Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: retinoblastoma • clinical laboratory testing • oncology