DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPTLC METHOD FOR QUANTIFICATION OF LURASIDONE HCL A.R. Polawar and M.C. Damle *

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1 Impact factor: /ICV: Pharma Science Monitor 5(3), Jul-Sep 2014 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES Journal home page: DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPTLC METHOD FOR QUANTIFICATION OF LURASIDONE HCL A.R. Polawar and M.C. Damle * Department of Quality Assurance, AISSMS College of Pharmacy, Pune, India. ABSTRACT Lurasidone is an atypical antipsychotic of benzisothiazol class used in the treatment of schizophrenia, an approach for the stress degradation was successfully applied for the development of stability indicating HPTLC method for the determination of Lurasidone HCl on the plates precoated with silica gel 60 F 254. The mobile phase used was Toluene: Methanol in the ratio of 9.5:0.5 v/v. The drug showed considerable absorbance at 231nm. Stress testing of Lurasidone Hydrochloride was carried out according to the international conference of harmonization (ICH) guideline Q1A (R2). The drug was subjected to acid, base, neutral hydrolysis, oxidation, thermal degradation and photolysis. The system showed a peak for Lurasidone HCl at Rf value of 0.48 ± The method was successfully validated according to ICH guidelines Q2R1. The data of linear regression analysis indicated a good linear relationship over the range of ng/band concentrations with correlation coefficient value greater than The accuracy of the method was established based on the recovery studies. The LOD and LOQ were and ng/band respectively. Under various stressed conditions, Lurasidone showed considerable degradation under alkali, acidic, oxidative and neutral hydrolytic condition. KEYWORDS: Lurasidone Hydrochloride, High Performance Thin Layer Chromatography (HPTLC), Stability-Indicating Method. INTRODUCTION Lurasidone is an atypical antipsychotic approved by the U.S. Food and Drug Administration (FDA) for treatment of Schizophrenia on October 28, 2010 [1]. Lurasidone Hydrochloride ( 3aR,4S,7R,7aS ) -{(1R,2R) 2 -[ 4 -( 1,2 - benzisothiazol-3-yl) piperazin 1 ylmethyl ] cyclohexylmethyl } hexahydro - 4, 7- methano -2H isoindole 1,3 - dione hydrochloride (Fig.1) is a azapirone derivative antipsychotic used in the treatment of schizophrenia [2]. The efficacy of Lurasidone Hydrochloride in schizophrenia is mediated through a combination of central dopamine Type 2 (D2) and serotonin Type 2 (5HT2A) receptor antagonism and it gives Antipsychotic Activity. Lurasidone is metabolized in the liver via the enzyme CYP3A44 [3]. This means that its plasma concentrations may be increased when combined with CYP3A4 inhibitors like ketoconazole or grape fruit juice, possibly leading to

2 Impact factor: /ICV: more side effects. As with other Atypical Neuroleptics, Lurasidone should not be used in elderly patients because it puts them at an increased risk for a stroke or transient ischemic attack [3,4]. Structure of Lurasidone Hydrochloride Literature search reveals following methods reported viz., simple spectrophotometric method for the estimation of Lurasidone in tablet dosage form (5,6), Simple RP-HPLC method for quantitative analysis of Lurasidone [7,8,9,10], LCMS method for quantification of Lurasidone in rat plasma and its application in pharmacokinetic studies [11] and Stability Indicating HPLC method for determination of Lurasidone in pharmaceutical dosage form [12] To the best of our knowledge, no stability indicating HPTLC method has been reported for Lurasidone Hydrochloride.The present work describes a simple stability indicating HPTLC method for the determination of Lurasidone HCl. Stability testing was carried out according to the international conference on harmonization (ICH) guidelines [13, 14]. The method was validated according to the ICH guidelines [15]. EXPERIMENTAL MATERIALS AND METHODS Chemicals and Reagents: Lurasidone HCl was provided as a gift sample by Wokhardt Pharmaceuticals, Aurangabad and used as such, without any further purification. Aluminum sheets precoated with silica gel (60 F 254, 20 cm 20 cm with 250 µm layer thickness) were purchased from E-Merck, Darmstadt, Merck (Germany). Methanol (AR grade), Toluene (AR grade) were purchased from S. D. fine chemical Laboratories, Mumbai. Hydrochloric acid (HCl), hydrogen peroxide (H 2 O 2, 30% v/v) and sodium hydroxide (NaOH) were purchased from LOBA CHEMIE PVT. LTD. Mumbai. Chromatographic conditions and instrumentation Chromatographic separation of drug was performed on Aluminum plates precoated with silica gel 60 F 254, (10 cm 10 cm with 250 µm layer thickness). Samples were applied on the plate as

