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Journal of Bioequivalence & Bioavailability
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A Validated Stability-indicating Liquid Chromatographic Method for Determination of Cabazitaxel-A Novel Microtubule Inhibitor

Mathrusri Annapurna Mukthinuthalapati1*, Venkatesh Bukkapatnam1 and Naga Supriya Grandhi2

1Department of Pharmaceutical Analysis & Quality Assurance, GITAM Institute of Pharmacy, GITAM University, Visakhapatnam, India

2Roland Institute of Pharmaceutical Sciences, Berhampur, Orissa

*Corresponding Author:
Mathrusri Annapurna Mukthinuthalapati
Department of Pharmaceutical Analysis & Quality Assurance
GITAM Institute of Pharmacy
GITAM University, Visakhapatnam, India
E-mail: [email protected]

Received Date: June 23, 2014; Accepted Date: July 28, 2014; Published Date: August 06, 2014

Citation: Mukthinuthalapati MA, Bukkapatnam V, Grandhi NS (2014) A Validated Stability-indicating Liquid Chromatographic Method for Determination of Cabazitaxel-A Novel Microtubule Inhibitor. J Bioequiv Availab 6: 134-138. doi: 10.4172/jbb.10000193

Copyright: © 2014 Mukthinuthalapati MA. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Cabazitaxel is used to treat advanced prostate cancer. Cabazitaxel is a semi-synthetic derivative of the natural taxoid 10-deacetylbaccatin III with potential antineoplastic activity. Cabazitaxel is also known as XRP6258, a semisynthetic taxane from a single diastereoisomer of 10-deacetyl baccatin III derived from the needles of various Taxus species. A stability-indicating high performance liquid chromatographic technique was developed for the determination of Cabazitaxel in bulk and pharmaceutical formulations. Chromatographic separation was performed on Shimadzu Model CBM-20A/20 Alite, using Zorbax SB-C18 column (150 mm×4.6 mm i.d., 3.5 μm particle size) with a mixture of 0.1% ortho phosphoric acid and methanol (20:80, v/v) as mobile phase with a flow rate of 1.0 ml/ min. Cabazitaxel was subjected to stress conditions (acidic, alkaline, oxidation photolytic and thermal degradations and the method were validated as per ICH guidelines.

Keywords

Cabazitaxel; RP-HPLC; Stability-indicating; ICH

Introduction

Abazitaxel is used to treat advanced prostate cancer that is no longer responding to hormone therapy. It is also being studied for use against other kinds of cancer. It interferes with microtubules, which are part of the internal structure that cells need when they are dividing. This leads to cell death. Because cancer cells divide faster than normal cells, they are more likely than normal cells to be affected by this drug. Cabazitaxel is a semi-synthetic derivative of a natural taxoid used for the treatment of hormone-refractory prostate cancer [1]. Unlike other taxane compounds, this agent is a poor substrate for the membraneassociated, multidrug resistance (MDR), P-glycoprotein (P-gp) efflux pump and may be useful for treating multidrug-resistant tumors. Cabazitaxel binds to and stabilizes tubulin, resulting in the inhibition of microtubule depolymerization and cell division, cell cycle arrest in the G2/M phase, and the inhibition of tumor cell proliferation.

Cabazitaxel has been approved in the US by the Food and Drug Administration (FDA) [2] in June 2010 and in Europe by the European Medicines Agency (EMA) in January 2011 in combination with prednisone for the treatment of patients with castration resistant metastatic prostate cancer whose disease progresses after docetaxel treatment [3], based on the results of the TROPIC trail investigating Cabazitaxel plus prednisone versus mitoxantrone plus prednisone following docetaxel failure [4]. Cabazitaxel chemically known as (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS-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- methanocyclodeca benzo [1,2-b] oxet-12-yl benzoate is chemotherapy drug with molecular formula C45H57NO14 and molecular weight 835.93 g/mol (Figure 1) [5,6]. Cabazitaxel penetrates the blood-brain barrier. Cabazitaxel is currently being investigated in the setting of metastatic breast cancer progressing after taxane or anthracycline based chemotherapeutic regimens [7,8].

bioequivalence-bioavailability-chemical-structure-cabazitaxel

Figure 1: Chemical structure of Cabazitaxel.

