Determination of Cabazitaxel (An Anti-prostate cancer agent) by reverse phase liquid chromatography

 

Mukthinuthalapati Mathrusri Annapurna*, Bukkapatnam Venkatesh,

Sunkara Mrunal Chaitanya

Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy, GITAM University, Visakhapatnam-530045, India

 

ABSTRACT:

Cabazitaxel is used in the treatment of hormone-refractory prostate cancer. It is a semisynthetic taxane that can be used as a precursor molecule obtained from yew tree needles. The authors have developed a stability indicating liquid chromatographic method for the determination of Cabazitaxel in pharmaceutical products. Shimadzu Model CBM-20A/20 Alite HPLC system (PDA detector) with Zorbax SB C₁₈ column (150 mm × 4.6 mm i.d., 3.5 µm particle size) was selected for the quantification of Cabazitaxel. A mixture of tetra butyl ammonium hydrogen sulphate and methanol was used as the mobile phase with a flow rate of 1.0 mL/min (UV detection at 210 nm). The proposed analytical method was statistically validated and forced degradation studies were conducted. Cabazitaxel has shown linearity over the concentration range 0.1-200 μg/mL with regression equation y = 26145x + 22943 (r²=0.9997) and considerable degradation was observed in acidic, alkaline and oxidation conditions. The method is specific as the resolution of Cabazitaxelwas good in presence of its degradation products. This validated stability indicating liquid chromatographic method can be used for the determination of Cabazitaxel in biological studies as well as for the pharmaceutical products.

 

 

 

INTRODUCTION:

Cabazitaxel¹(CBZT) is a taxane chemotherapy drug developed from the yew tree. Cabazitaxel (Fig 1) acts by stopping the division of cancer cells into two new cells and so blocks the growth of cancer. It is an advanced treatment of prostate cancer for men who are under hormone treatment and where docetaxel chemotherapy is not working. Clinically it was selected due to its poor affinity for P-glycoprotein, an ATP-dependent drug efflux pump.

 

Chemically it is known as (2aR, 4S, 4aS, 6R, 9S, 11S, 12S, 12aR, 12bS)- 12b-acetoxy- 9- ((2R, 3S)- 3- ((tert- butoxycarbonyl)amino)- 2- hydroxy- 3- phenyl propanoyl) oxy)- 11- hydroxy- 4,6- dimethoxy- 4a, 8, 13, 13- tetramethyl- 5- oxo- 2a, 3, 4, 4a, 5, 6, 9, 10, 11, 12, 12a, 12b- dodecahydro-1H- 7, 11- methanocyclodecabenzo [1, 2-b] oxet- 12- yl benzoate². European Medicines Agency has approved CBZT in January 2011 in Europe with prednisone as combination for the treatment metastatic prostate cancer which progresses even after docetaxel treatment³ based on the results of the TROPIC trail investigating CBZT plus prednisone versus mitoxantrone plus prednisone following docetaxel failure^4-5. CBZT is active in-vitro and in-vivo against taxane resistant cell lines^6-7. CBZT is presently investigated in the treatment of metastatic breast cancer progressing after taxane or anthracycline based chemotherapeutic regimens⁸.Cabazitaxel has been determined using LC-MS/MS in biological fluids such as dry blood spots, human plasma^9-11, HPLC^12-15 and spectroscopic techniques¹⁶. At present the authors have proposed a stability indicating RP-HPLC method for the determination of CBZT in presence of its degradants.

 

MATERIALS AND METHODS:

Instrumentation:

CBM-20A/20 Alite model HPLC system of Shimadzu make, equipped with SPD M20A prominence photodiode array (PDA) detector connected to the system Dell Optiplex 790 loaded with LC Solutions v2.0 is used for the integrating and processing of chromatograms (Injection volume was 20 µL). A reverse phase Zorbax SB C18 column of dimensions 150 mm × 4.6 mm i.d., 3.5 µm particle size was used as analytical column for the separation. All the analysis was done at 25ºC.

