Stress Degradation Studies on Tadalafil and Development of a Validated StabilityIndicating LC Assay for Bulk Drug and Pharmaceutical Dosage Form 2008, 67, 183–188
D. V. Subba Rao1,&, P. Radhakrishnanand1, V. Himabindu2 1
2
United States Pharmacopeia-India Private Limited, Reference Standard Laboratory, ICICI Knowledge Park, Turkapally, Shameerpet, Hyderabad 500078, India; E-Mail:
[email protected] Department of Chemistry, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad 500 072, India
Received: 25 July 2007 / Revised: 24 October 2007 / Accepted: 31 October 2007 Online publication: 12 December 2007
Abstract A stability-indicating HPLC assay method was developed for the quantitative determination of tadalafil in bulk samples and in pharmaceutical dosage forms in the presence of the degradation products. It involved a 250 mm · 4.6 mm, 5 lm C-18 column. The gradient LC method employs solution A and B as mobile phase. Solution A contains a mixture of buffer (phosphate buffer and tetra-n-butyl ammonium hydrogen sulfate) pH 2.5: acetonitrile (80:20, v/v) and solution B contains a mixture of water: acetonitrile (20:80, v/v). The flow rate was 1.0 mL min1 and the detection wavelength was 220 nm. The retention time of tadalafil is about 17 min. Tadalafil was subjected to different ICH prescribed stress conditions. Degradation was found to occur in hydrolytic and to some extent in oxidative stress conditions, while the drug was stable to photolytic and thermal stress. The drug was particularly labile under alkaline hydrolytic conditions. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The assay of stress samples was calculated against a qualified reference standard and the mass balance was close to 99.5%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and ruggedness.
Keywords Column liquid chromatography Stability-indicating assay Tadalafil
Introduction The present drug stability test guideline Q1A (R2) issued by international conference on harmonization (ICH) [1] Limited Short Communication DOI: 10.1365/s10337-007-0478-1 0009-5893/08/01
suggests that stress studies should be carried out on a drug to establish its inherent stability characteristics, leading to identification degradation products and hence supporting the suitability of
the proposed analytical procedures. It also requires that analytical test procedures for stability samples should be stability indicating and they should be fully validated. Accordingly, the aim of the present study was to establish inherent stability of tadalafil through stress studies under a variety of ICH recommended test conditions [1–3] and to develop a stability-indicating assay [4–6]. Tadalafil (Cialis), is chemically (6R,12aR)-2,3,6,7, 12,12a-hexahydro-2-methyl-6-(3,4-methylene dioxyphenyl) pyrazino (10 ,20 :1,6) pyrido-(3,4-b) indole-1,4-dione (Fig. 1) an oral treatment for erectile dysfunction, is a selective inhibitor of cyclic guanosine monophosphate (cGMP)specific phosphodiesterase type-5 (PDE-5). Through the inhibition on PDE-5, tadalafil increases the concentration of cyclic guanosine monophosphate (cGMP), producing smooth muscle relaxation and increased blood flow to the corpus cavernosum, thereby enhancing erectile response following appropriate sexual stimulation. This drug has been launched in more than 40 countries worldwide [7]. A few analytical methods have been reported for the estimation of tadalafil in formulation using HPLC [8–10] and capillary electrophoresis with UV detection [11, 12]. A high throughput validated
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Fig. 1. Structures and chemical name of tadalafil
analytical method for the quantitation of tadalafil in human plasma using LC-MSMS has been published [13]. So far, to our present knowledge no stability indicating HPLC assay method was developed. The aim of the present work is to develop a stability indicating LC method for tadalafil bulk drug and pharmaceutical dosage forms. More intensive stress studies in our laboratory were carried out on tadalafil. Accordingly, a stability-indicating method was developed, which could separate various degradation products.
Experimental Chemicals Samples and standards were supplied by Gansen Laboratories, Mumbai, India, commercially available 20 mg tadalafil tablets were purchased. The HPLC grade acetonitrile, analytical reagent grade sodium dihydrogen phosphate monohydrate and tetra-n-butyl ammonium hydrogen sulfate were purchased from Merck, Darmstadt, Germany; high pure water was prepared by using Millipore MilliQ Plus water purification system.
output signal was monitored and processed using empower software on Pentium computer (Digital Equipment Co), water baths equipped with MV controller (Julabo, Seelbach, Germany) were used for hydrolytic studies. Stability studies were carried out in a humidity chamber (Thermo Lab, India) and photo stability studies were carried out in a photostability chamber (Sanyo, Leicestershire, UK). Thermal stability studies were performed in a dry air oven (MACK Pharmatech, Hyderabad, India).
