INTRODUCTION:

Haloperidol is a dopamine inverse agonist of the typical antipsychotic class of medications that chemically belongs to butyrophenone group. It occurs as a white crystalline powder and chemically known as 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidyl]-1-(4-fluorophenyl)-butan-1-one with molecular weight of 375.86 g mol^-1. The chemical structure of haloperidol is shown in Figure 1. Its mechanism of action is mediated by blockade of D2 dopamine receptors in brain. Haloperidol is commonly used to treat moderate to severe psychiatric conditions including schizophrenia, manic states and medicament induced psychosis¹. It is also used to treat extreme behavior problems in children and to ease the symptoms of tourett’s syndrome. For the treatment of schizophrenia, the required dose of haloperidol was found to be 5-15 mg per day with an average of 10 mg per day. The dosage forms are typically tablets and injections. However, haloperidol produces extrapyramidal side effects including acute dystonic reactions, akathisia syndrome, drug induced Parkinsonism, bradykinesia and tardive dyskinesia².

Figure 1. Structure of haloperidol

In view of therapeutic importance, it has been planned to develop a sensitive analytical method for determination of haloperidol in solid lipid nanoparticles. Haloperidol has been determined in dosage forms using various analytical techniques such as high performance liquid chromatography³, high performance thin-layer chromatography⁴, 19F NMR spectroscopy⁵, square-wave adsorptive stripping voltammetry at a mercury electrode⁶, square-wave and cyclic voltammetry at hanging mercury drop electrode⁷, cyclic voltammetry at multi-walled carbon nanotubes-modified glassy carbon electrode⁸ Non aqueous titrimetric ⁹ and UV spectrophotometric ¹⁰ assay procedures have been described in official compendia.

UV Spectrophotometry is still popular because of the inherent simplicity, low cost, sensitivity, speed and reliability for determination of drugs in pharmaceutical preparations. The analytical procedures based on spectrophotometry are still being frequently published and Literature survey revealed that few spectrophotometric methods have been used to determine haloperidol in pharmaceutical preparations. These methods were based on the reaction with [Cr (NCS) 6]3-, [BiI6]3 and picric acid, chloranilic acid, and p-chloranil. Haloperidol showed absorption maximum at 245 nm in methanol - 0.1M HCl mixture (9:1) and hence it was determined in commercial dosage forms by UV spectrophotometry¹¹. Derivative spectrophotometric method has also been reported for quantitation of haloperidol in pharmaceutical preparations.

In this study, efforts were made to develop and validate a simple & sensitive UV spectrophotometric method for the estimation of haloperidol in prepared pharmaceutical formulations of solid lipid nanoparticles. The different analytical performance parameters such as linearity, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were determined according to ICH Q2 (R1) guidelines¹².

MATERIALS AND METHODS:

Instruments

A Shimadzu UV–Visible spectrophotometer (UV -1800, Shimadzu Corporation, Kyoto, Japan) was used for all absorbance measurements with one cm matched quartz cells and Shimadzu electronic balance (AUX 220, Shimadzu Corporation, Kyoto, Japan) was used for weighing of the sample.

Materials

Haloperidol was received as a gift sample from Vamsi Labs Ltd. Solapur, Maharashtra (India), Potassium dihydrogen phosphate was purchased from Qualigens fine chemicals, Mumbai (India), and Sodium hydroxide was purchased from Fisher scientific, Mumbai (India). All chemicals and reagents used were of analytical grade. Double distilled water was used to prepare solutions wherever required and it was filtered before use through a 0.22 µm membrane filter.

Preparation of phosphate buffer pH 7.4

The phosphate buffer pH 7.4 was prepared as per the specifications of Indian Pharmacopoeia¹³.

Preparation of 0.2 M potassium dihydrogen phosphate solution

27.218 gm of potassium dihydrogen phosphate was dissolved in 1000 ml of distilled water to produce 0.2M solution of potassium dihydrogen phosphate.

Preparation of 0.2 M NaOH solution

8 gm of sodium hydroxide was dissolved in 1000 ml of distilled water to produce 0.2M sodium hydroxide solution

Preparation of buffer

50 ml of 0.2 M potassium dihydrogen phosphate was placed in 200 ml of volumetric flask, and then pH was adjusted to 7.4 0 ± 0.05 by adding 39.1 ml of 0.2 M NaOH solution. Finally, the volume was made upto 200 ml with distilled water and then filtered through 0.22 μm membrane filter.

