INTRODUCTION:

Chemically lurasidone is [(3aR,4S,7R,7aS)-2-{(1R,2R)-2-[4-(1,2-benzisothiazol-3-yl) piperazin-1-ylmethyl] cyclohexylmethyl} hexahydro-4,7-methano-2H-isoindole-1, 3-dione hydrochloride and it is an azapirone derivative[1].The chemical structure of lurasidone hydrochloride (SM-13496) with its six chiral centers is shown in Fig.1.

Fig. 1: Chemical structure of Lurasidone

Lurasidone hydrochloride appears as a white to light yellow crystalline powder and is stable and its solubility in chloroform and acetonitrile. It is sparingly soluble in ethanol and it is slightly soluble in water and acetone[2]. The improvement of memory impairment due to MK-801 induction was found to be higher in lurasidone compared to other anti- psychotics and hence proved to be a clinically useful drug for cognitive impairments in treatment of schizophrenia. A thorough literature survey has revealed that UV spectroscopy [3], HPLC method for lurasidoine [4-5], UPLC, LC-MS, for its estimation in bulk, pharmaceutical dosage forms and biological samples. A simple and precise method was developed and validated by using a solvents comprising a mixture of methanol and water in the volume ratio of 70 :30 for method 1 and a mixture of acetonitrile and water in the volume ratio of 50:50 for method 2.

Instrumentation:

Double beam UV-Visible Spectrophotometer (Shimadzu-1800) connected to a computer loaded with Shimadzu UV Probe 2.33 software was used for all the spectrophotometric measurements in all proposed spectrophotometric methods.

Preparation of stock solution of Lurasidone:

Standard Lurasidone of 10mg was accurately weighed and transferred into a 10ml volumetric flask. About 5ml of methanol: water (70:30v/v) and acetonitrile: water (50:50v/v) is added and subjected to sonication and then the volume is made up to the mark with mobile phase to give a concentration of 1000μg/ml. From this stock solution 1ml was pipetted out and transferred into a 10ml volumetric flask and the volume was made up to the mark with solvent to give a concentration of 100µg/ml. Further dilutions were made to get a concentration of 10µg/ml of both methods.

Selection of analytical concentration ranges:

From the standard stock solutions of Lurasidone (100µg/ml), appropriate aliquots of 0.25ml, 0.5ml, 0.75ml, 1ml, 1.25ml, 1.5ml was pipetted out and transferred into a 10ml volumetric flasks and dilutions were made with to obtain working standard solutions with concentration ranging from 2.5 to 15µg/ml in both methods.

Preparation of sample solutions:

Twenty tablets of Lurasidone formulation were weighed and powdered. The powder equivalent to 10mg was calculated and transferred into a 10ml volumetric flask and 4ml of solvent methanol: water (70:30v/v) in method 1 and acetonitrile: water (50:50v/v) in method 2 is added and sonicated for 30min. The volume was shaken and made up to the mark with the respective solvent to obtain solutions of 1000µg/ml. The solutions was filtered through Whatmann filter paper (No. 41) and used for the estimation.

Validation parameters for Lurasidone:

Linearity, Range and Calibration:

The method was validated according to ICH guidelines [6]. The linearity of analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in the sample within the range. The range of the analytical method is the interval between the upper and lower levels that have been demonstrated to be determined within a suitable level of precision, accuracy and linearity. The method was found to be linear in the concentration range of 2.5-15µg/ml for both methods. And a 10 ml volume of lurasidone was adjusted to the mark to obtain concentrations of 2.5, 5, 7.5, 10, 12.5, 15µg/ml. Absorbance spectra of each solution against distilled water as blank were measured at 230nm and the graph of absorbance against concentration were plotted and the regression equation and correlation coefficient were determined for both the methods.

Precision:

The precision of an analytical method is the degree of agreement among individual test results, when the method is applied repeatedly to multiple sampling of homogeneous samples. It provides an indication of random error results and is expressed as relative standard deviation (%RSD).

