INTRODUCTION:

Haloperidol is a chemically 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperydyl)] -1-(4-flurophenyl)-butan-1-one. It is a class of antipsychotic medication that belongs to the butyrophenone category. It has a strong antipsychotic effect in the treatment of schizophrenia and, more especially, in the treatment of moderate to severe psychotic delirium.

Schizophrenia is one of the major causes of increased mortality from cardiovascular disease (CVD)^1-3. It contains mild antihistaminic and anticholinergic actions, therefore it is utilized as a cardiovascular and anticholinergic drug with very less side effects like hypotension, dry mouth, and constipation.^1-4 Trihexyphenidyl, widely known as Benzhexol, is an anti-muscarnic and anti parkinsonism agent. Trihexyphenidyl blocks efferent impulses in parasympathetically innervated sites such as smooth muscles, salivary glands, and eyes. The chemical name of trihexyphenidyl HCl is (±)-α-Cyclohexyl-α-phenyl-1-piperidinepropanol hydrochloride. Catalepsy, sedatives and anxiolytics are adverse effects of haloperidol and trihexyphenidyl hydrochloride^4-8. (Figure 1 and Figure 2). Both compounds are listed in different pharmacopoeias^9-11.

Figure 1 – Structure of Haloperidol (HAL)

Figure 2 – Structure of Trihexyphenidyl hydrochloride (THP)

Spectrophotometric technique^12-15, high performance liquid chromatography (HPLC)^16-24and liquid chromatography (LC) – Mass spectrometry (MS) have already been used to determine haloperidol alone²⁵. Liquid chromatography^26-27, ion pair extraction method²⁸, electron sputter ionization-Mass spectrometry²⁹ and electron impact ionization with a mass selective detector³⁰ were also used to determine trihexyphenidyl HCl. Also reported that both drugs could be analyzed simultaneously by Spectrophotometric technique³¹, reverse phase high performance liquid chromatography (RP-HPLC)^32-33 and RP-HPLC method³⁴. The current study proposes a quick, accurate, sensitive, and reproducible chromatographic method for simultaneous estimating Haloperidol and Trihexyphenidyl HCl from tablet formulations.

EXPERIMENTAL:

Instrumentation:

The HPLC system of Shimadzu LC-2010 CHT (Kyoto, Japan) having a Zorbax Eclipse XDB C₁₈ column (150 mm x 4.6 mm, 5 m) a quaternary gradient system. SPD-20A UV-Visible detector and auto sampler were included in the system (Shimadzu, Japan). LC solution was used to process the data.

Chemicals and reagents:

Man Pharmaceutical India in Gujarat, India, provided haloperidol reference standards, and Adlac Life Science Pvt Ltd provided trihexyphenidyl HCl (Ahmedabad, India). A tablet formulation comprising 1.5 mg of HAL and 2.0mg of THP was acquired from a local store in Ahmedabad, India. Acetonitrile and methanol (HPLC grade) were supplied by Rankem Chemicals Limited (Ahmedabad, India). Triethylamine and water (HPLC grade) were provided by Finar Chemicals Limited (Ahmedabad, India). Phosphate buffer (AR grade) was given by Oxford Laboratory (Maharashtra, India). All of the other reagents and substances used in this experiment were of analytical quality. The Milipore system was used to purify the water (Milipore Corp, Banglore, India).

Preparation of buffer: (0.05 M Phosphate Buffer):

Accurately weighed quantity of 6.8gms potassium dihydrogen phosphate was transferred in 1000mL beaker, dissolved in 600mL HPLC grade water and added 0.5mL triethylamine solution and diluted up to the mark with HPLC grade water. It was filtered through 0.45um membrane filter. Buffer pH was adjusted to 3.75 using orthophosphoric acid.

Preparation of mobile phase:

The mobile phase was prepared by mixing degassed 0.5 M phosphate buffer and acetonitrile in the ratio of 65:35; (v/v).

Preparation of stock and standard solutions:

By dissolving reference standards in methanol in separate volumetric flask, a standard stock solution containing 1000ug mL^-1 each of HAL and THP was prepared separately. 6mL HAL and 8 mL THP aliquots from the standard stock solutions of HAL and THP were transferred to a 50mL calibrated volumetric flask and the volume was made up to the mark using the same solvent to prepare a mixed standard preparation with a concentration of 120ug mL^-1 HAL and 160ug mL^-1 THP. By diluting the standard solutions to the appropriate volume with the same solvent, a calibration curve solution containing 6-60ug mL^-1 for HAL and 8-80 ug mL^-1 for THP was prepared.

