Development and Validation Of UV-Visible Spectroscopic Methods for The Quantification and Forced Degradation Profile of New Antipsychotic Drug Lumateperone in Bulk and Pharmaceutical Dosage Form

Lumateperone, sold under the brand name Caplyta, is an atypical antipsychotic medication of the butyrophenone class. It is approved for the treatment of schizophrenia as well as bipolar depression, Lumateperone was approved for medical use in the United States in December 2019 with an initial indication for schizophrenia and became available in February 2020. It has since demonstrated efficacy in bipolar depression and received FDA approval in December 2021 for depressive episodes associated with both bipolar I and II disorders. Lumateperone acts as an antagonist at 5-HT2A receptors and binds to several dopamine receptors (D1, D2, and D4) with moderate affinity. It has moderate serotonin transporter reuptake inhibition, which is partly responsible for its antidepressant effect in bipolar disorder and reduction of negative symptoms of schizophrenia.

Fig. 1 Structure of Lumateperone

Table 1. Drug profile

MATERIALS AND METHODS

Material

Drug sample used

Pharmaceutically pure sample of LUMATEPERONE TOSYLATE were obtained from Alkem Laboratories, Mumbai.

Fig. 2 Drug sample

Chemicals and Solvents:

All the chemicals and solvents used were of analytical grade. The solvent system used for the study composed of –

• Methanol
• Water

As a ratio methanol: water (70:30)

Pharmaceutical Formulation:

Manufacturing of immediate release LUMATEPERONE TABLETS (42mg) is carried out by the following formula for conducting 2 batches of 10, 50 tablets

Batch 1. LT1                                                         Batch 2. LT2

Fig. 3 Pharmaceutical formulation

Double beam UV-Visible Spectrophotometer Shimadzu 1900 using UV Probe Software. The spectra were recorded over range 200 - 400 nm against solvent in 1 cm Quarts cells.

Fig.4 Double Beam UV- Visible Spectrophotometer

Table 2. Formulation Table:

Fig.5: Drug & Excipients

Table 3. Quality control test for formulated tablets

Fig. 5 Dissolution test Graph

Method Development

In the present work, an attempt was made to develop and validate a simple, precise and accurate method for the estimation of LUMATEPERONE in pure bulk form and in pharmaceutical dosage form by UV - Visible Spectroscopy.

Selection of solvent system:

The solubility of drugs was determined in a variety of polar to non-polar solvents as per Indian Pharmacopeial standards. We performed trial and error method to select the best possible solvent which can dissolve both drugs and tablet formulation. By performing solubility studies of LUMATEPERONE in different solvents and its ratios, observing results we concluded best solvent system was Methanol: water in the ratios 70:30 respectively.

Preparation of Standard Stock Solution:

Accurately weighed 10 mg of Lumateperone were transferred into a clean & dried 100 ml volumetric flasks separately and then volume was made up to the mark with solvent Methanol: water (70:30) to get a standard concentration of 100 µg/ml. This standard stock solution (100 µg/ml) was further diluted with solvent system to obtain a series of dilution - 10, 20, 30, 40, and 50µg/ml for Lumateperone.

Fig.7 Stock & Dilutions of API

Preparation of Standard Test Solution

Accurately weighed 52 mg of tablet powder equivalent to 10 mg of lumateperone were transferred into a clean & dried 100 ml volumetric flasks separately and then volume was made up to the mark with solvent Methanol: water (70:30) to get a standard concentration of 100 µg/ml. This standard stock solution (100 µg/ml) was further diluted with solvent system to obtain a series of dilution - 10, 20, 30, 40, and 50µg/ml for Lumateperone.

Fig.8 Stock & Dilutions of Tablets

Determination of Maximum Wavelength (λmax)

The standard solution and also for test solution dilution having highest concentration was scanned at 200 nm to 400 nm with diluent as the blank to detect maximum wavelength.

Fig.9 Estimation of Maxima of Lumateperone

From the above (Figure-) spectra of Lumateperone standard solution wavelength maxima identified for quantification were 313 nm (λmax)

Fig. 10 Estimation of Maxima of Lumateperone

From the above (Figure-) spectra of Lumateperone Test solution wavelength maxima identified for quantification were 313 nm (λmax).

