INTRODUCTION:

Prochlorperazine Maleate, 2-Chloro-10-[3-(4-methyl-1-piperazinyl)propyl]-10H-phenothiazine is a 1^st- generation antipsychotic and also an antiemetic (figure 1)¹. There are several illnesses in which Prochlorperazine Maleate is approved by the FDA for use, including schizophrenia, schizoaffective disorder, and other conditions that exhibit symptoms of psychosis, as well as nausea and vomiting, (post-chemotherapy, post-radiation therapy, pre and post operative setting, and other conditions)^2-4. Non-FDA Indications include migraine and headaches. The drug is official in IP, BP and USP.

High Performance Liquid Chromatography (HPLC) is a technique in analytical chemistry used to separate, identify and quantify each component in a mixture. The separation of compounds mixture is based on differences in how evenly they are distributed between two phases: the stationary phase, which is enclosed inside columns, and the mobile phase, which is pumped through the columns at high pressures. The components having a higher affinity towards the stationary phase are held for a longer period of time and are eventually separated from those with a lower affinity towards the stationary phase. One of the most used analytical tool for quantifying diverse analytes in analytical chemistry is HPLC.^5-10 There is no HPLC method for Prochlorperazine Maleate assay that is official in IP. Although, a UV method for the assay of Prochlorperazine Maleate is available in IP¹¹ but our aim is to develop a method which is more precise, stability-indicating and robust. Literature survey revealed that many spectrophotometric, HPLC methods have been reported for Prochlorperazine in combination with other drugs.^12-21 On the basis of review, an appropriate RP-HPLC method is required for routine analysis of Prochlorperazine Maleate in tablet dosage form.

Figure 1: Structure of Prochlorperazine Maleate^1,11

MATERIAL AND METHODS:

Reagents and chemicals:

Working standard and commercial pharmaceutical tablets of Prochlorperazine Maleate I.P. (Batch no. SMA21002) were procured as a sample from Abbott Healthcare Pvt. Ltd. containing Prochlorperazine Maleate I.P. 5mg. Acetonitrile HPLC grade (Batch no. 296781102HS), Glacial acetic acid HPLC & spectroscopy grade (Batch no. 586751019LT) were procured from Finar Limited. Bio-AGE water purification system for Mili-Q water, Sodium acetate anhydrous was procured from Merck Specialities Private Limited. Membrane filters (PVDF) of 0.45μm was used for the mobile phase filtration and 0.45μm nylon filter was used for the sample solution.

Instrumentation:

The chromatographic separation was carried out by utilizing Thermofisher Scientific’s Dionex Ultimate 3000 UHPLC equipped with an inbuilt degasser, an autosampler, a column oven and UV/PDA detector. Chromeleon console 7.2 software was used to collate and data process. An Inertsil ODS-3 250×4.6mm, 5µm column was used for chromatographic separation and quantification at ambient temperature.

Chromatographic Conditions^20,21

The mixture of buffer pH 4.0 and Acetonitrile in 50:50 v/v (pH 4.0±0.05, adjusted with glacial acetic acid) was used as mobile phase with isocratic elution. For optimal separation Inertsil ODS-3 250 × 4.6mm, 5μm column was used. The mobile phase was pumped into the column at a flow rate of 1.5ml/min for a total run period of 8minutes. The injection volume was 20µl, the detection of effluent was performed at 254nm, and the separation was achieved at ambient temperature.

Buffer Solution (pH 4.0) Preparation:

Buffer solution was prepared by added about 4.115g Sodium acetate anhydrous in 900ml Milli-Q water. Mixed thoroughly to dissolve completely. Then, pH of solution was adjusted to 4.0±0.05 with glacial acetic acid. Made volume up to 1000ml with Milli-Q water. The buffer solution was filtered through 0.45µm filter vacuum filtration.

Mobile Phaseand Diluent Preparation:

Mobile phase was prepared by taking buffer pH4.0 (50%) and Acetonitrile (50%) volume then, placed it in an ultrasonic bath and degassed for 15-20 minutes. The mobile phase solution was also used as diluent for standard and sample preparation.

Preparation of Standard Solution:

Carefully weighed 50mg of Prochlorperazine Maleate working standard was transferred into 100ml amber coloured volumetric flask. Added about 70ml of diluent to it and sonicated to dissolve. With diluent made volume up to the mark and mix well. Then, 4ml of stock solution was pipette into a 20ml amber coloured volumetric flask and made volume up to the mark with diluent and mix.

Preparation of Sample Solution:

20 tablets were weighed and average weight was calculated i.e. 100.08 mg. Crushed them all into fine and uniform powder. Tablets powder equivalent to twiced the labelled claimed for Prochlorperazine Maleate (equivalent to 10mg) was precisely weighed and anxiously transferred it into 100ml amber coloured volumetric flask. Then, add about 30ml of diluent into volumetric flask and the solution was sonicated for about 20 minutes with intermitted shaking. With diluent made volume up to the mark. The solution was filtered with a 0.45µm nylon filter and the filtrate was collected after discarding the first few ml.

