Stress Degradation Studies on Dutasteride and Development of a Stability-Indicating HPLC Assay Method for Bulk Drug and Pharmaceutical Dosage Form 2008, 67, 841–845
D. V. Subba Rao1,2,&, P. Radhakrishnanand1,2 1
2
Reference Standard Laboratory, United States Pharmacopeia-India Private Limited, ICICI Knowledge Park, Turkapally, Shameerpet, Hyderabad 500 078, India; E-Mail:
[email protected] Department of Chemistry, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad 500 072, India
Received: 2 October 2007 / Revised: 25 December 2007 / Accepted: 30 January 2008 Online publication: 17 April 2008
Abstract A simple stability-indicating LC method has been developed for the quantitative determination of dutasteride in bulk drug samples and in pharmaceutical dosage forms in the presence of degradation products. The retention time of dutasteride is about 7 min. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Degradation was found to occur under hydrolysis and to a lesser extent under oxidation conditions but the compound was stable to photolytic and thermal stress. The assay of stress samples was calculated against a reference standard and the mass balance was found close to 99.3%. The developed method was validated with respect to linearity, accuracy, precision and ruggedness.
Keywords Column liquid chromatography Stability-indicating assay Stress studies Dutasteride
Introduction Dutasteride, (5a, 17b)-N-(2,5-bis(trifluoromethyl)phenyl)-3oxo-4-azaandrost-l-ene17-carboxamide (Fig. 1), is a potent and specific dual 5 alpha-reductase inhibitor for the treatment of benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) [1, 2]. It was
Limited Short Communication DOI: 10.1365/s10337-008-0584-8 0009-5893/08/05
approved in October 2002 by USFDA and has been approved in several countries [3, 4]. Dutasteride inhibits the conversion of testosterone to 5a-dihydro testosterone (DHT) [2]. DHT is the androgen primarily responsible for the initial development and subsequent enlargement of the prostate gland. DHT is converted
from testosterone by the enzyme 5a-reductase, which exists as two isoforms, Type 1 and Type 2. Type 1 5a-reductase is found primarily in the skin and liver, but has also been found in prostatic tissue in BPH. Type 2 5a-reductase is found in the prostate [2]. Rapid liquid chromatography– tandem mass spectrometry assay of dutasteride in human plasma [5] and mass spectral fragmentation reactions of a therapeutic 4-azasteroid and related compounds [6] have been reported. The current drug stability test guideline Q1A (R2) issued by the International Conference on Harmonization (ICH) [7] suggests that stress studies should be carried out on a drug to establish its inherent stability, leading to identification of degradation products and hence supporting the suitability of proposed analytical procedures. It also requires that analytical test procedures should be stability-indicating and that they should be fully validated. Accordingly, the aim of the present study was to establish the stability of dutasteride through stress studies under a variety of ICH-recommended test conditions [7–9] and to develop a stability-indicating assay method [10–12]. So far, to our knowledge no stabilityindicating LC assay method for dutasteride has been developed. The aim
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Intermediate precision of the method was evaluated by using a different system (Agilent 1100 series, Agilent Technologies, Waldbronn, Germany) with a diode array detector (DAD). The output signal was monitored and processed using Chemstation software (Agilent) on a Pentium computer.
Fig. 1. Chemical structure of dutasteride
of the present work was to develop a stability-indicating LC method for dutasteride in bulk drug and pharmaceutical dosage forms and capable of separating various degradation products.
Experimental Chemicals Samples and standard were supplied by Gansen Laboratories Limited; Mumbai, India; commercially available 0.5 mg dutasteride soft gelatin capsules (Dutas) were purchased. LC grade acetonitrile, analytical reagent grade sodium dihydrogen phosphate monohydrate and phosphoric acid were purchased from Merck, Darmstadt, Germany; high purity water was prepared with a Millipore Milli-Q plus system.
Equipment A Waters 2695 binary pump, auto sampler and a 2996 photo diode array detector were used. The output signal was monitored and processed using Empower software on a Pentium computer (Digital Equipment Co.), water baths equipped with MV controllers (Julabo, Seelabach, Germany) were used for hydrolysis studies. Stability studies were carried out in a humidity chamber (Thermo Lab, India). Thermal stability studies were performed in a dry air oven (MACK Pharmatech, Hyderabad, India).
