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2011 Asian J Pharm Biol Res

Original Research

Development and Validation of A Stability-Indicating High Performance Liquid Chromatography Assay for Aripiprazole in Bulk Drug Substance
Zarna R Dedania, Ronak Dedania, Navin Sheth, Balram Gajra, Jigar Patel
Veerayatan Institute of Pharmacy ,India Department of Pharmaeutical Sciences, Saurashtra University, Rajkot, Gujarat,India. E mail : zaroo229@yahoo.co.in
Abstract
Objective: To develop a validated stability-indicating assay method (SIAM) for aripiprazole after subjecting it to forced decomposition under hydrolysis, oxidation, photolysis and thermal stress conditions. Methods: The liquid chromatographic separation was achieved isocratically on a symmetry C18 column (5 m size, 250 mm 4.6 mm i.d.) using a mobile phase containing acetonitrile:methanol (80:20, v/v) at a flow rate of 1 ml/min and UV detection at 254 nm. Results: The method was linear over the concentration range of 10-60 g/ml (r = 0.998) with a limit of detection and quantization of 2.85 and 8.56 g/ml, respectively. The method has the requisite accuracy, specificity, sensitivity and precision to assay aripiprazole in bulk form and pharmaceutical dosage forms. Conclusion: Degradation products resulting from the stress studies did not interfere with the detection of aripiprazole and the assay is thus stability-indicating. Keywords:Thin layer chromatography, Densitometry, Analytical method validation, Amoxycillin trihydrate, Ambroxol Hydrochloride.

INTRODUCTION Aripiprazole, 7-[4-[4-(2, 3dichlorophenyl) piperazin-1-yl] butoxy]- 3,4dihydro- 1H-quinolin- 2-one (Figure. 1) [1] is an atypical antipsychotic medication used for the treatment of schizophrenia. It has also recently received FDA approval for the treatment of acute manic and mixed episodes associated with bipolar disorder. Aripiprazole appears to mediate its antipsychotic effects primarily by partial agonist at the D2 receptor. In addition to partial agonist activity at the D2 receptor, it is also a partial agonist at the 5-HT1A receptor, and like the other atypical antipsychotics, it displays an antagonist profile at the 5-HT2A receptor. Aripiprazole has moderate affinity for histamine and alpha adrenergic receptors and no appreciable affinity for cholinergic muscarinic receptors[2]. Several analytical methods have been reported in the literature for the routine quantitative assay of aripiprazole like high performance thin layer chromatography in bulk form and pharmaceutical dosage forms [3] . Capillary electrophoresis combined with high performance liquid chromatography (HPLC) [4], HPLC-diode array [5] detection , liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) [6], HPLC-MS [7], LC-MS/MS [8] from human plasma, gas chromatography-mass spectrometry (GC-MS) from blood [9], HPLC from rat plasma and brain [10], ultra performance liquid chromatography-mass

spectrometry from in-vitro samples . These methods [69] are complicated, costly and time consuming rather than a simple HPLC-UV method. Currently, most of the separations are performed by HPLC for reasons of robustness and familiarity of analysts with this technique. The previous published methods are not directly applicable for this issue and need more investigation for method development and validation. So an attempt has been made to develop simple, precise, accurate, specific, robust stability- indicating HPLC method for the quantitative determination to aripiprazole in pharmaceutical dosage forms and applied to the assay of aripiprazole in tablets and bulk form. MATERIALS AND METHODS Chemicals and reagents Aripiprazole working standard powder was provided as gift sample from Tripada Pharmaceuticals, Ahmedabad, India and was used without further purification. Arpizol tablets (Manufacturing date 11/2009 and Expiry date 04/2012) containing 20 mg aripiprazole as per label claim were obtained from local market. Methanol, acetonitrile, water were of HPLC grade and sodium hydroxide, hydrochloric acid, hydrogen peroxide were of Analytical grade obtained from E. Merck (India) Ltd., Mumbai. All chemicals were at least of analytical grade and used as received. HPLC instrumentation and conditions The present work was carried out on