Method Validation

Linearity & Range

Prepare a series of standard Lumateperone solutions and Test solution of lumateperone of having concentration of 10 µg/ml,20 µg/ml,30 µg/ml,40 µg/ml, and 50 µg/ml each. Measure the absorbance of each standard solution at the determined (λmax). Plot the absorbance values against the corresponding concentrations. A linear regression curve was constructed, the correlation coefficient (R²) and assessment value calculated. The correlation coefficient (R²) for Lumateperone Bulk formulation obtained is 0.994 & for pharmaceutical formulation obtained is 0.997.  The plot is a straight line and the results are tabulated in the Table.

Table 4. Linearity and Range

Fig.10 Linearity graph of Bulk formulation

Fig.11 Linearity graph of Pharmaceutical Formulation

Precision

Express the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision is usually expressed as the standard deviation (SD) or relative standard deviation (%RSD).

Repeatability

To check the degree of repeatability, standard solution containing Lumateperone were taken six times separately of the same concentration 10μg/ml and was analyzed. The standard deviation (S.D.) and Percent Relative Standard Deviation (% R.S.D.) was calculated and given in Table 5 & 6.

Table.5 Repeatability of API

Table.6 Repeatability of Tablets

Intra-day precision

Assessed by analysing multiple replicates of the same sample on the same day, under the same conditions. The % RSD due to Lumateperone concentration for samples for standard solution was found to be 1.825. The % RSD due to concentration for Lumateperone the assay meets the requirements. Results are tabulated in the Table 7.

Table 7. Intra Precision of API

The % RSD due to Lumateperone concentration for samples for standard solution was found to be 1.840. The % RSD due to concentration for Lumateperone the assay meets the requirements. Results are tabulated in the Table 8.

Table 8. Intra Precision of Tablets

Inter-day precision (Intermediate Precision)

Assessed by analysing multiple replicates of the same sample on different days, by different analysts, or using different equipment within the same laboratory. The %RSD due to Lumateperone concentration for the three samples was found to be 1.847% & 1.825%.

Table 9. Inter-day Precision of API

Table 10. Inter-day Precision of Tablets

Accuracy

This parameter determines the accuracy of the assay results under the same operating conditions test. A sample was constituted analysed for the accuracy with known quantity of standard samples of Lumateperone at 50%, 100%, 150% concentration levels and assayed as per the method stated under analytical Methods respectively. Three determinations were performed under each concentration levels respectively. Results are shown in Tables11,12,13. The % RSD due to recovery of Lumateperone at 50%,100%, 150% concentration levels were found to be 0.40%, 0.38% and 0.30% respectively. Nine sample preparations were analyzed according to the proposed method of analysis. The % RSD due to Lumateperone concentration for the assay meets the requirements and within 98.0% to 102%. Results are tabulated in the Table 11& 12.

Table 11. Accuracy and Recovery Results @ 50% Concentration level

Table12. Accuracy and Recovery Results @ 100 % Concentration level

Table13. Accuracy and Recovery Results @ 150% Concentration level

Robustness

Assess the capacity of the method to remain unaffected by small but deliberate variations in method parameters (e.g., wavelength, solvent composition, temperature). Make small changes to the method parameters and observe the effect on the absorbance of a standard solution. The method should remain reliable even with these small variations. The respective test assay results of Lumateperone having concentration as 10 μg/ml was illustrious. The result is expressed as shown in table 14 & 15.

Table 14. Robustness of API

Table 15. Robustness of Tablets

Ruggedness

The degree of reproducibility of test results obtained by the analysis of the same sample in different laboratories, by different analysts, using different instruments, different reagents, and/or different days. The respective test assay results of Lumateperone having concentration as 10 μg/ml was illustrious. The result is expressed as shown in table 16 & 17.

