Simultaneous determination of dacarbazine, its photolytic degradation product, 2-azahypoxanthine, and the metabolite 5-aminoimidazole-4-carboxamide in plasma and urine by high-pressure liquid chromatography

Abstract

A high-pressure liquid chromatographic method has been developed that allows for the simultaneous analysis of dacarbazine (DTIC), 5-aminoimidazole-4-carboxamide (AIC), and 2-azahypoxanthine (2-AZA) in plasma or urine. Plasma samples were prepared by ultrafiltration, whereas urine samples were filtered and diluted for analysis. Chromatography was done with a C18 ,uBondapak column along with gradient elution of the drugs. The mobile phase consisted of 100% 0.5 M sodium acetate (pH 7.0) and 25% acetonitrile in 0.05 M sodiumf acetate (pH 5.5) with detection at 280 nm. Linearity was observed up to 500 ,ug/ml for DTIC and up to 53 ,ug/ml for AIC and 2-AZA. The assay methodology was reproducible, with a lower limit of detection of 5.0, 0.5, and 0.5 ,Lg/ml for DTIC, AIC, and 2-AZA, respectively. Interday and intraday coefficients of variation ranged between 4 to 14% and 2 to 16%, respectively. The analytical method was applied to the analysis of plasma and urine samples resulting from the isolation perfusion chemotherapy of an extremity with 57 mg of DTIC per kg in a patient with melanoma.The pharmacokinetics of dacarbazine (DTIC), a cytotoxic agent, has recently been studied in animals and humans (1-5) by a specific high-pressure liquid chromatographic (HPLC) assay. Assays used in these studies involved different chromatographic conditions for the determination of DTIC and its metabolite, 5-aminoimidazole-4-carboxamide (AIC). In addition, neither of the methods uses an internal standard. The methods also describe the analysis of 2-azahypoxanthine (2-AZA), a photolytic degradation product of DTIC, but again, the analysis is a separate assay with either DTIC or AIC and never with all three being simultaneously determined, which indeed would be the most efficient methodology.To facilitate pharmacokinetic studies in animals and humans, a gradient elution HPLC assay for the simultaneous determination of DTIC, AIC, and 2-AZA in plasma and urine was developed that uses an internal standard.Sodium acetate and phosphoric acid were obtained from J. T. Baker Chemical Co., Phillipsburg, N.J. Acetonitrile (HPLC grade) and 3-methylxanthine (internal standard) were purchased from Fisher Scientific Co., Fairlawn, N.J., and Aldrich Chernical Co., Inc., Milwaukee, Wis., respectively. DTIC, AIC, and 2-AZA were obtained from Miles Pharmaceuticals, West Haven, Conn. The DTIC was dessicated for 2 h over phosphorus pentoxide under vacuum before being used. Subsequently, all three compounds were stored at 22°C in a dessicator until used. DTIC solutions were kept covered with aluminum foil whenever they were handled to prevent exposure to UV light and possible degradation. The detector output was monitored with a model 3390 integrator-plotter (Hewlett-Packard Co., Rockville, Md.) set at an attenuation of 6 and a chart speed of 0.2 cm/mm. Analysis was done by gradient elution with solvent systems designated as solvent A, which was 100% 0.5 M sodium acetate adjusted to pH 7.0 with 10% concentrated phosphoric acid in Water and a solvent system, a...

Electrochemical Behaviour of the Anticancer Dacarbazine‐Cu2+ Complex and Its Analytical Applications

“…The mean percentage recovery showed no significant matrix interference ( Table 4). The proposed procedure is hence more simple, faster, more sensitive and more selective, and exhibits a lower limits of detection (6.12 10 À10 M) and limit of quantification (2.04 10 À9 M) as compared to the procedures described previously [6][7][8][9][10].…”

“…The first method for determination of dacarbazine was described by Loo and Stasswender [5] and involved decomposition by ultraviolet irradiation to produce 5-diazo-imidazole-4-carboxamide which was derivatized with Bratton-Marshall reagent to produce an azo dye, which was measured at 540 nm. High-performance liquid chromatography [6][7][8] was applied in the reverse-phase mode with a C-18 column, methanol/acetate buffer as mobile phase, and ultraviolet detection at 300 nm for determination of dacarbazine. The electrochemical behaviour of dacarbazine was studied and electroanalytical methods have been applied for determination of dacarbazine [9,10].…”

Unravelling seemingly complex chemistry of reactions using electrospray ionization mass spectrometry

“…Dacarbazine, 13 (m/z 183), is an antineoplastic agent which is used in the treatment of malignant melanoma and is also administered to the patients with advanced Hodgkin's disease. 126 Dacarbazine is sensitive to light and it rapidly decomposes to 5-diazoimidazole-4-carboxamide and subsequently to 2-azahypoxanthine, 14 (m/z 138). Thus, studies involving the photochemical stability of 13 are of interest during its production and formulation.…”

Determination of 5-aminoimidazole-4-carboxamide in human plasma by ion-pair extraction and LC–MS/MS

“…Detection of AICA in biological fluids by high-performance liquid chromatography (HPLC) with ultraviolet detection [9,10] has been reported, but it lacked sensitivity. In addition, a capillary electrophoretic method for screening of patients with AICA-ribosiduria has been reported [11].…”