# Comprehensive Stability Analysis of Haloperidol: Insights from Advanced Chromatographic and Thermal Analysis

Khadidja Djilali, Rachida Maachi, Hocine Boutoumi, Sabrina Lekmine, Zohra Ait Mesbah, Sabry M. Attia, Noureddine Nasrallah, Rachida Bouallouche, Jie Zhang, Hichem Tahraoui, et al.

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Khadidja Djilali, Rachida Maachi, Hocine Boutoumi, Sabrina Lekmine, Zohra Ait Mesbah, et al. Comprehensive Stability Analysis of Haloperidol: Insights from Advanced Chromatographic and Thermal Analysis. Processes, 2025, 13 (3), pp.904. 10.3390/pr13030904. hal-05023826

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Article

# Comprehensive Stability Analysis of Haloperidol: Insights from Advanced Chromatographic and Thermal Analysis

Khadidja Djilali<fnref n="1" />, Rachida Maachi<fnref n="1" />, Hocine Boutoumi<fnref n="2" />, Sabrina Lekmine<fnref n="3" />, Zohra Ait Mesbah<fnref n="4" />, Sabry M. Attia<fnref n="5" /> ID, Noureddine Nasrallah<fnref n="1" />, Rachida Bouallouche<fnref n="1" />, Jie Zhang<fnref n="6" /> ID, Hichem Tahraoui<fnref n="7" />,<fnref n="8" /> ID and Abdeltif Amrane<fnref n="8" />,* ID

<figure><img src="image_2.png" /></figure>

check for
updates

Academic Editors: Alina Pyka-Pajak
and Paolo Trucillo

Received: 30 December 2024

Revised: 3 March 2025

Accepted: 17 March 2025

Published: 19 March 2025

**Citation:** Djilali, K.; Maachi, R.; Boutoumi, H.; Lekmine, S.; Ait Mesbah, Z.; Attia, S.M.; Nasrallah, N.; Bouallouche, R.; Zhang, J.; Tahraoui, H.; et al. Comprehensive Stability Analysis of Haloperidol: Insights from Advanced Chromatographic and Thermal Analysis. *Processes* 2025, 13, 904. https://doi.org/10.3390/pr13030904

**Copyright:** © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
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(https://creativecommons.org/licenses/by/4.0/).

**Abstract:** In this study, we thoroughly investigated the stability of haloperidol using a comprehensive set of chromatographic and thermal analyses. Various stress conditions were examined, including exposure to oxidizing agents (such as hydrogen peroxide), dry heat, photolytic conditions, and acid and alkaline hydrolysis. Significant degradation was observed in acidic and alkaline environments, leading to the formation of degradation by-products, specifically DPA, DPB, DPC, and DPD for acidic and basic conditions. In contrast, haloperidol demonstrated robust stability under photolytic, oxidative, and dry-heat conditions. For the analysis of the drug and its degradation products, a C-18 column was employed, coupled with a mobile phase consisting of methanol and a phosphate buffer (pH = 9.8) in a 90:10 (v/v) ratio. The analytical method was rigorously validated according to ICH Q2 (R1) guidelines, ensuring its accuracy and reliability. This method exhibited excellent linearity within a concentration range of 1 to 50 µg/mL, with an R<fnref n="2" /> of 0.999. Additionally, this method is applicable to commercial formulations, without the need for prior extraction. LC-MS/MS analysis revealed distinct *m/z* values and fragmentation spectra corresponding to the degradation products, including an impurity not documented in the European Pharmacopoeia monograph for the drug. Three additional degradation products were identified based on *m/z* values and base fragments. Thermal analyses, including thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and differential thermal analysis (DTA), provided further evidence of the active ingredient's thermal stability, with a melting temperature of approximately 150 °C. These results collectively offer valuable insights into the degradation behavior of haloperidol, providing critical implications for its pharmaceutical quality and integrity under various environmental conditions.

<footnote n="1">¹ Laboratory of Reaction Engineering, Department of Mechanical and Process Engineering, University of Science and Technology Houari Boumediene, Algiers-Bab Ezzouar 16111, Algeria; khadidjadjilali@gmail.com (K.D.); racha.maachi@gmail.com (R.M.); nas_nour@yahoo.fr (N.N.); r.bouallouche@mesrs.dz (R.B.)</footnote>

<footnote n="2">² Laboratoire de Génie Chimique, Département de Génie des Procédés, Faculté de Technologie, Université Saad Dahlab, Blida 1, BP270, Blida 09000, Algeria; ybentoumi@ymail.com</footnote>

<footnote n="3">³ Biotechnology, Water, Environment and Health Laboratory, Abbes Laghrour University, Khenchela 40000, Algeria; sabrina.lekmin.400@gmail.com</footnote>

<footnote n="4">⁴ Laboratory of Energy Processes and Nanotechnology, Blida University 1 rue de Soumaa, BP 270, Blida 09000, Algeria; zohraait.mesbah@gmail.com</footnote>

<footnote n="5">⁵ Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia; attiasm@ksu.edu.sa</footnote>

<footnote n="6">⁶ School of Engineering, Merz Court, Newcastle University, Newcastle upon Tyne NE1 7RU, UK; jie.zhang@newcastle.ac.uk</footnote>

<footnote n="7">⁷ Laboratoire de Biomatériaux et Phénomènes de Transport (LBMPT), Université de Médéa, University of Médéa, Nouveau Pôle Urbain, Médéa 26000, Algeria; hichemm.tahraouii@gmail.com</footnote>

<footnote n="8">⁸ Ecole Nationale Supérieure de Chimie de Rennes (ENSCR), CNRS, ISCR-UMR6226, University de Rennes, F-35000 Rennes, France</footnote>

<footnote>* Correspondence: abdeltif.amrane@univ-rennes.fr</footnote>
**Keywords:** haloperidol; validation; stability; stress conditions; HPLC; LC-MS/MS; TGA/DSC/DTA

# 1. Introduction

Ensuring the accurate composition of drugs is a paramount concern in the pharmaceutical industry [1,2]. The reliability and accuracy of the active pharmaceutical ingredients (APIs) declared by manufacturers are crucial factors in determining the efficacy and safety of medical treatments [3–5]. Various analytical techniques are utilized to verify the qualitative and quantitative compositions of drugs. These methods play a vital role in quality control during drug production, the evaluation of generic alternatives, and the identification of counterfeit products [6,7]. Among the preferred techniques is high-performance liquid chromatography (HPLC) coupled with UV-Vis detection. Additionally, more advanced methods, such as liquid chromatography combined with mass spectrometry (LC-MS), are employed to precisely identify active pharmaceutical ingredients. Furthermore, thermal analysis techniques like thermogravimetric analysis (TGA) can be used for comprehensive characterization [8,9].