Preparation of test solution:

In a mortar, accurately weighed twenty tablets were taken and finely powdered. A tablet powder containing 1.5mg HAL and 2.0mg THP was precisely weighed and transferred in a 100mL volumetric flask. After adding 50 mL of methanol, the solution was sonicated for 10 minutes with an ultrasonicator. The volume was adjusted up to 100mL using the same solvent. A 0.45µm membrane filter was used to filter the mixture. HAL and THP were present in this solution at concentrations of 15 ug mL^-1 and 20ug mL^-1, respectively.

Validation of the method:

The developed method was validated in accordance with the International Conference on Harmonization (ICH) standards³⁵. To evaluate system performance, the system suitability parameters were evaluated. To determine system precision, six replicate injections of standard preparations were used. The number of theoretical plates and asymmetry were calculated.

Specificity:

Peak purity profiling studies were used to determine the method's specificity. The purity of the drug was determined by examining the spectrum at the beginning, middle, and end of the peak. Using software, the peak purity was determined.

Linearity:

Peak area vs. concentration of HAL and THP were plotted on calibration graphs, and the regression equation was calculated. The calibration graphs for HAL and THP were plotted over six different linear concentrations, ranging from 6 to 60ug mL^-1 for HAL and 8 to 80ug mL^-1 for THP. Under the operating chromatographic condition described above, aliquots (20ul) of each solution were injected [Number of replicates (n = 6)].

Accuracy:

Recovery studies, or the external standard addition method, were used to confirm the accuracy of the method. At three different levels, a known amount of standard was added to the pre-analyzed sample. Each test was carried out three times.

Precision:

By analyzing a mixed standard solution of HAL and THP at concentrations of 15-45ug mL^-1 and 20-60ug mL^-1, respectively, three times on the same day and three times on different days, the proposed method's intraday and interday precision were determined. A relative standard deviation is used to express the results.

Limit of detection (LOD) and limit of quantitation (LOQ):

The signal-to-noise (S/N) ratios of 3:1 and 10:1 were calculated using International Conference on Harmonization guidelines 24 to determine the limits of detection (LOD) and quantitation (LOQ) of HAL and THP³⁵.

Robustness:

The method's robustness was determined by analyzing the test solution after making small but deliberate changes to the analytical conditions such as flow rate (±0.2mL min-1), buffer pH (±0.05), mobile phase composition (35±5 %), and column temperature (±2°C).

Analysis of HAL and THP in tablet dosage form:

The signal of sample solution was recorded at 210nm using the method mentioned above for quantification of HAL and THP. Using peak area values in the calibration graph's regression equation, the amount of HAL and THP in the sample solution was calculated.

RESULTS AND DISCUSSION:

HAL and THP in tablet dose form have been determined using an RP-HPLC technique. A chromatographic technique for the measurement of HAL and THP in fixed dosage form was developed. The reverse-phase liquid chromatography method was chosen based on their structural formula.

For the RP-HPLC method, various columns were available. The Zorbax Eclipse XDB C₁₈ column (150 mm x 4.6 mm, 5 um) was chosen over the other column because the method's primary goal was to resolve both compounds. Good peak symmetry, better repeatability, lower back pressure, high resolution power, and a high degree of retention were all proved by the column. The temperature in the column was kept constant at 30^oCelsius.

To develop a suitable method for the simultaneous estimation of HAL and THP, a variety of mobile phases were tried. The optimal separation of the drugs from their excipients was achieved using 0.05 M phosphate buffer: acetonitrile: triethylamine (65:35:0.5 v/v). Different solvents were tried with the buffer to improve separation, but acetonitrile performed better than the others. To optimize the separation condition, the composition of the mobile phase was changed. After analyzing the dosage form with a PDA detector, the optimum mobile phase was determined to be 0.05 M phosphate buffer: acetonitrile: triethylamine (65:35:0.5 v/v).

