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INTRODUCTION
Lumateperone is an atypical antipsychotic drug derived from butyrophenone and is used to treat schizophrenia and manic illness (Chavan and Dolas, 2024). chemically, it is 1-(4-fluorophenyl)-4- [(10R, 15S). -4-methyl-1, 4, 12-triazatetracyclo [7.6.1.05, 16.010, 15] hexadeca-5, 7, 9(16)-trien-12-yl] butan-1-one (Corponi et al., 2019). Lumateperone is a second-generation antipsychotic drug administered orally that reaches peak plasma concentrations within 1-2 hr (Depietro, 2023). It is a revolutionary investigational drug that affects serotonin, dopamine and glutamate levels in mental condition (Dhami et al., 2021). It shows its activity as a serotonin receptor antagonist and exhibits a decreased affinity for dopamine receptors (Edinoff et al., 2020; Gamel et al., 2021). Lumateperone has a very high binding affinity (60-fold) for the serotonin receptor compared to the Dopamine (D2) receptor. It also acts on serotonin transporters to reduce serotonin levels, resulting in sleep disorders and schizophrenia (Giulio et al., 2023; Howell et al., 2015).
High-Performance Thin-Layer Chromatography (HPTLC) is a very effective and advanced analytical approach. It has several benefits, including low operating costs and good sample performance, which have made it a standard analytical technique in the modern period. The main superiority of HPTLC in contrast to High-Performance Liquid Chromatography (HPLC) is that HPTLC processes several samples with the least amount of mobile phase, which lowers processing time and cost per study (ICH Q1A R2). The literature provides a number of analytical techniques used to estimate lumateperone, RP-HPLC (Kamboj et al., 2017; Maini et al., 2021) and stability-indicating investigations (McIntyre et al., 2023). According to the presented data, no literature exists for lumateperone estimation using the HPTLC approach; thus, there is a need to develop a low-cost, sensitive HPTLC method for lumateperone. The current study aimed to develop and validate (ICH Q1A R2 guideline., 2003) a new stability-indicating HPTLC method for lumateperone, as well as to characterize the degradation products produced during the stability study using mass spectrometry.
MATERIALS AND METHODS
Reagents and Chemicals
Lumateperone (purity 98.26%) was procured from Sun Pharmaceutical, Mumbai. Lumateperone tablet (42 mg) were formulated in college laboratories. Analytical grade solvents and reagents (Loba Chemie Pvt. Ltd.,) were used.
Instruments
The sample applicator made up of Camag Linomat V, with pressure 3.5 bar, having dimensions of 360 mM×510 mM×410 mM (Width×Length×Height). The stationary phase comprised silica gel (60) F254 plates supplied by Merck, Mumbai, India, with a Camag 100 μL syringe (Hamilton, Switzerland). Development chamber made up of a camag twin glass chamber (10×10 cm and 10×20 cm).
Chromatographic Conditions
The prewashed (methanol) plates were used and dried for 5 min at 100ºC in the oven. The mobile phase was developed by trying different mobile phases, comprising ethyl acetate, methanol, toluene, and ammonia in a ratio of 4:2:4:0.1 v/v. Samples were spotted using a Camag Linomat sample applicator and a microliter syringe to create 6-mM-wide bands on precoated silica plates. The plates were dried using an air dryer at 252 nm.
Standard stock solution
The accurately weighed 4.2 mg of Lumateperone was taken into volumetric flask (10 mL) with methanol. Then it was sonicated for 15 min, the further solutions were prepared to get 420 µg/mL concentration.
Optimization of HPTLC method
The HPTLC conditions were optimized with efficient separation of Lumateperone, Mobile phase, comprised of ethyl acetate: methanol: toluene: ammonia (4:2:4:0.1 v/v) since it was determined to have the optimum system suitability characteristics, Rf value was 0.61 at a wavelength of 252 nm.
Formulated Tablets Analysis
The twenty tablets of Lumateperone, having 42 mg of API per tablet, were taken. The average weight of the tablet was determined and then it was crushed into the powder. The tablet weight equivalent to 42 mg of lumateperone was transferred in a 10 mL volumetric flask containing 4 mL of methanol. It underwent sonication for 20 min and was diluted to make the volume 420 µg/mL using methanol. After sonication, the solution was carefully filtered using 0.45 µM Whatman filter paper. The 0.6 µL spots were applied on a TLC plate (pre-coated) with the mobile phase; the TLC plates were developed and analyzed.