This study focuses on haloperidol, a molecule belonging to the class of basic butyrophenes, commonly used as a neuroleptic agent. Haloperidol is prescribed for the treatment of acute and chronic psychoses and psychotic disorders, such as schizophrenia, manic states, and delusions [10,11]. Widely used in many countries, it is considered equally effective compared to other low-potency psychotropic drugs, including chlorpromazine and thioridazine [12]. Chemically known as 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidinyl]-1-(4-fluorophenyl)-1-butanone, haloperidol is a white, crystalline powder with a molecular weight of 375.9 g/mol. It has a significant pKa value, typically reported to be around 8.3, which corresponds to the phenolic hydroxyl group. This group plays a crucial role in determining the solubility and ionic form of haloperidol, factors that are essential for its absorption and bioavailability in the body. The phenolic hydroxyl group, consisting of a hydroxyl group (-OH) attached to a benzene ring, is directly connected to the main aromatic ring of the molecule. The chemical structure of haloperidol, highlighting the phenol hydroxyl group responsible for its pKa value, is illustrated in Figure 1 [13]. Although haloperidol exhibits low solubility in water [14,15], it can dissolve in the presence of lactic acid or tartaric acid [14,16–18].

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Chemical structure of haloperidol.</figcaption></figure>
Haloperidol is available in tablet, oral, and injectable forms, so it is crucial to ensure the long-term stability of these formulations [16]. Numerous studies have focused on the determination of haloperidol in pharmaceutical forms, particularly in solid formulations. The most commonly used analytical methods include HPLC, UV-visible spectrophotometry, and derivative spectrophotometry [5,15,19,20], often combined with extraction or complexation steps [21–30]. One research team prepared a haloperidol solution with a concentration of 2 mg/mL using purified water, adding a small excess of lactic acid to ensure stability. The solution was then protected from exposure to sunlight or daylight to maintain its integrity [17,31]. The results demonstrated that haloperidol is sensitive to light, as the solutions stored in clear glass vials showed discoloration within hours and developed a greyish precipitate over several weeks [16]. In contrast, the solutions stored in amber glass vials remained unchanged, with no discoloration or precipitation observed even after eighteen months. Further investigations in 1981 confirmed that haloperidol solutions containing lactic acid at pH 3 remained stable for up to 5 years at room temperature, 2 years at 40 °C, and 6 months at 60 °C. However, exposure to natural light caused cloudiness, discoloration, and a reduction in haloperidol content [18]. In 1983, a new HPLC method was developed to identify haloperidol and detect its degradation products, revealing its instability under high temperatures, light exposure, and varying pH conditions [32,33]. By 1988, researchers had examined various physicochemical properties of haloperidol, particularly regarding the effects of oxygen and light. Their findings over a two-year stability period indicated that oxygen had minimal impact on haloperidol solutions stored in amber glass [34]. In 2002, an HPLC method for haloperidol determination was introduced, enabling effective separation of its degradation products. This method involved the use of an octadecylsilane stationary phase column under isocratic conditions, with degradation experiments conducted using hydrochloric acid, sodium hydroxide, and hydrogen peroxide, leading to the identification of major degradation products [35]. Additionally, in 2003, a rapid and sensitive HPLC technique was developed and validated for the simultaneous determination of haloperidol and its degradation products (Cis-PFBoxide, hydroxy-PFB, 2H-PFB, and trans-PFBoxide) using a porous graphitic carbon (PGC) column [36]. The extensive body of literature highlights haloperidol’s susceptibility to hydrolysis, oxidation, photolysis, and heat, emphasizing the need for careful handling to protect the drug from these degrading factors, whether in liquid or solid form. Various chromatographic techniques, such as UPLC, RP-HPLC, capillary electrophoresis, and thin-layer chromatography, have been employed to evaluate the stability of haloperidol and its formulations. However, there remains a significant gap in fully characterizing and identifying its degradation products [37,38].

The inconsistencies in findings regarding storage conditions, packaging, and duration underscore the need for a more in-depth investigation into the stability of haloperidol. However, little research has been conducted on its quantitative analysis. In this study, we aimed to explore the degradation behavior of haloperidol under hydrolytic, oxidative, thermal, and photolytic conditions while also characterizing the resulting degradation products using a chromatographic approach combined with mass spectrometry. There is a pressing need to develop a rapid and straightforward HPLC method for haloperidol analysis. Although numerous studies have documented HPLC and LC-MS/MS methods for various drugs, there is a lack of stability-indicating methods specifically designed for haloperidol. Recent recommendations highlight the importance of developing HPLC methods for active pharmaceutical ingredients that ensure effective separation between the drug and its degradation products; established methodologies are available for this purpose [39,40]. Additionally, a recent study provides a critical review of the use of HPLC-MS in this context [41]. Therefore, the primary objective of this study was to introduce a new and rigorously validated stability-indicating HPLC method for the quantitative
determination of haloperidol, addressing degradation under acidic, basic, oxidative, thermal, and light-induced conditions. The chemical structures of the degradation products formed under stress conditions were thoroughly investigated using LC-MS/MS, while thermal analysis via TGA/DTA/DSC was employed to confirm the thermal stability of haloperidol. The insights gained from the thermal stability studies and understanding the thermal decomposition pathways are essential for advancing our theoretical knowledge of haloperidol's chemical properties. Furthermore, these findings could have practical applications in optimizing the production, processing, and storage of haloperidol. Notably, no prior research has explored the thermal stability and decomposition of haloperidol. This study aims to address this gap by investigating the thermal decomposition mechanisms of haloperidol under nitrogen atmospheres using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Another goal of this research was to utilize the proposed HPLC method to monitor the stability of haloperidol under various stress conditions. It is important to note that forced degradation studies lack standardized protocols [42]. In accordance with ICH guidelines, we conducted a comprehensive assessment of haloperidol's forced degradation under diverse hydrolytic, oxidative, photolytic, and thermal conditions [43].