The flow rate was set at 1.2 mL min^-1 because it was found to best resolve the HAL and THP peaks and provide complete separation. HAL and THP retention times were found to be 2.7±0.2 and 3.8±0.2 minutes, respectively. The wavelength of HAL and THP was discovered to be 210nm, which provided a good response at the specified concentration. All of the peaks were well defined and free of tailing under the described experimental conditions. As part of the method robustness testing, the effects of small deliberate changes in the mobile phase composition, pH, and flow rate were evaluated (figure 3).

Figure 3 – Chromatogram of Haloperidol (HAL) and Trihexyphenidyl hydrochloride (THP) with detection at 210 nm

Method validation:

The method was evaluated to ensure that it was suitable for its intended purpose and had adequate validation characteristics.

Linearity and range:

The correlation coefficient was used to evaluate the calibration curve that was created. The method was found to be linear over the concentration ranges of HAL (6-60g mL-1) and THP (8-80g mL-1). Table 1 shows the parameters for the regression analysis.

Table 1 – Data indicating linearity of the proposed method

No.	Parameter	HAL	THP
1	Linearity range	6-60	8-80
2	Regression equation	Y = 46397.6600 x + 8507.1144	Y = 25348.0191x + 2723.3086
3	Slope	46397.6600	25348.0191
4	Intercept	8507.1144	2723.3086
5	Correlation (R²)	0.9990	1.000

Accuracy:

The recovery experiments were carried out using the standard addition method. The mean recoveries obtained for Haloperidol were 99.10-100.7% and 98.7-100.6% for Trihexyphenidyl HCl. Table 2 shows the results of accuracy.

Method precision:

Intraday RSD values for HAL and THP were found to be 0.105 – 0.929 and 0.646 – 0.805, respectively. For inter-day, the RSD values of HAL and THP were found to be 0.357 – 1.054 and 0.662 – 0.911, respectively. Table 2 shows the results of method precision.

LOD and LOQ:

HAL and THP LOD values were found to be 0.015g mL^-1 and 0.048g mL^-1, respectively. HAL and THP LOQ values were found to be 0.046g mL^-1 and 0.148g mL^-1, respectively. Table 2 shows the LOD and LOQ results.

Table 2 – Summary of validation parameters

S. No.	Parameters	Results
S. No.	Parameters	HAL	THP
1.	Accuracy (% recovery)	99.10 - 100.7 %	98.7-100.6%
2	Precession (% RSD)	Intra-day 0.105 – 0.929 Inter-day 0.357 – 1.054	Intra-day 0.646 – 0.805 Inter-day 0.662 – 0.911
3	LOD	0.015 µg mL^-1	0.048 µg mL^-1
4	LOQ	0.046 µg mL^-1	0.148 µg mL^-1
5	Specificity	Specific	Specific
6	Robustness Temperature(30 ± 2^oC) Flow rate (1.2 ±0.2 mL/min) Mobile phase (35 ± 5 %) ACN: Buffer; 35:65, v/v pH (3.75 ± 0.05 )	0.155 – 0.155 0.058 – 0.294 0.101 – 0.101 0.102 – 0.155	0.058 – 0.153 0.058 – 0.117 0.058 – 0.117 0.058 – 0.058

Assay of the tablet dosage form:

The validated method proposed was successfully applied to determine HAL and THP in tablet dosage form. The results obtained for HAL and THP were comparable to the labeled amounts. Table 3 shows the results of the assay.

Table 3 – Assay of tablet dosage form

Dosage form	Label claim	% Assay ± SD (n=6)
Tablet	HAL: 1.5mg	100.10±0.219
Tablet	THP: 2.0mg	99.95±0.020

CONCLUSION:

A newly developed and validated analytical method can be used for simultaneous determination of HAL and THP in combined tablet dosage form and also applied for assay of their dosage form. The presented method is simple, economical, accurate, precise, rapid, and specific for determining Haloperidol and Trihexyphenidyl hydrochloride in tablet dosage form at the same time. Hence, it can be recommended for routine quality control analysis.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

We would like to acknowledge the contribution of the Man Pharmaceutical Ltd and Adlac Life Sciences Pvt. Ltd. for providing Active pharmaceuticals ingredients.

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Received on 23.12.2021 Modified on 08.03.2022

Asian J. Pharm. Ana. 2022; 12(4):253-257.

DOI: 10.52711/2231-5675.2022.00041