INTRODUCTION:

Pimozide is an antipsychotic drug belongs to the diphenylbutylpiperidine derivative and dopamine antagonist. The chemical structure of Pimozide was presented in figure 1. It was approved in 1985 by FDA for treatment of schizophrenia, chronic psychosis, Tourette’s syndrome and resistant tics^1-4. The Pimozide is availableas oral dosage forms works by selectively inhibiting the type 2 dopaminergic receptors in the central nervous system (CNS), thereby decreasing the dopamine neurotransmission and reducing the occurrence of motor and vocal tics and delusions of parasitosis^5-9.

Figure 1: Chemical structure of Pimozide

The chemical formula of Pimozide is 3-[1-[4,4-bis (4-fluorophenyl) butyl] piperidine-4-yl]-1H-benzimidazol-2-one with molecular weight of 461.5 g/mol. In accordance to the literature available, there is no LC-MS method was reported for stress studies. Various analytical methods like RP-HPLC^10-15, HPTLC^16-17, LC-MS^18-20and UV spectrophotometry^21-25 have been reported for Pimozide analysis and other compounds. Hence the present study is aimed to develop a LC-MS method to study the behaviour of Pimozide at various stress studies. The stability indicating method provides a background for the pre formulation studies, stability studies, proper storage requirements etc.

MATERIALS AND METHODS:

Instrumentation:

A HPLC (Agilent Technologies) equipped with quaternary G1311 pump, COLCOM G1316A thermostat column temperature control, thermostatic auto sampler G 1329A with sample volume of 0. 1 – 1500µL and variable programmable UV detector G 1314 A is used for separation. The LC is operated and integrated with Agilent chemstation LC software. Waters ZQ mass detector (Model LAA 1369) with waters empowers system used for molecular mass analysis. The mass spectra were taken in ESI (Turbo Ion Spray) positive mode within mass range of 40-1000a.m.u. and analysed in the Quadra pole analyser with MS tune of 3.0 (V) at a source temperature of 320⁰C.

Chemicals and solvents:

The Pimozide standard drug was procured from Johnson and Johnson Pvt. Ltd., Mumbai, Maharashtra, India. The HPLC grade chemicals of acetonitrile, methanol and water were purchased from Merck chemicals private limited, Mumbai. The other analytical grade chemicals used for stress studies like hydrochloric acid (HCl), Sodium hydroxide (NaoH) and Hydrogen peroxide (H₂O₂) are purchased from Thermo fisher scientific India private limited, Mumbai.

Preparation of stock and working solutions:

The initial Pimozide standard stock solution was prepared by weighing of 100mg of drug into 100ml of volumetric flask and dissolved with methanol. The solution was sonicated for 15 minutes for proper dilution of drug and flittered through 0.45µ Millipore filter and final volume makeup with methanol. A series of standard working solutions were prepared by diluting the stock solution with methanol to obtain desired concentration.

Method development and validation:

The LC-MS method was developed for and quantification of Pimozide and its degradation products. The mobile phase solvents like methanol, water and acetonitrile with different compositions were tested to achieve acceptable peak shape and ionization. Among the tested combination of solvents methanol and acetonitrile (65:35 v/v) gives better results. In addition to the mobile phase- associated factors, use of an efficient analytical column, which can also allow high throughput separation, was preferred to increase total versatility of the method. For this Waters make C18 column bonded with monolithic silica (150mm X 4.5mm, 5µm) with ambient temperature was utilized. The mobile phase flow rate and UV wavelength were optimized and finally the separation was carried out in isocratic elution mode at 1.0ml/min with UV detection at 230nm. The mass spectrometer parameters like source temperature, voltage of source like cone voltage, capillary voltage and nitrogen gas flow rate were optimized and spectral results were observed at positive ion mode. The LC-MS method was validated with respect to in accordance with ICH Q2 (R1) guidelines. The specificity of the method was studied for standard PMZ drug and its stress degradation products under various stress conditions.