## 2. Materials and Methods

### 2.1. Instruments and Equipment

For this research, we employed a Hewlett Packard 1100 series chromatograph (manufactured in the USA), which included a G1312A binary pump, a G1322A degasser, a G1315A UV detector, and a G1316A column furnace with a manual injector (Agilent Technologies, USA). The HPLC system was controlled, and data acquisition and chromatogram generation were managed using ChemStation software version B.04.03, Agilent Technologies, Santa Clara, CA, USA. Additionally, a Shimadzu 8040 UPLC-ESI-MS-MS system (produced in Kyoto, Japan) was utilized, featuring ultra-high sensitivity along with UFMS technology and equipped with a Nexera XR LC-20AD binary pump Kyoto, JP. Thermogravimetric analysis (TGA) was conducted using a thermal analyzer (Model SDT Q600, TA Instruments, New Castle, DE, USA) for all three experiments. The analysis covered a temperature range from 20 °C to 500 °C, with a heating rate of 5 °C/min, using aluminum crucibles containing 10 mg of the sample. Nitrogen was used as purge gas, and the resulting data were processed using the TA Instruments Universal Analysis software.

### 2.2. Materials

Haloperidol was kindly supplied as a gift sample by the GROUPE SAN-TE-ALGERIE laboratory in Algeria, with a purity of 99%.

HPLC-grade methanol, monobasic phosphate, lactic acid, and sodium hydroxide were procured from SIGMA-ALDRICH (USA). Hydrochloric acid was sourced from BIOCHEM (Germany), while hydrogen peroxide was obtained from SCHARLAU (Spain). Purified water and methylparaben were supplied by PUBCHEM (Germany).

### 2.3. Methods

Lactic acid was chosen as the solubilizing solvent for haloperidol based on bibliographic data indicating its low solubility in water (0.1 mg/mL) and methanol (approximately 16.7 mg/mL at 25 °C) as well as its favorable solubility in ethanol, methylene chloride [14,15,19], and lactic acid [18].
### 2.3.1. Chromatographic Conditions

Preliminary tests were conducted to fine-tune critical parameters and enhance the reproducibility of retention times. The composition of the mobile phase was explored by testing different ratios (80:20, 90:10, and 95:5) of methanol to the phosphate buffer to achieve optimal separation of degradation products. In addition to pH 9.8, more moderate pH values (7.4 and 8.0) were also evaluated, allowing for the confirmation that pH 9.8 provided superior peak resolution and effective separation. A flow rate of 1 mL/min was chosen after comparing 0.8 mL/min, 1 mL/min, and 1.2 mL/min, as this rate offered the best compromise between analysis time and separation efficiency.

The chromatographic conditions employed in the analysis included a stationary phase comprising a C18 column (250 × 4.6 mm, 5 µm) and a mobile phase. The composition of the mobile phase was precisely defined in the software to ensure accurate control of the mixture. It consisted of methanol and potassium phosphate buffer (pH 9.8) in a 90:10% v/v ratio. The potassium phosphate buffer solution was prepared following the protocol established in our earlier study [31], where 100 mg of monobasic phosphate was dissolved in 1000 mL of ultrapure water, adjusted to pH 9.8 using 1M NaOH, and filtered through a 0.45 µm syringe filter. Consequently, the mobile phase comprised 90% methanol and 10% potassium phosphate buffer by volume. For every 100 mL of mobile phase, 90 mL corresponded to methanol, and 10 mL corresponded to potassium phosphate buffer. This specific ratio was meticulously chosen to optimize the separation of sample components. The analysis was performed at room temperature with a flow rate of 1 mL/min, and eluent detection was conducted at 248 nm.

### 2.3.2. Forced Degradation Studies

Forced degradation studies were carried out on haloperidol with the aim of achieving a degradation rate of 5–20%. Acidic and alkaline hydrolysis were performed at 70 °C under controlled pH conditions for 7 days. Oxidative degradation was induced by exposing haloperidol to 0.3% and 3% *v/v* $H_2O_2$ at 60 °C in the absence of light for 7 days. Photolytic stress was applied by exposing haloperidol to UV light using a UV lamp (Ultraviolet light UVA 24 W (Philips PL/L, Amsterdam), 270 nm, 20 mW/cm$^2$) for 48 h. Additionally, dry-heat studies were conducted by subjecting haloperidol to temperatures of 60 °C and 80 °C for 15 days in a solid-state hot-air oven. After exposure, the drug samples were cooled and analyzed to ensure a final drug concentration of 20 µg/mL had been reached. Degradation was evaluated based on a reduction in the peak area of haloperidol and/or the appearance of additional peaks.

### 2.3.3. Analysis of Haloperidol via HPLC and Validation of the Method

Analytical methods are frequently validated in accordance with the ICH Q2 (R1) guidelines [44]. In this research, a haloperidol solution at a concentration of 100 µg/mL was analyzed using HPLC, with 1% lactic acid serving as the dissolution solvent and the mobile phase acting as the blank, measured at a wavelength of 248 nm. The linearity of the calibration curve was assessed across a concentration range of 1 µg/mL to 50 µg/mL using haloperidol standard solutions. These standards were prepared by diluting the 100 µg/mL haloperidol stock solution with methanol as the diluent. The peak areas of the standard solutions, covering concentrations from 1 µg/mL to 50 µg/mL, were measured at a maximum wavelength (λmax) of 248 nm. The haloperidol quantification method was validated according to ICH guidelines [44,45], focusing on parameters such as linearity, precision (evaluated through repeatability and intermediate precision), and accuracy.

The linearity of the calibration curve was confirmed by a very high correlation coefficient (R = 0.999) between absorbance and standard concentrations. Repeatability was
assessed by determining intra-day variation for three concentrations (1, 5, and 25 µg/mL), with three repetitions for each concentration on the same day. Intermediate precision was evaluated by inter-day variation for the same three concentrations (1, 5, and 25 µg/mL), with two repetitions for each concentration analyzed over three different days.

Accuracy, which reflects the systematic error of an assay method, was evaluated at three different concentration levels (1, 5, and 25 µg/mL) once linearity was established [31,46]. Variations in the organic concentration percentage, flow rate, and wavelength of the system were intentionally introduced under the ideal chromatographic conditions described above, with the parameters recorded. The effectiveness of the proposed method was confirmed by successfully separating haloperidol and its degradation products, with no peaks observed in blank assays during the retention time of haloperidol and its degradation products.