 

Materials and reagents:

Reference standard of CBZT (purity >99%) was obtained from Dr. Reddy's Laboratories Ltd (Visakhapatnam, India) as gift samples.  Jevtana^® (Sanofi aventis, Malaysia) is the brand name which is available for CBZT as infusion (Label claim: 60 mg). All chemicals used were of analytical grade and used as received.

 

Tetra butyl ammonium hydrogen sulphate buffer (10mM) solution was prepared by transferring accurately 3.3954 g of TBAHS into a 1000mL volumetric flask and dissolved in HPLC grade water. The resulting solution was sonicated for half an hour and made up to volume with HPLC grade water and filtered.

 

The stock solution of CBZT was prepared by accurately transferring 25 mg of CBZT in to a 25 mL volumetric flask and dissolved in methanol (1000 μg/mL) and further dilutions were made from the stock solution and filtered through 0.45 μm membrane filter before injecting in to the HPLC system.

 

Optimized chromatographic conditions:

The analysis was carried out using a mixture of tetra butyl ammonium hydrogen sulphate solution and methanol (20:80, v/v) as mobile phase (Isocratic mode) with a flow rate 1.0 mL/min on Zorbax SB C18 column (150 mm × 4.6 mm i.d., 3.5 µm particle size) (UV detection at 210 nm). All chromatographic conditions were performed at ambient room temperature (25°C ± 2°C).

 

 

Method validation:

Linearity:

A series of Cabazitaxelsolutions (0.1–200 μg/mL) were prepared from the stock solution and 20 µL of each solution was injected in to the HPLC system and the respective chromatograms were recorded. A calibration curve was drawn by plotting the concentration of the drug solutions on the x-axis and the corresponding peak area on the y-axis and the resulting linear regression equation so obtained was used for the determination of assay of marketed formulations. The limit of quantification and limit of detection were based on the standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in ICH guidelines¹⁷. Sensitivity of the method was established with respect to limit of detection (LOD) and LOQ for analytes.

 

Precision:

The method precision was expressed in terms of repeatability. The intra-day and inter-day precision studies were performed by analyzing the samples of CBZT at three different concentration levels (20, 50 and 100 μg/mL) and the % RSD of the three assay values (n=3) was calculated. The inter-day precision study was conducted on three different days i.e. day 1, day 2 and day 3 whereas the intra-day precision study was conducted on the same day at different intervals of time.

 

Accuracy:

The accuracy of the assay method was evaluated in triplicate spiked at three pre-defined concentration levels (80, 100 and 120%), and the percentage recoveries were calculated. Standard addition and recovery experiments were conducted to determine the accuracy of the method for the quantification of CBZT in the drug product. The study was carried out in triplicate at 18, 20 and 22 μg/mL. The percentage recovery was calculated in each case.

 

Robustness:

The robustness of the assay method was established by varying the chromatographic conditions which included flow rate (0.9 and 1.1 mL/min), percentage of methanol in the mobile phase (78 and 82%) and wavelength (208 and 212 nm). Robustness of the method was studied using three replicates at a concentration level of 100 μg/mLof CBZT.

 

Forced degradation studies:

Forced degradation studies were performed to evaluate the ability of the method to separate the drug substance from the degradation products and specificity of the method¹⁸. Forced degradation studies were conducted at an initial concentration of 1 mg/mL of CBZT and which is refluxed at 80ºC for 20 min and then further diluted with mobile phase. 

To perform the acidic degradation, the drug solution (1.0 mg/mL CBZT) was exposed to acidic degradation with 0.1 M hydrochloric acid maintained at 80ºC for 20 min in a thermostat. The stressed sample was cooled, neutralized with 0.1 N sodium hydroxide and then diluted to required concentration with mobile phase and 20 µL of the solution and was analyzed by the HPLC method developed.

 

For performing alkaline degradation, the drug solution (1.0 mg/mLCBZT) was treated with 0.1 N sodium hydroxide maintained at 80ºC for 20 min in a thermostat. The solution was cooled, neutralized using 0.1 N hydrochloric acid and then diluted as per the requirement and 20 µL of the solution and was analyzed by the HPLC method developed.