Repeatability:

Repeatability expresses the analytical variability under the same operating conditions over a short interval of time. The repeatability studies were carried out by selecting 5µg/ml as the standard concentration and repeating it for six times for both methods.

Intermediate precision:

Variation of results within the same day (intra-day) and variation of results between days (inter-day) were analysed for both methods. Intra-day precision was determined by analyzing lurasidone for 6 times on the same day at 230nm. Inter-day precision was determined by analyzing 3 concentrations of lurasidone on the preceding day at 230nm and %RSD was calculated. For method 1 - 5, 10,15µg/ml was analysed and for method 2 - 2.5, 5, 7.5 µg/ml were analysed.

Accuracy:

Accuracy is the closeness of the results obtained by the method to the true value. Recovery studies were carried out at 50%, 100% and 150% by adding known amount of standard drug solutions for method 1 & method 2 i.e. ( 2.5, 5, 7.5 µg/ml) to the sample solutions whose concentration is maintained constant, i.e. 5µg/ml. The %recovery was calculated.

Ruggedness:

The solutions were prepared and analysed with change in the analytical conditions like different instrument and different analyst. Standard concentrations of 5µg/ml were carried used to carry out the ruggedness studies for both methods.

Limit of detection (LOD) and Limit of quantification (LOQ):

The sensitivity of the proposed method for the measurement of lurasidone was estimated in terms of Limit of detection (LOD) and Limit of quantification (LOQ). The LOD and LOQ were calculated by using the slope and SD of response (intercept). The mean slope value and the SD of response were obtained from the calibration curve. The LOD and LOQ calculations were done and reported.

Application of the proposed method for pharmaceutical formulation:

The solution was filtered through whatmann filter paper No.41. 0.5 ml this solution was transferred to 10ml volumetric flask and final volume was made with 0.1N HCL. It gives 0.5µg/ml. It was scanned on a spectrophotometer in the UV range 200–400 nm. The spectrum was recorded at 280 nm against blank solution of 0.1N HCL. Determine the amount of % lurasidone Hydrochloride in tablet according to the following formula.

AT x WS × Sample D.F x Average Weight

% Assay = --------------------------------------------------------x PR

AR x Standard D.F x WT× LA

Where,

WS = weight of standard; WT = weight of sample Where, AT = Absorbance of lurasidone hydrochloride in the test solution , AR = Absorbance of lurasidone in the standard solution , Std. D.F = Standard dilution factor, Sample D.F = Sample dilution factor PR = Purity of working standard [%], LA = Labeled amount of lurasidone.

RESULTS AND DISCUSSION:

The method was validated according to ICH guidelines [6] in order to determine the linearity, precision, accuracy and ruggedness of the method [7].

Method:

The standard stock solution of lurasidone 10µg/ml concentration was scanned from 200-400nm and the absorption spectras were recorded at 230nm wavelength in UV spectrophotometer using methanol: water (70:30) and acetonitrile: water (50:50) as solvents individually.

Linearity:

The absorbance of the solutions of lurasidone was determined at a wavelengths of 230nm. The correlation coefficient was found to be 0.999 and the regression equation was found to be Y=0.061x + 0.007 for method 1 and Y=0.086x + 0.022 for method 2. The calibration curves and overlay spectra’s of lurasidone was shown in Fig 1a 1b, Fig 2a and Fig 2b.

Precision:

Repeatability was determined for all the drugs and % RSD was found to be less than 2 which was found to be 1.5, 0.41-1.69 and 0.39-1.11 respectively and are presented in Table 1.

Intermediate Precision (reproducibility):

The precision of the developed method was expressed in terms of percent relative standard deviation (% RSD). These results show reproducibility of the assay. The %

RSD values were found to be less than 2 that indicate this method precise for the determination of the pure form. The interday and intraday precision results were mentioned in Table 2 and Table 3.