100 mL volumetric flask, 70 mL of acetonitrile: water (1:1, v/v) added and kept on rotatory shaker for 10 min to disperse the material completely and sonicated for 10 min and diluted to 100 mL (1,000 lg mL1). The resulting solution was centrifuged at 3,000 rpm for 5 min. About 10 mL of supernant solution was taken and diluted to 100 mL with acetonitrile: water (1:1, v/v) (100 lg mL1). This was filtered using a 0.45 l (nylon 66-membrane filter.
Chromatographic Conditions
Stress Studies
The chromatographic column used was a Waters Symmetry shield RP-18, 250 mm · 4.6 mm i.d with 5 lm particles. Mobile phase A contains a mixture of buffer and acetonitrile in the ratio of 80:20 (v/v). Buffer consists of 10 mM sodium dihydrogen phosphate monohydrate and 5 mM of tetra-n-butyl ammonium hydrogen sulfate, pH adjusted to 2.5 using diluted phosphoric acid (1 in 10). The mobile phase B contains a mixture of water and acetonitrile in the ratio of 20:80 (v/v). The flow rate of the mobile phase was 1.0 mL min1. The HPLC gradient program was set as: time (min)/% solution B: 0/20, 5/20, 30/90, 32/90, and 35/20. The column temperature was maintained at 27 C and the detection was monitored at a wavelength of 220 nm. The injection volume was 10 lL.
Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities [4]. 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. All stress decomposition studies were performed at an initial drug concentration of 0.1 mg mL1 (100 lg mL1). Acid hydrolysis was performed in 0.1 N HCl at 27 C for 48 h and in 1N HCl at 70 C for 3 h. The study in basic solution was carried out in 0.1 N NaOH at 27 C for 48 h. For study in neutral solution, drug dissolved in water was heated at 70 C for 3 h. Oxidation studies were carried out at room temperature in 6 and 20% hydrogen peroxide for 48 h. Photo degradation studies were carried out according to option 2 of Q1B in ICH guidelines [3]. Samples were exposed to light for an overall illumination of 1.2 million lux hours and an integrated near ultraviolet energy of 200 watt hm2. The drug powder was exposed to dry heat at 40 C for 10 d and at 60 C for 7 d. Samples were withdrawn at appropriate time and subjected to HPLC analysis after suitable dilution (0.1 mg mL1).
Preparation of Stock Solutions A stock solution of tadalafil standard and sample (1.0 mg mL1) was prepared by dissolving an appropriate amount in acetonitrile: water (1:1, v/v). Working solutions of 0.1 mg mL1were prepared from above stock solution for assay determination.
Preparation of Sample Solution
Equipment The LC system used was a Waters 2695 binary pump plus auto sampler and a 2996 photo diode array detector. The
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Twenty tablets were weighed and the content transferred into a clean and dry mortar, grinded well. Then an equivalent to 100 mg of drug was transferred to a
Method Validation Precision
The precision of the assay method was evaluated by carrying out six indepen-
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dent assays of tadalafil (100 lg mL1) test samples against qualified reference standard. The percentage of RSD of six assay values was calculated. The precision was also checked at different concentrations (50 and 150 lg mL1). The intermediate precision of the method was evaluated by different analyst and by using different instrument from the same laboratory. Linearity
Linearity test solutions were prepared from stock solution at six concentration levels from 25 to 150% of analyte concentration (10, 25, 50, 100, 125 and 150 lg mL1). The slope, Y-intercept and correlation coefficient were calculated. Accuracy
The accuracy of the method was evaluated in triplicate at three concentration levels, i.e. 50, 100 and 150 lg mL1 in bulk sample. The percentages of recoveries were calculated. Specificity and Selectivity
The specificity of the method was established through study of resolution factors of the drug peak from the nearest resolving peak, and also among all other peaks.
Results of Forced Degradation Studies
Degradation was not observed in tadalfil during stress conditions like photo, thermal degradation and water hydrolysis. Tadalafil was degraded into 1.04 RRT imp at 3% level and the assay was about 96% in 1N HCl after 3 h reflux. In 0.1 N NaOH after 48 h treatment at room temperature tadalafil was degraded into 1.04 RRT impurity at about 50% level and the assay was about 49%. The impurity which was formed at RRT 1.04 in both acid and base degradation was the isomer of tadalafil (6S,12aR)-2,3,6,7, 12,12a-hexahydro-2-methyl-6-(3,4-methylene dioxyphenyl) pyrazino (10 ,20 :1,6) pyrido-(3,4-b)indole-1,4-dione. This was confirmed with spiking analysis with qualified reference standard of ((6S, 12aR)2,3,6,7,12,12a-hexahydro-2-methyl-6(3,4-methylene dioxyphenyl) pyrazino (10 ,20 :1,6) pyrido-(3,4-b) indole-1,4dione), spectral matching and with mass spectroscopy. Them mass number of peaks at 1.04 RRT observed is 389. Mild degradation (about 7%) was observed after 48 h in 20% hydrogen peroxide for tadalafil and the assay was found to be about 93.5%. Peak purity test results confirm that the tadalafil peak is homogeneous and pure in all the analyzed stress samples of tadalafil. Mass balance (% assay + % degradants + % impurities) is calculated for each stress sample. The mass balance of stressed samples was close to 99.5%.