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Chromatographic Conditions A 3 lm, 100 · 4.6 mm Phenomenex Luna C-18, column was used for separations. The mobile phase contained a mixture of buffer and acetonitrile in the ratio of 40:60 (v/v). The buffer consisted of 10 mM sodium dihydrogen phosphate monohydrate, pH adjusted to 3.0 using diluted phosphoric acid (1 mL phosphoric acid in 10 mL water). The flow rate of the mobile phase was 1.0 mL min 1. The column temperature was maintained at 27 C and the eluent was monitored at a wavelength of 210 nm. The injection volume was 10 lL.
Preparation of Stock Solutions Stock solutions of dutasteride standard and samples (1.0 mg mL 1) were prepared by dissolving appropriate amounts in acetonitrile. Working solutions of 0.1 mg mL 1 were prepared from the stock solution for assay determination.
Preparation of Sample Solution Twenty soft gelatin capsules were weighed and the contents were ground in a clean dry mortar. Then equivalent to 10 mg of drug was transferred to a 100 mL volumetric flask, 80 mL of acetonitrile added and the flask placed on a rotatory shaker for 10 min to disperse the material completely, sonicated for 10 min. The contents were then diluted to 100 mL (1,000 lg mL 1). The resulting solution was centrifuged at 3,000 rpm for 5 min. After centrifuging,
the supernatant solution was filtered using a 0.45 lm nylon 66-membrane filter. This solution was used for analysis.
Stress Studies/Specificity Stress testing of the drug substance can help to identify the likely degradation products, which can in turn help establish the degradation pathways and the intrinsic stability of the molecule. Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities [10]. All stress decomposition studies were performed at an initial drug concentration of 1 mg mL 1 (1,000 lg mL 1). Acid hydrolysis was performed in 1N HCl at 27 C for 48 h and in 1N HCl at 80 C for 3 h. The study in basic solution was carried out in 1 N NaOH at 27 C for 48 h and in 1 N NaOH at 80 C for 3 h. For study in neutral solution, the drug dissolved in water and acetonitrile (6:4, v/v) was heated at 80 C for 3 h. Oxidation studies were carried out at room temperature in 6% hydrogen peroxide for 48 h and in 6% hydrogen peroxide at 80 C for 2 h. Photodegradation studies were carried out according to Option 2 of Q1B in ICH guidelines [9]. Samples were exposed to light for an overall illumination of 1.2 million lux hours and an integrated near ultraviolet energy of 200 W h m2 . The drug powder was exposed to dry heat at 60 C for 10 days. Samples were withdrawn at appropriate times and subjected to LC analysis after suitable dilution (0.1 mg mL 1).
Method Validation Precision
The precision of the method was evaluated by carrying out six independent assays of a sample of dutasteride (100 lg mL 1) against a reference
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standard. The percentage RSD of the six assay values obtained was calculated. The precision was also checked at different concentrations (50 and 150 lg mL 1). The intermediate precision of the method was evaluated by different analysts and by using different instruments in the same laboratory. Linearity
Linearity test solutions were prepared from stock solution at six concentration levels from 10 to 150% of analyte concentration (10, 25, 50, 100, 125 and 150 lg mL 1). The slope, Y-intercept and correlation coefficient were calculated on an intra-day and an inter-day basis (n = 3). The %RSD for slope and Y-intercept were also calculated. Accuracy
The accuracy of the method was evaluated in triplicate at three concentration levels, i.e. 50,100 and 150 lg mL 1 using the bulk sample. The percentage recoveries were calculated. Selectivity
The selectivity of the method was established from the resolution of the drug peak from the nearest peak and also among all the other peaks. Solution Stability and Mobile Phase Stability
The solution stability of dutasteride was carried out by leaving the test solution in a tightly capped volumetric flask at room temperature for 48 h. The solution was assayed at 6 h intervals to the end of the study period, using a freshly prepared standard solution of dutasteride for comparison each time. The mobile phase stability was also investigated by assaying the freshly prepared sample solutions against freshly prepared standard solutions at 6 h intervals up to 48 h. Mobile phase composition and preparation was kept constant during the study period. The % RSD of the assay of dutasteride was calculated during the duration of the
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mobile phase and solution stability experiments.
grated near ultraviolet energy of 200 watt h m2 in a photostability chamber, no degradation was observed.