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e ISSN: 2231-2218

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isocratic high pressure liquid chromatography TM consist of pump (Cyberlab , USA) with universal loop injector (Rheodyne) of injection capacity 20 l. Detector consists of photodiode array detector; the reversed phase column use was RPC18 (5 m size, 250 mm4.6 mm i.d.) at ambient temperature. Separation was achieved using a mobile phase consisting of acetonitrile:methanol (80:20, v/v) at a flow rate of 1 ml/min. The eluted compounds were monitored at 254 nm. The column was maintained at ambient temperature and an injection volume of 20 l was used. The mobile phase was filtered through 0.45 micron membrane filter and ultrasonicated for 10 min prior to use. For analysis of forced degradation samples, the photodiode array detector was used in scan mode with a scan range of 200400 nm. Peak homogeneity was expressed in terms of peak purity values, and was obtained directly from spectral analysis report obtained using the instrument software. Preparation of stock and standard solutions A 1 mg/ml stock solution of aripiprazole was prepared in HPLC grade acetonitrile. Sub stock solution was prepared from stock solution by diluting 10 ml standard stock solution up to 100 ml to get 100 g/ml. Aliquots of the standard sub-stock solution were transferred into 10 ml volumetric flasks and the solutions were made up to volume with mobile phase to give final concentrations of 10, 20, 30, 40, 50 and 60 g/ml. Each 20 l standard solution was injected into the column after filtration using 0.2 micron membrane filter. Preparation of tablets for assay Twenty tablets were weighed, crushed and mixed in a mortar with pestle. A portion of powder equivalent to the weight of one tablet was accurately weighed and transferred into each of six 100 ml volumetric flasks and made up to the volume with mobile phase and mixed well. The volumetric flasks were sonicated for 20 min to effect complete dissolution of the aripiprazole and the solutions were then made up to the volume with mobile phase. Suitable aliquots of solution were filtered through a 0.45 micron nylon filter. A 2 ml quantity of the filtered solution was transferred to a 10 ml volumetric flask and made up to the volume with mobile phase to yield concentration 40 g/ml in the range of linearity previously described. Forced degradation studies of API n order to determine whether the analytical method and assay were stabilityindicating, aripiprazole tablets and aripiprazole API powder were stressed under various conditions to conduct forced degradation studies [12]. Regulatory guidance in ICH Q2A, Q2B, Q3B and FDA 21 CFR section 211 all require the development and validation of stabilityindicating potency assays. Unfortunately, the current guidance documents do not indicate detailed degradation conditions in stress testing. However, the forced degradation conditions, stress agent concentration and time of stress were selected based on trial and error method, so that the degradation of drug preferably remained in between 10% and complete degradation. As aripiprazole is practically insoluble in water and is readily soluble in acetonitrile, acetonitrile was used as co-solvent in all studies. All solutions prepared for use in forced degradation studies were prepared to yield final concentrations of 20g/ml. Acid degradation studies A 100 mg quantity of aripiprazole sample was taken into a 100 ml round bottom flask, 10 ml of 0.1M hydrochloric acid solution was added, and contents were mixed well and kept for constant stirring for 24 h at room temperature. 2 ml of this solution was taken in 100 ml volumetric flask and neutralized with 2 ml of 0.1M sodium hydroxide and then diluted to 100 ml with diluent. Alkali degradation studies A 100 mg quantity of aripiprazole sample was taken into a 100 ml round bottom flask, 10 ml of 0.1M sodium hydroxide solution was added, and contents were mixed well and kept for constant stirring for 24 h at room temperature. 2 ml of this solution was taken in 100 ml volumetric flask and neutralized with 2 ml of 0.1M hydrochloric acid and then diluted to 100 ml with diluent. Oxidation A 100 mg quantity of aripiprazole sample was taken in 100 ml round bottom flask, 10 ml of

