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
Zy. Wu et al., 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, Journal of chromatography B. Biomedical applications, 673(2), 1995, pp. 267-279
Citations number
29
Categorie Soggetti
Chemistry Analytical","Biochemical Research Methods
Journal title
Journal of chromatography B. Biomedical applications
A sensitive, specific and rapid reversed-phase high-performance liquid
chromatographic (HPLC) assay was developed for the quantitation of me
lphalan and its hydrolysis products in samples from the isolated perfu
sion of human and rat limbs. Samples of perfusate, plasma and tissue w
ere analysed, following methanol precipitation, using a phenyl column
and fluorescence detection. Dansyl-arginine (38 mu g ml(-1)) was emplo
yed as the internal standard. Good resolution was observed allowing qu
antitation of melphalan, monohydroxymelphalan (MOH) and dihydroxymelph
alan (DOH) in perfusate and plasma and melphalan in tissue. The mean r
ecoveries 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 mu g ml(-1), for DOH in the concentration range 0.5-30.0 mu g ml
(-1) and for MOH in the range 1.4-25.1 mu g ml(-1), in perfusate and p
lasma. The lower limits of quantitation of melphalan, MOH and DOH in p
erfusate 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 concen
trations were all less than 5% and the inter-assay C.V.s were less tha
n 9%. An ultra-filtration study to determine the protein binding of me
lphalan and the hydrolysis products showed that the unbound fractions
(f(u)) of melphalan in buffer containing dextran and bovine serum albu
min were 0.873 and 0.521, respectively. The assay was used to quantita
te melphalan and its hydrolysis products in samples from isolated perf
usions in the human limb and rat hindlimb.