Assay of fluconazole by megabore capillary gas-liquid chromatography with nitrogen-selective detection

Abstract

A megabore column gas-liquid chromatographic method which uses nitrogen-phosphorus detection was developed for the analysis of fluconazole in plasma, serum, cerebrospinal fluid, or urine. The assay was linear from 0.2 to 200 ,Ig/ml and had an average coefficient of variation of 7%. The suitability of the assay for pharmacokinetic studies was demonstrated.Fluconazole 858; 2-(2,4-difluorophenyl)-1,3-bis (lH-1,2,4-triazol-1-yl)-2-propanol; Fig. 1] is a triazole compound with potent antifungal activity which is currently being evaluated in clinical trials (5). It is well absorbed after oral administration (4) and shows good penetration into cerebrospinal fluid (1, 3). Early analytical methods for assay of this compound in biological fluids, designed to support preliminary animal and human studies, were effective but rather complex. Both gas-liquid chromatographic and highperformance liquid chromatographic (HPLC) approaches were used (2, 3, 6). However, with this compound now in clinical trials, a simpler and more rapid and reliable analytical method was desirable for therapeutic drug monitoring. We therefore developed the following method, which uses megabore column gas-liquid chromatography with nitrogenselective detection. MATERIALS AND METHODSInstrumentation. A Varian 6000 (Varian Instruments, Walnut Creek, Calif.) gas chromatograph with a nitrogen-phosphorus detector operating at an applied voltage of 5 to 10 mV was employed. A fused silica DB-5 megabore column (45 m long, inner diameter of 0.55 mm; J. W. Scientific, Folsom, Calif.) with a 1-,um-thick bonded liquid phase was used. The instrument conditions were as follows: injector temperature, 225°C; detector temperature, 275°C; column temperature, 1900C.Reagents. Fluconazole and internal standard UK-54,373 Standards and controls. Plasma standards were prepared by adding appropriate amounts of fluconazole in methanol to tubes that had been washed in a chromic acid bath and then sonicated in water purified by ion exchange, carbon filter, and 0.45-,um filter. The methanol was removed either by blowing nitrogen or air into the tubes at room temperature or by vacuum. ,ug/ml were employed in each group of urine specimens analyzed. Controls of 2 and 7 ,ug of fluconazole per ml, prepared in the same manner as were the standards, were also included in each group of serum or urine specimens assayed.Assay procedure. To assay plasma, serum, or cerebrospinal fluid, 0.5 ml of specimen was pipetted into a tube fitted with a Teflon-lined cap. One level spoonful of a Coors 01 spatula filled with NaHCO3-Na2CO3 (2:1) was added to the specimen to adjust the pH to 9.0 and to provide a salting-out effect. A 10-ml sample of extracting solvent (methylene chloride containing 0.1875 ,g of the UK-54,373 internal standard per ml) was added to the tube, and the mixture was mechanically shaken for 15 min. The tube was subsequently centrifuged in a floor model centrifuge (model CU-5000; International Equipment Co., Needham Heights, Mass.) for 5 min at 2,000 rpm. The upper aqueous layer was ...

Novel Spectrophotometric Methods for the Determination of Fluconazole in the Presence of Its Oxidative Degradation Product

“…This can be summarized as the following: The literature survey reveals several analytical methods used for quantitative determination of FLZ in body fluids and pharmaceutical preparations. These methods include high-performance liquid chromatography [2][3][4][5][6][7][8] , liquid chromatography-tandem mass spectrometry [9][10][11] , gas chromatography [12][13][14][15] ,micellar electrokinetic capillary chromatography 16 and bioassay 17 .…”

Comparison of microbiological assay and HPLC-UV for determination of fluconazole in capsules

“…It is important to mention that capsules with dosage lower than that recommended are ineffective and compromise patient health. Studies focused on fluconazole determination in pharmaceutical formulation by bioassay are scarce, where the majority of papers are related to its study in clinical samples using a HPLC method (blood, serum, saliva or skin) (Harris et al, 1989;Rex et al, 1991;GarciaHermoso et al, 1995;Koks et al, 1996;Hülsewede and Dermoumi, 1996;Cohen et al, 1997;Moraes et al, 1999;Abdel-Moety et al, 2002;Mathy et al, 2003;Marchetti et al, 2003;Porta et al, 2005;Kim et al, 2007;Lima et al, 2009). …”

“…The most commonly described assay for fluconazole content in capsules are high performance liquid chromatography (HPLC), gas liquid chromatography and spectrophotometry (Harris et al, 1989;Rex et al, 1991;Moraes et al, 1999;Porta et al, 2005;Kim et al, 2007). These methods require expensive equipment and considerable technician time (Rex et al, 1991).…”

Treatment of exogenous Candida endophthalmitis in rabbits with oral fluconazole

“…The samples of aqueous and vitreous humors were stored at Ϫ70ЊC. Fluconazole levels were determined by a gas-liquid chromatography assay as previously described (21). 5FC levels were determined by a bioassay as previously described (5).…”