Fig1a. Overlay spectra and Fig 1b. Linearity graph of Lurasidone at 230nm using methanol: water (70:30v/v)

Fig 2a. Overlay spectra and Fig 2b. Calibration curve of Lurasidone at 230nm using acetonitrile: water(50:50v/v)

Table 1. Repeatability studies of Lurasidone

Conc [µg/ml]	Method 1			Method 2
Conc [µg/ml]	Absorbance at 230 nm	Absorbance Mean ±S.D(n=6)	%RSD	Absorbance at 230 nm	Absorbance Mean ±S.D(n=6)	%RSD
5	0.311	0.312±0.007	1.5	0.382	0.385±0.002	0.55
5	0.315			0.384
5	0.31			0.385
5	0.312			0.385
5	0.312			0.387
5	0.315			0.388

Table 2. Intermediate precision for methanol : water (70:30)

Conc taken (μg/mL)	Intra-day precision			Inter-day precision
	Absorbance	Absorbance mean ±S.D(n=3)	%RSD n=3	Absorbance	Absorbance mean ±S.D (n=3)	%RSD n=3
5	0.320	0.314±0.002	1.69	0.308	0.312±0.001	1.13
	0.31			0.315
	0.312			0.312
10	0.62	0.626±0.006	0.85	0.621	0.618±0.007	0.49
	0.628			0.618
	0.630			0.615
15	0.922	0.926±0.004	0.410	0.918	0.922±0.014	0.396
	0.928			0.923
	0.929			0.925

Table 3. Intermediate precision for acetonitrile water (50:50)

Conc taken (μg/mL)	Intra-day precision			Inter-day precision
	Absorbance	Absorbance mean ±S.D(n=3)	%RSD n=3	Absorbance	Absorbance mean ±S.D(n=3)	%RSD n=3
5	0.320	0.314±0.002	1.69	0.308	0.312±0.001	1.13
	0.31			0.315
	0.312			0.312
10	0.62	0.626±0.006	0.85	0.621	0.618±0.007	0.49
	0.628			0.618
	0.630			0.615
15	0.922	0.926±0.004	0.410	0.918	0.922±0.014	0.396
	0.928			0.923
	0.929			0.925

Accuracy:

Accuracy is determined by performing recovery studies at 3 levels in which known amount of analyte shall be added and recovery shall be carried out in three replicates of each concentration level and the % recovery was calculated. The accuracy results are shown in Table 4.

Ruggedness:

Ruggedness Studies was performed by two different analysts and two different instruments and the results of the study and % RSD obtained was less than 2 which is within the acceptance limits. And %RSD were found to be in the range of 1.12-1.38 and 0.811-1.12. respectively for methanol: water (70:30v/v) and of 0.649-1.06 and 1.06-1.23 for acetonitrile: water(50:50v/v) was reported in Table 5 and Table6.

Limit of Detection and Limit of Quantification:

The parameters LOD and LOQ were determined on the basis of response and slope the regression equation . LOD and LOQ values are 0.69 and 2.1 for method 1 0.6 and 1.85 for method 2.

Application of the proposed method for pharmaceutical formulation:

The results of %purity was found to be 100.06 and 100.26 which are shown in Table 7.

Table 4. Determination of accuracy results for Lurasidone

Spiked level (% )	Formulation conc (µg/ml)	Pure drug conc (µg/ml)	Method 1			Method 2
			Amount recovery (µg/ml)	% Mean recovery ±SD	% RSD	Amount recovery (µg/ml)	% Mean recovery ±SD	%RSD
50	5	2.5	7.48	100.6±0.59	0.591	7.49	100.5±0.67	0.66
	5	2.5	7.55			7.53
	5	2.5	7.57			7.59
100	5	5.0	10.02	100.4±0.42	0.424	9.8	100.3±0.7	0.69
	5	5.0	10.10			10.1
	5	5.0	10.08			10.03
150	5	7.5	12.53	100.1±0.33	0.336	12.56	100.4±0.41	0.409
	5	7.5	12.57			12.49
	5	7.5	12.45			12.59