Very few analytical methods have been reported for the determination of Cabazitaxel such as spectroscopic techniques [9], HPLC [10,11], LC-MS/MS [12-14] in biological fluids. At present the authors have proposed a stability indicating RP-HPLC method for the determination of Cabazitaxel in presence of its degradation products.

Experimental

Chemicals and reagents

Cabazitaxel standard (purity 99%) was obtained from Dr. Reddy’s Laboratories Ltd. (Visakhapatnam, India). Methanol (HPLC grade), Polysorbate 80, Ethanol (HPLC grade), Sodium hydroxide (NaOH) and Hydrochloric acid (HCl), orthophosphoric acid and Hydrogen peroxide (H2O2) were purchased from Merck (India). All chemicals were of analytical grade and used as received.

Cabazitaxel is available as infusion with brand name Jevtana® (Sanofi-Aventis, Malaysia) with label claim of 60 mg of drug (60 mg cabazitaxel in 1.5 mL polysorbate 80 with diluent approximately 5.7 mL of 13% (w/w) ethanol in water for injection).

Instrumentation

Chromatographic separation was achieved by using Zorbax SBC18 column (150 mm×4.6 mm i.d., 3.5 μm particle size) for HPLC system of Shimadzu Model CBM-20A/20 Alite, equipped with SPD M20A prominence photodiode array detector (maintained at 25 ºC) and LC Solutions 1.25 software.

Chromatographic conditions

Isocratic elution was performed using 0.1% orthophosphoric acid: methanol (20:80, v/v) as mobile phase. The overall run time was 10 min. with flow rate 1.0 ml/min with UV detection at 210 nm. 20 μL of sample was injected into the HPLC system.

Preparation of stock solution

Cabazitaxel stock solution (1000 μg/ml) was prepared by weighing accurately 25 mg of Cabazitaxel in a 25 ml volumetric flask with mobile phase. Working standard solutions were prepared on daily basis from the stock solution with mobile phase and filtered through 0.45 μm membrane filter prior to injection.

Method validation

The method was validated for system suitability, linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, selectivity and robustness [15].

Linearity test solutions for the assay method were prepared from a stock solution at different concentration levels (0.1–200 μg/ml) of the assay analyte concentration and 20 μL of each solution was injected in to the HPLC system and the peak area of the chromatogram obtained was noted. The calibration curve was plotted by taking the concentration on the x-axis and the corresponding peak area on the y-axis. The data was treated with linear regression analysis method.

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 guidelines Q2 (R1) [15].

Precision was evaluated at three concentration levels (20, 50 and 100 μg/ml) against the qualified reference standard. The intra-day precision study was carried out by doing 3 independent assays for each concentration of Cabazitaxel and the mean as well as the % RSD were calculated for each concentration. The inter-day precision study was performed on three different days i.e. day 1, day 2 and day 3 at three different concentration levels (20, 50 and 100 μg/ml) and each value is the average of three determinations. The% RSD of three obtained assay values on three different days was calculated.

Standard addition and recovery experiments were conducted to determine the accuracy of the method. The study was carried out in triplicate at three different levels (80, 100 and 120%) and the percentage recovery was calculated. The accuracy study was performed by adding known amount of the standard (8, 10 and 12 μg/ml) to the samples which was prepared in our laboratory using the excipients. The study was carried out in triplicates and the percentage recoveries were calculated according to table 3.

The robustness of the assay method was established by introducing small changes in the HPLC conditions which included wavelength (208 and 212 nm), percentage of methanol in the mobile phase (78 and 82%) and flow rate (0.9 and 1.1 ml/min) and each value (Table 4) is the average of three determinations. Robustness of the method was studied for 100 μg/ml of Cabazitaxel.

As the marketed formulations were not available in India the drug was mixed with some of the common excipients used in the laboratory and extracted with the mobile phase. The percentage recovery was calculated from the calibration curve.