Selection of Wavelength of Detection:

Prochlorperazine Maleate standard solution (100µg/ml) was scanned and spectrum was observed by using UV spectrophotometer at 200-400nm.

Description of Final Method:

The final method for analyzing Prochlorperazine Maleate was described based on the results of the optimization of chromatographic conditions.

Method Validation^22,23

The parameters listed below were used to validate the method.

System Suitability:

System suitability was checked by the drug of concentration 100.0μg/ml. The majority of the characteristics were evaluated using USP criteria, such as peak area, theoretical plates, plate height, and tailing factor.

Precision:

In Intraday precision (Repeatability), Prochlorperazine Maleate six sample solutions were prepared freshly and injection of 20μl from each sample was injected in chromatographic system. In interday precision (Ruggedness), different analyst conducted test as intraday precision on a different day and different system. 5 injections of Prochlorperazine Maleate working standard and injection of 20μl from six sample solutions were injected in another chromatographic system.

Linearity:

It was performed by preparing different concentrations of the drug solution. The different concentrations of Prochlorperazine Maleate were prepared for linearity in the range of 50-150μg/ml. For each concentration level, 20µl of the soln. was injected and a calibration curve was obtained by graphing the peak area v/s the drug concentration.

Specificity:

For an assay, specificity implies that the signal recorded is from the substance of interest, without any interference from excipients, degradation products, or contamination. After each injection, standard solution and sample solution chromatograms should have similar retention times. At the retention time of the analyte peak, the blank chromatogram should show no signal and there is no interference from blank. As a result, the procedure is specific.

Robustness:

Changes in buffer pH, wavelength, flow rate, etc. were used to calculate robustness, and the %RSD was obtained. Small alterations in the above parameter can be used to determine robustness, and the method's resistivity was developed as well. The modification in parameters could result in considerable variations in peak area, retention time, and RSD.

Solution stability:

Sample and std. solutions were prepared and evaluated for solution stability by analysed at different time interval. Solution stability studies were performed for 32 hrs and %RSD of area was calculated. The solution was injected at regular intervals for 32hrs.

Filter compatibility:

Filtration of samples is frequently required to prevent undissolved drug materials from entering the analytical sample system and dissolving further. The compatibility of 0.45µm nylon and 0.45µm PVDF filters was investigated. The sample solution was filtered and the assay value %difference was calculated for each centrifuged and filtered sample. The %difference in assay value of the filtered sample against centrifuged sample was calculated.

Accuracy:

Recovery studies were conducted using the standard addition approach to ensure the method's reliability and accuracy. The accuracy studies were conducted by adding Prochlorperazine Maleate sample drug in the range of 50% - 150% in triplicate. Data from nine measurements at three concentration levels, covering the whole concentration range was obtained.

Forced degradation studies:

Forced degradation studies help to identify chemical behavior of the drug substance and provide data to support identification of possible degradants (impurities) and degradation pathway. Further forced degradation studies are crucial for the development of stability-indicating methods. Forced degradation studies were carried out by exposing sample to acid degradation, base degradation, peroxide degradation, thermal and photolytic degradation.

RESULT AND DISCUSSION:

Detection of Wavelength:

For the development of a new method, the results suggest that a 100ppm standard solution had a λmax at 257.30nm. UV spectra of Prochlorperazine Maleate is shown in Figure 2.

Figure 2: Spectra of Prochlorperazine Maleate

Figure 3: HPLC Chromatogram of Prochlorperazine Maleate

Method development:

To resolve the Prochlorperazine Maleate peak, several mobile phase mixtures were attempted. The optimum mobile phase containing buffer pH4.0: Acetonitrile 50:50v/v (pH 4.0±0.05, adjusted with glacial acetic acid) was selected because it could resolve the peak of Prochlorperazine Maleate. Determination was achieved with UV detection at 254nm on the basis of peak area at 1.5ml/min flow rate. A typical HPLC chromatogram achieved during Prochlorperazine Maleate determination is shown in Figure 3.

Method validation:

The proposed method was validated as per ICH (International Conference on Harmonization) guidelines for parameters such as specificity, accuracy, precision, filter compatibility, linearity, robustness, solution stability etc.

1. System suitability:

The system suitability parameters like symmetry of the retention time, tailing factor, theoretical plates were optimized in order to devise and develop system suitability conditions. The obtained system suitability results were 8430 number of theoretical plates, tailing factor of 1.25 and relative standard deviation (for 5 replicate injections) 0.16% as presented in Table 1.