3 Impact factor: /ICV: a band with 4 mm width using Camag 100 μl sample syringe (Hamilton, Switzerland) with a Linomat 5 applicator (Camag, Switzerland). The mobile phase was composed of Toluene: Methanol (9.5:0.5 v/v). 20 cm 10 cm CAMAG twin trough glass chamber was used for linear ascending development of TLC plate under 15 min saturation conditions and 10 ml of mobile phase was used per run, migration distance was 90 mm. Densitometric scanning was performed using Camag TLC scanner 3 in the range of nm, operated by wincats software (Version 1.4.3, Camag), slit dimensions were 3.00 x 0.45 mm and Deuterium lamp was used as a radiation source. Selection of detection wavelength From the standard stock solution further dilutions were done using methanol and scanned over the range of nm and the spectra was obtained. It was observed that the drug showed considerable absorbance at 231 nm. Fig. 1: UV Spectrum of Lurasidone HCl (100µg/ml) Preparation of Standard stock solution Standard stock solution of Lurasidone HCl was prepared by dissolving 10 mg of drug in 10 ml of methanol to get concentration of 1000µg/ml. From the standard stock solution, working standard solution was prepared to contain 100µg/ml of Lurasidone HCl. Preparation of sample solution of blend Strength of marketed tablets: 40mg of Lurasidone Hydrochloride/tablet. Preparation of blend: Blend containing 40mg Lurasidone Hydrochloride was prepared by spiking drug into blank blend (58mg starch: lactose (1:1), 2mg Magnesium Stearate). Mixing was done by geometric addition method. Blend equivalent to 200mg of Lurasidone HCl was prepared to satisfy dose/tablet of marketed formulation approximately. Preparation of test solutions: Blend equivalent to 40 mg Lurasidone HCl was weighed and dissolved in 20ml methanol. This solution was centrifuged, filtered and volume was made up to 20ml with methanol which contains 2mg/ml Lurasidone HCl (A). From solution A, Further dilution was made with

4 Impact factor: /ICV: methanol to get a concentration of 100μg/ml of Lurasidone HCl (B), 10µL of the resultant solution was then applied at TLC plate and densitogram was developed. Densitogram: Solution of Lurasidone HCl (100µg/ml) was prepared. 6µl (600ng/band) of solution was applied on preactivated TLC plate with the help of Hamilton syringe (100μl), using Linomat5 sample applicator. The development chamber was saturated with mobile phase for 15 min. The spotted plate was placed in the saturated chamber and developed up to 90 mm distance. The plate was dried and was scanned over 90 mm distance at 231nm. The retention factor (Fig. 3.2) was found to be: Lurasidone HCl = 0.48± 0.03 Fig. 2: Densitogram of standard solution of Lurasidone HCl STRESS DEGRADATION STUDY OF BULK DRUG Stress degradation studies were carried under condition of acid/ base/ neutral hydrolysis, oxidation, dry heat and photolysis. For each study, samples were prepared as follows 1. The blank subjected to stress in the same manner as the drug solution 2. Lurasidone HCl working standard solution subjected to stress condition. Dry heat and photolytic degradation were carried out in solid state. 6µL of the resultant solution was then applied at TLC plate and densitogram was developed Degradation under alkali catalyzed hydrolytic condition To 1 ml of 1000 µg.ml -1 solution of Lurasidone HCl, 1mL of 0.1 N NaOH was added. The volume was made up to 10 ml with methanol. The above solution was kept for 4 hours at room temperature. Degradation under acid catalyzed hydrolytic condition

