High-performance liquid chromatographic assay for the measurement of melphalan and its hydrolysis products in perfusate and plasma and melphalan in tissues from human and rat isolated limb perfusions |
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| Institution: | 1. Department of Medicine, University of Queensland, Princess Alexandra Hospital, Woolloongabba, Qld. 4102, Australia;2. Conjoint Endocrine Laboratory, Royal Brisbane Hospital, Herston, Qld. 4029, Australia;3. Department of Surgery, University of Queensland, Princess Alexandra Hospital, Woolloongabba, Qld. 4102, Australia;1. N.D. Zelinsky Institute of Organic Chemistry RAS, Moscow, Russia;2. Institute of Physical and Organic Chemistry at Southern Federal University, Rostov on Don, Russia;3. University of Rennes, CNRS, ISCR (Institut des Sciences Chimiques de Rennes), UMR 6226, F-35000 Rennes, France;1. Ghent University, Department of Chemistry, Atomic & Mass Spectrometry – A&MS Research Unit, Campus Sterre, Krijgslaan 281-S12, 9000 Ghent, Belgium;2. University of Alicante, Department of Analytical Chemistry, Nutrition and Food Sciences, P.O. Box 99, 03080 Alicante, Spain;3. Ghent University, Department of Organic and Macromolecular Chemistry, Campus Sterre, Krijgslaan 281-S4-bis, 9000 Ghent, Belgium;1. Department of Chemistry, Cotton University, Guwahati 781001, Assam, India;2. Departament de Química, Universitat de les Illes Balears, Crta de Valldemossa km 7.7, 07122 Palma de Mallorca (Baleares), Spain;3. Department of Chemical Sciences, Tezpur University, 784028 Assam, India;1. Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX 76019, USA;2. Life and Health Sciences Department, University of North Texas at Dallas, Dallas, TX 75241, USA |
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| Abstract: | A sensitive, specific and rapid reversed-phase high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of melphalan and its hydrolysis products in samples from the isolated perfusion of human and rat limbs. Samples of perfusate, plasma and tissue were analysed, following methanol precipitation, using a phenyl column and fluorescence detection. Dansyl-arginine (38 μg ml?1) was employed as the internal standard. Good resolution was observed allowing quantitation of melphalan, monohydroxymelphalan (MOH) and dihydroxymelphalan (DOH) in perfusate and plasma and melphalan in tissue. The mean recoveries of melphalan, MOH and DOH from perfusate and plasma were all 100 ± 10%. The recovery of melphalan in tissue was 93.5%. A linear response was demonstrated for melphalan in the concentration range 1.8–56.8 μg ml?1, for DOH in the concentration range 0.5–30.0 μg ml?1 and for MOH in the range 1.4–25.1 μg ml?1, in perfusate and plasma. The lower limits of quantitation of melphalan, MOH and DOH in perfusate and plasma were 1.4, 2.4 and 1.2 ng on column, respectively, and 7.2 ng of melphalan on column in tissue. Intra-assay coefficients of variation (C.V.) for melphalan, MOH and DOH, at low and high concentrations were all less than 5% and the inter-assay C.V.s were less than 9%. An ultra-filtration study to determine the protein binding of melphalan and the hydrolysis products showed that the unbound fractions (fu) of melphalan in buffer containing dextran and bovine serum albumin were 0.873 and 0.521, respectively. The assay was used to quantitate melphalan and its hydrolysis products in samples from isolated perfusions in the human limb and rat hindlimb. |
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