Assays for the quantification of melphalan and its hydrolysis products in human plasma by liquid chromatography-tandem mass spectrometry.

Two high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assays are described for the quantification of melphalan in human plasma. N-phenyldiethanolamine was tested as internal standard. The first assay consisted of a protein precipitation by cold methanol and a reversed-phase HPLC whereas the second one was based on a solid phase extraction and a hydrophilic interaction chromatography. Both provided a very satisfactory mean extraction yield with a small volume of sample. The first method was simple, rapid and used as a routine assay. The second one was developed in order to determine melphalan hydrolysis products and to avoid scarce cases when interferences from biological matrix alter the quantification of melphalan using the first method. The two assays were linear and sensitive in the range of 1-500ng/mL for the first one and in a range of 25-2000ng/mL for the second one. Concentrations out of the range fixed with the first method were also validated. The procedure was reliable with precision and accuracy below 10%. All compounds were detected after positive mode electrospray ionization in selected reaction monitoring mode. These new analytical procedures were developed for melphalan pharmacokinetic studies or therapeutic drug monitoring.

Simultaneous quantification of 5-FU, 5-FUrd, 5-FdUrd, 5-FdUMP, dUMP and TMP in cultured cell models by LC-MS/MS.

To specifically quantify several metabolites of 5-fluorouracil (5-FU) and two endogenous monophosphate nucleotides, we developed an original method based on a liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay allowed the determination of: (i) the intracellular production of 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP) from 5-FU or 5-fluoro-2'-deoxyuridine (5-FdUrd), (ii) the impact of 5-FdUMP concentration on the intracellular 2'-deoxyuridine-5'-monophosphate (dUMP)/thymidine-5'-monophosphate (TMP) ratio, and (iii) the secretion extent of 5-FdUMP and 5-FU from human cultured cells by ABC transporters. Under our experimental conditions, cells were incubated with 5-FU or 5-FUrd. Then, cellular proteins were precipitated by methanol. This procedure provided high extraction recovery. In addition, to measure 5-FU and 5-FdUMP secretion from cells, we carried out quantification of these molecules in culture medium. Media were either directly injected (5-FU) or underwent a solid phase extraction using Oasis Wax extraction cartridge (5-FdUMP). Separation of analytes was performed on a dC18 Atlantis 3.5microm, (100mmx2.1mm i.d) column with isocratic mode using ammonium formate buffer/methanol/water (5/5/90, v/v) as mobile phase. The run time did not exceed 6.2min. The analytes were ionized in an electrospray interface under negative ion mode. We validated the method over a range of 2.5-150ngmL(-1) according to the compounds. Intra- and inter-assay variability was lower than 10% over seven days. All compounds were stable in cells or in culture medium when samples were stored at -20 degrees C for at least two weeks, and after three freeze-thaw cycles. No matrix effect was observed in both media.