 

To perform oxidation degradation, the drug solution (1.0 mg/mLCBZT) was treated with 30% H₂O₂ for 20 min in a thermostat maintained at 80ºC. Then it was cooled and then diluted with mobile phase as per the requirement and 20 µL of the solution and was analyzed by the HPLC method developed.

 

Photolytic degradation was induced by exposing drug solution (1.0 mg/mLCBZT) to UV light (365 nm) for 5hours, then diluted with mobile phase as per the requirement and 20 µL of the solution and was analyzed by the HPLC method developed.

 

Assay of Cabazitaxel:

The marketed formulation of Cabazitaxel i.e. Jevtana^® (Sanofi aventis, Malaysia) (Label claim 60 mg) is not available in India market. So the formulation was prepared with in the laboratory with the available excipients and then the drug was extracted with the mobile phase as per the method optimized. The extracted solution was further diluted as per the requirement and 20 µL of each solution was injected in to the HPLC system and the percentage recovery was calculated.

 

RESULTS AND DISCUSSION:

The main purpose of the development of new chromatographic method to obtain sufficient selectivity of the drug in a short separation time giving symmetrical well defined peaks and allowing good resolution of CBZT and its degradants within a reasonable run time.

 

Method optimization:

Initially to investigate the chromatographic separation was done on Zorbax SB C18 column (150 mm × 4.6 mm i.d., 3.5 µm particle size) as stationary phase. Several modifications of the mobile phase conditions and the flow conditions were performed in order to study the possibilities of changing the selectivity of the chromatographic system. In order to study the effect of the organic modifier ratio on selectivity retention time the mobile phase with different ratios of organic phase (60, 70 and 80 %). TBAHS: methanol (40:60, v/v) was used as the mobile phase with 0.8 mL/min^-1 as flow rate in which a broad peak was obtained which is not satisfactory. The ratio of mobile phase was then changed to 30: 70, v/vand where a sharp peak was eluted at 6.35 mins with tailing. Then the mobile phase composition was modified as 20:80, v/v where the drug peak eluted was sharp and symmetrical with short analytical retention time less than 5 mins (4.27 ± 0.03 min) and therefore the final mobile phase ratio chosen was 20:80, v/v (UV detection at 210 nm). The representative chromatogram of CBZT was shown in Fig. 3A.

 

Method Validation:

The method was validated for system suitability, linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, selectivity and robustness (ICH guidelines, 2005) and stability of the solution was also determined (ICH guidelines, 2003).

 

Linearity:

The linearity of the test method established for CBZT by standard addition method for calibration which shows over a concentration range of 0.1–200 µg/mL (Table 1) with % RSD 0.142-0.510 and the calibration curve was clearly shown in Fig 2. The linear regression equation was found to be y = 26145x -22943 with correlation coefficient 0.9997. The LOQ and LOD were found to be 0.0881 µg/mL and 0.0289 µg/mL

 

Table.1. Linearity of Cabazitaxel(CBZT)

*Mean of three replicates

 

Precision:

The intra-day precision of the method was expressed by analyzing triplicates of each at three different concentration levels (10, 50 and 100 µg/mL) on the same day. The inter-day precision was determined by conducting triplicates of each at three different concentration levels (10, 50 and 100 µg/mL) on three different days. The % RSD for intra-day precision was found to be 0.32-0.45 whereas the inter-day precision was found to be 0.97-1.12 and also with very low values of SEM (< 0.65) for both intra-day and inter-day precision (Table 2).

 

Accuracy:

The method accuracy was proved by the recovery test. A known amount of CBZT standard
(10 µg/mL) were spiked to aliquots of sample solutions and then diluted to yield the total concentrations of 18, 20 and 22 µg/mL as described in Table 4. The resultant % RSD was found to be 0.13-0.48 (<2.0 %) with very low values of SEM (<0.2723) and the % recovery was found to be 98.83-99.46% (Table 3).

