Quantifi cation of 4-Oxiranyl Methoxy-9h-Carbazole a Genotoxic Impurity in Carvedilol Drug Substances by Lc-Ms

4-Oxiranylmethoxy-9H-Carbazole is generally used as an important intermediate in the preparation of pharmaceutical compounds such as antihipertensives. However, this 4-Oxiranylmethoxy-9H-Carbazole is found to be a genotoxic product. This Genotoxic compounds affects the human genes, and the presence of these genotoxins in the pharmaceutical compounds can be controlled. In current regulatory practice (ICH Guidelines 2002), genotoxic compounds are usually considered to operate by a non-threshold mode of action and, thus any level of exposure carries at least theoretically a risk. This precautions view implies that pharmaceutical measurements should be guided by the so called ‘ALARA’ principle (As Low As Reasonably Achievable), i.e., where avoidance is not possible, genotoxic impurities must be kept to a low level. However, the draft guidelines from European agency (Guidelines on the limits of Genotoxic impurities CPMP/SWP/5199/02, EMEA/CHMP/QMP/251344/2006, EMEA/CHMP/SWP/431994/2007) and feedback from the US food and drug administration (USFDA) (USFDA, Guidance for Industry 2008) to pharmaceutical industries via drug application has enabled the pharmaceutical industries to establish interim strategies. Generally the daily intake of these genotoxins has been limited to a daily dose of 1.5 g/day. Therefore it is preferable for the potential genotoxins to be controlled during the synthesis; where the levels cannot be controlled and no safety data yet exists it may be preferable for the pharmaceutical company to change the route of synthesis of the drug substances. As 4-Oxiranylmethoxy-9HCarbazole is also a genotoxic compound, the regulatory team may be expected to estimate the levels of 4-Oxiranylmethoxy-9H-Carbazole to be controlled to 15 ppm in the drug substance. It was felt necessary to develop simple, sensitive validated method for 4-Oxiranylmethoxy9H-Carbazole.


Introduction
4-Oxiranylmethoxy-9H-Carbazole is generally used as an important intermediate in the preparation of pharmaceutical compounds such as antihipertensives. However, this 4-Oxiranylmethoxy-9H-Carbazole is found to be a genotoxic product. This Genotoxic compounds affects the human genes, and the presence of these genotoxins in the pharmaceutical compounds can be controlled. In current regulatory practice (ICH Guidelines 2002), genotoxic compounds are usually considered to operate by a non-threshold mode of action and, thus any level of exposure carries at least theoretically a risk. This precautions view implies that pharmaceutical measurements should be guided by the so called 'ALARA' principle (As Low As Reasonably Achievable), i.e., where avoidance is not possible, genotoxic impurities must be kept to a low level. However, the draft guidelines from European agency (Guidelines on the limits of Genotoxic impurities CPMP/SWP/5199/02, EMEA/CHMP/QMP/251344/2006, EMEA/CHMP/SWP/431994/2007) and feedback from the US food and drug administration (USFDA) (USFDA, Guidance for Industry 2008) to pharmaceutical industries via drug application has enabled the pharmaceutical industries to establish interim strategies. Generally the daily intake of these genotoxins has been limited to a daily dose of 1.5 g/day. Therefore it is preferable for the potential genotoxins to be controlled during the synthesis; where the levels cannot be controlled and no safety data yet exists it may be preferable for the pharmaceutical company to change the route of synthesis of the drug substances. As 4-Oxiranylmethoxy-9H-Carbazole is also a genotoxic compound, the regulatory team may be expected to estimate the levels of 4-Oxiranylmethoxy-9H-Carbazole to be controlled to 15 ppm in the drug substance. It was felt necessary to develop simple, sensitive validated method for 4-Oxiranylmethoxy-9H-Carbazole.

