Determination of Related Substances in Cabazitaxel Using Reverse Phase-liquid Chromatography Method

The present paper describes the reverse phasehigh performance liquid chromatographic method and was validated as per ICH guidelines for the determination of related substances in Cabazitaxel. RP-Liquid chromatography technique was performed with pH 3.0 phosphate buffer and acetonitrile as mobile phase at a flow rate of 0.8 mL/min. on Waters 2489 UV 2695 pump, Waters 2998 PDA 2695 pump Software Empower2 photodiode array detector using Zorbax SB C18 column with UV detection at 220 nm. Linearity was observed in the concentration range of Cabazitaxel LOQ–0.10% (R2 = 0.9998), the concentration range of CBZM01 impurity 0.03–0.225% (R2 = 0.9997), the concentration range of CBZM02 impurity 0.03–0.225% (R2 = 0.9997), the concentration range of CBZN09 impurity 0.03–0.225% (R2 = 0.9998). Limit of detection (%) and the limit of quantitation (ng/mL) were found to be CBZM01 impurity 0.002% and 73ng/mL, CBZM02 impurity 0.002 % and 71ng/mL, CBZN09 impurity 0.002 % and 6ng/mL and Cabazitaxel 0.002 % and 0.008 % respectively. The percent recovery was in good agreement with the labeled amount in the dosage forms and hence, the method is specific, simple, reproducible and accurate for the determination of Cabazitaxel.


Chemicals used
Orthophosphoric acid, Acetonitrile HPLC grade and water were obtained from Merck,India.All chemicals were of an analytical grade and used as received.

Preparation of mobile phase-A
1mL of H 3 PO 4 is added to 1000mL of water and the solution is properly mixed.Filtered through 0.45µ membrane filter paper and degassed.

Preparation of mobile phase-B
Acetonitrile used as mobile phase-B.

Preparation of Diluent:
600mL Acetonitrile and 400mL water are mixed to prepare one litre of diluents and the solution is properly mixed.blank, CBZM01,CBZM02,CBZM09 and spiked solution were shown in Figure 1.5-1.9.
The above study reveals that all the known impurities of Cabazitaxel are adequately

System precision
System suitability was performed by analyzing the reference solution six times.%RSD for replicate injections of each component from reference solution was calculated.Preparations of Cabazitaxel, CBZM01, CBZM02 and CBZN09 at concentrations of 0.15% of the nominal concentration of sample required by the method were analyzed in triplicate for each solution.Results of system suitability were presented in the Table 1.2-1.3.

Limit of Detection (LOD) Detection limit of Cabazitaxel
Stock solution of CBZ standard at concentration level 1.0 mg/mL was prepared by weighing accurately about 25.029mg of CBZ standard and was transferred into a 25mL volumetric flask, dissolved in diluent and filled up with diluent to the volumetric mark (Table 1.4.).The stock solution was diluted 100times by dispensing 250µL into a 25mL volumetric flask, completed with diluent.This CBZ solution was further diluted into a 25mL volumetric flask, dosing 50µL.The final concentration (% level) of CBZ was (0.002 %).Details of CBZ detection limit was incorporated in the Table 1.5.

Detection limits of the specified impurities
The stock solutions of individual specified impurities at concentration level 1.0mg/mL were prepared (10.000 mg of specified impurity standard was weighed into a 10mL volumetric flask, resolved.Hence, the method is selective for the determination of related substances in Cabazitaxel.Retention time and relative retention time for known impurities were listed in the Table 1.1.of the specified impurity was 16.5-18.3ng/mL(0.002 %).The limit of detection values obtained for each impurity and Cabazitaxel are within the acceptance criteria.Detection limit evaluation of CBZ01, CBZ02 and CBZ09 were mentioned in the Table 1.7-1.9.

Limit of quantitation Quantitation limit of CBZ
The stock solutions of individual specified impurities at concentration level 1.0mg/mL were prepared( about 10.000 mg of specified impurity of the specified impurity was 16.5-18.3ng/mL(0.002 %).Specified impurities mixed solution of the proper concentration was analysed three times.Results of CBZ quantitation limit was mentioned in the Table 2.0.

Quantitation limits of the specified impurities
Quantitation limit of the specified impurities (CBZM01, CBZN09 and CBZM02) was estimated based on the detection limit results.Quantitation limit was verified by six injections of each specified impurity solution on the estimated concentration which would result in required S/N <8-15>.The limit of quantitation values obtained for each impurity and Cabazitaxel are within the acceptance criteria.Determined quantitation limit for CBZM01 is 73ng/ mL (0.008 %), for CBZM02 is 71ng/mL (0.008 %) and for CBZN09 is 66ng/mL (0.007 %).The detailed results regarding quantitation limit of various impurities were listed in the Table 2.1-2.3.

Linearty and Range
The linearity is determined by injecting the solutions in duplicate containing known impurities and Cabazitaxel ranging from 0.05 to 1.13% and impurities ranging from 0.05% to 0.22% of the specified limit.Regression analysis was performed and correlation coefficient was determined (Table 2.4 and Figure 2.0).Response factor for each impurity was determined with respect to Cabazitaxel.
The linearity range as the range for determining the impurities were reported.The details of the results were presented in the Table 2.5-2.7 and Figure 2.1-2.3 show the line of best fit for peak area versus concentration for each impurity.The linearity results for Cabazitaxel and all the impurities in the specified concentration range are found satisfactory, with a correlation coefficient greater than 0.99.

Accuracy
Analyses performed triple injections performed on 0.10%, 0.15% and 0.22% of the nominal concentration, respectively) were used.From linear regressions, theoretical contents corresponding to the peak areas obtained from the triple analyses and deviations of these values from the actual concentrations were calculated.The percentage recovery values obtained for each impurity are in the range of about 97.3-101.8,which are within the specified criteria.Summary of % recovery of CBZ and its impurities were listed in the Table 2.8-3.1.

RESULTS AND DISCUSSION
A simple, economic, accurate and precise reverse phase liquid chromatography method was successfully developed using Zorbax SB C18 (100 mm×3.0 mm, 1.8µm particle size).Injection volume of 3μL is injected and eluted with the mobile phase eluent-A: pH 3.0 phosphate buffer and eluent-B: acetonitrile, which is pumped at a flow rate of 0.8 mL/min.Detection, was carried out at 220 nm.
For Selectivity, the chromatograms were recorded for standard and sample solutions of Cabazitaxel and its related substances.Selectivity studies reveal that the peak is well separated from each other.Therefore the method is selective for the determination of related substances in Cabazitaxel.
The limit of detection (LOD) and limit of quantitation (LOQ) for CBZM01 impurity 0.002% and 0.008%, CBZM02 impurity 0.002% and 0.008%, CBZN09 impurity 0.002% and 0.007% and Cabazitaxel 0.002% and 0.008% respectively.Using the optimized chromatographic conditions, the retention times (in min) of impurities were 6.297 for CBZM01 impurity, 17.417 for CBZM02, 10.669 for CBZN09 and 11.021 for Cabazitaxel.The linearity results for Cabazitaxel and all the impurities