Forced degradation studies

Forced degradation studies were performed to evaluate the stability indicating properties and specificity of the method [16]. All solutions for stress studies were prepared at an initial concentration of 1 mg/ml of Cabazitaxel and refluxed for 30 min at 60ºC and then diluted with mobile phase. 1.0 mg/ml Cabazitaxel solution was exposed to acidic degradation with 0.1 M HCl for 20 min at 60ºC the stressed sample was cooled, neutralized and diluted with mobile phase. Similarly stress studies were conducted in alkaline conditions with 0.01 M NaOH at 60ºC for 20 min. and neutralized after cooling with proper dilution with mobile phase. Oxidative stress studies were performed using 30% H2O2 and thermal stress studies were conducted in thermostat at 60ºC for 20 min. 20 μL solution of each of these solutions which were exposed to forced degradation studies were injected in to the HPLC system and the chromatograms were recorded from which the percentage recovery as well as the degradants were studied.

Results and Discussion

Method optimization

Initially the stressed samples were analyzed using a mixture of 0.1% ortho phosphoric acid: methanol (45:55, v/v) with a flow rate of 1.0 ml/min in which the peak shape was not symmetrical. Then the mobile phase composition was slightly modified with 0.1% ortho phosphoric acid: methanol (20:80, v/v) where a sharp peak was eluted at 4.241 ± 0.02 min without tailing (UV detection 210 nm) which was taken as the best chromatographic response for the entire study. The typical chromatogram of the blank as well as the drug solution was shown in Figure 3 respectively. The performance characteristics of the present stability indicating liquid chromatographic method was compared and discussed with the previously published methods in Table 1.

bioequivalence-bioavailability-calibration-curve-cabazitaxel

Figure 2: Calibration curve of Cabazitaxel.

bioequivalence-bioavailability-chromatograms-cabazitaxel-oxidative

Figure 3: Typical chromatograms of blank [A], Cabazitaxel (100 μg/ml) [B], acidic [C], alkaline [D], oxidative [E], thermal [F] and photolytic [G] degradations.

Mobile phase/Reagent λ(nm) Linearity(μg/ml) Remarks Reference
Phosphate buffer: Acetonitrile (30:70, v/v) 230 0.1-150 HPLC [10]
Sodium acetate buffer: Acetonitrile (30:70, v/v) 234 0.1-250 HPLC [11]
Ammonium hydroxide and methanol (83:17, v/v) (pH 3  ±  0.1) 275 2-20 LC-MS [12]
Acetonitrile: Ammonium acetate (80:20, v/v) 236 2.49-99.60 (LC-MS/MS) (dried blood spots) [13]
Acetonitrile: Ammonium formate (gradient) 362 (10-100) 103 (LC-MS) Human plasma [14]
o-phosphoric acid : Methanol (20: 80, v/v) 210 0.1-200 Stability indicating HPLC (PDA detector) Present work

Table 1: Comparison of the previously published liquid chromatographic methods with the present method.

Method validation

Cabazitaxel has shown linearity 0.1-200 μg/ml (Table 2) (% RSD 0.11-0.54) with linear regression equation

Conc. (mg/ml) *Mean peak area ± SD *RSD (%)
0.1 2552 ± 587 0.23
1 22629 ± 70.15 0.31
5 111635 ± 112.80 0.11
10 207597 ± 871.91 0.42
20 410587 ± 1149.64 0.28
50 1085699 ± 4885.65 0.45
100 2109177 ± 4218.35 0.20
150 3156873 ± 9786.31 0.31
200 4198887 ± 22673.99 0.54

Table 2: Linearity of Cabazitaxel.

y=21017x+3853.9 (r2=0.9999)

The LOQ was found to be 0.0967 μg/ml and the LOD was found to be 0.0319 μg/ml (Figure 2).

The % RSD range was obtained as 0.23-1.43 and 0.21-0.51 for intraday and inter-day precision studies respectively and 98.95-99.43% of recovery was observed in the accuracy studies with % RSD 0.28-0.60 (<2.0%) (Table 3) indicating that the method is precise and accurate.