Validation of Method
The method was validated using ICH Q2R1 guideline for analysis of the following parameters.
Linearity and Range
For the linearity, a series of Lumateperone solutions from 84 -504 ng/band were prepared from standard and applied on a TLC plate. Following that, TLC plates were activated, dried and analysed using identical chromatographic parameters. The curve was plotted by using Lumateperone concentrations versus peak area.
Precision
The precision study was determined by repeating the sample analysis with different time intervals: intraday precision (same day) and inter-day precision (next day). The % RSD was determined.
Accuracy
The method's accuracy was performed by spiking the dosage form with Lumateperone standard and analyzing the recovery at 3 concentrations: 80%, 100% and 120%. It was investigated three times; the accuracy was expressed as the % recovery.
Detection Limit and Quantification Limit
The detection and quantification limit of proposed method were performed using the formula from the slope and the standard deviation of the intercept. Formula for calculation for LOD and LOQ is given below.
Where σ=Standard deviation of calibration curve.
S=Slope of the calibration curve.
Robustness
Robustness was assessed by making a few minor, deliberate adjustments to the chromatographic conditions. Saturation time of 5, 10 and 15 min, mobile phase composition and variation in development chamber volume of mobile phase. The % RSD was calculated for all parameters with changes in Rf values and peak area.
Specificity
Specificity was carried out by examining standard lumateperone and lumateperone samples taken from the dosage form; the method's specificity was ascertained by comparing the reflected band's absorbance spectra shown in (Figure 1a and 1b) for specificity and overlay of Lumateperone standard and formulated Lumateperone tablet. Summary of all validation parameter is provided in Table 1.
| Sl. No. | Specifications | Validation Results |
|---|---|---|
| 1 | Linearity (n=6) | 84 to 504 ng/band |
| 2 | Regression Equation | y=4.1883x+94.253 |
| 3 | Correlation coefficient (R²) | 0.9994 |
| 4 | Accuracy (n=3) | % Recovery |
| 80% | 101.3 | |
| 100% | 101.8 | |
| 120% | 102 | |
| 5 | Precision (ng/band) Intra-day (n=3) | Mean±%RSD |
| 168 | 897.9±0.44 | |
| 252 | 1248.9±0.78 | |
| 336 | 1557.2±0.56 | |
| Precision (ng/band) Inter-day (n=3) | ||
| 168 | 897.9±0.42 | |
| 252 | 1245.8±0.72 | |
| 336 | 1541.3±0.47 | |
| 6 | LOD (ng/band) | 3.22 ng/band |
| 7 | LOQ (ng/band) | 9.78 ng/band |
| 8 | Robustness (n=3) | % RSD |
| Mobile phase | 0.51±0.32 | |
| Saturation time | 0.45±0.35 | |
| Development chamber volume of mobile phase | 0.63±0.38 |
Degradation Studies
Lumateperone was exposed to stress degradation in acidic, basic, oxidative, neutral, thermal and photolytic conditions.
Acidic Condition
The 4.2 mg of Lumateperone API was weighed accurately with 3 mL (0.01 N) HCl. The mixture was kept for 20 min at room temperature and then it was neutralized with (0.01 N) NaOH. The volume was made up to the mark with methanol to achieve the concentration of 420 µg/mL.
Basic Condition
The 4.2 mg of Lumateperone was weighed and 3 mL of (0.01 N) NaOH was added. Then it was kept aside for 10 min at room temperature. Methanol was added to the solution to dilute it further to make the concentration of 420 µg/mL.
Oxidative Condition
The 4.2 mg of Lumateperone was weighed, 3 mL of 3% hydrogen peroxide was added. Then it was kept aside for 30 min at room temperature. Methanol was added to the solution to dilute it further to the mark (concentration of 420 µg/mL).
Neutral Condition
The 4.2 mg of Lumateperone was weighed and 3 mL of water was added. Then it was kept aside for 30 min at room temperature. Methanol was added to the solution to dilute it further to the mark (concentration of 420 µg/mL).