Forced degradation study:

Stress studies were performed in order to know the effect of acidic, alkali, oxidation, thermal stress and UV light on Pimozide. The acidic hydrolysis was performed by adding 5ml of 1M HCl to the 5ml standard stock solution at room temperature and incubated for 30 minutes. After incubation aliquots of the stress samples were neutralized with appropriate volumes of 1M NaoH and were diluted to the 100ng/mL concentration and filtered prior to LC-MS analysis. Alkali hydrolysis was carried out by adding 5ml of 1M NaOH to the 5ml of standard stock solution at room temperature and incubated for 30 minutes. After incubation aliquots of the alkali s tress samples were neutralized with appropriate volumes of 1 M HCl. The stress samples were diluted to the 100ng/mL concentration and filtered prior to LC-MS analysis. For oxidative degradation study 5ml stock solution was mixed with 5ml of 30% H₂O₂ at room temperature and incubated for 30 minutes. Thermal stress was performed by stock solution was subjected to incubation at 60⁰C in water bath for 30 minutes. The UV stress study was performed by exposing the standard stock solution to UV light for 30 minutes.

RESULT AND DISCUSSION:

Under developed conditions, the drug was detected at 3.16min after injection. The standard chromatograms is presented in figure 2. The mass spectral analysis is performed after several optimized conditions like source voltage (3V), capillary voltage (2.8kV), and nitrogen gas flow maintained at 570L/hr. Electrospray was used as the ionization technique in the positive mode. Fragmentation reaction of the most abundant ion in the MS spectrum (m/z 465, figure 2) used for the quantification of Pimozide. In the positive ESI conditions, Pimozide produced predominantly protonated molecule at m/z 462.1 in Q1 full scan mass spectra. The mass spectrum of Pimozide produces several fragmentsat m/z of 462.1, 364.8, 328.4, 217.5 and 145.3, however the fragment at m/z 217.5 is used for its diagnosis. The product ion spectra was given in figure 3, where [M+H]+ of each compound was selected as precursor ion. The transition m/z 462.1 → 364.8→ 217.5 was considered for quantification analysis due to its more stable and less disturbed.

Figure 2 (A): Chromatogram of Pimozide Standard

Figure 2 (B): Mass spectrum of Pimozide Standard

The proposed method conditions were investigated for validation parameters such as specificity, linearity, precision, robustness, LOD and LOQ. The linearity of the proposed method was established at seven linear concentration levels in the range of 0.1-300ng/mL and is presented in figure 3. The precision of the proposed method was evaluated at two levels, i.e. repeatability (intraday) and interday precision. It was checked by calculating the %RSD of six replicate determinations by injecting standard solutions containing 50ng/ml. The % RSD was found within the limits viz. 1.62 and 1.43. Similarly, the ruggedness of the proposed method also studied by varying the analyst and injecting the standard solutions. The recovery of the proposed method was evaluated using standard addition method by spiking the drug and its impurity concentration separately to the standard solution at 50%, 100% and 150% levels. The robustness of the method also evaluated by calculating the % of recovery of chromatograms achieved at deliberate modification of the proposed conditions in the flow rate of mobile phase and temperature of the column and pH of the mobile phase. LOQ of the method was found lowest calibration concentration i.e 0.1ng/ml and LOD under the proposed method was found to be 0.03 ng/ml.μg/ml. The summery of validation results were presented in table 1 and these results indicates the sensitivity of the method. For mass spectrometric analysis, the mass detector was operated within the mass range from m/z 100 to m/z 800 using an electrospray ionization in positive ion mode (ESI+).