Solution Stability and Mobile Phase Stability
The solution stability of tadalafil was carried out by leaving the test solution in a tightly capped volumetric flask at room temperature for 48 h. The same sample solution was assayed for a 6 h interval up to the study period against freshly prepared standard solution of tadalafil. The mobile phase stability was also carried out by assaying the freshly prepared sample solutions against freshly prepared standard solutions for a 6 h interval up to 48 h. Mobile phase prepared was kept constant during the study period. The percentage of RSD of assay of tadalafil was calculated for the study period during mobile phase and solution stability experiments.
Limited Short Communication
Method Development and Optimization of Stability-Indicating Method
The method was optimized to separate major degradation products formed under various stress conditions. The main target of the chromatographic method is to get the separation for closely eluting degradation products, mainly the degradation product at 1.04 RRT from the tadalafil peak. The degradation samples were run using different stationary phases like C18, Cyano, C8 and different mobile phases containing buffers like phosphate, sulphate and acetate with different pH (2–8) and using organic modifiers like acetonitrile and methanol
in the mobile phase. But the separation was satisfactory in the adopted chromatographic conditions only. It indicated that the gradient method with ion pair mobile phase was successful in separating drugs and all chromophoric degradation products. There was no interference of excipients with degradation peaks and tadalafil peak. Analysis was performed for different batches of bulk drug samples (n = 3) and for pharmaceutical dosage forms (n = 3). The assay results for bulk samples were 99.78% (PP002), 99.82% (PP004) and 99.63% (PP005) whereas for drug product, 99.20% (TDKH0016), 99.28% (TDKH0022), 99.10% (TDKH0036) which show all the results are well within the limit. Accelarated and longterm stability study results as per ICH Q1A (R2) for tadalafil [1] were generated for 3 months and the results are well within the limits.
Validation of Developed Stability-Indicating Method
The percentage RSD values for the precision study were <0.8% (intra-day precision) and 0.4% (inter-day precision) confirming good precision of the method. Linear calibration plot for this method was obtained over the calibration ranges tested, i.e. 10–150 lg mL1 and the correlation coefficient obtained was greater than 0.999. The results show that an excellent correlation existed between the peak area and concentration of the analyte. The slope and Y-intercept of the calibration curve were 57,180 and 178,222 respectively. The percentage recovery of tadalafil in bulk drug samples ranged from 96.4 to 99.6% and in pharmaceutical dosage forms from 98.6 to 99.2%. Excellent recoveries were made at each added concentration. The solution stability and mobile phase stability experiment data confirms that sample solutions and mobile phase used during the 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
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Tadalafil Tadalaf il - 17.706
(a) 0.20 0.18
0.16
Tadalafil
0.14
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0.04 0.02 0.00 0.00
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Tadalafil in 1N HCl Reflux for 3 h (Acid Deg) 3.149
Tadalafil - 17.919
0.08
2.357
(b) 0.10 Tadalafil stressed with 1N HCl for 3 hours reflux.
AU
0.06
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19.957 20.363 20.817
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Fig. 2. Typical chromatogram of (a) tadalafil sample and (b, c, d) stressed tadalafil samples
>2.6 from the nearest resolving peak (RRT * 1.04). Intermediate precision was performed to confirm that separation was satisfactory under conditions mentioned above. The resolution between the drug peak and the nearest resolving peak (RRT * 1.04) was >2.6 indicating that
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the method remains selective for all components under the tested conditions.
Conclusions The gradient RP-LC method proved to be simple, linear, precise, accurate and
specific. The method was completely validated showing satisfactory data for all the method validation parameters tested. The developed method is stability-indicating and can be used for the analysis of the drug and degradation products in stability samples by the industry.
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in 20% H2O2 for 48 h (Peroxide Deg) Tadalaf il - 17.684
(c) 0.20 Tadalafil 0.18
0.16
Tadalafil stressed with 20%H 2O2 for 48 hours
0.14
AU
0.12
0.10 0.08
13. 586
12.479
9.953
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7.699
6. 138 6. 455
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18.389
Tadalafil in 0.1 N NaOH for 48 h (Base Deg) 2.493
(d) 0.20 0.18
Tadalafil stressed with 0.1N NaOH for 48 hours
Tadalafil - 17. 671
0.16 0.14 0.12
AU
0.10 0.08
2. 117
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2.971 3.312 3.700
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Fig. 2. continued
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