### 2.3.4. Application of the Method to the Analysis of a Finished Product

The approach described in this research was utilized to examine samples of a 0.2% oral solution of haloperidol (ISOPERIDOL®) obtained from pharmacies. For the preparation of the injection sample solution, 0.5 mL of the final product was mixed with 25 mL of methanol. This mixture was then filtered using a 0.22 µm filter and subsequently analyzed using HPLC.

### 2.3.5. Characterization of Degradation Products Using LC-MS/MS

Samples exposed to stress conditions (1 N HCl, 1 N NaOH for 7 days, and 48 h of UV light irradiation) were analyzed using LC-MS/MS. Positive electrospray ionization (ESI) was conducted with the following parameters: a CID gas pressure of 230 KPs, a conversion dynode voltage of -6.00 kV, an interface temperature maintained at 350 °C, a desolvation line (DL) temperature of 250 °C, a nebulizing gas flow rate of 3.00 L/min, a thermal block temperature of 400 °C, and a drying gas flow rate of 15.00 L/min. The analysis was performed using a mass spectrometer. Sample separation was achieved via HPLC with a mobile phase suitable for mass spectrometry, consisting of solvent A (20% water and 0.1% formic acid) and solvent B (80% methanol). The LC-MS/MS data obtained were subsequently used for structural identification.

### 2.3.6. Analysis of Haloperidol via TGA/DTG/DSC

The thermal stability of haloperidol was evaluated using TGA over a temperature range of 20 °C to 500° C. The experiments were performed using a thermal analyzer (model SDT Q 600-TA, New Castle, DE, USA). Samples were heated from 20 °C to 500 °C at a rate of 5 °C/min in aluminum crucibles containing 10 mg of the sample. Nitrogen was used as the purge gas, and the data were analyzed using TA Instruments' Universal Analysis software. During the heating process, any endothermic or exothermic reactions were monitored. The variations relative to the reference were recorded and translated into a specific thermograph corresponding to the thermal process.

## 3. Results and Discussion

### 3.1. *Calibration Curve and Linearity*

The linearity of the calibration curve was confirmed by the high correlation coefficient ($R = 0.999$) between absorbance and the standard concentrations.

### 3.2. *Chromatographic Conditions for Analysis*

To ensure accurate analysis of haloperidol and its degradation products, a series of experiments were conducted to identify the most suitable mobile phase. The goal was to
achieve optimal retention of haloperidol while effectively separating it from its degradation products. Various mobile phase combinations were evaluated, each aimed at delivering the desired chromatographic performance. Under the selected conditions, haloperidol was adequately retained, with a peak retention time of 3.3 ± 0.05 min.

The system's suitability was confirmed to be acceptable under these conditions. The optimized chromatographic method successfully separated excipients and degradation products from the haloperidol peak, a process that is essential for ensuring precise quantification of haloperidol without interference from other sample components. Additionally, all the eluents were efficiently detected at the optimal wavelength of 248 nm, allowing for accurate identification and quantification of haloperidol and its related compounds. Figure 2 illustrates the chromatogram obtained using these optimized conditions, highlighting the method's effectiveness in accurately analyzing haloperidol samples while maintaining system suitability and peak resolution [35,47–50].

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Chromatogram of haloperidol standard solution (50 µg/mL).</figcaption></figure>

### 3.3. Forced Degradation Studies

Understanding the degradation pathways of haloperidol is crucial for evaluating its stability and developing effective formulation strategies. In this study, forced degradation experiments were performed to assess its behavior under hydrolytic, oxidative, thermal, and photolytic stress conditions (Table 1).

Haloperidol underwent hydrolytic degradation in both acidic and alkaline environments, revealing its sensitivity to changes in pH. When exposed to oxidative stress via hydrogen peroxide, degradation products were formed, confirming its vulnerability to oxidation. Thermal degradation was observed at higher temperatures (60 °C and 80 °C), indicating its instability under heat. Furthermore, exposure to UV light caused photolytic degradation, underscoring the importance of protecting this drug from light in formulations. These results provide valuable insights into the primary degradation pathways of haloperidol, offering essential information for enhancing its stability and guiding the development of appropriate pharmaceutical formulations and packaging.
<table><caption>**Table 1.** Forced-degradation results for haloperidol.</caption><thead><tr><th>Forced Degradation Conditions</th><th>Percentage Degradation (%)</th><th>Degraded Concentration (µg/mL)</th></tr></thead><tbody><tr><td>0.1 N HCl, 60 °C, 7 days</td><td>16</td><td>3.2</td></tr><tr><td>1.0 N HCl, 60 °C, 7 days</td><td>25</td><td>5</td></tr><tr><td>0.1 N NaOH, 60 °C, 7 days</td><td>16</td><td>3.2</td></tr><tr><td>1.0 N NaOH, 60 °C, 7 days</td><td>17</td><td>3.4</td></tr><tr><td>H<sub>2</sub>O<sub>2</sub> 0.3%, 25 °C, 7 days</td><td>0.00</td><td>0.00</td></tr><tr><td>H<sub>2</sub>O<sub>2</sub> 0.3%, 60 °C, 7 days</td><td>0.00</td><td>0.00</td></tr><tr><td>H<sub>2</sub>O<sub>2</sub> 3.0%, 25 °C, 7 days</td><td>0.00</td><td>0.00</td></tr><tr><td>H<sub>2</sub>O<sub>2</sub> 3.0%, 60 °C, 7 days</td><td>0.00</td><td>0.00</td></tr><tr><td>Haloperidol powder, 60 °C, 15 days</td><td>0.00</td><td>0.00</td></tr><tr><td>Haloperidol powder, 80 °C, 15 days</td><td>0.00</td><td>0.00</td></tr><tr><td>Haloperidol solution, 60 °C, 15 days</td><td>10.00</td><td>2.00</td></tr><tr><td>Haloperidol solution, 80 °C, 15 days</td><td>17.00</td><td>3.40</td></tr><tr><td>Haloperidol powder, UV light, 48 h</td><td>0.00</td><td>0.00</td></tr><tr><td>Haloperidol solution, UV light, 48 h</td><td>13</td><td>2.6</td></tr></tbody></table>

### 3.4. Method Validation

The calibration curve exhibited excellent linearity across the concentration range of 1.0–50 µg/mL, with a correlation coefficient (R²) of 0.999. The relative standard deviation (RSD) was consistently below 2%, demonstrating the method’s precision (Table S1, Figure 3A). Furthermore, the residual plot (Figure 3B) displayed a random distribution around the zero line, confirming the absence of systematic errors and aligning with ICH guidelines. This streamlined presentation focuses on the key findings, as requested by the reviewer, omitting repetitive methodological details.