5 Impact factor: /ICV: To 1 ml of 1000 µg.ml -1 solution of Lurasidone HCl, 1mL of 1N HCL was added. The volume was made upto 10 ml with methanol. The above solution was kept for overnight at room temperature. Degradation under neutral hydrolytic condition To 1 ml of 1000 µg.ml -1 solution of Lurasidone HCl, 1mL of distilled water was added. The volume was made upto 10 ml with methanol. The above solution was kept for overnight at room temperature. Degradation under oxidative condition To 1 ml of 1000 µg.ml -1 solution of Lurasidone HCl, 1 ml of 3% H 2 O 2 was added. The volume was made upto 10 ml with methanol. The above solution was kept for overnight at room temperature. Degradation under dry heat Dry heat studies were performed by keeping drug sample in oven (80 0 C) for a period of 6 hours. Photo-degradation studies The photo degradation study of the drug was carried out by exposing the drug to UV light providing illumination of NLT 200 watt hr/m 2, after UV light exposure drug color was changed and showed higher degradation, hence fresh drug was exposed to cool white fluorescence light of NLT 1.2million Lux-Hr. 1. B)

6 Impact factor: /ICV: C) D) E) F) Fig.3: Representative Densitogram of Alkali (A), Acid (B), Oxidative (C), Neutral (D), Dry Heat (E), Photolytic (F) degradation of Lurasidone HCl 600ng/band respectively.

7 Impact factor: /ICV: TABLE 1: SUMMARY OF STRESS DEGRADATION OF LURASIDONE HCL Stress Degradation Condition Percent recovery (%) Percent degraded (%) Peak purity r(s, m) r(m, e) Initial Base (0.1 N NaOH, kept for 4hrs) Acid (0.1 N HCl, Kept for overnight) H 2 O 2 3% (kept for overnight) Water (kept for overnight) Heat dry (80 0 C, 6 hrs) Photo stability (UV, 200 watt hrs/square meter Florescence, 1.2 million Lux. Hrs) RESULTS This optimized method led to the retention Factor of 0.48 ± 0.02 for Lurasidone HCl Validation of Analytical Method The method was validated as per ICH Q2 (R1) guidelines. Specificity The specificity of the method was ascertained by peak purity profiling studies. The peak purity values were found to be more than 0.998, indicating the non interference of any other peak of degradation product or impurity. Linearity From the standard stock solution (1000µg/ml) of Lurasidone HCl, solution was prepared containing 100µg/ml of Lurasidone HCl. This solution was further used for spotting. Five replicates per concentration were spotted. The linearity (relationship between peak area and concentration) was determined by analyzing five solutions over the concentration range of ng/band for Lurasidone HCl Fig.4.The results obtained are shown in Table 2, the peak

8 Impact factor: /ICV: areas were plotted against the corresponding concentrations to obtain the calibration curve as shown in Fig.5 for Lurasidone HCl. Fig. 4: Densitogram of linearity of Lurasidone HCl ( ng/band) TABLE 2: LINEARITY STUDY OF LURASIDONE HCL Concentrations of Lurasidone HCl (ng/band) Replicate Peak Area Mean Std.dev %RSD