Results and Discussion Thermal Degradation Degradation in Acidic Solution In 1 N HCl at room temperature no major degradation was observed but the drug gradually underwent degradation with time on heating at 80 C, forming degradation products at RRTs of 0.15, 0.20, 0.47 and 0.67 (Fig. 2b).
Degradation in Basic Solution In 1 N NaOH at room temperature after 48 h, no major degradation was observed but the drug gradually underwent degradation with time on heating at 80 C in 1N NaOH forming degradation products at RRTs of 0.19, 0.40, 0.47 and 0.67 (Fig. 2c).
Oxidative Conditions The drug was exposed to 6% hydrogen peroxide at room temperature for 48 h. Mild degradation was observed but the drug gradually underwent greater degradation with time on heating at 80 C in 6% hydrogen peroxide forming products at RRTs of 0.39, 0.42 and 0.91 (Fig. 2d).
Degradation in Neutral Aqueous Solution No major degradation product was observed after 48 h at room temperature. The drug was also stable in water on heating at 80 C for 3 h (Fig. 2e).
Photolytic Conditions The drug was stable against the effect of photolysis. When the powdered drug was exposed to light for an overall illumination of 1.2 million lux h and an inte-
When the drug powder was exposed to dry heat at 60 C for 10 days, no decomposition was observed. Results of the assays of all the stressed samples were calculated using a reference standard of dutasteride. Considering the purities from the chromatograms of the stressed samples, mass balance (% assay + % degradants + % impurities) was calculated for each sample; the mass balance of the samples was close to 99.3%. No degradants were observed after 15 min over the extended run time of 60 min for any of the dutasteride stressed samples.
Method Development and Optimization of Stability-Indicating Method The main target was to obtain separation for closely eluting degradation products, mainly the degradation product at 0.91 RRT, from the dutasteride peak. The degradation samples were run on C18, Cyano and C8 columns and with mobile phases containing various buffers with different pH (2–8) and using acetonitrile or methanol in the mobile phase. Phosphate buffer with pH 3.0 and methanol (50:50, v/v) at 1.0 mL min 1 was chosen for initial trail with a 250 mm · 4.6 mm ID column and 5 lm C18 stationary phase. When stressed samples were injected the resolution between nearest impurity peak (0.91 RRT) and dutasteride was good but the retention time of dutasteride was *35 min and the tailing of the peak was also high (*1.6). Similar results were obtained with a 250 mm · 4.6 mm ID and a 5 lm C8 column. To reduce the retention time and tailing of the dutasteride peak, column length and particle size were decreased
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(a)
Dutasteride - 7.239
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0.08 0.06 0.04 0.02 0.00 6.00
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4.287 4.849
1.459
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Dutasteride stressed with 1N HCl for 3 hours reflux.
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2.606 2.919
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Dutasteride - 7.227
0.100 0.090 0.080 0.070
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Dutasteride stressed with 6%H2O2 for 2 hours reflux
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12.797
11.928
9.750
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5.839 6.133
4.434
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4.030
2.418
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2.821 3.033 3.262 3.416 3.571
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0.000 -0.010 2.00
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Dutasteride stressed with water for 3 hours reflux
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0.874 0.986 1.146
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0.00 2.00
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Time (min)
Fig. 2. Typical chromatograms of dutasteride and stressed dutasteride samples
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(Phenomenex Luna C18, 100 mm · 4.6 mm i.d with 3 lm particles). When stressed samples were injected in this column the resolution between the 0.91 RRT peak and dutasteride was greater than 2.0, tailing was reduced to *1.3 and retention time was found to be about 25 min. To further improve retention time and symmetry, methanol was replaced with acetonitrile (buffer: acetonitrile 50:50 v/v), to give tailing of dutasteride of *1.1, a retention time of approximately 12 min and resolution between the 0.91RRT peak and dutasteride was greater than 2.0. Resolution between the 0.91RRT peak and dutasteride was less than 2.0 with buffer: acetonitrile 30:70, v/v. Optimum results (Rs between 0.91RRT peak and dutasteride > 