Table 5. Ruggedness of Lurasidone for different analyst

Conc (µg/ml)	Method 1				Method 2
	Analyst 1		Analyst 2		Analyst 1		Analyst 2
	Mean Absorbance	%RSD	Mean Absorbance	%RSD	Mean Absorbance	%RSD	Mean Absorbance	%RSD
5	0.314±0.004	1.12	0.331±0.004	1.3	0.379±0.004	1.06	0.387 ±0.002	0.649
5
5

Table 6. Ruggedness of Lurasidone for different instrument

Conc (µg/ml)	Method 1				Method 2
	Absorbance of instrument I (Shimadzu)		Absorbance of instrument II (Lab India)		Absorbance of instrument I (Shimadzu)		Absorbance of instrument II (Lab India)
	Mean Absorbance	%RSD	Mean Absorbance	%RSD	Mean Absorbance	%RSD	Mean Absorbance	%RSD
5	0.314±0.004	1.12	0.326±0.002	0.811	0.379±0.004	1.06	0.387±0.002	0.64

CONCLUSION:

Regression equation like slope (b), intercept(a) and correlation coefficient (R²) using the method of least squares were calculated and are presented in Table 8. The results show that the methods are reasonably precise. The developed UV Spectrophotometric method was found to be simple, economic, easy, accurate, precise, reproducible and highly sensitive and can be used for routine estimation of lurasidone in bulk and formulations.

Table 8. Summary of UV Spectrophotometric Method for Estimation of Lurasidone

PARAMETERS	METHOD 1	METHOD 2
λmax (nm)	230	230
Beers law limit µg/ml	2.5-15µg/ml	2.5-15µg/ml
Correlation coefficient ( r²)	0.9995	0.9993
Molar absorbivity (l mol^-1cm^-2)	0.103 x 10⁴	0.105 x 10⁴
Sandell’s sensitivity	0.0001	0.0002
Regression equation (y=mx+c)	Y=0.061x+ 0.007	Y=0.086x+ 0.022
Slope (m)	0.061	0.086
Intercept(c)	0.007	0.022
Accuracy	100.1-100.6	100.3-100.5
Precision (%RSD)	0.39-1.69	0.54-1.62
LOD (µg/ml)	0.697	0.613
LOQ (µg/ml)	2.11	1.85

ACKNOWLEDGEMENTS:

The authors are grateful to Malla Reddy College of Pharmacy for providing necessary research facilities to carry out the research work and to MSN Drugs, India for providing the gift sample of the drug.

REFERENCES:

1. Meltzer HY, “Lurasidone in the treatment of schizophrenia: a randomized, double-blind, placebo- and Olanzapine-controlled study.”, Department of Psychiatry, Vanderbilt University School of Medicine, Nashville, Tenn.

2. Leslie Citrome “Lurasidone for Schizophrenia: A Brief Review of a New Second-Generation Antipsychotic” New York University School of Medicine, Department of Psychiatry, 2010.

3. Muvvala S Sudhir and Ratnakaram V Nadh, 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) 609.

4. Nirav K. Joshi, Nehal J. Shah, Development and validation of RP-HPLC method for estimation of lurasidone hydrochloride: A novel antipsychotic drug in bulk drug and pharmaceutical, pharma science monitor, (3), 2012.

5. Katasani Damodar Srinu Bhogineni, Bala Ramanjaneyulu, RP-HPLC method development and validation for the analyisis of lurasidone in pharmaceutical dosage forms. Drug Invention Today Year: 2012 3 (12).

6. Mali Nikita, Patel Jignesh, Patel Mandev, 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),44- 50.

7. Validation of Analytical Procedures: Methodology, ICH Harmonized Tripartite Guidelines, 1996, pp 1-8.

Received on 07.04.2015 Accepted on 22.04.2015

Asian J. Pharm. Res. 5(2): April-June 2015; Page 102-107

DOI: 10.5958/2231-5691.2015.00015.5