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3% hydrogen peroxide solution was added and contents were mixed well at room temperature. 2 ml of this solution was diluted to 100 ml with diluent immediate after preparation. Temperature stress studies A 1 g quantity of aripiprazole sample was taken in to a Petri dish and kept in oven at 80 C for 24 h. A 10 mg quantity of this sample was taken in to a 100 ml volumetric flask, dissolved in diluent and diluted to volume with diluent. 2 ml of this solution was taken in 10 ml volumetric flask and then diluted to 10 ml with diluent. Photo stability A 1 g quantity of aripiprazole sample was taken in to a Petri dish and kept in photostability chamber/200Wh/m2 in UV light and 1.2million lx h in visible light for 24 h. A 10 mg quantity of this sample was taken in to a 100 ml volumetric flask, dissolved in diluent and diluted to volume with diluent. 2 ml of this solution was taken in 10 ml volumetric flask and then diluted to 10 ml with diluent.

Table 1. Precision study

Table 2. Recovery study

* Mean%SD for three determinations.

Table 3. System suitability parameters

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Figure 1 Figure 2 Figure 1 :Structural formula for Aripiprazole

Figure 2 (A)

Figure 4

Figure 5

Figure 2 (B)

Figure 2 (C)

Figure 2 (D)

Figure 2 (E)

Figure 2(A) :Typcal HPLC chromatogram of Acid Hydorlysis dergaded active pharmaceutical Ingredient (API).Figure 2 (B) : Typical HPLC Chromatogram of base hydrolysis-degraded API. Figure 2 (C) : Typical HPLC Chromatogram of oxidative-degraded API. Figure 2 (D) : Typical HPLC Chromatogram of Thermal degraded API.Figure 2 (E) : Typical HPLC Chromatogram of Photodegraded API.

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Figure 3(A)

Figure 3(B)

Figure 3(A) HPLC Chromatogram of Aripiprazole standard soulution (20 g/ml).Figure 3(B) HPLC Chromatogram of Aripiprazole tablets
RESULTS AND DISCUSSION HPLC method development and optimization , A RP-C18 column (250 mm 4.6 mm i.d. 5m particle size), maintained at ambient temperature ? (25 C) was used for the separation and the method validated for the determination of aripiprazole in pharmaceutical dosage forms. The stressed samples were initially analyzed using a mobile phase consisting of acetonitrile:methanol (60:40, v/v) at a flow rate of 1.5 ml/ min and UV detection at 254 nm. Under these conditions, the peak shape was not optimal. An improvement was observed in the peak shape, by changing ratio acetonitrile:methanol (90:10, v/v) but tailing was observed. Eventually, a mobile phase consisting of acetonitrile:methanol (80:20, v/v) at a flow rate of 1 ml/min provided the best chromatographic response and was used for further studies. Validation The method was validated with respect to parameters including linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, specificity, robustness, solution stability and system suitability. Linearity The calibration curves constructed for aripiprazole were linear over the concentration range of 10-60 g/ml. Peak areas of aripiprazole were plotted versus concentration and linear regression analysis performed on the resultant curve. Typically, the regression equation for the calibration curve was found to be y = 5421x + 30239 with 0.998 regression coefficient. LOQ and LOD The LOQ and LOD were determined based on signal-to-noise ratios, using an analytical response of 10 and three times the background noise, respectively [13]. The LOQ and LOD were found to be 8.65and 2.85 g/ml respectively. Precision Table 1 provides data obtained from the precision experiments. The R.S.D. values for intraday and interday precision were <0.88% and <1.09%, respectively, thereby indicating that the method was sufficiently precise. A similar qualitative separation of the drug was obtained even on analysis on a different chromatographic system on a different day, indicating that the method has sufficient intermediate precision. Accuracy Percentage recovery was calculated from differences between the peak areas obtained for fortified and unfortified solutions. As shown from the data in Table 2, excellent recoveries were made at each added concentration of drug. Specificity Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities and degradation Products. The specificity of the developed LC method was checked in the presence of its degradation products. The method was found to be specific to the drug. Specificity was established by determination of purity of the drug peak using a PDA detector. Also, the resolution factor of the drug peak was determined with respect to the nearest resolving peaks. Robustness The robustness was illustrated by getting the retention time, when mobile phase flow rate (0.2 ml/min), organic solvent ratio (5%) and