9 Impact factor: /ICV: y = 4.887x R² = Series1 Linear (Series1) Fig.5: Calibration curve for Lurasidone HCl Range Lurasidone HCl = ng/band Assay Spiked blend analysis was carried out as mentioned under section test solution Procedure. Analysis was repeated six times. Sample solution was spotted and area was recorded. % assay was determined from linearity equation. (fig.4) TABLE 3: ASSAY OF SPIKED BLEND Sr. No. Peak area Of Amount Recovered % Recovery Lurasidone (ng/band) Mean %RSD Accuracy To check accuracy of the method, recovery studies were carried by spiking the drug to the blank blend (excipient). Blend A, B, C were prepared containing 20mg, 40mg, 60mg of Lurasidone HCl respectively and test solutions of each blend were prepared to get final concentrations

10 Impact factor: /ICV: (800µg/ml, 1000µg/ml and 1200µg/ml) of Lurasidone HCl respectively These solutions were spotted on the TLC plate. The drug concentrations were calculated by using linearity equation of Lurasidone HCl. The results obtained are shown in Table 4. Level (%) TABLE 4: RECOVERY STUDIES OF LURASIDONE HCL Precision The intra-day precision study of Lurasidone HCl was carried out by estimating the peak responses six times on the same day with 600ng/band concentration and inter-day precision study of Lurasidone HCl was carried out by estimating the peak responses six times on three different days with 200,400,600ng/band concentration and % RSD value obtained TABLE 5: INTER-DAY PRECISION OF LURASIDONE HCL Concentration (ng/band) Therapeautic Conc. (ng/band) Area Area of LURA Mean Area Recovered Conc. (ng/band) SD % Recovery % RSD TABLE 6: INTRADAY PRECISION STUDY OF LURASIDONE HCL 600ng/band Replicate Intraday Mean Area Std. Dev % RSD 1.387

11 Impact factor: /ICV: Limit of detection and quantification (LOD and LOQ) From the linearity data the limit of detection and Quantitation was calculated, using the following formula. LOD= 3.3 σ/ S and LOQ = 10 σ/ S σ = standard deviation of the response S = slope of the calibration curve of the analyte. LOD=15.92ng/ band LOQ= ng/band Robustness Robustness of the method was determined by carrying out the analysis under conditions during which mobile phase ratio,chamber saturation time were altered, Time was also changed from spotting to development and development to scanning and the effects on the area were noted. It was found that method is robust. TABLE 7: ROBUSTNESS STUDY Sr. Parameters Robust condition % RSD No. Saturation time (15min) 13min ± 2 min. 17min 0.4 Mobile phase 2.03 Toluene: Methanol (9.7: 0.5 v/v) composition 2. Toluene: Methanol Toluene: Methanol (9.3: 0.5 v/v) 0.6 (9.5:0.5 v/v) Time from spotting to After 30min development(immediate) After 2hrs Time from development to scanning(immediate) After 30min After 2hrs 0.66

12 Impact factor: /ICV: Summary of validation study TABLE NO. 8 SUMMARIES OF VALIDATION PARAMETERS Sr. No. Validation parameters Lurasidone HCl Linearity Equation, (r 2 ), Range Y= 4.887x r 2 = ng/band Precision (% RSD) Interday Intraday %1.376 %1.387 Accuracy % Recovery Limit of Detection 15.92ng/band 5. Limit of Quantitation ng/band 6. Specificity Specific 7. Robustness Robust DISCUSSION The analytical method was developed by studying different parameters. First of all, maximum absorbance was found to be at 231nm. The stationary phase used for study of Lurasidone HCl was Aluminum plates precoated with silica gel 60 F 254, (10 cm 10 cm with 250 µm layer thickness. Different ratios of mobile phase constituents were studied, mobile phase with Toluene: Methanol in the ratio of 9.5:0.5v/v was chosen due to good symmetrical peak. Retention Factor was 0.48 ± Stress degradation study of Lurasidone HCl was carried out according to ICH guidelines Q1A (R2). It was observed that Lurasidone showed considerable degradation under alkali, acidic, oxidative and neutral hydrolytic condition. Validation was carried out as per ICH guidelines Q2 (R1). The percent recovery was found to be Both Intraday and Interday precision was found to be well within range. In specificity study, there was no interference by degradant, impurity and excipients. Detection and Quantification limit was found to be and 48.25ng/band respectively. The analytical method was found linear over the range of ng/band. CONCLUSION The developed method was found to be simple, sensitive, selective, accurate, and repeatable for analysis of Lurasidone HCL in the blend without any interference from the excipients. The

