New Validated Stability-Indicating RP-HPLC Method for The Determination of Docetaxel and Its Related Substances

 

S. Hemchand*¹, R. Ravi Chandra Babu¹, Mukthinuthalapati Mathrusri Annapurna²

¹GITAM Institute of Science, GITAM (Deemed to be University), Visakhapatnam, India                         

²GITAM Institute of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, India

 

ABSTRACT:

Docetaxel is used for the treatment of castration resistant prostate cancer. Docetaxel is a semi synthetic chemo therapeutic agent extracted from the leaves of Taxus baccata. Two process related substances and four impurities were separated and quantified using Sunfire C18 column (250 x 4.6 mm, 5µm particle size) on gradient mode (40 ± 5°C). A mixture of acetonitrile: 0.01 % Acetic acid was used as mobile phase and a mixture of acetonitrile: methanol (1:1, v/v) was used as diluent and the compounds were monitored at 230 nm. Forced degradation studies were performed and the method was validated as per ICH guidelines.

 

 

INTRODUCTION:

Docetaxel is a white crystalline powder (C₄₃H₅₃NO₁₄) with molecular weight 807.8 g/mol and it is slightly soluble in water. Docetaxel (Figure 1) is a butoxy carbonyl amino-2-hydroxy-3-phenylpropionate derivative,  extracted from the leaves of Taxus             baccata^1-2. Docetaxel acts by two mechanisms - Inhibition of micro tubular depolymerization and Attenuation of the effects of bcl-2 and bcl-xL gene expression. Taxane-induced microtubule stabilization arrests cells in the G (2) M phase of the cell cycle and induces bcl-2 phosphorylation, thereby promoting a cascade of events that ultimately leads to apoptotic cell death³. Docetaxel is approved by the US FDA for castration resistant prostate cancer treatment. It acts against a variety of tumors ^4-6. Due to excessive activation of PI3K/AKT signaling in castration resistant prostate cancer cells ^7-8 resistance is developed against Docetaxel.

 

Figure 1: Chemical structure of Docetaxel

 

Docetaxel was determined in biological samples^9-14 using liquid chromatographic methods and mass spectral techniques. Rao et al., published one stability indicating method¹⁵ and Naresh et al established an assay method for parenterals¹⁶. Dev et al., developed a mass spectroscopic technique for the determination of Docetaxel and the impurities were isolated by using Medium Pressure Liquid Chromatography¹⁷ (MPLC) and characterized by using NMR and FTIR. In the present study the authors have proposed a gradient liquid chromatographic method for the assay of Docetaxel and its related substances along with four impurities using Waters Alliance 2695 series HPLC system with 2998 photodiode array detector. Forced degradation studies were performed and they were quantified using the established method and the method was validated as per ICH guidelines^18-19. The prominent features of the previously published methods were compared with the present proposed method in Table 1.

 

Table 1: Review of published methods

 

MATERIALS AND METHODS:

Accurately about 10 mg of Docetaxel anhydrous was weighed and transferred in to a 10 mL volumetric flask. Add 7 mL of diluent (Acetonitrile: Methanol) (1:1, v/v) and sonicated for 1min to dissolve and dilutee to the mark with diluent and mixed and the solution is stable for 3 days at 2-8°C temperature.

 

Chromatographic conditions

Docetaxel and its process related substances such as 2’-Epi-Docetaxel and 10 Deacetyl baccatin-III along with Impurity A, Impurity B, Impurity C, Impurity D were separated on gradient mode and quantified using Sunfire C18 column (250 x 4.6 mm, 5µm particle size) column for separation and quantification using a mixture of Acetonitrile and 0.01% Acetic acid with flow rate of 1.2 mL/min (at 27°C). Methanol: water (1:1) was used as diluent. Waters Alliance 2695 series HPLC system with 2998 photodiode array detector and the detector was monitored at 230 nm. The gradient program was given in Table 2.

 

Table 2: Gradient program

 

Method validation¹⁸

Linearity, Precision, Accuracy and Robustness

Linearity solutions were prepared with concentration LOQ to 0.60% w.r.t working conc. spiked with all four impurities and 10 µl was injected in to the system and the calibration curves were drawn. For method precision study six replicate sample solutions of Docetaxel at a concentration of 1.0 mg/mL in diluent containing 0.10% of Impurity D; 0.3% of Impurity B and Impurity C; 0.5% of Impurity A w.r.t. the sample concentration was prepared. Each spiked sample solution was injected once and calculated the % RSD for content of Impurity A, Impurity B, Impurity C, Impurity D, Single unknown impurity and total impurities. Intermediate precision of the method can be determined on different day, by different analyst, different lots of reagents, different column and using different equipment. Accuracy study was performed by spiking the Docetaxel solution with the related substance and there by the percentage recovery values were calculated. Three different sample solutions (1.0 mg/mL) of Docetaxel containing Impurity A, Impurity B, Impurity C and Impurity D at LOQ level were prepared at four concentration levels i.e. 50%, 100%, 120% and 150% w.r.t. the specification limit of working concentration and injected each solution once.  From the corrected area of Impurity A, Impurity B, Impurity C and Impurity D, % recovery was calculated. Robustness of the method was evaluated by deliberately altering the method conditions from the original method parameters and verifying compliance of the system suitability requirements.