<figure><img src="image_5.png" /><figcaption>**Figure 3.** (A) Linearity of the method. (B) Residual graph of the linearity of the method.</figcaption></figure>
The validation results for the proposed HPLC method for haloperidol analysis are summarized in Table S2. These results are consistent with those reported in the literature [37] and meet the criteria outlined in the ICH guidelines [44]. Consequently, the method can be considered reliable for the determination of haloperidol in liquid dosage forms [19,47,51].

### 3.5. Application of the Method to the Analysis of Haloperidol in a Finished Product (ISOPERIDOL) (2 mg/mL Solution)

When the sample solution, prepared via the simple dilution of the finished product, was injected, two clear chromatographic peaks were observed: one at approximately 3.30 min, corresponding to haloperidol, and another at 2.11 min, representing methylparaben, as shown in Figure 4. These compounds were efficiently eluted and separated, with a resolution greater than 2.

<figure><img src="image_6.png" /><figcaption>**Figure 4.** Chromatogram of the sample (0.2% haloperidol oral solution).</figcaption></figure>

The accurate quantification of haloperidol in the final product is crucial to ensure the product's quality and adherence to regulatory standards. The reported concentration of (2 ± 0.54) mg/mL, derived from three replicate measurements, demonstrates the high precision of the analytical method used. The percentage estimate of 100.5% indicates that the product complies with the strict requirements of the British Pharmacopoeia, which typically mandates that the concentration of active pharmaceutical ingredients should fall within the range of 95% to 100%. This level of accuracy and compliance highlights the reliability of the analytical method and ensures the efficacy and safety of the finished product for its intended use [19,37].

The HPLC method presented in this study is a dependable approach for evaluating the content of haloperidol in oral solutions. Its specificity and robustness make it highly suitable for quality control applications in pharmaceutical laboratories. A significant advantage of this method is its simple mobile-phase composition, which contrasts with the more complex formulations often described in the literature and pharmacopeias. This streamlined approach improves analytical efficiency and laboratory practicality, making it a valuable and effective alternative for pharmaceutical quality control. In summary,
this method combines simplicity and reliability, proving to be highly useful in quality assurance protocols.

### 3.6. Characterization of Degradation Products Using LC-MS/MS

LC-MS/MS analysis in ESI+ mode was employed to characterize the degradation products of haloperidol by detecting their molecular mass (m/z) values and fragmentation patterns. This approach facilitated the identification of key degradation products, as summarized in Table 2, and provided valuable insights into their structural elucidation. The results, depicted in Figure S1, enhance our understanding of haloperidol's degradation pathways, aiding in the optimization of its stability for pharmaceutical formulations.

<table><caption>**Table 2.** Potential degradation products of haloperidol.</caption><thead><tr><th>Name</th><th>Molecular Formula</th><th>Structural Form and Chemical Name</th><th>Molecular Weight (g/mol)</th></tr></thead><tbody><tr><td>Haloperidol</td><td>C<sub>21</sub>H<sub>23</sub>ClFNO<sub>2</sub></td><td>[Chemical Structure Image]</td><td>376</td></tr><tr><td>Degradation Product I</td><td>C<sub>21</sub>H<sub>25</sub>Cl<sub>2</sub>FNO<sub>2</sub></td><td>[Chemical Structure Image]</td><td>411.5</td></tr><tr><td>Degradation Product II</td><td>C<sub>26</sub>H<sub>32</sub>ClF<sub>2</sub>O<sub>3</sub></td><td>[Chemical Structure Image]</td><td>476</td></tr><tr><td>Degradation Product III</td><td>C<sub>26</sub>H<sub>33</sub>Cl<sub>2</sub>FN<sub>2</sub>O<sub>3</sub></td><td>[Chemical Structure Image]</td><td>511.5</td></tr><tr><td>Degradation Product IV</td><td>C<sub>21</sub>H<sub>23</sub>ClFNO<sub>3</sub></td><td>[Chemical Structure Image]</td><td>392</td></tr></tbody></table>

The results of the haloperidol analysis conducted using LC-MS/MS are presented in Figure 5. The *m/z* value for haloperidol in the reported methods was found to be 376.
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Mass spectrum of haloperidol.</figcaption></figure>

### 3.6.1. Acidic Stress: HCl 1N

The mass spectra of the haloperidol solution under acidic conditions, presented in Figures S2–S4, provide critical insights into its degradation behavior. By analyzing the mass-fragmentation patterns, degradation signals were identified, confirming the formation of degradation products. Under acidic stress, the degradation product [A] was detected with a significant signal at *m/z* = 411. Additionally, a key fragment with a mass-to-charge ratio of *m/z* = 362.5 was observed, further corroborating the presence of this degradation product.

In the case of degradation product [B], illustrated in Figure S3, multiple fragments were observed in the mass spectra. A notable signal corresponding to this product was identified at *m/z* = 476. It is hypothesized that degradation product [B] may originate from a synthetic impurity. Importantly, this impurity has not been previously reported or documented in the literature. The detection of this previously unidentified impurity highlights the necessity of comprehensive characterization and analysis in pharmaceutical research. Understanding the nature and sources of such impurities is essential for ensuring the quality, safety, and efficacy of pharmaceutical formulations.

The mass spectra of degradation product [C], shown in Figure S4, revealed several fragments, including a prominent signal at *m/z* = 511.5. A fundamental fragment with a mass-to-charge ratio of *m/z* = 453.6 was also observed. These findings contribute to a deeper understanding of the degradation pathways of haloperidol under acidic conditions. Identifying and characterizing degradation products such as [A] and [C] are vital for evaluating the stability and integrity of haloperidol formulations.

