Validated RP-HPLC Method For Analysis of Aripiprazole in A Formulation
Validated RP-HPLC Method For Analysis of Aripiprazole in A Formulation
Validated RP-HPLC Method For Analysis of Aripiprazole in A Formulation
net
Introduction
Aripiprazole, (7-[4-[4-(2, 3-dichlorophenyl)-1-piperazinyl] butoxy]-3, 4-dihydrocarbostyril (Figure 1), is a psychotropic agent belonging to the chemical class of benzisoxazole derivatives and is indicated for the treatment of schizophrenia1,2. In the references, gas chromatography-mass spectrometry3, LCMS/MS4, capillary electrophoresis6, methods are reported for the analysis of aripiprazole in biological fluids. HPLC is the technique that most commonly used for the determination of aripiprazole in plasma5-9, UPLC-MS in in vitro samples10 and RP-HPLC method in bulk drug and solid dosage forms by internal standard method11 also reported. In this paper we describe a simple, accurate, sensitive and validated RP-HPLC method for analysis of aripiprazole in tablet formulation. This method has been successfully used for quality-control analysis of drugs and for other analytical purposes.
828
R. KALAICHELVI et al.
Experimental
A reference standard of aripiprazole was obtained from Orchid Healthcare (Chennai, India). A pharmaceutical product containing the same drug (30 mg per tablet), obtained from the same laboratory and was used in the experiments. Acetonitrile (HPLC grade; ACN) and water of HPLC grade were from Qualigens (Bombay, India) and orthophosphoric acid and triethanolamine (analytical reagent grade) were purchased from SD Fine Chemicals (Bombay, India). De-ionized water was used throughout the experiment. Before use, mobile phase was filtered through a 0.45 m cellulose acetate filter from Millipore (USA). Whatman no. 41 filter papers (obtained commercially) were used for preparation of sample solutions.
Calibration
Calibration plots were constructed by analysis of appropriate working solutions (concentration 20, 30, 40, 50 and 60 g mL-1) of aripiprazole in the mobile phase and plotting concentration against peak-area response for each injection. Unknown samples were quantified by reference to these calibration plots.
Sample preparation
Twenty tablets were weighed and powdered. An amount of powder equivalent to 50 mg aripiprazole was accurately weighed and transferred to a 50 mL volumetric flask. Mobile phase (25 mL) was added and the mixture was sonicated for 10 min for complete extraction of the drug and the solution was diluted to volume with mobile phase. The solution was centrifuged at 4000 rpm for 10 min and the clear supernatant was collected and filtered through a 0.2 m membrane filter. From this solution 2 mL was taken and diluted to 50 mL with mobile phase, to furnish a 40 g mL-1 solution, of which 10 L was injected for HPLC analysis.
Statistical calculations
Standard regression curve analysis was performed by use of Microsoft (USA) Office Excel 2003 software, without forcing through zero. Means and standard deviations were calculated by use of SPSS software version 9.5 (SPSS, Cary, NC, USA). Homoscedasticity for the calibration plots was tested by using GraphPad Prism software, demo version.
829
Chromatography
Symmetrical peaks were obtained for aripiprazole. Typical chromatograms obtained from a blank and from a solution of the drug are illustrated in Figure 2(a&b). The retention time of aripiprazole was 3.8 min and the overall chromatographic run time was 8.0 min.
Voltag mv Voltage,mv
Min
Figure 2(a)
mv Voltage,
Min
Figure 2(b) Figure 2. (a) Typical chromatograms obtained from blank and (b) aripiprazole solution.
830
R. KALAICHELVI et al.
Concentration, g/mL
Accuracy
Recovery studies were performed in triplicate after spiking raw material in volumetric flasks with amounts of aripiprazole equivalent to 80, 100 and 120% of the standard concentration of aripiprazole (40 g mL-1) as in the analytical method. The results obtained (Table 1) indicate that recovery were excellent, not less than 99% and that relative standard deviations also less than 2%. Table 1. Accuracy of the method. Mean amount Spike Concentration Recovery, RSD, recovered, Drug -1 level, % added, g mL %, n = 3 %, n = 3 -1 g mL , n=3 80 32 32.24 100.75 0.86 Aripiprazole 100 40 39.93 99.82 0.95 tablets 120 48 48.02 100.04 0.97
Precision
Intra-day precision was calculated from results obtained from five-fold replicate analysis of samples at three different concentrations on the same day. Inter-day precision was calculated from results from the same samples analyzed on five consecutive days. The results obtained are listed in Table 2.
