/ml for itraconazole; and 0.05 – 10 mcg/ml for hydroxyitraconazole. The inter-day coefficient of variations (CVs) ranged from 4.0% to 5.5% (n=18), and the intra-day coefficient of variations (CVs) ranged from 1.9% to 3.8% (n = 20). The recoveries were between 95% and 103% for three concentrations tested. Preliminary data suggests the assay is very accurate. No carryover effect was observed.
Conclusion:
Many clinical and reference laboratories use different sample extraction and detection methods for the quantitation of individual antifungal agents. By harmonizing the extraction procedures and quantitating multiple analytes in a single analytical run, our method offers the benefit of cutting labor and costs substantially. In contrast to the seven-minute ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method published by Dr. Oscar Marchetti [1], our three-minute HPLC-MS/MS method cuts the analytical time by more than half and only requires standard high-performance liquid chromatography instrument commonly seen in clinical laboratories. Our simple and fast sample workup/analysis method makes it possible for dosing adjustment within short intervals on a daily basis. The minimal serum volume required for the assay is only 100 µl, which makes the method particularly suitable for neonates and infants.
In summary, we have developed an accurate, simple, fast, and cost-effective method to simultaneously quantify four antifungal agents (voriconazole, posaconazole, fluconazole, and itraconazole) and two metabolites (voriconazole-N-oxide and hydroxyitraconazole) in human serum. This method will be used for routine therapeutic drug monitoring aimed at maximizing the real-time efficacy and safety of single-drug antifungal regimens and combination salvage therapies.
References & Acknowledgements:
[1] Decosterd LA et al. Antimicrob Agents Chemother. 2010.
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