Determination of wavelength of maximum absorption

A standard stock solution of haloperidol (100 μg/ml) was prepared by dissolving 10 mg of drug in 10 ml of methanol in a 100 ml of volumetric flask and then volume was made upto mark with phosphate buffer (pH 7.4). 2 ml of this stock solution was then diluted up to 10 ml with the same diluent to obtain 20 μg/ml haloperidol solution. An UV spectroscopic scanning (200– 400 nm) was carried out with 20 μg/ml drug solution to determine the maximum absorption (ʎ max) for the detection haloperidol using same diluent as blank.

Preparation of calibration curve

Haloperidol (10 mg) was dissolved in 10 ml of methanol in a 100 ml volumetric flask and then volume was made upto mark with 100 ml with phosphate buffer (pH 7.4) to obtain the stock solution (100 μg/ml). The dilutions of this stock solution were made by diluting the required aliquot with phosphate buffer to obtain standard solutions in the range of 2- 20 μg/ml. The absorbance of the resultant solutions was determined at a λmax of 247. 5 nm

Validation Procedure

Method was validated according to ICH Guidelines in terms of linearity, range, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ)¹².

Linearity and range

Linearity is the ability of the method to obtain test results that are directly proportional to analyte concentration within a given range. The range of an analytical method is the interval between the upper and lower concentration of analyte for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.

To study the linearity, serial dilutions of haloperidol were suitably prepared in the concentration range of 2 - 20 μg/ mlin phosphate buffer (pH 7.4). The absorbance of each solution was scanned at 247.5 nm using same diluent as blank. Calibration curve was constructed by plotting concentration versus absorbance on x and y axis respectively. Linearity was determined by regression equation. This experiment was repeated 3 times.

Range is established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy and precision when applied to samples containing amount of analyte within or at the extremes of the specified range of the analytical procedure.

Precision

The precision was determined at two levels as per ICH, Q2 (R1) suggestions i.e. repeatability and intermediate precision.

Repeatability of drug sample was determined as intraday variation (3 concentrations/3 replicates each, three times a day / a minimum of 9 determinations covering the specified range for the procedure) whereas intermediate precision was determined by interday variation (for three different days) for the determination of haloperidol at three different concentration levels of 6, 12 and 18 μg/ml in triplicate. The % RSD was calculated for absorbance to obtain the intraday variation and interday variation.

Accuracy as recovery studies

Accuracy is the closeness of the test results obtained by the analytical method to the true value. The method was further validated to check the sensitivity of the method to estimate haloperidol in the presence of excipients. The accuracy of the method was evaluated by standard addition method. The pre-analysed samples of haloperidol (8 μg /ml) were spiked with the extra 50%, 100% and 150 %, of the standard drug and the mixtures were analysed by the proposed method. The experiment was performed in triplicate. The % recovery and % RSD of each sample were calculated at each concentration level.

Limit of detection and limit of quantitation

The limit of detection (LOD) is defined as the lowest concentration of an analyte that can be detected, but not necessarily quantified as an exact value.The limit of quantitation (LOQ) is the lowest concentration of an analyte that can be quantitatively determined with acceptable precision and accuracy under the stated operational conditions of the method.

LOD and LOQ of the drug were calculated using the following equations as per ICH guidelines.

LOD = 3.3 × σ/S.................. (1)

LOQ = 10 × σ/S................... (2)

Where σ = the standard deviation of the response; S= the slope of the regression line.

Effect of storage on stability of drug solution

The haloperidol solution was prepared in selected medium and then it was divided into two portions. Each solution was stored at different places in the laboratory i.e. under dark condition at room temperature and day light condition in the laboratory (on the bench top) for short term stability study (48 h). Fresh sample and samples stored for 48 h were analysed by UV spectrophotometer at ʎmax of 247.5 nm.

RESULTS AND DISCUSSION:

Wavelength of maximum absorption

The wavelength of maximum absorption (ʎmax) was found to be 247.5 nm in selected medium. It was also observed that there was no change in the λmax of the drug in this concentration range (2-20 μg/ml) as shown in Figure 2 by overlay spectra of drug.

Preparation of calibration curve

Calibration curve was prepared in the concentration range of 2- 20 μg/ml to determine haloperidol quantitatively for routine analysis in the prepared formulations of solid lipid nanoparticles. Various parameters like Cumulative amount of drug release, entrapment efficiency and drug loading were determined by calibration curve but here data is not shown. Calibration curve data and calibration curve are shown in Table 1 and Figure 3 respectively.