### 3.6.2. Basic Stress NaOH 1N

The mass spectra of the haloperidol solution exposed to basic conditions, as shown in Figures S5–S7, offer important insights into its degradation behavior. These spectra enable the identification of degradation signals through their mass-fragmentation patterns, characterized by their mass-to-charge ratios (*m/z*). For degradation caused by basic conditions, the resulting degradation product [D] exhibited several fragments. A significant signal corresponding to this degradation product was detected at *m/z* = 392. Additionally, a fundamental fragment with a mass-to-charge ratio of *m/z* = 363.1 was observed alongside the main degradation product.
Degradation product [B], observed under basic stress conditions, displayed multiple fragments in the mass-spectra analysis. Specifically, the signal corresponding to this degradation product was identified at *m/z* = 476.

Similarly, another degradation product, degradation product [B], that formed under basic stress conditions showed multiple fragments in the mass spectra. The signal associated with this degradation product was detected at *m/z* = 511.5, and a fundamental fragment was observed at *m/z* = 453.6.

This detailed analysis provides valuable insights into the degradation pathways of haloperidol under basic conditions, underscoring the importance of monitoring degradation products to ensure the stability and efficacy of pharmaceutical formulations.

### 3.6.3. Photolytic Stress

The mass spectra of the haloperidol degradation product formed under photolytic conditions are shown in Figure S8. Degradation signals were identified based on their mass-fragmentation patterns (*m/z*). Specifically, degradation product [B], resulting from photolytic stress, displayed multiple fragments, with the primary signal detected at *m/z* = 476. This analysis enhances our understanding of the photolytic degradation pathways of haloperidol and underscores the importance of ensuring proper storage conditions to preserve its stability.

The active ingredient, haloperidol, remained stable under UV light radiation. The chromatogram shows two peaks: the main peak corresponds to the predominant active ingredient, haloperidol, while the second peak represents impurity [B]. This impurity, detected in all the stability tests under HCl and NaOH conditions, is not documented in the literature and is likely a byproduct formed during the synthesis of haloperidol. Based on the LC-MS results, four degradation products were identified under various hydrolytic stress conditions using the forced degradation protocol. One degradation product, labeled [C], was common to both acidic and basic stress conditions. Under basic stress, haloperidol degrades and forms cis-4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidinyl]-1-(4-fluorophenyl)-1-butanone N-oxide (Product [D]). However, in the literature, product [D] is described as an oxidative degradation product [32,52]. Similar findings regarding the formation of N-oxide derivatives under such conditions have been reported for other nitrogen-containing heterocyclic compounds [18,53].

The separation of degradation products from the haloperidol peak and its potential impurities demonstrates the stability-indicating capability of the method [54]. The proposed new stability-indicating method was validated for specificity, precision, accuracy, limit of quantification, limit of detection, linearity, robustness, and ruggedness, following International Conference on Harmonization (ICH) guidelines.

The validated RP-HPLC method effectively detected most degradation products formed during the forced degradation studies, confirming its reliability and robustness for stability-indicating purposes.

### 3.7. Haloperidol Analysis via TGA/DTG/DSC

The thermal stability of haloperidol was evaluated under conditions ranging from 20 to 500 °C with thermo-analytical data collected at a heating rate of 5 °C/min, as illustrated in Figure 6.

The thermal analysis results for haloperidol confirm its stability up to 230 °C, as evidenced by the TGA, which shows no mass loss within this range. The DSC thermogram reveals a melting point at 149.5 °C, indicating the transition from solid to liquid form without decomposition [38]. Beyond 230 °C, haloperidol undergoes thermal degradation through a single-step mechanism, with complete decomposition occurring at 341.5 °C, leaving
ing a residual mass of approximately 1% [18,55]. The DTG curve supports this behavior, displaying a distinct decomposition phase between 230 °C and 341.5 °C. The DSC thermogram also shows an endothermic peak at 149.5 °C, corresponding to the melting process, with a heat of fusion of 151.69 J/g [13,56]. These findings confirm the thermal stability of haloperidol up to 230 °C and provide critical insights into its degradation pathway under heat stress.

<figure><img src="image_8.png" /><figcaption>**Figure 6.** Thermal analysis curves for haloperidol (heating rate: 5 °C/min).</figcaption></figure>

## 3.8. Comparative Analysis of the Stability of Haloperidol and the Analytical Methods

The comparison table (Table 3) highlights the significant differences in the analytical methods used for haloperidol quantification. Different stationary phases, including C18, CN, monolithic silica, and carbon columns, influence the retention time and sensitivity of the analyses. C18-based columns are most commonly used due to their efficiency in retaining haloperidol while ensuring stable peak separation. Non-porous and monolithic silica columns, while allowing rapid analysis, can be more expensive and require specialized equipment.

The composition of the mobile phase plays a crucial role in the efficiency of the separation. In most methods, a combination of a phosphate buffer and organic solvents such as methanol, acetonitrile, or tetrahydrofuran is used. The pH of the mobile phase significantly affects the retention time and peak resolution. Lower pH values (2.0–3.0) improve separation but may shorten column lifetime, while neutral to high pH values (6.5–9.8) improve stability and reduce interference from degradation products. Some methods involve the use of gradient elution, which provides superior peak resolution but results in longer analysis times compared to isocratic elution.

