Identification of Major Degradation Products of Ketoconazole

Analytical methods were developed for the identification of major degradation products of Ketoconazole, an antifungal agent. The stressed degradation of Ketoconazole drug substance was performed under acid, base, thermal, photo and oxidative stress conditions. The major degradation was observed under acid, base and oxidative stress conditions. The degradation study was performed on Inertsil ODS-3V, length 100 X diameter 4.6 mm, particle size 3 μm column using gradient method. These degradants were identified by LC-MS technique.


Introduction
Ketoconazole is an antifungal drug approved by the US FDA in 1981. Only a few analytical methods for the determination of the drug in biological samples and in the presence of other drugs have been reported [1][2][3][4][5][6][7][8][9][10][11][12]. The photodegradation behavior of Ketoconazole has been reported by Staub et al [13]. The drug substance is official in Ph. Eur. but the specified impurities are not mentioned. The present study deals with understanding the degradation behavior of Ketoconazole by subjecting it to acid, base, aqueous, thermal, photo and oxidative stress conditions. Furthermore, the two major degradation impurities observed under stressed condition were identified by LC-MS techniques, elemental analysis, NMR, and their structures were justified through mechanistic explanation. Sci Pharm. 2011; 79: 817-836

Material and reagents
Ketoconazole drug substance was obtained from Sharon Biomedichem (Navi Mumbai, India). All the chemicals and reagents, hydrochloric acid, sodium hydroxide, hydrogen peroxide (30 %), tetrabutylammonium hydrogen sulphate, acetonitrile and methanol were used of analytical grade, while a millipore milli Q plus water purification system (Milford, USA) was used to prepare distilled water (>18 μΩ).

Instruments
Integrated HPLC system, Ultimate 3000 manufactured by Dionex (Germany) was used for method development and method validation. This system consisted of a quaternary gradient pump, auto sampler, column oven and a photodiode array detector. PC installed Chromeleon software was used to record and to integrate the chromatograms. The analysis was carried out at ambient temperature. LCMS system, Agilent ion trap 6310 was used for mass fragmentation analysis. NMR experiments were recorded on Bruker 500 Mz spectrometer. Photostability studies were performed in a photostability chamber from Thermolab (India).

Analytical HPLC conditions
Inertsil ODS (Length: 100 mm, Diameter: 4.6 mm, Particle size: 3 μm) analytical column was used as a stationary phase. The flow rate was 2.0 ml min −1 and the detector was set at 220 nm. The volume of the sample solution injected was 10 μl. The gradient mobile phase consisted of Mobile phase A {(Acetonitrile: 3.4 g/l solution of tetrabutylammonium hydrogen sulphate (5:95 V/V)}: and Mobile phase B {(Acetonitrile: 3.4 g/l solution of tetrabutylammonium hydrogen sulphate (50:50 V/V)}. A membrane filter of 0.45 μm porosity was used to filter and degas the mobile phase. (Gradient program as mentioned in Tab. 1).

Tab. 1.
Mobile Phase gradient for HPLC chromatographic method

Stress degradation of drug substance
Stress studies were carried out under acid, base, thermal, photo and oxidative stress conditions.

Acid Hydrolysis
250.0 mg of test sample + 2ml 1N HCl into 25 ml volumetric flask. Sample heated on boiling water bath at 100 deg, withdrawn at 2 min and 8 min, respectively, then neutralized with 1N NaOH solution and make up the volume to 25 ml with methanol. Pipette out 4 ml into 50 ml volumetric flask and dilute to volume with methanol.
One unknown degradation impurity was observed under acidic condition (Table 3 and figure 1c). In figure 1c, the main degradation product is unknown impurity at RRT 0.80. One unknown degradation impurity was observed under basic condition which is the same as observed under acidic condition (Table 4 and figure 1d). In figure 1d, the main degradation product is unknown impurity at 0.80. One unknown degradation impurity was observed under oxidative stress condition and it is different from the impurity observed under acidic/ basic condition (Table 5 and figure 1e). In figure 1.e, the main degradation product is unknown impurity at RRT 0.72.

Thermal
Test sample of Ketoconazole was subjected to thermal degradation by exposure to oven at 105°C for 24h and 60°C at 5 days and 10 days. 250.0 mg test sample of Ketoconazole were dissolved and diluted with methanol to 25 ml. Pipette out 4 ml into 50 ml volumetric flask and dilute to volume with methanol.

Photolysis
About 250.0 mg test sample of Ketoconazole is kept for UV degradation for 24hours at 254 nm wavelength and then dissolved and diluted with methanol to 25 ml. Pipette out 4 ml into 50 ml volumetric flask and dilute to volume with methanol. The drug substance was found stable under photo and thermal stress conditions as shown in below ( Table 6, figure 1f and 1g).

Elemental analysis
Elemental analysis (CHNO) of Ketoconazole, Hydrolysis degradent and Oxidative degradent performed and results shown below Table 7

NMR analysis
NMR analysis of Ketoconazole and the oxidative degradent were performed and the results are shown in Table 9.

Structure of elucidated compounds
Hence, the formation of the oxidative degradation product from the drug as shown below is only due to the N-oxide formation at the piperazine ring. The lone pair at the nitrogen of the piperazine ring is more prone for oxidation to form an N-oxide. However, out of two nitrogen atoms, the electron pair on the nitrogen attached to the carbonyl group is participating in resonance delocalization with this group. Hence, the most possible N-Oxide at the nitrogen is at the one attached to the phenolic group (scheme 1).

Conclusions
The Stress degradation on Ketoconazole was carried out under different acid, base, thermal, photo and oxidative stress conditions. The drug was found susceptible to acid, base and oxidative stress degradation. The unknown degradation products formed in the oxidative and hydrolysis stressed sample were identified using LC-MS and elemental analysis (CHNO). The investigations of oxidative and hydrolysis degradent will help to take proper care during selection of excipients in formulation, storage, packaging and handling of the drug product.