 

Forced degradation studies¹⁹

The HPLC method used for the determination of assay content and related substances of Docetaxel anhydrous, is proposed for assigning degradation profile of Docetaxel in acid, base stress condition using HCl and NaOH and oxidative stress using 30% H₂O₂ Saturated Cu(OAC)₂0.2M Cu(OAC)₂ and 0.2M KMnO₄.

 

Acid hydrolysis

50.26 mg of Docetaxel sample weighed accurately and 2.5 mL of 1N HCl was transferred in to a 50 mL volumetric flask, diluted to volume with diluent and then mixed well.  This solution was kept for 6 hrs at room temperature and then injected in to the system.

 

Base hydrolysis

50.45 mg of Docetaxel sample was weighed accurately, transferred into 50 mL of volumetric flask dissolved in 10 mL of diluent and then 0.25 mL of 0.1N NaOH solution was added, diluted with diluent and mixed well.  This solution was kept for 6 hrs at room temperature and then injected in to the system.

 

Oxidation

Docetaxel was subjected to oxidation using H₂O₂, saturated Cu(OAC)₂ solution, 0.2M Cu(OAC)₂ solution and 0.2M KMnO₄ solution for identifying the impurities in different environments.

 

52.77 mg of Docetaxel sample was weighed, 5 mL diluent was added and mixed, diluted to volume with 30% H₂O₂. This solution was refluxed for 4 hrs at 60°C temperature and then injected in to the system.

 

48.05 mg of Docetaxel sample was weighed, and 2.0 mL of saturated Cu(OAC)₂ solution was added in a 50mL volumetric flask, diluted to volume with diluent and mixed. This solution was kept for 1 hour at room temperature and then injected in to the system. Saturated solution of Cu(OAC)₂ was prepared in methanol and acetonitrile mixture (50:50, v/v).

 

50.17 mg of Docetaxel sample was weighed, and 2.0 mL of 0.2M Cu(OAC)₂ solution was added in a 50mL volumetric flask, diluted to volume with diluent and mixed. This solution was kept for 30 min at room temperature and then injected in to the system.

 

50.29 mg of Docetaxel sample was weighed, and 2.0 mL of 0.2M KMnO₄ solution was added in a 50mL volumetric flask, diluted to volume with diluent and mixed. This solution was kept for 30 min at room temperature and then injected in to the system.

 

Assay of Docetaxel injection

Docetaxel is available with brand names Docecad (Cadila Pharmaceuticals Ltd., India) and Docetere (Dr. Reddy’s Laboratories, India) (Label claim: 20 mg/0.5 ml, 80 mg/2 ml and 120 mg/3 ml) Taxotere (Sanofi Aventis Pharma, India) (Label claim: 20 mg/1 ml and 80 mg/4 ml) as solution for injection. Gift samples of Docetaxel was obtained from Dr. Reddy’s Laboratories, India.

 

RESULTS AND DISCUSSION:

A stability indicating RP-HPLC (gradient mode) method was developed and validated for the separation and quantification of Docetaxel and its related substances using Sunfire C18, (250 x 4.6 mm, 5 µm) column with flow rate 1.2 ml/min within a run time of 60 mins. The injection volume was 10 µL and the column temperature was 40 ± 5°C and the Sample solutions were maintained at 5 ± 3°C.

 

Method validation

Good linearity response was obtained for Docetaxel, Impurity A, Impurity B, Impurity C and Impurity D over the concentration ranges of LOQ to 0.60% w.r.t. the working concentration (Table 3). Correlation coefficients for Docetaxel, Impurity A, Impurity B, Impurity C and Impurity D were 0.9999, 0.9991, 0.9999, 0.9999 and 1.0000 respectively (Table 4). The RRF, LOD and LOQ (Figure 2) for Docetaxel, Impurity A, Impurity B, Impurity C and Impurity D were calculated by slope ratio (Docetaxel to Impurity) method. The Relative response factor will be considered as 1.68 for Impurity A, 1.05 for Impurity B, 1.07 for Impurity C and 0.83 for Impurity D for routine analysis and in validation calculations. The RSD at LOQ level for Docetaxel, Impurity A, Impurity B, Impurity C and Impurity D is found to be 5.8%, 5.5%, 8.3%, 11.0% and 9.4% respectively indicating that it passed the acceptance criteria and the method is precise (Table 5). The RSD of peak areas at LOQ level should not be more than 15.0% for each analyte.