Figure2. Overlay UV spectra of haloperidol in Phosphate buffer (pH 7.4)

Table1. Calibration curve data for haloperidol in phosphate buffer (pH 7.4)

Concen-tration (µg/ml)	Mean absorbance at 247.5 nm ± SD (n = 3)	Regressed absorbance	Equation of Line
2	0.103±0.006	0.1013	Equation of Line y = 0.0417x + 0.0179 Correlation coefficient R² = 0.9994 Slope m = 0.0417 Intercept c = 0.0179
4	0.178±0.010	0.1847
6	0.261±0.011	0.2681
8	0.362±0.002	0.3515
10	0.44±0.006	0.4349
12	0.515±0.004	0.5183
14	0.607±0.004	0.6017
16	0.691±0.002	0.6851
18	0.768±0.002	0.7685
20	0.846±0.003	0.8519

Figure3. Calibration curve of haloperidol in phosphate buffer (pH 7.4)

Table2. Precision of proposed method

Concentration (μg /ml)	Repeatability (intraday precision)		Intermediate precision (interday)
Concentration (μg /ml)	Mean absorbance at 247.5 nm± SD (n=3)	RSD(%)	Day	Mean absorbance at 247.5 nm ± SD (n=3)	RSD (%)
6	0.264±0.005	1.9	1	0.263±0.004	1.34
			2	0.262±0.005	1.81
			3	0.263±0.004	1.53
12	0.515± 0.002	0.4	1	0.516±0.002	0.40
			2	0.517±0.002	0.39
			3	0.513±0.001	0.19
18	0.767±0.002	0.3	1	0.768±0.003	0.40
			2	0.764±0.001	0.15
			3	0.766±0.004	0.47

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

LOD and LOQ of this method were determined by the standard deviation method. The value of LOD and LOQ were found to be 0.225 and 0.681 μg/ml respectively.

Effect of storage on stability of drug solution

The data of drug absorbance of a constant drug concentration (8 µg/ml) from (1) fresh drug solution (2) drug solution stored in dark for 48 h (3) drug solution stored in day light condition for 48 h was subjected to one way ANOVA. It was found that the values of drug absorbance were not significantly different (p< 0.05). The results indicated that the drug was stable in selected medium after storage up to 48h and was not affected by light and dark cycles. The results of effect of storage on stability of drug solution are shown in Table 4 and the results of all validation parameters are shown in Table 5.

Table5. UV spectrophotometric parameters of haloperidol

S. No.	Parameters	Results
1.	Absorption maxima (nm)	247.5
2.	Linearity range (μg/ml)	2-20
3.	Regression equation	y = 0.0417x + 0.0179
4.	Slope	0.0417
5.	Intercept	0.0179
6.	Correlation coefficient (R²)	0.9994
7.	Recovery (%)	99.00-100.6
8.	LOD (μg/ml)	0.225
9.	LOQ (μg/ml)	0.681

CONCLUSION:

The results and the statistical parameters demonstrated that the proposed UV spectrophotometric method is simple, rapid, specific, accurate and precise. Therefore, this method can be used to determine haloperidol quantitatively for routine analysis in the prepared formulations of solid lipid nanoparticles without interference of commonly used excipients and related substances. The results of stability indicated that the drug was stable in selected medium after storage up to 48h.

ACKNOWLEDGEMENTS:

The author expresses deep appreciation and thanks to the Management of ITS Pharmacy College, Muradnagar, Ghaziabad, UP, India for their valuable cooperation in the present research work. Authors are also gratified to Vamsi Labs Ltd. Solapur, Maharashtra, India for providing the gift sample of haloperidol.

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Received on 17.03.2014 Modified on 01.05.2014

Research J. Pharm. and Tech. 7(6): June, 2014; Page 638-642

% of standard spiked to the sample	Concentration			% of drug recovered	%RSD
% of standard spiked to the sample	Sample (μg /ml)	Total including spiked sample(μg /ml)	Spiked sample Determined (μg /ml) ± SD (n = 3)	% of drug recovered	%RSD
50	8	12	11.88±0.16	99.00	1.31
100	8	16	I6.10±0.11	100.6	0.68
150	8	20	19.89±0.11	99.45	0.55

Concentration (µg/ml)	Mean absorbance at 247.5 nm ± SD (n=3)
Concentration (µg/ml)	Fresh drug solution	Drug solution stored in dark	Drug solution stored in day light condition
8	0.374 ±0 .007	0.360 ± 0.002	0.363± 0.008