 

Table.2. Precision and accuracy studies of CBZT

*Mean of three replicates

 

Table.3. Robustness study of CBZT

*Mean of three replicates

 

Forced degradation studies:

The stability indicating capability of the method was established from the separation of CBZT peak from the degraded products. Typical chromatograms obtained following the assay of stressed samples are shown in Fig. 3B-3F. A slight decomposition (< 20 %) was observed when CBZT drug was exposed to acidic (19.59 %), oxidative (4.93 %), thermal (2.79 %) and U.V (9.68 %) conditions. The drug was so sensitive towards the alkaline environment (50.79 %) and the carbonyl and hydroxyl groups may be responsible for the alkaline degradation of the drug molecule. During the acidic degradation study a degradant peak observed at 1.637 min which was separated from the drug peak with a resolution of 10.187.

 

Table.4. Forced degradation studies of CBZT

*Mean of three replicates

Table 4. continued

 

 

Fig 1: Chemical structure of Cabazitaxel (CBZT)

 

Fig 2: Calibration curve of Cabazitaxel (CBZT)

 

Fig 3: Typical chromatograms of Cabazitaxelstandard (100 μg/mL) [A]Acidic [B] Alkaline [C] Oxidative [D] Thermal [E] Photolytic [F] degradations

During the alkaline degradation study various degradant peaks were observed at 2.286, 2.031, 2.545 min and the drug peak is properly resolved with a resolution of 6.731.  In photolytic degradation study a degradant peak observed at 1.555 min without interfering with the CBZT drug peak with resolution of 10.82.

 

In oxidative degradation study an extra peak observed at 1.544 (peroxide peak) with a resolution of 10.489 from the drug peak. From the overall forced degradation studies it indicates that the method is specific with better resolution (Table 5).

 

The 3D chromatograms have been collected for the degradation studies and shown in Fig 4. In which the UV spectra of CBZT peak and its degradation peaks along with the retention time which witness the selection of the wavelength.  The peak purity evaluation data shows that there is no peak interference with CBZT peak (Fig 5). The 3D chromatograms witness the selectivity of detection wavelength and the detection of the drug and its degradation products.

The system suitability parameters for all the degradation studies were shown in Table 5. The number of theoretical plates (N) is used to determine the performance and effectiveness of the column. The efficiency of a column can be measured by the number of theoretical plates per meter. It is a measure of band spreading of a peak. Smaller the band spread, higher is the number of theoretical plates, indicating good column and system performance. The theoretical plates were found to be more than 2000, the capacity factor was found to be less than 2 and the tailing factor was less than <1.5 indicating that the method is more selective and specific.

 

The present stability-indicating method for the determination of CBZT in pharmaceutical formulations is specific as the drug peak was well separated even in the presence of degradation products with a very good resolution. It was observed that CBZT is more sensitive towards the alkaline environment during the forced degradation studies.

 

 

Fig 4: 3D chromatograms of Cabazitaxel standard (100 μg/mL) [A] Acidic [B] Alkaline [C] Oxidative [D]

Thermal [E] Photolytic [F] degradations

 

CONCLUSION:

The proposed stability-indicating HPLC method was validated as per ICH guidelines and applied for the determination of Cabazitaxel in pharmaceutical dosage

 

forms and can be successfully applied to perform long-term and accelerated stability studies of CBZT formulations.

ACKNOWLEDGEMENT:

The authors are grateful to M/s GITAM University, Visakhapatnam for providing the research facilities. The authors have no conflict of interest.

 

REFERENCES:

1.        O’Neil MJ, The Merck Index, Merck Research Laboratories, Whitehouse Station, NJ, 2006.

2.        Cheetham P, Petrylak DP, Tubulin-targeted agents including Docetaxel and Cabazitaxel. Cancer Journal, 19(1), 2013, 59-65.

3.        Assessment Report for Jevtana (Cabazitaxel), Procedure No.: EMEA/H/C/002018, European Medicines Agency, London, 2011.

4.        De Bono JS, Oudard S, Ozguroglu M, Hansen S, Machiels JP, Kocak I, Gravis G, Bodrogi I, Mackenzie MJ, Shen L, Roessner M, Gupta S, Sartor AO, Prednisone plus Cabazitaxel or mitoxantrone for metastatic castration-resistant prostate cancer progressing after docetaxel treatment: a randomised open-label trial. The Lancet, 376, 2010, 1147-1154.

