Kenneth Setchell (Presenter)
Cincinnati Children’s Hospital Medical Center
Bio: Kenneth D. Setchell, PhD, joined the tenured faculty of the Department of Pediatrics, University of Cincinnati College of Medicine in 1984, having moved from the Clinical Research Centre of the Medical Research Council, (UK) where he previously held a tenured scientific position. In 1973 he obtained a PhD in steroid biochemistry from the University of London. He was then awarded a fellowship from The Royal Society for post-doctoral studies at the Karolinska Institute in Stockholm, Sweden (1974-1975) in the application of mass spectrometry to clinical and biomedical problems specifically relating to the steroid hormone field. He returned to the United Kingdom to a scientific position in the Division of Clinical Chemistry at the Medical Research Council's Clinical Research Centre where he continued his research in the field of steroids, expanding into cholesterol and bile acid metabolism as it related to gastrointestinal diseases. His parallel research discoveries of the first mammalian lignans later led to the discovery of soy isoflavones in humans, their association with soy intake, and established his research in the area of bioactive plant constituents and human nutrition and disease.
Authorship: Kenneth D.R. Setchell* Junfang Zhao, Chandra Sharat, Parinda A. Mehta, Alexander A. Vinks
Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
| Short Abstract Paper Spray (PS) ionization is a new technique that generates gas phase ions directly from dried blood spots or other fluids without the need for sample pretreatment or chromatography. A simple and rapid PS-MS/MS method is described for measuring the chemotherapeutic drug melphalan in blood from patients undergoing HSCT. This novel methodology has excellent linearity over a large dynamic range, high precision and accuracy, and yielded comparable results to a conventional LC-MS/MS method. The clinical application of this methodology has permitted the determination of melphalan pharmacokinetics in real-time enabling personalized dose adjustments to be made to avoid the potential toxicity of this drug. |
Long Abstract
Introduction
Melphalan is a chemotherapeutic drug commonly used in hematopoietic stem cell transplantation (HSCT) for patients with a variety of hematological and immunological disorders. However, the toxicity of high dose melphalan can be profound, especially for infants and small children where the optimal therapeutic range has yet to be accurately established. Personalized drug dosing would allow administration of the optimal dose to ensure engraftment of transplanted cells, while minimizing the toxicity associated with this drug. Paper Spray (PS) ionization is a novel method that permits rapid quantitative analysis of pharmaceutical drugs by mass spectrometry directly from biological samples without the need for prior sample preparation or separation. In this study, we developed and validated a rapid PS-MS/MS method suitable for measuring melphalan in small samples of blood, and we compared this method with our conventional LC-ESI-MS/MS method. We evaluated the feasibility of this novel approach for real-time pharmacokinetics (PK) in order to select appropriate dosage regimen for pediatric patients undergoing HSCT.
Methods
Whole blood samples were obtained from patients receiving melphalan during HSCT at timed intervals post administration to determine each patient’s pharmacokinetic profile. From this profile the optimal dose for each patient was determined. The study was approved by the Cincinnati Children’s Hospital Medical Center Investigational Review Board. Melphalan was measured by Paper Spray ionization MS and compared with a validated electrospray ionization tandem MS method. All experiments were performed using an automated Paper Spray ion source (Prosolia, Inc. Indianapolis, IN) interfaced with a TSQ Quantum Ultra mass spectrometer (Thermo Scientific, San Jose, CA) operated in selected reaction monitoring (SRM) mode. XCalibur software was used to control the MS and to process data. Specific precursor/product ions were simultaneously monitored for melphalan and its stable isotopically-labelled internal standard (D8-melphalan) by collision-induced dissociation (CID). Blood samples were prepared by spiking known amounts of melphalan standards and the internal standard into drug free human blood to establish a calibration curve and quality control samples. A small amount of patient’s blood (12μL) equilibrated with D8-melphalan solution was deposited on the Paper Spray cartridge and dried. A small volume of solvent optimized to efficiently extract the drug was applied to the paper. A stepwise high voltage (3-5kV) was then applied inducing an electrospray ionization at the tip of the paper; the solvent evaporates from the droplets generating gas phase ions of the analyte, which then can be detected by a mass spectrometer. The analysis time for each sample was about 3 minutes with essentially no prior sample preparation.
Results and Discussion
This method has excellent limits of detection (25 ng/mL) for the measurement of melphalan and linearity of response over a wide dynamic range (50 – 50,000 ng/mL). The calculated inter-assay accuracies (% bias) ranged from -2.5 – 5.3% for all concentration with an overall inter-batch precision lower than 9.8% for the whole blood samples used as Quality Controls. Similar values were obtained for the intra-assay accuracy (<8%) and precision (0.4 – 1.1%) for all QC samples. PS-MS/MS measurement of blood melphalan concentrations showed an excellent correlation (R2=0.959, n=62) with our conventional LC-MS/MS methods, but the time for analysis was significantly shorter with PS-MS/MS approach. Non-compartment pharmacokinetic analysis was conducted using WinNonlin version 6.4 (Pharsight, Certara L.P) for 5 young children patients with age of 1.5 -16.8 years both for the test dose (10% of full dose) and full dose. Trapezoidal area under the curves (AUCs) were calculated and the results showed a high linear correlations between the PS-MS/MS and LC-MS/MS methods (R2=0.981). The calculated mean AUCs in patients given full doses was 293±89 min* ug/mL by PS method and was 256±63 min* ug/mL by LC method (n=5), while the calculated AUCs in patients given test doses were 24.4±13.9 min*ug/mL by PS method and 25.9±12.6 min*ug/mL by LC method (n=5).
Conclusion
Our results show the successful clinical application of this novel, simple and rapid PS-MS/MS method for measuring melphalan PK in whole blood especially from patients undergoing HSCT where accurate optimization of drug dosing is important to avoid the toxic effect of this therapeutic drug. This assay enables the clinician to develop a personalized dosing strategy for Melphalan. This pharmacokinetically guided individualized dosing strategy will help to improve overall HSCT outcomes for these patients by balancing toxicity and efficacy of melphalan.
References & Acknowledgements:
Acknowledgement
We thank Thermo Scientific for the mass spectrometry support, and Prosolia, Inc. for providing the paper spray automated ion source and paper spray cartridges.
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