2.6), tailing factor of *1.1 and retention time around 7 min, were obtained when buffer: acetonitrile (40:60, v/v) was used as the mobile phase. The effect of buffer pH was also studied under the above conditions and it was found that when pH decreased, the symmetry of the dutasteride peak was improved and retention reduced. The results indicated that the 3 lm Phenomenex Luna C-18, 100 mm · 4.6 mm i.d column and an isocratic eluent of pH 3.0 phosphate buffer: acetonitrile (40:60, v/v) was successful in separation of the drug and all degradation products. Under the above conditions, results were as follows, retention time of dutasteride was around 7 min, with a tailing factor of 1.1, number of theoretical plates (N) for the dutasteride peak was 11656 and % RSD for 5 replicate injections was 0.2%. Interference of excipients (gelatin, glycerin and ferric oxide) was also checked by injecting sample solutions of these materials. There was no interference of excipients with degradation peaks or the dutasteride peak. Peak purity of stressed samples of dutasteride was checked by using a 2996 photodiode array detector of Waters. The purity angle was within the purity threshold limit obtained in all stressed samples, demonstrating analyte peak homogeneity. Peak purity results for degraded peaks from the PDA detector confirm that the degradant peaks were
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homogeneous and pure in all the analyzed stress samples. Analysis was performed for different batches of bulk drug samples (n = 3) and for pharmaceutical dosage forms (n = 3). The results for bulk samples was found to be 99.80% (DU009), 99.62% (DU012) and 99.53% (DU015) and for drug product, 99.35% (AV70021), 99.21% (AV70026), 99.48% (AV70029). All these results are well within the required limits. Accelerated and long term stability study results as per ICH Q1A (R2) for dutasteride were generated for 6 months and the results were well within the limits.
Validation of Developed Stability-Indicating Method The % RSD values were <0.2% for intra-day precision and 0.4% for interday precision, confirmed good precision of the method. A linear calibration plot for this method was obtained over the calibration range 10–150 lg mL 1 and the correlation coefficient obtained was greater than 0.999, showing an excellent correlation between the peak area and concentration of the analyte. The intraday slope and Y-intercept of the calibration curve were 26575 and 1375
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respectively and the inter-day slope and Y-intercept of the calibration curve were 26583 and 3330. The percentage recovery of dutasteride in bulk drug samples ranged from 101.8 to 103.1% and in pharmaceutical dosage forms it ranged from 99.2 to 100.6%. Excellent recoveries were made at each added concentration. The solution stability and mobile phase stability experiments data confirmed that sample solutions and mobile phase used during assay were stable up to 48 h. Figure 2 shows that the method was sufficiently specific to the drug. The resolution factor for the drug peaks was >2.6 from the nearest impurity peak (RRT *0.91). Intermediate precision was performed to confirm that separation was satisfactory. The resolution between the drug peak and the peak at RRT *0.91 was >2.6 indicating that the method remains selective for all components under the tested conditions.
Conclusions The isocratic RP-LC method developed proved to be simple, linear, precise, accurate and specific. The method was completely validated showing satisfactory data for all the parameters tested.
The method is stability-indicating and can be used for the analysis of the drug and degradation products in stability samples.
References 1. Gisleskog PO, Hermann D, HammarlundUdenaes M, Karlsson MO (1999) Br J Clin Pharmacol 47:53–58 2. Evans HC, Goa KL (2003) Drugs Aging 20:905–916 3. Gaines KK (2003) Urol Nurs 23:218–240 4. Brown CT, Nuttall MC (2003) Int J Clin Pract 57:705–709 5. Ramakrishna NVS, Vishwottam KN, Puran S, Koteshwara M, Manoj S, Santosh M (2004) J Chromatogr B 809:117–124 6. Burinsky DJ, Williams JD, Thornquest AD, Sides SL (2001) J Am Soc Mass Spectrom 12:385–398 7. ICH (2003) Stability Testing of New Drug Substances and Products Q1A (R2), International Conference on Harmonization, IFPMA, Geneva 8. Singh S, Bakshi M (2000) Pharm Tech On-line 24:1–14 9. ICH (1996) Photo stability Testing of New Drug Substances and Products Q1B, International Conference on Harmonization, IFPMA, Geneva 10. Bakshi M, Singh S (2002) J Pharm Biomed Anal 28:1011–1040 11. Singh S, Singh B, Bahuguna R, Wadhwa L, Saxena R (2006) J Pharm Biomed Anal 41:1037–1040 12. Carstensen JT, Rhodes CT (eds) (2000) Drug Stability: Principles and Practices, 3rd edn. Marcel Dekker, Inc., New York
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