5.        Zafar Iqbal Malik, Cabazitaxel: Evidence and clinical experience. British Journal of Medical and Surgical Urology, 4(1), 2011, S14–S20.

6.        Pivot X, Koralewski P, Hidalgo JL, Chan A, Goncalves A, Schwartsmann G, Assadourian S, Lotz JP, A multicenter phase II study of XRP6258 administered as a 1-h I.V. infusion every 3 weeks in taxane-resistant metastatic breast cancer patients.  Annals of Oncology. 19, 2008, 1547-1552.

7.        Mita AC, Denis LJ, Rowinsky EK, de Bono JS, Goetz AD, Ochoa L, Phase I and pharmacokinetic study of  XRP6258 (RPR 116258A), a novel taxane, administered   as a 1-h infusion every 3 weeks in patients with advanced  solid tumors. Clin. Cancer Res. 15, 2009, 723-730.

8.        Villanueva C, Awada A, Campone M, Machiels JP, Besse T, Magherini E, Dubin F, Semiond D, Pivot X, A multicentre dose-escalating study of Cabazitaxel (xrp6258) in combination with capecitabine in patients with metastatic breast cancer progressing after anthracycline and taxane treatment: A phase I/II study.European J. Cancer. 47, 2011, 1037-1045.

9.        JagannathPatro V, Nageshwara Rao R, Tripathy NK, LC-MS/MS determintion of Cabazitaxel in rat whole blood on dry blood Spots. Open Access Sci. Reports. 1, 2012, 1-4.

10.     Kort A, Hillebrand MJX, Cirkel GA, Voest EE, Schinkel AH, Rosing H, Schellens JHM, Beijnen JH, Quantification of Cabazitaxel, its metabolite docetaxel and the determination of the demethylated metabolites RPR112698 and RPR123142 as docetaxel equivalents in human plasma by liquid chromatography tandem mass spectrometry. J. Chromatogr.  B. 925, 2013, 117-123.

11.     Peter de Bruijn., Anne-Joy M de Graan, Annemieke Nieuweboer, Ron HJ Mathijssen, Mei-Ho Lam, Ronald de Wit, Erik AC Wiemer, Walter J Loos,. Quantification of Cabazitaxel in human plasma by liquid chromatography/ triple-quadrupole mass spectrometry: A practical solution for non-specific binding. J. Pharm. Biomed. Anal. 59 2012, 117–122.

12.     Mathrusri Annapurna M, Pramadvara K, Venkatesh B, Sowjanya G, Stability-indicating RP-HPLC method for the determination of Cabazitaxel. Indo American J. Pharm. Res. 3(11), 2013, 9262-9269.

13.     Mathrusri Annapurna M, Venkatesh B, Naga Supriya G, A validated stability-indicating liquid chromatographic method for determination of Cabazitaxel -A novel microtubule inhibitor. J. Bioequivalence andBio Availability, 6(4), 2014, 134-138.

14.     Mathrusri Annapurna M, Venkatesh B, Pramadvara K and Hemchand S, Development and validation of a stability-indicating liquid chromatographic method for the assay of Cabazitaxel. Chemical Science Transactions, 3(2), 2014, 854-860.

15.     Chengyan Li, Gongjian Lan, Jinyuan Jiang, Mingjie Sun, Taijun Hang, Development and validation of a stability-indicating HPLC method for the determination of the impurities in Cabazitaxel. Chromatographia, 78, 2015, 825-831.

16.     Gudisa Kishore, New spectrophotometric methods for the quantitative estimation of Cabazitaxel in formulations. Int. J. Res. Rev. Pharm. Appl. sci. 2, 2012, 950-958.

17.     ICH validation of analytical procedures: text and methodology Q2 (R1), International Conference on Harmonization, 2005.

18.     ICH stability testing of new drug substances and products Q1A (R2), International Conference on Harmonization, 2003.

 

 

 

Accepted on 29.04.2017           © RJPT All right reserved