Table 1. Result of systems suitability study

S. No.	Parameters	Values	Values obtained
1.	Theoretical plates	Not less than 2000	8430
2.	Tailing factor	Not more than 2.0	1.25
3.	Relative standard deviation (for 5 replicate injections)	Not more than 2.0%	0.16%

2. Accuracy:

The test was carried out by spiking the standard concentration of the drug at three distinct levels 50%, 100% and 150%. % recovery and the total % RSD were calculated by the proposed method. % RSD and % recovery in each spiked level for the drug developed method was calculated as shown in Table 2. The drug's % RSD was discovered to be less than 2 and % recovery was discovered in range 98-102 %, which is within the acceptable range. The results of the recovery clearly depict that the method is accurate.

3. Robustness:

Changes in buffer pH, wavelength, flow rate, etc. were used to calculate robustness, and the %RSD was obtained. Small alterations in the above parameter can be used to determine robustness, and the method's resistivity was developed as well. Table 3 shows the findings, which were acquired in the same order as the optimised results. The %RSD was within the acceptable range, i.e. less than 2. None of these variables had a considerable impact on the retention time of drug suggesting that the presented method could be considered as robust.

Table 2. Result of Accuracy Results

S. No.	Level	Preparation	Amount added (ppm)	Amount recovered (ppm)	% Recovery	% RSD
1.	50%	1	5	4.99	99.97	0.12
		2	5	5.01	100.20
		3	5	5.008	100.17
2.	100%	1	10	9.93	99.31	0.29
		2	10	9.98	99.80
		3	10	9.98	99.85
3.	150%	1	15	14.88	99.21	0.03
		2	15	14.88	99.18
		3	15	14.87	99.14

Table 3. Result of Robustness

S. No.	Ideal	Wavelength (252 nm)	Wavelength (256 nm)	Flow rate (1.35 ml/min)	Flow rate (1.65 ml/min)	pH-3.5	pH-4.5
1.	4.238	4.305	4.310	4.778	3.942	3.948	5.177
2.	4.245	4.305	4.308	4.778	3.940	3.950	5.180
3.	4.248	4.305	4.308	4.777	3.940	3.950	5.183
4.	4.250	4.305	4.308	4.777	3.938	3.950	5.185
5.	4.252	4.305	4.307	4.775	3.938	3.948	5.188
Mean	4.246	4.305	4.308	4.777	3.939	3.949	5.183
SD	0.005	0.005	0.001	0.001	0.001	0.001	0.004
%RSD	0.12	0.00	0.02	0.02	0.04	0.02	0.08

4. Specificity:

There were no peaks acquired at the retention time of Prochlorperazine Maleate in the HPLC chromatograms of blank and sample solution, as shown in Figure 4 and 5. In their equivalent approach (Figure 6), the standard solution for the drug showed a symmetric peak with a defined retention period, a clear baseline, and no interference. This shows that the proposed method is specific.

Table 4. Result of Specificity

S. No.	Type	Retention Time (min)	Interference
1.	Blank	-	No interference observed
2.	Standard solution	4.1	No interference observed
3.	Sample solution	4.1	No interference observed

Figure 4: HPLC Chromatogram of Blank

Figure 5: HPLC Chromatogram of Prochlorperazine Maleate Std.

Figure 6: HPLC Chromatogram of Prochlorperazine Maleate Sample

5. Filter compatibility:

Compatibility of 0.45µm nylon filter and 0.45µm PVDF filter were studied. Sample solutions were filtered and analysed and the variation in the assay value was calculated and tabulated in table 5. After the analysis, it was found that both nylon and PVDF filters were suitable for filtration.

Table 5. Result of Filter compatibility

S. No	Sample Type	Prochlorperazine Maleate
S. No	Sample Type	% Assay	% Difference from centrifuged sample
1.	Centrifuged	101.82	-
2.	0.45µ Nylon filter	100.26	1.56
3.	0.45µ PVDF	103.09	1.27

6. Linearity and range:

The method's linearity was established throughout a concentration range of 50-150 % (or 50-150 µg/ml). From the std. stock solution, aliquots of 50%, 75%, 100%, 125%, 150% were prepared. For each conc. level 20 µl of the soln. was injected and a calibration curve was obtained by graphing the peak area v/s the drug concentration, which showed a linear relationship over a conc. range of 50-150 µg/ml. The regression equation was found to be y =663.71x+903.27. The correlation coefficient was found to be, r²= 0.9999. The calibration curve and observation were shown in the table 6.

Figure 7: Calibration Curve for the HPLC method (x axis shows the concentration in µg/ml and y axis shows area of the peak)

7. Precision:

The obtained %RSD values of inter and intra precision were 0.38 and 0.25 respectively. The precision studies results reported in %RSD, which meet the ICH guidelines permitted standards and shows strong repeatability, suggesting that the developed method has excellent precision. The result of Intra-day precision (Repeatability) and inter-day precision (Ruggedness) are given in table 7.