Sensitivity varies widely among methods, as demonstrated by the limits of detection (LOD) and quantitation (LOQ). The most sensitive approaches, such as those using nonporous silica and monolithic silica columns, achieve detection limits in the nanogram-per-milliliter range. In contrast, traditional methods using C18 columns typically provide LOQs in the microgram-per-milliliter range. In contrast, the LC-MS/MS in our study is distinguished by an exceptionally low LOD of 1 ng/mL, making it particularly suitable for the detection of trace amounts of degradation products.
<table><caption>**Table 3.** Table comparing results between the current study and the literature.</caption><thead><tr><th>Methodology Used</th><th>Stationary Phase</th><th>Mobile Phase</th><th>λ (nm)</th><th>Retention Time (min)</th><th>LOD</th><th>LOQ</th><th>Pharmaceutical Form</th><th>Strengths</th><th>Limitations</th><th>Reference</th></tr></thead><tbody><tr><td>HPLC, UV-Vis, spectrofluorimetry</td><td>Various columns</td><td>Various solvents</td><td>Variable</td><td>Variable</td><td>Variable</td><td>Variable</td><td>Solid and liquid forms</td><td>Covers multiple analytical techniques</td><td>Limited stability studies</td><td>[37]</td></tr><tr><td>HPLC</td><td>C18 (250 × 4.6 mm, 5 µm)</td><td>Methanol/tetrabutyl ammonium sulfate (55:45)</td><td>254 nm</td><td>7</td><td>0.90 µg/mL</td><td>2.75 µg/mL</td><td>Oral solution</td><td>Good sensitivity</td><td>Longer retention time</td><td>[47]</td></tr><tr><td>HPLC</td><td>ODS-A (150 × 4.6 mm, 3 µm)</td><td>TBA sulfate/acetonitrile/isopropanol (gradient)</td><td>230 nm</td><td>37.9</td><td>0.137 µg/mL</td><td>0.458 µg/mL</td><td>Injection</td><td>Highly sensitive</td><td>Very long analysis time</td><td>[57]</td></tr><tr><td>HPLC</td><td>ODS (33 × 4.6 mm, 1.5 µm)</td><td>Phosphate buffer/TEA/acetonitrile (77:23:10)</td><td>220 nm</td><td>1.3</td><td>1 ng/mL</td><td>---</td><td>Tablet</td><td>Ultra-fast, high sensitivity</td><td>LOQ has not been determined</td><td>[58]</td></tr><tr><td>HPLC</td><td>RP-18 (25 cm × 4.6 mm)</td><td>Phosphate buffer/acetonitrile/tetrahydrofuran/TEA (63:34:3:0.1)</td><td>246 nm</td><td>9.3</td><td>15 ng/mL</td><td>50 ng/mL</td><td>Tablet</td><td>High accuracy</td><td>Longer analysis time</td><td>[35]</td></tr><tr><td>HPLC</td><td>C18 (250 × 4.6 mm, 5 µm)</td><td>Methanol/acetonitrile (50:50)</td><td>244 nm</td><td>2.23</td><td>0.40 µg/mL</td><td>1.20 µg/mL</td><td>Tablet</td><td>Good sensitivity</td><td>Stability not studied</td><td>[48]</td></tr><tr><td>HPLC</td><td>Monolithic silica (100 × 4.6 mm)</td><td>Phosphate buffer/acetonitrile (70:30)</td><td>230 nm</td><td>4.26</td><td>1 ng/mL</td><td>3 ng/mL</td><td>Injection</td><td>Very sensitive</td><td>High column cost</td><td>[59]</td></tr><tr><td>HPLC</td><td>Carbon (100 × 4.6 mm, 7 µm)</td><td>Tetrahydrofuran/water/trichloroacetic acid (55:45)</td><td>254 nm</td><td>4.25</td><td>0.1 µg/mL</td><td>---</td><td>Tablet</td><td>Good separation</td><td>LOQ has not been determined</td><td>[36]</td></tr><tr><td>HPLC</td><td>XDB C18 (50 × 4.6 mm, 1.8 µm)</td><td>Organic phase/phosphate buffer/acetonitrile (gradient)</td><td>230 nm</td><td>3.77</td><td>1.16 µg/mL</td><td>3.86 µg/mL</td><td>---</td><td>Good sensitivity</td><td>More complex method</td><td>[49]</td></tr><tr><td>HPLC</td><td>CN (30 cm × 3.9 mm)</td><td>Tetrahydrofuran/water/phosphoric acid (40:60)</td><td>254 nm</td><td>5.4</td><td>---</td><td>---</td><td>---</td><td>Standard method</td><td>LOD and LOQ not specified</td><td>[32]</td></tr><tr><td>HPLC</td><td>C18 (250 × 4.6 mm, 5 µm)</td><td>Methanol/phosphate buffer/TEA (50:50:0.2)</td><td>254 nm</td><td>---</td><td>---</td><td>---</td><td>Solution</td><td>Good accuracy</td><td>Incomplete information</td><td>[60]</td></tr><tr><td>HPLC</td><td>C8 (150 × 4.6 mm, 5 µm)</td><td>Acetonitrile/tetramethylammonium perchlorate (pH 2.8)</td><td>230 nm</td><td>6.4</td><td>---</td><td>---</td><td>Solution</td><td>Good separation</td><td>LOD and LOQ not specified</td><td>[61]</td></tr><tr><td>HPLC (green chromatography)</td><td>C18 (250 × 4.6 mm, 5 µm)</td><td>Methanol/phosphate buffer (pH 9.8) (90:10)</td><td>248 nm</td><td>&lt;4</td><td>0.40 µg/mL</td><td>1.20 µg/mL</td><td>Solution and powder</td><td>Eco-friendly, validated fast method</td><td>Lacks impurity characterization</td><td>[31]</td></tr><tr><td>HPLC, LC-MS/MS, TGA/DSC/DTA</td><td>C18 (250 × 4.6 mm, 5 µm)</td><td>Methanol/phosphate buffer (pH 9.8) (90:10)</td><td>248 nm</td><td>4.2</td><td>1 ng/mL</td><td>3 ng/mL</td><td>All forms</td><td>Comprehensive stability study</td><td>Needs large-scale validation</td><td>In this study</td></tr><tr><td>HPLC, LC-MS/MS, TGA/DSC/DTA</td><td>C18 (250 × 4.6 mm, 5 µm)</td><td>Methanol/phosphate buffer (pH 9.8) (90:10)</td><td>248 nm</td><td>4.2</td><td>1 ng/mL</td><td>3 ng/mL</td><td>All forms</td><td>Comprehensive stability study</td><td>Needs large-scale validation</td><td>In this study</td></tr></tbody></table>
The pharmaceutical form analyzed also influences the choice of method. Tablets and oral solutions require robust methods with high accuracy, while injectable formulations require ultra-sensitive detection to identify analytes at low concentrations. The green chromatography approach developed by Djilali et al. (2025) offers a rapid and environmentally friendly alternative, making it ideal for routine quality control applications [31]. On the other hand, traditional HPLC methods remain reliable but may lack the advanced ability to differentiate impurities and degradation byproducts.

Among the methods compared, this study demonstrates a distinctive advantage in integrating HPLC with LC-MS/MS and thermal analysis techniques (TGA/DSC/DTA). This comprehensive approach allows for a more in-depth study of haloperidol's stability under stress conditions. While green chromatography is preferred for routine analyses due to its speed and durability, the methodology used in this study excels in characterizing degradation products and ensuring pharmaceutical integrity.