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column temperature (2 ?C) were deliberately varied. Solution stability The solution stability of aripiprazole in diluents was determined by leaving 0.1 mg/ml sample solution in a tightly capped volumetric flask at room temperature for 48 h and measuring the amount for every 6 h. The solution stability of aripiprazole assay (in diluents) was determined by leaving 0.1 mg/ml solution in tightly capped volumetric flasks at room temperature for 48 h during which they were assayed at 6 h intervals and comparing the results with those obtained from freshly prepared solution. The mobile phase was prepared at the beginning of the study period and not changed during the experiment. The %RSD values for solution stability experiments were calculated and found to be 1.22% and 1.35% for purity and assay methods, respectively. All the samples were found to be stable up to 48 h. System suitability The system suitability test was applied to a representative chromatogram to check the various parameters such as column efficiency, resolution, precision and peak tailing. The result obtained is shown in Table 3. All these parameters were evaluated with the background of regulatory requirements, which suggests good chromatographic condition. Stability studies All stressed samples in both solid and solution state remained colorless. HPLC studies on ariprazole under different stress conditions using acetonitrile:methanol (80:20, v/v) as the solvent system suggested the following degradation behavior as shown in Figure 2. After 24 h storage at room temperature in acidic and alkaline condition drug was found to be degraded 27.48% and 8% respectively. Under storage at room temperature and in 3% H2O2 around 52.90% of drug was degraded immediate after preparation. A 24.73% thermal degradation was seen on exposure of solid aripiprazole powder at 80 C for 24 h and 20.29% degradation was seen on exposure of solid aripiprazole powder to light in a photostability chamber for 24 h. It was not the intention of the study to identify degradation products, but merely to show that they would not interfere if and when present. Degradation products did not appear on chromatograms, indicating homogenous peaks and thus establishing the selectivity of assay method. The indication is that the drug was degraded to nonchromophoric products. The stability of the stock solution was determined by quantitation of aripiprazole and comparison to freshly prepared standard. No significant change (<2%) was observed in stock solution response, relative to freshly prepared standard. Assay The proposed method was applied to the determination of aripiprazole in Arpizol. A typical chromatogram obtained following the assay of aripiprazole tablets is depicted in Figure 3.The result of these assays yielded 100.04% (%R.S.D. = 1.25%) of labelled claim for the tablets. The results the excipients used in these dosage forms. CONCLUSION A validated stability-indicating HPLC analytical method has been developed for the determination of aripiprazole in API. The results of stress testing undertaken according to the International Conference on Harmonization (ICH) guidelines reveal that the method is selective and stability-indicating. The proposed method is simple, accurate, precise, specific, and has the ability to separate the drug from degradation products and excipients found in the tablet dosage forms. The method is suitable for the routine analysis of aripiprazole in either bulk API powder or in pharmaceutical dosage forms. The simplicity of the method allows for a p p l i cat i o n i n l a b o rato r i es t h at l a c k sophisticated analytical instruments, such as LCMS or GCMS. These methods are complicated and costly rather than a simple HPLC-UV method. In addition, the HPLC procedure can be applied to the analysis of samples obtained during accelerated stability experiments to predict expiry dates of pharmaceuticals. ACKNOWLEDGMENTS The authors are grateful to Veerayatan Institute of Pharmacy, Jakhania and Saurashtra University, Rajkot, India for providing the facilities to carry t h e e x p e r i m e n t a s w e l l a s Tr i p a d a Pharmaceuticals, Ahmedabad, India for providing gift samples of aripiprazole REFERENCES 1. http://en.wikipedia.org/wiki/Aripiprazole, 06/02/2009. 2 . http://www.drugbank.ca/drugs/DB012

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