Thermal Condition
The 4.2 mg of lumateperone was weighed and lumateperone was heated to 40ºC in an oven. Following a 5-min duration, the API was dissolved with methanol and then it was diluted to the desired level (420 µg/mL).
Photolytic degradation
The 4.2 mg of lumateperone was weighed and then it was kept under UV light for a day. Following a 24 hr. Then it was dissolved in methanol and diluted to the desired quantity (420 µg/mL). From the above-prepared sample, 0.6 µL was applied on HPTLC plates to achieve a 252 ng/band concentration (Nakka et al., 2024; Rathod, 2023).
Preparative isolation and characterization of degradation products
Lumateperone (252 ng/band) was spotted on a 0.6 µL TLC plate for the acidic, alkaline and thermal degradation investigation. Plates were prepared and mobile phase was optimized. Study results using mass spectroscopy revealed degradation products. The identification of every divided fragment in the degradation sample is aided by MS examinations.
Tablet Formulation
The marketed preparation of lumateperone was not available in market. So, the tablet was formulated in college laboratory using 42 mg Lumateperone by wet granulation method.
RESULTS
Wavelength Selection
The UV spectra of Lumateperone was recorded in methanol and maximum absorbance was seen at 252 nm.
Chromatographic system
To analyze the Lumateperone (bulk dosage form and degradation samples) the optimized mobile phase was used i.e. ethyl acetate: methanol: toluene: ammonia (4:2:4:0.1v/v). The plate was developed in a mobile phase (optimized). Good peak shape and resolution between the sample and degradation products were obtained, with an acceptable Rf =0.61.
Validation
Summary of developed validation parameters
The presented HPTLC technique is simple, accurate and robust for analysis of Lumateperone and it was shown based on results of validation parameters; it proved the reliability of the method. Summary of validation is provided in Table 1.
Analysis of formulated tablet
The Lumateperone drug concentration was determined between range 98.9 to 100.8% in the formulation trials as shown in Table 2. This method can be used for routine drug estimation.
| Sl. No. | Quantity of API taken (ng/band) | Peak area | Quantity of API recovered (ng/band) | % Recovery |
|---|---|---|---|---|
| 1 | 252 | 1240.2 | 249.4 | 98.9 |
| 2 | 252 | 1263.5 | 254.1 | 100.8 |
| 3 | 252 | 1239.1 | 249.1 | 98.8 |
| 4 | 252 | 1244.8 | 252.3 | 99.3 |
| 5 | 252 | 1258.6 | 254.2 | 100.4 |
| 6 | 252 | 1264.8 | 251.7 | 100.8 |
| Mean | 1251.75 | 251.74 | 99.89 | |
| Standard Deviation (SD) | 11.6 | |||
| % Relative Standard Deviation (% RSD) | 0.93 |
Stress degradation studies
The stress degradation analyses were determined by acid, alkali, oxidative, thermal, photolytic and neutral degradation of the Lumateperone standard. well-separated, pure Lumateperone bands with a specific Rf value were discovered. Table 3 presents the results and Figure 2 displays a densitogram of deterioration at 252 nm. It was shown that the drug degraded in almost every kind of stress, including alkali, hydrolytic, oxidative and acidic stressors. The significant percentage of degradation was observed in photolytic stress.
| Sl. No. | Type of degradation | Stressors condition | Rf value of degradants | % Degradation | % Recovered |
|---|---|---|---|---|---|
| 1 | Acidic degradation | 0.01 N HCl, for 20 min at room temperature. | 0.8 | 19.95 | 80.05 |
| 2 | Alkaline degradation | 0.01N NaOH for 15 min at room temperature. | 0.58 | 26.28 | 73.72 |
| 3 | Oxidative degradation | 3% H2O2, for 30 min at room temperature. | 0.79 | 13.8 | 86.2 |
| 4 | Thermal degradation | 40ºC in oven for 15 min | 0.78 | 21.9 | 78.1 |
| 5 | Photolytic degradation | Exposure to UV light for 24 hr. | 0.81 | 11.9 | 88.9 |
| 6 | Neutral degradation | H2O for 30 min, at room temperature. | 0.15 | 10.6 | 89.4 |
Characterization of Degradation Products
The studies were performed in various stress conditions and degradation products thus obtained were characterized by mass spectrometry. Lumateperone has an m/z value of 393.5.