Validated RP-HPLC Method for Analysis Table 2. Intra-day and inter-day precision of the method. Concentration Added, g mL-1 32 40 48 Intra-day precision RSD, Mean amount found, %, n = 5 g mL-1, n = 5 32.04 0.98 40.12 0.95 47.98 1.01
831
Inter-day precision Mean amount RSD, Found, g mL-1, n = 5 %, n = 5 32.61 0.99 39.99 1.07 48.12 0.96
Specificity
The specificity of the method was tested by chromatographing a mixture of commonly used tablet excipients, for example starch, lactose and magnesium stearate (blank placebo) and comparing the chromatogram with that obtained from a mixture of drug and the same additives (placebo). The chromatograms obtained (Figures 4 & 5) showed separation of the analyte from the excipients was complete, i.e. there was no interference from the excipients under the chromatographic conditions used for the analysis.
Voltage,mv
Min
Voltage, mv
Min
Stability
The stability of aripiprazole in solution was checked by determining the percentage deviation of the amounts present in solution after 72 h at room temperature in comparison with the amount at zero time. The results obtained after 72 h showed no significant variation; the percentage deviation was less than 2% of the initial amount. This is indicative of good stability of each component in the mixture over a period of 72 h.
832
R. KALAICHELVI et al.
Conclusion
This RP-HPLC method for analysis of aripiprazole in formulations is very simple, sensitive, and accurate. The run time is 8 min only; so many samples can also be processed and analyzed in a short period of time. The procedure described is suitable for the routine estimation of aripiprazole in pharmaceutical formulations.
References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Kane J M, Carson W H, Saha A R, McQuade R D, Ingenito G G, Zimbroff D L and Ali M W, J Clin Psychiatry, 2002, 63, 763-71. http://www.rxlist.com/aripiprazole. Hui C H, Chin H L, Tsuo H L, Tsung M H, Hsien J C, Yu C W and Ying L T, J Chromatogr B., 2007, 856, 5761. Masanori K, Yasuo M, Yukihiro H and Takahiko O, J Chromatogr B., 2005, 822, 294299. Frederique L, Kayssa D, Khalid T, Linda K, Sophie B, Pascal P and Marie L P, J Chromatogr B, 2008, 867, 1519. Musenga A, Saracino M A, Spinelli D, Rizzato E, Boncompagni G, Kenndler E and Raggi M A, Anal Chim Acta, 2008, 612, 204-211. Lancelin F, Djebrani K, Tabaouti K, Kraoul L, Brovedani S, Paubel P and Piketty M L, J Chromatogr B Analyt Technol Biomed Life Sci., 2008, 867, 15-9. Shimokawa Y, Akiyama H, Kashiyama E, Koga T and Miyamoto G, J Chromatogr B Analyt Technol Biomed Life Sci., 2005, 821, 8-14. Kirchherr H and Khn-Velten W N, J Chromatogr B Analyt Technol Biomed Life Sci., 2006, 843, 100-113. Li K Y, Zhou Y G, Ren H Y, Wang F, Zhang B K and Li H D, J Chromatogr B; Analyt Technol Biomed Life Sci., 2007, 850, 581-585. Vjayakumar M and Muley P R, The Indian Pharmacist, 2005, 4, 71-75. Snyder L R, Kirkland J J and J L Glajch (Eds) Practical HPLC Method Development, WileyInterscience, New York, 1988, 402. International Conference on the Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH) Q2B. Validation of Analytical Procedures, Methodology, 1996, 1-8. US Pharmacopoeial Convention, United States Pharmacopoeia, 28th Edn, US Pharmacopoeial Convention, Rockville, MD, 2005,1196-1198. FDA: Guidance for Industry, Analytical Procedures and Methods Validation, 2000.
14. 15.