Sample, chemicals and reagents
Carvedilol was synthesized from the CRD research department in APL Research centre, (A Division of Aurobindo Pharma Ltd.) (Bachupally, Quthubullapur, Hyderabad-90, INDIA). Acetonitrile (HPLC grade), Ammonium acetate (BDH grade), High pure water was prepared by using the Milli-Q plus purifier.         quadrupole mass spectrometer (API 2000, PE SCIEX) coupled with Shimadzu HPLC equipped with SPD 10 A VP UV-VIS detector and LC 10 AT VP pumps. Analyst software was used for data acquisition and data processing. The turbo ion spray voltage was maintained at -4.5 Kv and temperature was set at 375°C. The auxiliary gas and sheath gas used was high pure Nitrogen. Zero air was used as Nebulizer gas. The analysis was carried out using YMC PACK C8 250 X 4.6 mm column with 5 m particle diameter with mobile phase consisting of a mixture of 0.01M Ammonium acetate pH-5 in water and acetonitrile in the ratio of 15:85 v/v. The flow rate was 1ml/min with flow rate split down to 0.2 ml/min in to the LC-MS system. The column was monitored at 55°C.The injection volume was 20 l. Electrospray ionization in negative mode was used with a multiple reaction monitoring (MRM) mode was used as MS method for quantification of 4-Oxiranylmethoxy-9H-Carbazole in drug substances. In this method 4-Oxiranylmethoxy-9H-Carbazole is monitored by its molecular ion value of 238.10 (M-H) and its daughter ion 181.0 with focusing potential-270, declustering potential -25, entrance potential -10 , and the curtain gas flow 25 (psi) respectively.

Results and Discussion
The main target of LC-MS/MS method was to quantification of 4-Oxiranylmethoxy-9H-Carbazole impurity in the Carvedilol active ingredient. During the method development we used different reversed phase stationary and mobile phase was used and finally chromatographic separation was achieved on a YMC PACK C8 250*4.6,5mic column (YMC) in isocratic mode using with 0.01M Ammonium acetate pH-5 in water and Acetonitrile in the ratio of 15:85 v/v. The flow rate was 1.0 ml/min split down to 0.2 ml/min in to LC-MS system. Linearity of 4-Oxiranylmethoxy-9H-Carbazole: By selecting ion monitoring, the linearity of 4-Oxiranylmethoxy-9H-Carbazole was satisfactorily done a series of solutions were prepared using 4-Oxiranylmethoxy-9H-Carbazole at concentration levels from around detection level to 150% and the concentration levels are 22.5 (g/g), 18 (g/g), 15 (g/g), 12.5 (g/g), 6 (g/g), 1.5 (g/g), 1 (g/g) and 0.2 (g/g) respectively. The peak area versus concentration data was done by linearity plot slop, intercept, and residual sum of squares analysis. The calibration curve was given based on response over the concentration range for 4-Oxiranylmethoxy-9H-Carbazole. The correlation coefficients for 4-Oxiranylmethoxy-9H-Carbazole were 0.999. Linearity of the 4-Oxiranylmethoxy-9H-Carbazole spectrogram was shown in the Figure 3 and the results are tabulated in Table 1.

Limit of detection (LOD) and Limit of Quantification (LOQ) for 4-Oxiranylmethoxy-9H-Carbazole:
The LOD and LOQ values of 4-Oxiranylmethoxy-9H-Carbazole were predicted from the linearity data. Each predicted concentration was verified for precision by preparing the solutions at about predicted concentration and injecting each solution six times for LC-MS/MS study and the predicted concentration for LOQ was 2.68 (g/g) and LOD was 0.825 (g/g). The   spectrograms are shown in Figure 4 and Figure 5 ( Figure 5 is included in supplementry data). Further the results are tabulated in Table 2.
Recovery of 4-oxiranylmethoxy-9H-carbazole from the API: The accuracy of the method was evaluated in sample solutions were prepared in triplicate by spiking 4-Oxiranylmethoxy-9H-Carbazole at LOQ level to 150% with Carvedilol drug substance and injected each solution in to LCMS as per methodology. The percentage of recovery was calculated. A satisfactory value of 4-Oxiranylmethoxy-9H-Carbazole (96.10%, 97.70% and 101.5%) was found. At such low levels these recoveries and % RSD were satisfactory. Accuracy at LOQ to 150% level spectrogram is shown in Figure 6 and Figure 7 ( Figure  6 and Figure 7 is included in supplementry data), further the results are tabulated in Table 3.

Conclusion
LC-MS/MS method has been suitable for quantification of 4-Oxiranylmethoxy-9H-Carbazole for highly sensitive method of analysis with limit of detection 0.825 ppm. The methodology has one of the restrictions of 4-Oxiranylmethoxy-9H-Carbazole in Carvedilol sample.