 

LOD for Impurity A

LOQ for Impurity A

LOQ for Docetaxel, Impurity B, Impurity C and Impurity D

Figure 2: Chromatograms observed for Docetaxel, Impurity B, Impurity C and Impurity D(LOQ and LOD)

In method precision the RSD of Impurity A, Impurity B, Impurity C, Impurity D, Single unknown impurity and total impurities from the six sample preparations was found to be 1.6%, 2.0%, 3.3%, 0.0%,0.0% and 0.0% respectively indicates that the acceptance criteria (Less than 10%) was satisfactory. In intermediate precision the RSD of Impurity A, Impurity B, Impurity C, Impurity D, Single unknown impurity and total impurities from the six sample preparations was found to be 1.3%, 2.0%, 1.2%, 4.3%, 0.0% and 0.0% respectively indicates that the acceptance criteria (Less than 10%) was satisfactory (Table 6). Accuracy study was performed at LOQ level (Table 7) and the recovery obtained was 100.0% - 107.6% for Impurity A, 91.7% -94.4% for Impurity B, 85.3% - 90.9% for Impurity C and 107.5% - 116.3% for Impurity D. The RSD of the recovery obtained in the range of 0.9%-2.6% (Table 8) and the acceptance criteria is < 10.0%. The effect of flow rate, column temperature, mobile phase composition on system suitability were summarized in Table 9. Docetaxel was well separated from its related substances and proven that the method is specific (Figure 3). The USP resolution between 2’-Epi-Docetaxel and Docetaxel should not be less than 1.3 and that of the tailing factor should not be more than 1.5 for Docetaxel peak.

 

 

Table 3: Linearity of Docetaxel and its related substances (LOQ to 0.60% w.r.t working conc.)

 

Table 4: Linearity (Regression equations) of related substances

 

Table 5: Precision study of Docetaxel and its related substances (LOQ to 0.60 % w.r.t. working Conc.)

Table 6A: Method Precision study of Impurities

 

Table 7: Accuracy at LOQ level of related substances

 

Table 8: Accuracy of related substances at LOQ level

 

Table 9: Robustness of Docetaxel and its related substances

 

Chromatogram of Docetaxel with its related substances

Lower flow rate (1.1 mL/min)

Higher flow rate (1.3 mL/min)

Higher column temperature (45°C)

Lower column temperature (35°C)

Mobile phase variation (Lower Organic)

Mobile phase variation (Higher Organic)

Figure  3: Robustness study of Docetaxel and its related substances

 

          

Forced degradation studies

Docetaxel was subjected to forced degradation studies and the assay results along with the peak purity and purity threshold values were shown in Table 10 and the resultant chromatograms obtained during the degradation study were shown in Figure 4. Assay of Docetaxel is being decreased by 5.6% from the initial value after 6 hours in 0.05N HCl stressed condition. In related substances, Impurity B and single unknown impurity at relative retention time (RRT) 0.15 increased by 0.25% and 2.83% from initial respectively. Assay of Docetaxel is being decreased by 9.1% from the initial value after 6 hours in 0.0005N NaOH stressed condition. In related substances, 10- Deacetyl baccatin- III, Impurity C and single unknown impurity at RRT-0.42 increased by 1.72%, 8.04 and 0.15% from initial respectively. Assay of Docetaxel increased by 2.9% from the initial value after 4 hours reflux in 30% H₂O₂ stressed condition. In related substances, Impurity B and single unknown impurity at RRT-0.67 increased by 0.13% and 0.11% from initial respectively. Assay of Docetaxel decreased by 57.7% from the initial value after 1 hour in saturated Cu(OAC)₂ stressed condition. In Related substances, Impurity B, Impurity C, Impurity D and single unknown impurity at RRT-0.34 increased by 32.08%, 0.45%, 17.83% and 3.62% from initial respectively. Assay of Docetaxel decreased by 4.2% from the initial value after 30 minutes in 0.2M Cu(OAC)₂ stressed condition. In Related substances, Impurity B, Impurity C and Impurity D increased by 2.24%, 0.03% and 0.08% from initial respectively. Assay of Docetaxel decreased by 13.9% from the initial value after 30 minutes in 0.2M KMnO₄ stressed condition. In Related substances, 10-Deacetyl baccatin III, Impurity B, Impurity C, Impurity D and single unknown impurity at RRT-0.62 increased by 0.74%, 0.12%, 10.18%, 0.87% and 0.60% from initial respectively. The peak purity of Docetaxel 10-Deacetyl baccatin III, Impurity B, Impurity C and Impurity D was less than the peak threshold (Figure 5).