Figure 3: Calibration curve graph of Pimozide standard

Table 1: Summery of method validation results

S. No.	Method Parameter	Result
1	Retention time of PMZ	3.2minutes
2	Linearity range	0.1 - 300 ng/mL
3	Intraday precision (RSD)	1.62
4	Interday precision (RSD)	1.43
5	Ruggedness (RSD)	1.76
6	Robustness (% of Change)	0.26-1.45
7	Recovery (%)	98.18-99.66
8	Limit of detection	0.03 ng/mL
9	Limit of Quantitation	0.1 ng/mL

Degradation behaviour of Pimozide:

Degradation of Pimozide was observed under all tested stress conditions by subjecting the stress samples into LC/MS analysis. The results of the stress conditions were presented in table 2. High degradation was observed under acidic (figure 4) and UV light conditions (figure 8) where more than 10% of the drug was degraded. Very less reduction in Pimozide quantity was found in alkali (figure 5), peroxide (figure 6) and thermal (figure 7) stress conditions compared with other. The formation of degradation compounds was found high in alkali condition even the quantity of drug was reduced lesser. Three degradation compounds were formed and identified at alkali condition. In presence of oxidizing reagent, the drug was found moderately stable, under UV stress condition also, three degradation compounds were identified. The drug was found stable at thermal stress condition where only 6.03% of was reduced and only one additional degradation peak was identified.

Figure 4 (A): Chromatogram of Pimozide under acidic stress condition

Figure 4 (B): Mass spectrum of Pimozide under acidic stress condition

Figure 5 (A): Chromatogram of Pimozide under alkali stress condition

Figure 5 (B): Mass spectrum of Pimozide under alkali stress condition

Figure 6 (A): Chromatogram of Pimozide under Peroxide stress condition

Figure 6 (B): Mass spectrum of Pimozide under Peroxide stress condition

Figure 7 (A): Chromatogram of Pimozide under Thermal stress condition

Figure 7 (B): Mass spectrum of Pimozide under Thermal stress condition

Figure 8 (A): Chromatogram of Pimozide under UV light stress condition

Figure 8 (B): Mass spectrum of Pimozide under UV light stress condition

Table 2: Results of forced degradation studies of Pimozide under different stress conditions

S. No.	Stress Condition	No. of additional detections	Peak Area	% Obtai ned	% degrad ation
1	Acidic	2	230233	89.36	10.64
2	Alkali	3	239965	93.13	6.87
3	Peroxide	2	241030	93.55	6.45
4	Thermal	1	242133	93.97	6.03
5	UV Light	3	228950	88.86	11.14

The conversion of carbonyl group into alcohol was observed in all the stress degradation conditions at a retention time of 1.4 min and the conversion was confirmed by observing mass spectral line with m/z value 462.1 (M+1). The breakage of heterocyclic ring and the formation of aromatic amine compound was observed in acid degradation and the compound was detected at aretention time of 6.7 min and was identified at m/z 345 (M+1). The breakage of N-C-N bond in the heterocyclic nitrogen ring with carbonyl group was observed in base degradation study and the compound was observed ata retention time of 4.4 min and was confirmed by m/z 463.5. Similar type of breakage was observed in UV light degradation studies and similar mass fragmentation was observed in UV light degradation also. The breakage of halogen containing aromatic ring was observed in acid degradation and the compound was observed at a retention of 5.6 min and was confirmed by m/z 480 (M+1). Similar type of compound was observed inperoxide degradation study also. Difluoro phenyl benzene compound was observed at 6.7 min in acidic and UV degradation studies and the mass fragmentation observed confirmed the structure of the compound and was identified at m/z 246(M+1).

CONCLUSION:

In the present study novel LC-MS method for qualitative and quantitative analysis of Pimozide and its degradation products was described. The proposed method was successfully separated all the degradation products and is validated according to the ICH guidelines and found within acceptable limit in precision, recovery, ruggedness and robustness studies. The range (linearity) of analysis was wide (0.1 to 300ng/mL) and lowest in concentration. The LOD (0.03 ng/mL) of the method proves that the method sensitivity. Stress degradation conditions under acidic, alkali, oxidation, thermal and UV light were studied and results reported that drug showed stable behaviour towards thermal stress conditions and found sensitive towards acidic, UV light conditions and moderate stable under alkali, oxidative stress conditions. The developed method was proven to be simple, sensitive, precise and specific. Hence it is recommended as a suitable method for industrial application for stability studies of Pimozide.

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Received on 09.12.2020 Modified on 14.09.2021

Research J. Pharm. and Tech. 2022; 15(6):2433-2438.

DOI: 10.52711/0974-360X.2022.00405