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Jonathan W. Kim, Ludmila Alexandrova, Emilia DeMarchis, Diana Lee, Allis Chien; Lc-ms/ms Quantification Of Melphalan Plasma Levels In Children Undergoing Selective Intra-arterial Infusion Of Chemotherapy For Retinoblastoma. Invest. Ophthalmol. Vis. Sci. 2012;53(14):468.
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Melphalan is an alkylating agent with effective tumoricidal properties but also severe systemic side effects. A recent clinical trial has demonstrated promising results in retinoblastoma patients when melphalan is infused selectively into the ophthalmic artery. A minority of subjects developed neutropenia, which suggests that systemic diffusion of the drug does occur. Developing a reliable systemic assay to determine plasma levels of melphalan following ophthalmic artery infusion is critical in optimizing the benefits of this treatment.
We utilized high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the melphalan levels in human plasma. Melphalan and the internal standard (N-phenyldiethanolamine (N-PEA)) were purchased from Sigma (Steinhein, Germany) and drug-free normal human plasma was obtained from a regional blood bank. Stock solutions of Melphalan 2 mg/mL solution in dimethyl sulfoxide (DMSO) and the internal standard (IS) 1 mg/mL in ethanol were both stored at -80°C. Two concentration ranges (range 1: 2 - 400 ng/mL and range 2: 20 - 4000 ng/mL) were tested in order to develop a calibration curve. The extraction procedure for the concentration ranges 1 and 2 utilized 100 uL and 50 uL plasma respectively. Blank human plasma was spiked with appropriate calibration solutions of melphalan and internal standard. A methanol/acetonitrile protein precipitation was performed and the samples were analyzed with an LC-MS/MS assay.
Mass chromatograms for the range 1 provided eight calibration points: 2ng/mL; 4; 10; 20; 50; 100; 200; 400 ng/mL. A calculated ratio of the peak intensities for both melphalan and the internal standard were then used to generate a calibration curve. Similarly, mass chromatograms for range 2 generated eight calibration points: 20 ng/mL; 40; 80; 200; 400; 1000; 2000; 4000 ng/mL. A calculated ratio of the peak intensities for both melphalan and the internal standard were then used to generate a second calibration curve (figure 2). Weighting of 1/x 2 was used to fit the data to a linear least-squared regression curve, where x represents concentration (ng/mL). The linear detection response was defined for concentrations within the range of 2 to 400 ng/mL and within the range of 20 to 4000 ng/mL.
A LC-MS/MS method for determination of melphalan levels in human plasma has been developed, and calibration curves for range 1 and range 2 were generated by using a 100µL plasma aliquot and a 50µL plasma aliquot, respectively. Ongoing aspects of the project include assay validation to demonstrate accuracy, reproducibility and stability of melphalan in human plasma.
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