RESUMO
Studies with WR-2721 and related compounds have been hindered by the lack of a suitable assay for the drug and its major metabolites. We have developed a chromatographic method which requires no derivatization for the separation and detection of WR-2721, the free thiol, its symmetrical disulfide and other mixed disulfides. Our procedure involves ion-pairing for separation of ionizable compounds by causing polar molecules to become more lipophilic and hence separable using reverse phase HPLC. Detection is based upon liquid scintillation counting of S-35 incorporated during the synthesis of the parent compound. This method requires no pre-column preparation of samples and, by detecting the S-35 label, eliminates the chance that a coeluting species could interfere with detection, as might occur with post-column derivatization. Chromatography was done using a 10 micron C8RP column and 35% MeOH/65% 0.0113M NaH2PO4, 0.005 M hexanesulfonate, pH 5.9, flowing at 1 ml/min. Half-minute fractions were collected into scintillation vials for counting. Retention volumes for the various compounds were: column breakthrough (3.5 ml), WR-2721 (4.5 ml), WR-1065 (9 ml), and WR-33278 (24 ml). This analytical technique employing radiotracers can be used to study radioprotective mechanisms by time dependent measurements of the tissue distribution and chemical form of labeled drug. Such chemical information can then be correlated with biological measures of radiation protection.
Assuntos
Amifostina/análise , Compostos Organotiofosforados/análise , Protetores contra Radiação/análise , Amifostina/sangue , Amifostina/líquido cefalorraquidiano , Amifostina/urina , Animais , Cromatografia Líquida de Alta Pressão/métodos , Masculino , Mercaptoetilaminas/sangue , Mercaptoetilaminas/líquido cefalorraquidiano , Mercaptoetilaminas/urina , Camundongos , Ratos , Ratos Endogâmicos F344 , Saliva/análise , Glândula Submandibular/análise , Radioisótopos de EnxofreRESUMO
A liquid chromatographic (LC) procedure using alumina as stationary phase in both the pre- and the analytical column, is reported for the determination of WR1065, the active metabolite of the amino- and thiol-containing anticancer drug WR2721. After pre-column derivatization of the thiol group, the analyte is determined by LC with diode laser induced fluorescence detection in the near-infrared. Selective removal of excess label is achieved by means of column switching; it allows the detection of 5 x 10(-9) M WR1065 in water and 10-fold diluted, deproteinated plasma samples. The detection limit is determined by the derivatization reaction and not by the fluorescence detection of the labelled analyte. Endogeneous thiols do not interfere.
Assuntos
Antineoplásicos/análise , Mercaptoetilaminas/análise , Protetores contra Radiação/análise , Antineoplásicos/sangue , Antineoplásicos/urina , Cromatografia em Camada Fina , Humanos , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Lasers , Mercaptoetilaminas/sangue , Mercaptoetilaminas/urina , Espectrometria de Fluorescência , TemperaturaRESUMO
The radioprotective compound WR2721 is a thiophosphate, which, when administered orally, is activated at the acid pH of the stomach to its free thiol (MDP). The free thiol is a mucolytic compound which acts via the reduction of disulfide bonds of mucin molecules. An equimolar mixture of MDP and cysteine, in urine at pH 6.0 and 37 degrees C, when oxidized by molecular oxygen, preferentially forms the soluble mixed disulfide between MDP and cysteine. The disulfide cystine will undergo thiol-disulfide interchange with MDP; as a result, cystine crystals are effectively dissolved. Moreover, in the presence of catalytic amounts of free thiol, the disulfide of MDP will undergo thiol-disulfide interchange with cystine to dissolve cystine crystals. The mixed disulfide of MDP with cysteine is soluble in urine at pH 6.0 and 37 degrees C to at least 100 mg/ml. Chromatographic procedures which permit the analysis of MDP and its mixed disulfide derivatives as MDP-sulfonic acid are described. By these procedures, it was demonstrated that 20% of a single oral dose of WR2721 was excreted as MDP derivatives in the urine of normal volunteers. These procedures will permit the evaluation of WR2721 in the treatment of cystinuria.