 

Figure 4A: Chromatogram of Blank

Figure 4B: Chromatogram of Docetaxel (As such sample)

Figure 4C: Chromatogram of 0.05N HCl  blank

Figure 4D: Chromatogram of Docetaxel during acidic degradation (0.05N HCl)

Figure 4E: Chromatogram of 0.0005 NaOH blank

Figure 4F: Chromatogram of Docetaxel during basic degradation (0.0005 NaOH)

Figure 4G: Chromatogram of 30% H2O2 blank

Figure 4H: Chromatogram of Docetaxel during oxidative degradation (30% H2O2/reflux 4 hrs)

Figure 4I: Chromatogram of Saturated Cu(OAC)2 blank

Figure 4J: Chromatogram of Docetaxel during oxidative degradation (Saturated Cu(OAC)2)

Figure 4K: Chromatogram of Docetaxel during oxidative degradation (0.2M Cu(OAC)2 )

Figure 4L: Chromatogram of Docetaxel during oxidative degradation (0.02M KMnO4)

Table 10: Degradation results of Docetaxel Degradation condition Assay (%) Total impurities (%) Purity angle Purity threshold Docetaxel 98.4 0.22 0.140 0.655 Acidic degradation (0.05N HCl) 92.6 4.15 0.146 0.560 Basic degradation (0.0005N NaOH) 89.1 10.30 0.105 0.487 Oxidative degradation 30% H2O2, reflux 4 hrs, 60°C Saturated Cu(OAC)2, 1 hr, room temp. 0.2M Cu(OAC)2, 30 min, room temp. 0.2M KMnO4, 30 min, room temp.   101.3 40.7 94.0 84.5   0.51 54.14 2.56 13.18   0.060 0.126 0.152 0.171   0.298 0.335 1.012 0.413 ** If purity angle < purity threshold, the peak is considered spectrally pure

Purity plot of Docetaxel (As such sample)
Purity plot of Docetaxel (0.05N HCl)	Purity plot of Docetaxel (0.0005N NaOH)
Purity plot of Docetaxel (30% H2O2/reflux 4hrs)	Purity plot of Docetaxel (Saturated Cu(OAC)2)
Purity plot of Docetaxel (0.2M Cu(OAC)2)	Purity plot of Docetaxel (KMnO4)
Purity plot of Impurity C (0.0005N NaOH)	Purity plot of 10-Deacetyl baccatin III (0.0005N NaOH)
Purity plot of Impurity B (30% H2O2/reflux 4hrs)	Purity plot of Impurity B (Saturated Cu(OAC)2)
Purity plot of Impurity D (Saturated Cu(OAC)2)	Purity plot of Impurity C (Saturated Cu(OAC)2)
Purity plot of Impurity C (0.2M Cu(OAC)2)	Purity plot of Impurity A (0.2M Cu(OAC)2)
Purity plot of Impurity B (0.2M Cu(OAC)2)	Purity plot of Impurity D (0.2M Cu(OAC)2)
Purity plot of Impurity C (KMnO4)	Purity plot of Impurity D (KMnO4)
Figure 5: Purity plots of Docetaxel during forced degradation studies

 

Assay of Docetaxel

Docetaxel injections of two different brands (Cadila Pharmaceuticals Ltd., India and Dr. Reddy’s Laboratories, India) (Label claim: 20 mg/0.5 ml) were analyzed and found that Docetaxel has shown 99.65-99.85 purity and there is no interference of excipients (Table 11).

 

_{Table 11: Assay of} Docetaxel _injection

* Mean of three replicates

 

Specificity

Specificity of the method was determined by injecting the analyte spiked with all the known components expected to be present in the drug substance. Separate solutions of diluent, Docetaxel, known impurities such as Impurity A, Impurity B, Impurity C and Impurity D along with other related substances 2’-Epi-Docetaxel and 10- Deacetyl baccatin- III was injected the resolution, retention time, relative response factor were shown in Table 12 and the corresponding chromatograms and peak purity plots were shown in Figure 6 and Figure 7.

 

 

Table 12: Specificity

 

Docetaxel (As such solution)	Docetaxel (Specificity solution)
Impurity A	Impurity B
Impurity C	Impurity D
2’-Epi-Docetaxel	10-Deacetyl baccatin-III
Figure 7: Purity plots of Docetaxel and its related substances (Specificity)

 

CONCLUSIONS:

A simple and new gradient stability indicating RP-HPLC method was developed for the determination of Docetaxel and its related substances. The method was validated (ICH guidelines) by linearity, precision, accuracy and robustness and this method is highly helpful for the identification and quantification of impurities and related substances in injections and the proposed method is specific and the system suitability parameters are within acceptable criteria.

 

ACKNOWLEDGEMENT:

The authors are grateful to Dr. Reddy’s Laboratories, India for providing the gift samples of Docetaxel and its related substances. The authors declare no conflict of interest.

 

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Accepted on 06.08.2019          © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(9):4165-4181.