The acidic-degraded solution of the drug was characterized using mass spectrometry, shown in Figure 3a, revealing two degradants. Lumateperone, with an m/z value of 393.5, undergoes cleavage at Fluorophenyl butan-1-one (C11H13FO) and (C11H12FO) to yield an imine derivative as degradation product 1 (DP1) with an m/z value of 213 (M+H=214.3) and an iminium Derivative (DP2) with an m/z value of 214 (M+=214.8). The degradation pathway is shown in Figure 3b.
The alkali-degraded solution of the drug shown in Figure 4a reveals one degradation product of Lumateperone, with an m/z value of 393.5, which undergoes removal of the amino piperidine ring (C11H9N2) to yield Degradant Product 3 (DP3), the ammonium derivative, which has an m/z value of 224 (M+=224.6). The degradation pathway is shown in Figure 4b.
The thermally degraded solution of the drug shown in the spectra of Figure 5a reveals the Degradation Product (DP4) lumateperone with an m/z value of 393 that undergoes removal of 4-fluorophenyl and carbonyl group (C8H6FO) to yield a stable fragment iminium derivative, i.e., DP4, with an m/z value of 256.18 (M+H=257.6). Degradation pathway illustrated in Figure 5b. The summary of Lumateperone and its degradation products are shown in Table 4
| Sl. No. | Name of Compound | Molecular weight in g/mol | Molecular formula | Name of compound (IUPAC) |
|---|---|---|---|---|
| 1 | Lumateperone | 393 | C24H28FN3O | 1-(4-Fluorophenyl)-4-[(10R,15S)-4-methyl-1,4,12-triazatetracyclo [7.6.1.05,16.010,15] hexadeca-5,7,9(16)-trien-12-yl] butan-1-one. |
| 2 | DP 1 | 214 | C13H15N3 | Imine Derivative |
| 3 | DP 2 | 214 | C13H16N+3 | Iminium Derivative |
| 4 | DP3 | 224 | C13H19FN+O | Ammonium Derivative 4-(N-ethyl-N-methylammonium)-1-(4-fluorophenyl) butan-1-one. |
| 5 | DP 4 | 257 | C16H15N+3 | Iminium Derivative |
The greenness of the above method was assessed using the AGREE program, which offers reliable and precise findings for method greenness. Following the input of all 12 green chemistry principles into software on a 0 to 1 scale, which is indicated by a red, yellow, or green color, the graph is generated automatically by the software. The entire result is displayed centrally, i.e., 0.72, with a value closer to one and a darker green signifying how much greener and more eco-friendly the process is (Figure 6). The estimated score of 0.72 indicates that the analyzed method is more ecologically friendly (Shivaji, 2023).
DISCUSSION
As there are no reported HPTLC methods available for Lumateperone estimation, hence the new HPTLC method development and validation of Lumateperone was performed according to ICH Q2R1 guidelines. The detailed investigation of degradation under various stress conditions was performed and degradants were characterized by mass spectrometry.
The results of proposed methods show the mobile phase was optimized, ethyl acetate: methanol: toluene: ammonia (4:2:4:0.1 v/v) at the 252 nm absorbance. RF value was obtained at 0.61. Linearity was performed between a range of 84-504 ng/band, with R2=0.9994; recovery was achieved between 98.8% and 100.8%.
The stability results show the drug is susceptible to acidic, basic, alkaline, photolytic, thermal and neutral conditions. (Table ) The degradation product obtained DP1 as an imine derivative at 214 m/z value, DP2 as 4-(N-ethyl-N-methylammonium)-1-(4-fluorophenyl) butan-1-one at 224 m/z value and DP4 as an iminium derivative at 257 m/z value were identified and distinguished by HR-MS with their proposed drug degradation pathways.
CONCLUSION
A new, robust, sensitive and precise HPTLC method of lumateperone was optimized and validated using ICH Q2R1 guidelines. Also, the mass spectra of lumateperone with its degradation products under different stress conditions were analyzed with the degradation pathways using mass spectrometry. The above methods found the potential green method for the quality analysis for lumateperone tablet formulation.