Junfang Zhao (Presenter)
Cincinnati Children’s Hospital Medical Center
Authorship: Junfang Zhao1, Nicholas Edward Manicke2, Chandra Sharat1, Parinda A. Mehta1, Alexander A. Vinks1, Kenneth D.R. Setchell1
1 Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. 2 Indiana University-Purdue University Indiana
PaperSpray (PS) is an ionization technique that generates gas phase ions directly from a dried blood spot or other fluids without the need for sample pretreatment and chromatography. We describe a simple and rapid PS-MS/MS method for real-time measurement of the chemotherapeutic drug melphalan in blood from patients undergoing hematopoietic stem-cell transplantation. Our aim was to determine the optimum target range for melphalan in the pediatric population. PS-MS/MS had excellent linearity of response over large dynamic range, high precision and accuracy. In a pharmacokinetic study of melphalan PS-MS/MS correlated well with ESI-LC-MS/MS but had the advantage of speed of analysis.
Melphalan is a chemotherapy 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 is profound, especially for infants and small children and the optimal therapeutic range has yet to be accurately established. Personalized drug dosing would allow administration of the optimal chemotherapy dose to ensure engraftment of transplanted cells, while minimizing the toxicities. PaperSpray is a novel ionization method that allows rapid quantitative analysis of pharmaceutical drugs by mass spectrometry directly from biological samples without the need for prior sample preparation or separation. We report, our experience with the feasibility of this novel approach for determining melphalan pharmacokinetics (PK) in pediatric patients undergoing HSCT.
Blood samples from patients receiving melphalan for their HSCT were measured for PK by the novel PaperSpray technique and compared with a validated electrospray ionization tandem MS method. All experiments were performed using an automated PaperSpray ion source (Prosolia, Inc. Indianapolis, IN) interfaced with a TSQ Quantum Ultra mass spectrometer (Thermo Scientific, San Jose, CA). Data collection was executed in selected reaction monitoring (SRM) mode, observing the desired fragment intensities for the melphalan and it’s internal standard ([2H8]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. A small amount of patient’s blood (12ìL) was first deposited on Paper Spray cartridge and after the blood spot was dried under a stream of nitrogen gas, a small volume (ca. 80ìL) of solvent, selected to efficiently extract the drug, was applied to the paper. A stepwise high voltage (3-5kV) was applied to the paper, inducing an electrospray at the sharp tip of the paper; the solvent evaporates from the droplets generating gas phase ions of the analyte molecules. The entire analysis for each sample was about only 1 min with essentially no prior sample preparations other than addition of internal standard to the blood sample.
Results and Discussion
This method had excellent limits of detection (25 ng/mL) 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 concentrations 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=0.95) with our conventional LC-MS/MS methods, but the time for analysis was significantly shorter with PS-MS/MS approach due to the elimination of sample preparation steps. The pharmacokinetic parameters, AUC(221 ± 78 ìg/mL*min by PS and 197 ± 62 ìg/mL*min by ESI ), t1/2 (42 ± 9 min by PS and 47 ± 3 min by ESI),and Cmax (4701 ± 1575 ng/mL by PS and 4353 ± 1781 ng/mL) were estimated using a nocompartmental pharmacokinetic model and our preliminary melphalan pharmacokinetic studies for 4 young children patients showed the good linear correlations between the PS and LC methods.
We describe for the first time a novel, simple and rapid PS-MS/MS method for measuring melphalan PK in whole blood from patients undergoing HSCT. This methodology was found to be simple, fast and ideally suited for obtaining melphalan levels in small samples of blood directly from pediatric patients with reporting of values immediately back to the physician to allow for real-time dose adjustment. Validation of this assay will enable the clinicians to develop a personalized dosing strategy for Melphalan. This pharmacokinetically guided individualized dosing strategy will be tested in our next study and improve overall HSCT outcomes for these patients by balancing toxicity and efficacy of Melphalan.
References & Acknowledgements:
IP Royalty: no
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