Table 16. Ruggedness of API

Table 17. Ruggedness of Tablets

Limit of Detection(LOD)

The lowest amount of analyte in a sample that can be detected but not necessarily quantitated as an exact value. It can be calculated using the formula: LOD = 3.3 × (SD/Slope), where SD is the standard deviation of the response (y-intercepts of regression lines or standard deviations of blank determinations) and Slope is the slope of the calibration curve.

LOD = 3.3 × (0.002/0.025) = 0.264 μg/ml. ( for API)

LOD= 3.3 ×(0.001/ 0.032) = 0.103 μg/ml.(for tablets)

Limit of Quantitation (LOQ)

The lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. It can be calculated using the formula: LOQ = 10 × (SD/Slope), where SD is the standard deviation of the response (y-intercepts of regression lines or standard deviations of blank determinations) and Slope is the slope of the calibration curve.

LOQ = 10 × (0.002/0.025) = 0.8 μg/ml.(for API)

LOQ = 10 × (0.001/0.032) = 0.312μg/ml.(for Tablets)

Assay of Lumateperone in its formulation

For analysis of commercial formulations of Tablets, 10 tablets were weighed, powdered and accurately weighed the equivalent to 10mg of Lumateperone, which was transferred into 100 ml volumetric flask and make up to 100ml with methanol: water (70:30), filtered and further diluted with methanol: water (70:30) to get the concentrations within the linearity range 10-50 μg/ml and measured at 313 nm for Lumateperone. Then the amount of drug present in the formulations was calculated. The results were shown in Table 18.

Table 18. Results of Assay

Derivative spectroscopy 

Fig. 12 Smoothing of lumateperone spectra

First Derivative: The first derivative spectrum shows a zero crossing at the λmax? of the original peak. It also exhibits a maximum and a minimum at the inflection points of the zero-order band.

Fig.13 1^st order derivative

Second Derivative: The second derivative spectrum typically displays a negative peak at the λmax? of the original band and positive satellite peaks on either side. The sharpness of this negative peak can provide a more accurate determination of λmax?, especially for broad bands.

Fig. 14 2^nd order derivative

STRSS DEGRADATION STUDIES:

Acidic Degradation:

5ml of stock solution of Lumateperone, and 5 ml of 2 N HCl were added in 10 ml of volumetric flask and the volumetric flask was kept at 80?C for 3-hour reflux and left them for the 30 minutes. Afterwards the absorbance of solution was analyses separately at wavelength max 313nm given

Fig.15 Graph of acidic degradation

Alkali Degradation

5 ml of stock solution of Lumateperone and 5 ml of 2 N NaOH was added in 10 ml of volumetric flask and the volumetric flask was kept at 80 c for 3 Hours reflux and left them for the 30 minutes. Afterwards the absorbance of solution was analyses separately at wavelength max 313nm.

Fig.16 Graph of Alkali Degradation

Dry Heat Induced Degradation:

Lumateperone sample was taken in a Petri plate and exposed to a temperature of 50°C for 3 hours in an oven. After 3 hours, 10 mg of the sample was diluted with methanol: water (70:30) to 10 ml. From this solution, dilution was done to achieve the appropriate concentration (5μg/ml)

Fig.17 Graph of dry heat induced degradation

Oxidative Degradation

Applying 3% of hydrogen peroxide (H2O2) at room temperature (25°C) and left them for the 3 hour. Afterwards the absorbance of solution were analyses separately at wavelength max 313nm. (Graph)

Fig.18 Graph of oxidative degradation

Sunlight Degradation

Lumateperone sample was taken in a Petri plate and exposed to sunlight for 3 hrs. 10 mg of the sample was diluted with methanol: water (70:30) up to 10 ml. From this solution, dilution was done to achieve the appropriate concentration (5μg/ml)

Fig. 19 Graph of Sunlight degradation

RESULT AND DISCUSSION:

The method discussed in the present work provides a simple, stable, rapid, accurate, precise, reliable, less expensive (Economical), time saving and convenient method for the analysis of LUMATEPERONE using U.V. Spectrophotometry. The results were shown in Table 19.

Table 19. Result of API & Tablets

Table 20. degradation study results