Conc. (μg/ml) Intra-day precision Inter-day precision
*Mean peak area± SD (% RSD) *Mean peak area ± SD (% RSD)
20 410508.33 ± 961.20 (0.23) 410331.33 ± 867.24  (0.21)
50 1069037.33 ± 15310.37 (1.43) 1084725.33 ± 5525.22 (0.51)
100 2114961.00 ± 9778.37 (0.46) 2110788.33 ± 5377.64 (0.25)
Accuracy
Spiked conc. (μg/ml) Total conc. (μg/ml) Mean peak area ±  SD (% RSD) Drug Found (μg/ml) % Recovery*
8 (80 %) 18 378301.00 ± 1042.36 (0.28) 17.82 98.98
10 (100 %) 20 421803.00 ± 1587.67 (0.38) 19.89 99.43
12 (120 %) 22 461385.33 ± 2765.64 (0.60) 21.77 98.95

Table 3: Precision and accuracy studies of Cabazitaxel.

The robustness of an analytical procedure refers to its ability to remain unaffected by small and deliberate variations in method parameters and provides an indication of its reliability for routine analysis. The results obtained (Table 4) from assay of the test solutions were not affected by varying the conditions and were in accordance with the results for original conditions. The % RSD was less than 2.0% (0.19-1.66) indicating that the proposed method is robust.

Parameter Condition *Mean peak area ± SD (% RSD) *Assay (%)
Flow rate
(± 0.1 ml/min)
0.9
1.0
1.1
2102325.00 ± 14560.41 (0.69) 99.86
Detection
wavelength
(± 2 nm)
208
210
212
2103174.33 ± 3932.91 (0.19) 99.90
Mobile phase
composition
(± 2 %, v/v)
18:82
20:80
22:78
2093672.33 ± 34806.61(1.66) 99.45

Table 4: Robustness study of Cabazitaxel.

As the marketed formulation was not available the drug was mixed with different excipients available in the laboratory and then extracted using the mobile phase. The proposed method was applied to the laboratory prepared formulation and the percentage recovery was calculated as 97.84.

Forced degradation studies

The stability indicating capability of the method was established from the separation of Cabazitaxel peak from the degraded samples. The typical chromatograms obtained from the forced degradation studies were shown in Figure 3. Cabazitaxel has shown 19.48% degradation with degradant at 1.736 min during acidic stress indicating that the drug is sensitive towards acidic environment. The amino group present in the drug structure may be highly responsible for this degradation. Cabazitaxel has undergone complete decomposition with degradants 1.667 min, 1.992 min and 2.571 min at during alkali stress indicating that the drug is very much sensitive towards alkaline condition. In other degradations the drug has undergone decomposition slightly (<15.0%). Decomposition was seen on exposure of Cabazitaxel solution to acidic (19.48%), alkaline (99.72%), oxidative (0.17%) thermal (1.03%) and photolytic (3.02%) conditions (Table 5). It is concluded that Cabazitaxel is very much sensitive towards alkaline conditions.

Stress Condition *Mean peak area *Drug recovered (%) *Drug decomposed (%) Theoretical plates Tailing factor
Standard (Untreated) 2109177 100 - 3517.939 1.064
Acidic 1698271 80.52 19.48 3392.511 1.039
Alkaline 5807 0.28 99.72 - -
Oxidative 2105689 99.83 0.17 3415.160 1.038
Thermal 2087474 98.97 1.03 3521.359 1.053
Photolytic 2045504 96.98 3.02 3477.122 1.049

Table 5: Forced degradation studies of Cabazitaxel.

The present stability-indicating method for the determination of Cabazitaxel in pharmaceutical formulations is specific because the drug peak was well separated even in the presence of degradation products. The system suitability parameters for the Cabazitaxel peak shows that the theoretical plates were more than 2000 and the tailing factor was less than 2 (or <1.5-2.0) (Table 5).

Conclusion

The proposed stability-indicating liquid chromatographic method for the determination of cabazitaxel is economical as acetonitrile was replaced with methanol and the method can be applied for the assay of pharmaceutical dosage forms. Cabazitaxel is very much sensitive towards the alkaline conditions and it can be very easily undergoes decomposition.

Acknowledgements

The authors are grateful to University Grants Commission, New Delhi for their financial support and to M/s GITAM University, Visakhapatnam for providing the research facilities.

References

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