**4. Discussions**

The primary innovation of this research lies in the thorough characterization of haloperidol's stability under various stress conditions, employing both chromatographic and thermal analyses. This study expands on existing knowledge by identifying specific conditions under which haloperidol undergoes significant degradation, particularly emphasizing its stability under dry-heat and photolysis conditions, while also noting notable degradation under acidic and alkaline environments. Additionally, the use of advanced analytical techniques such as LC-MS/MS enabled the precise identification of degradation products, including some not listed in the European Pharmacopoeia monograph for the respective medicinal product. This detailed characterization of degradation products enhances our understanding of the potential degradation pathways of haloperidol, essential for ensuring its quality and stability as an active ingredient in pharmaceutical formulations. In summary, this study significantly contributes to our understanding of haloperidol's stability, highlighting its behavior under various environmental stresses and providing a precise analytical methodology for assessing its stability in pharmaceutical formulations.

A novel RP-HPLC method was developed for the analysis of haloperidol, utilizing a mobile phase composed of methanol and a phosphate buffer (90:10 ratio, pH = 9.8) with isocratic elution. The method was rigorously validated in accordance with industry regulatory standards, confirming its high linearity, accuracy, sensitivity, and specificity. The calibration curve for haloperidol in a standard solution exhibited excellent linearity across a concentration range of 1–50 µg/mL, with a correlation coefficient of 0.999 and a mean relative standard deviation (RSD) below 2%. This method was successfully applied to determine the dosage (2 mg/mL) of haloperidol in commercial liquid samples without the need for extraction or pre-treatment steps.

This study outlines the development and validation of an HPLC method designed to accurately quantify haloperidol in bulk formulations while accounting for potential degradation products. This stability-indicating isocratic HPLC method was carefully developed to evaluate haloperidol content under various stress conditions, including exposure to acidic, basic, oxidative, thermal, and UV light environments. Using this method, we effectively separated the drug from its degradation products. Notably, haloperidol in powder form demonstrated resilience against dry-heat and photolytic stress, while its solution counterpart was susceptible to degradation under hydrolytic and photolytic conditions, though it remained stable under oxidative stress. Based on these findings, the industrial manufacturing process for haloperidol solution could be revised by eliminating the nitrogen bubbling stage and potentially using buffer solutions to stabilize the pH.
Additionally, we propose optimizing the dry pharmaceutical formulation, which could
offer greater stability given the thermal stability of haloperidol powder.

Linearity testing confirmed a reliable range of 1 to 50 µg/mL for haloperidol. Validation of the HPLC method demonstrated high specificity, linearity, precision, and accuracy. Mass spectrometry facilitated the characterization of degradation products, revealing different compounds depending on the applied stressors. Acidic stress led to the formation of DPA, DPB, and DPC, while basic stress resulted in DPB, DPC, and DPD. Photolytic stress introduced a new compound, DPB, identified as an impurity in haloperidol synthesis. Further analysis using TGA/DTG/DSC confirmed haloperidol's stability under thermal stress, with a melting temperature of approximately 150 °C. Although the primary goal of the forced-degradation study was to assess haloperidol's stability rather than identify specific degradation products, it was evident that pH significantly influenced haloperidol's stability in solutions. The simplicity of the proposed HPLC method allows for the straightforward determination of haloperidol content in pharmaceutical oral solutions at a concentration of 0.2%, requiring only simple dilution without prior sample extraction or treatment. Furthermore, this method shows promise for conducting stability studies on haloperidol.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pr13030904/s1, Figure S1: Possible degradation products of haloperidol. Figure S2. Mass spectrum of haloperidol degradation product [A] (haloperidol hydrochloride) and its fragmentation products. Figure S3. Mass spectrum of haloperidol degradation product [B] (Haloperidol impurity) and its fragmentation products. Figure S4. Mass spectrum of haloperidol degradation product [C] (haloperidol hydrochloride) and its fragmentation products. Figure S5. Mass spectrum of haloperidol degradation product [D] (haloperidol-N-Oxide) and its fragmentation products. Figure S6. mass spectrum of haloperidol impurity [B]., Figure S7. Mass spectrum of haloperidol degradation product [C] (haloperidol hydrochloride) and its fragmentation products. Figure S8. Mass spectrum of haloperidol degradation product [B] (haloperidol impurity) and its fragmentation products. Table S1. Results of the method linearity study. Table S2. Results of validation method.

**Author Contributions:** Conceptualization, K.D., R.M., H.B., S.L., Z.A.M., S.M.A., N.N., R.B., J.Z., H.T. and A.A.; data curation, K.D., S.M.A., H.T. and A.A.; formal analysis, K.D., R.M., H.B., S.L., Z.A.M., S.M.A., N.N., R.B., J.Z., H.T. and A.A.; funding acquisition, S.M.A.; investigation, K.D., R.M., Z.A.M., S.M.A., N.N., R.B., J.Z., H.T. and A.A.; methodology, K.D., R.M., H.B., S.L., Z.A.M., S.M.A., N.N., R.B., J.Z., H.T. and A.A.; project administration, R.M., N.N., R.B., J.Z., H.T. and A.A.; resources, K.D., R.M., H.B., S.L., Z.A.M., S.M.A., N.N., R.B., J.Z., H.T. and A.A.; software, K.D., R.M., S.M.A., N.N., J.Z., H.T. and A.A.; supervision, R.M., N.N., J.Z., H.T. and A.A.; validation, K.D., R.M., H.B., S.L., Z.A.M., S.M.A., N.N., R.B., J.Z., H.T. and A.A.; visualization, K.D., R.M., H.B., S.L., Z.A.M., S.M.A., N.N., R.B., J.Z., H.T. and A.A.; writing—original draft, K.D.; writing—review and editing, R.M., H.B., S.L., Z.A.M., S.M.A., N.N., R.B., J.Z., H.T. and A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** King Saud University, RSPD2025R748

**Data Availability Statement:** The data that support the findings of this study are available within the article.

**Acknowledgments:** The authors wish to extend their sincere gratitude to the Center for Scientific and Technical Research in Physical and Chemical Analysis (CRAPCC) in Algeria for allowing to use instruments for this work. The authors also extend thanks to the University of Saad Dahlab Blida 1 (USDB), and the University of Science and Technology Houari Boumediene, Algiers-Bab Ezzouar, Algeria, for providing the facilities and resources used in this research work. The authors acknowledge and extend their appreciation to the Researchers Supporting Project Number (RSPD2025R748), King Saud University, Riyadh, Saudi Arabia for funding this study.
**Conflicts of Interest:** The authors declare that there are no known competing financial or personal interests that could have appeared to influence this research work.

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