Purpose: Melphalan is a biofunctional alkylating chemotherapeutic used in the treatment of numerous cancers (e.g. myeloma, ovarian carcinoma, and breast cancer). Melphalan’s inherent instability drives spontaneous hydrolysis at room temperature. Therefore, development of a robust method that can rapidly analyze samples is needed to produce reliable results. In this study, we developed an extraction and LC-MS/MS method to quantitate both total and free melphalan to support clinical analysis.
Methods: In order to assess free melphalan, plasma ultrafiltrate was prepared using 10kD centrifugation tubes. Melphalan and the added internal standard were extracted from human plasma (total melphalan) and human plasma ultrafiltrate (free melphalan), using a protein precipitation extraction method with cold methanol. The LC-MS/MS analysis was carried out on a Sciex API-4000 mass spectrometer coupled with a Shimadzu LC-20 HPLC System. The chromatographic separation was achieved on a reversed phase column (Venusil XPB C8 (L), 5µ, 50 x 2.1 mm). The mass spectrometer was operated in positive electrospray ionization mode. The MRM transitions were monitored a m/z 305.2 - > 246.2 for melphalan and 313.1 - > 254.2 for melphalan-d8 (IS).
Results: Excellent linearity using a 1/concentration2 weighting was obtained for both total melphalan (10.0 - 15,000 ng/mL) and free melphalan (1.00 - 1,500 ng/mL). The correlation coefficients for total and free melphalan were ≥ 0.9960. The inter-assay precision, as measured by covariance (CV), ranged from 4.1% to 13.2% for total melphalan and 1.9% to 7.4% for free melphalan. The inter-assay accuracy, as measured by relative error (RE), ranged from -8.2% to -1.7% for total melphalan and -5.0% to 2.0% for free melphalan. The 3.5 minute LC-MS/MS method allowed for up to 97 injections for total melphalan (CV 2.0% to 6.1%, RE -7.3% to -2.5%). Free melphalan was validated for up to 112 injections (CV 1.5% to 3.1%, RE -2.7% to -2.2%). Stability during the extraction process was established for 6 hours. Stability during analysis was established for 27 hours. The validated method translated well to clinical analysis which was confirmed by 100% of tested samples meeting the incurred sample reanalysis acceptance criteria.
Conclusion: A fast extraction method for both total and free melphalan from human plasma and human plasma ultrafiltrate, respectively, as well as a quick LC-MS/MS method was demonstrated using a Sciex API‑4000. This method allows for rapid translation of study samples into data to support clinical analysis of this versatile chemotherapeutic.