Double-salting out assisted liquid-liquid extraction (SALLE) HPLC method for estimation of temozolomide from biological samples.

The role of temozolomide (TMZ) in treatment of high grade gliomas, melanomas and other malignancies is being defined by the current clinical developmental trials. Temozolomide belongs to the group of alkylating agents and is prescribed to patients suffering from most aggressive forms of brain tumors. The estimation techniques for temozolomide from the extracted plasma or biological samples includes high-performance liquid chromatography with UV detection (HPLC-UV), micellar electrokinetic capillary chromatography (MKEC) and liquid chromatography coupled to mass spectroscopy (LC-MS). These methods suffer from disadvantages like low resolution, low sensitivity, low recovery or cost involvement. An analytical method possessing capacity to estimate low quantities of TMZ in plasma samples with high extraction efficiency (%) and high resolution with cost effectiveness needs to be developed. Cost effective, robust and low plasma component interfering HPLC method using salting out liquid-liquid extraction (SALLE) technique was developed and validated for estimation of drug from plasma samples. The extraction efficiency (%) with conventional LLE technique with methanol, ethyl acetate, dichloromethane and acetonitrile was found to be 5.99±2.45, 45.39±4.56, 46.04±1.14 and 46.23±3.67 respectively. Extraction efficiency (%) improved with SALLE where sodium chloride was used as an electrolyte and was found to be 6.80±5.56, 52.01±3.13, 62.69±2.11 and 69.20±1.18 with methanol, ethyl acetate, dichloromethane and acetonitrile as organic solvent. Upon utilization of two salts for extraction (double salting liquid-liquid extraction) the extraction efficiency (%) was further improved and was twice of LLE. It was found that double salting liquid-liquid extraction technique yielded extraction efficiency (%) of 11.71±5.66, 55.62±3.44, 77.28±2.89 and 87.75±0.89. Hence a method based on double SALLE was developed for quantification of TMZ demonstrating linearity in the range of 0.47-20 µg/ml. The LOQ and LOD for the developed method were 0.4 µg/ml and 0.1 µg/ml, respectively. Thus, plasma non-interfering SALLE-HPLC method that is precise, robust, accurate, specific and cost effective for estimation of temozolomide from plasma samples was developed and validated.

5-(3-Hydroxymethyl-3-methyl-1-triazeno imidazole-4-carboxamide is a metabolite of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DIC, DTIC NSC-45388).

The cancer chemotherapeutic drug, 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide (DIC, DTIC, NSC-45388), is metabolised in rats to a structurally related product which was detected by thin-layer chromatography. The novel metabolite has a lower mobility and a colour reaction that is indistinguishable from the parent compound. The metabolite is not retained on an anionic exchanger which is inconsistent with the expected covalent binding of the drug to endogenic anionic substrates (e.g. glucuronic acid). Since both DIC and the metabolite yielded 5-[(4-ethylamino-1-napthyl)-azo]imidazole-4-carboxamide through release of 5-diazoimidazole-4-carboxamide, followed by coupling with N-ethyl-1-napthylamine, no biotransformation (hydroxylation) of the imidazole moiety of the injected DIC had occurred. By corollary, the lowered chromatographic mobility of the metabolite was explicable by the introduction of a polar but non-acidic function into the terminal dimethylamino group of the triazene side-chain. The metabolite was identified as 5-(3-hydroxymethyl-3-methyl-1-triazeno)imidazole-4-carboxamide by co-chromatography with an authentic sample of HMIC and by its methylating capacity for nucleophilic substrates.