8. Forced degradation studies:

In forced degradation studies, it was found that the principal peak did not have any impurity peaks, and it was also observed that the sample was highly sensitive to base, UV light and peroxide exposure. The percentage degradations determined by the method are given in table 8.

Table 7. Result of Intra-day precision (Repeatability) and Inter-day precision (Ruggedness)

S. No.	Injection	% Assay (Intra-Day)	%Assay (Inter-Day)
S. No.	Injection	Prochlorperazine Maleate	Prochlorperazine Maleate
1.	Injection 1	100.91	99.01
2.	Injection 2	100.83	99.10
3.	Injection 3	100.46	98.50
4.	Injection 4	101.51	98.65
5.	Injection 5	100.52	98.75
6.	Injection 6	100.69	98.54
	Mean	100.82	98.76
	%RSD	0.38	0.25

Table 8. Result of Forced degradation for Prochlorperazine Maleate Sample

S. No.	Degradation type	Concentration of reagent	Time	Degradation
1.	Acid	5 ml of 5N HCl	1 hr	2.09%
2.	Base	5 ml of 5N NaOH	1 hr	29.46%
3.	Peroxide	1 ml of 30 % H₂O₂	30 min	5.54%
4.	Thermal	60ºC	24hrs	0.17%
5.	UV(unexposed)	NA	7 days	0.42%
6.	UV (exposed)	NA	7 days	8.88%
7.	Control sample	NA	NA	NA

9. Solution stability:

The standard and sample solution’s stability was evaluated by assessing samples at ambient temperature for a specified time interval up to 32 hours. When solution was kept at ambient temperature for 32 hours, the % area difference values at different time intervals were found to be < 2 of the initial zero time interval solution, showing that the solutions were stable.

Table 9. Result of Solution Stability for Prochlorperazine Maleate (standard solution)

S. No.	Time	Area	% Area Difference
1.	Initial	3906864.00	0.00
2.	After 4 hr	3904444.00	0.00
3.	After 8 hr	3899055.00	0.00
4.	After 12 hr	3887953.00	0.00
5.	After 16 hr	3830539.00	0.02
6.	After 20 hr	3875967.00	0.01
7.	After 24 hr	3815050.00	0.02
8.	After 28 hr	3774240.00	0.03
9.	After 32 hr	3749127.00	0.04

Table 10. Result of Solution Stability for Prochlorperazine Maleate (sample solution)

S. No.	Time	Area	% Area Difference
1.	Initial	3954447.00	0.00
2.	After 4 hr	3977572.00	-0.01
3.	After 8 hr	3966760.00	0.00
4.	After 12 hr	3977862.00	-0.01
5.	After 16 hr	3981662.00	-0.01
6.	After 20 hr	4032743.00	-0.02
7.	After 24 hr	4004024.00	-0.01
8.	After 28 hr	4011988.00	-0.01
9.	After 32 hr	3979701.00	-0.01

CONCLUSION:

Hence, it can be concluded that the proposed RP-HPLC method is simple, precise, and accurate for the assay of Prochlorperazine Maleate in tablet dosage form. The % RSD for intra-day and inter-day precision was less than 2%. As per the validation results the approach was precise, inexpensive, isocratic, specific, time efficient, robust, and degradation study shows drug was highly base, peroxide and light sensitive. Furthermore, the HPLC method allows for faster estimation of Prochlorperazine Maleate without the interference of excipients, with a run time of only eight minutes; the proposed method can also be utilized for routine quality control of tablet dosage forms containing Prochlorperzine Maleate.

CONFLICT OF INTERESTS:

Declare none.

ACKNOWLEDGEMENTS:

The author wishes to thanks Indian Pharmacopoeia Commission (IPC), Ghaziabad and Sanskar College of Pharmacy and Research, Ghaziabad for providing required lab facilities with enthusiastic environment and unconditional support during research work.

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Received on 16.07.2022 Revised on 23.04.2024

Accepted on 13.02.2025 Published on 01.07.2025

Available online from July 05, 2025

Research J. Pharmacy and Technology. 2025;18(7):2991-2997.

DOI: 10.52711/0974-360X.2025.00428

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

S. No.	Range (in %)	Conc. (µg/ml)	Peak area 1	Peak area 2	Average area
1.	50%	50	34237.1990	34244.2560	34240.7275
2.	75%	75	50624.0250	50627.9360	50625.9805
3.	100%	100	66958.9840	66900.1760	66929.5800
4.	125%	125	84083.9210	84147.3480	84115.6345
5.	150%	150	100464.4730	100454.9660	100459.7195
Slope=663.71
Intercept = 903.27
R²=0.9999