13 Impact factor: /ICV: results indicated the suitability of the method to study stability of Lurasidone HCl under various forced degradation conditions like hydrolysis, dry heat and photolytic degradation ACKNOWLEDGEMENT The authors are thankful to Wokhardt Pharmaceuticals, Aurangabad for providing working standard of Lurasidone Hydrochloride. Authors are also thankful to the Principal and Management, AISSMS College of Pharmacy, Pune for providing required facilities for research work. REFERENCES 1. FDA approves Latuda to treat schizophrenia in adults. USFDA 2010; Retrieved October 29, [Accessed on: Tuesday, January 28, 2014] Available at [Accessed on: Tuesday, January 28, 2014] 3. Ishiyama T, Tokuda K, Ishibashi T, Ito A, Toma S, Ohno Y: Lurasidone (SM-13496), a novel atypical antipsychotic drug, reverses MK-801-induced impairment of learning and memory in the rat passive-avoidance test. Eur J Pharmacol. 2007; 572 (2-3): Dainippon Sumitomo Pharma: Lurasidone Demonstrated Efficacy in Treating Patients with Schizophrenia in Pivotal Phase III Study [Accessed on: Tuesday, January 28,2014] Available at 5. Muvvala SS and Ratnakaram VN: Simple and Validated Ultraviolet Spectrophotometric Method for the Estimation of Lurasidone in Bulk Form. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2013; 4(1): Mali N, Patel J, Patel M: Validated Spectrophotometric Methods for the Estimation of Lurasidone Hydrochloride in Bulk and Pharmaceutical Dosage Forms. International Journal of Research in Pharmacy and Science 2012; 2(2): Polawar AR and Damle MC: Development and validation of RP-HPLC method for estimation of Lurasidone hydrochloride in bulk and pharmaceutical dosage form. IJRPC 2014; 4(2): Katasani D, Srinu B, Bala R: RP-HPLC Method Development and Validation for the Analysis of Lurasidone in Pharmaceutical Dosage Forms. Drug Invention Today 2011; 3(12): Joshi NK and Shah NJ: Development and Validation of RP-HPLC method for estimation of Lurasidone hydrochloride: a novel antipsychotic drug in bulk and pharmaceutical dosage form. Pharma Science Monitor 2012; 3(4):

14 Impact factor: /ICV: Patel Krunal Y, Patel Satish: A Development and validation of reverse phase High Performance Liquid Chromatography Method for Estimation of Lurasidone Hydrochloride in synthetic mixture, IJPDA; 2(3): Koo TS, Kim SJ, Lee J, Ha DJ, Baek M, Moon H: Quantification of Lurasidone, an atypical antipsychotic drug, in rat plasma with high-performance liquid chromatography with tendem mass spectrometry. Biomed Chromatogr. 2011; 25: Chhabdal PJ, Balaji M, Srinivasarao V, Appa KMC: Development and validation of stability indicating method for determination of Lurasidone in bulk drug and pharmaceutical dosage form by HPLC. IJPRD. 2013; 5(9): ICH, Q1A (R2): Stability Testing of New Drug Substances and Products, ICH Harmonized Tripartite Guideline, Geneva Switzerland, ICH, Q1B: Stability Testing: Photostability Testing Of New Drug Substances and Products, ICH Harmonized Tripartite Guideline, Geneva Switzerland, ICH Q2 (R1): Validation of Analytical Procedures: Text and Methodology, ICH Harmonized Tripartite Guideline, Geneva Switzerland, For Correspondence M.C. Damle mcdamle@rediffmail.com