INTRODUCTION:

Pregabalin (PGB), (S)-3-(aminomethyl)-5-methylhexanoic acid (Figure 1), is an antiepileptic drug and structurally related to the neurotransmitter gamma aminobutyric acid (GABA). It is also used in neuropathic pain and generalized anxiety disorder. The system of activity of pregabalin varies from the activity of other antiepileptic and pain-relieving drugs. Pregabalin acts on voltage-gated calcium channel and binds to alpha2-delta subunit at neuronal ending of pre synaptic region of spinal cord and brain.

Regulates hyper excited neurons through authoritative to presynaptic neurons of voltage-gated calcium channel switch minimize calcium flood in to presynaptic terminals.

Over the top arrival of excitatory neurotransmitters (e.g., glutamate, substance P, nor adrenaline) diminishes the decreased calcium stream. It accordingly mitigates neuropathic pain and is liable for Pregabalin's anxiolytic, anticonvulsant, and pain-relieving capacity.^1-5

Figure 1:Structure of Pregabalin

Currently there are no official UV-Visible spectrophotometric methods for estimation of Pregabalin in any Pharmacopoeia. But several reports are reported in literatures for Pregabalin determination based on chromatographic and spectroscopic methods^6-15. These methods may involve tedious procedures including extraction methods, pre and post column derivatization. But routine analysis of the drug in pure and dosage forms in research laboratories and pharmaceutical industries require relatively uncomplicated and cost effective methods like UV or visible spectroscopy or fluorimetry. Pregabalin has a poor UV–Visible absorbance profile and the reported visible methods includes tedious procedures, we found it very pertinent to develop and validate a simple visible spectroscopic method for the routine analysis of Pregabalin in its pure and dosage form.

MATERIALS AND METHODS:

Materials:

Pure drug sample of Pregabalin is kindly gifted by Rubicon Pharma Ltd, Mumbai and Pregabalin Capsule was obtained from local pharmacy. A SHIMADZU model PHARMASPEC-1800 UV-Visible double beam spectrophotometer with 1cm matched quartz cell was used for recording spectra and absorbance measurements. And Deluxe pH meter was used to adjust the pH of buffer. All reagents used were of analytical grade. Bromophenol blue were purchased from Qualigens Fine Chemicals, Mumbai. Potassium hydrogen phthalate and Hydrochloric acid were purchased from Spectrum Reagents and Chemicals Pvt. Limited, Edayar, Cochin.

Method development and optimization:

Selection of solvent:

The solvent for a sample is selected in such a way that solvent neither absorbs in the region of measurement nor affect the absorption of sample. Various solvents were tried, out of which, water was selected as it is freely soluble in water.

Stock solution of Pregabalin:

50mg of Pregabalin was dissolved in distilled water and made up to 100ml with Distilled water (500μg/ml).

Selection of reagent:

Bromophenol Blue (BPB) was selected as the reagent for the method. The selection of reagent was based on the physical and chemical characteristics of the drug and the drug have primary amino group.

Preparation of reagents:

Stock solution of Bromophenol Blue (1000μg/ml): 50mg of Bromophenol blue was weighed and dissolved in 10 ml Ethanol and make up to 50ml with Distilled water. From this working standard solutions were prepared (150, 200 and 250µg/ml.)

Preparation of Acid phathalate buffer:

Placed 25ml of 0.2 M Potassium Hydrogen Phthalate in a 100ml standard flask, added specified volume of 0.2 M HCl and make up with Distilled water as per IP to get the buffer with pH 2.4, 3 and 3.6. Measured the pH.

Selection of analytical wavelength:

Pregabalin solutions were suitably prepared and added Acid phthalate buffer of pH 3 and Bromophenol Blue (BPB). Solution was scanned in the range 400-800nm. Absorption maximum (λmax) was obtained at 592nm. (Figure 2).

Optimization of reaction conditions:

The condition in which reagents reacts with Pregabalin was investigated. All reaction conditions were optimised at room temperature.

Optimization of reagents concentration:

Optimization of concentration of Buffer (pH 3) and Bromophenol Blue (200μg/ml) required to achieve maximum sensitivity of the developed complex was ascertained by adding different volumes 1, 2 and 3ml of Acid phthalate buffer (pH 2.5, 3 and 3.5) and 0.8,1 and 2ml of BPB reagent (150, 200 and 250µg/ml) to a constant concentration of pure drug solution (50µg/ml). Maximum absorbance of stable colour complex was found at 2ml of acid phthalate Buffer of pH3 and 2ml of BPB reagent of 200µg/ml.

Optimized Method:

Aliquot of the standard drug solution was pipetted into separate 10ml standard flasks containing of 2ml Acid Phthalate Buffer (pH 3). Mixed well and 2ml BPB reagent was added and made up to volume with Distilled water. Absorbance was measured against blank at 592nm for Pregabalin.

Analysis of formulation:

Twenty capsules were weighed accurately and the average weight was calculated. An accurately weighed quantity of powder equivalent to 50mg of Pregabalin transferred into 100ml standard flask. Add 50ml of Distilled water and sonicated for 5 minutes. Dissolved the contents and made up the volume with Distilled water. The solution was then filtered through Whatman filter paper. Appropriate aliquots within the Beer’s law limit (70µg/ml of Pregabalin) were analyzed by the proposed method. Aliquot of the sample solute on is pipette into 10ml standard flask containing 2ml Acid Phthalate Buffer (pH 3). Mixed well and 2ml BPB reagent was added and made up to volume with Distilled water. Absorbance was measured against blank at 592nm (Figure 3). The concentration of Pregabalin present in the formulation was calculated.

Method validation^16-18

Linearity and Range:

Five concentrations of the standard Pregabalin (50, 60, 70, 80 and 90µg/ml) were prepared and the regression coefficient were found out (Figure 4).

Accuracy:

The accuracy of the method was determined by recovery experiments. A known quantity of the pure drug was added to the pre-analyzed sample formulation at 50%, 100% and 150% levels. The recovery studies were carried out three times and the percentage recovery and percentage relative standard deviation of the percentage recovery was calculated.

Precision:

In order to determine the precision of the proposed method, pure drug solutions (Pregabalin) at a particular concentration level (within the working range) were prepared and analyzed in three replicates during the same day (intra-day precision) and on three consecutive days (inter-day precision). Percentage relative standard deviation (%RSD) for intra-day and inter-day indicates repeatability and usefulness of the proposed methods in the routine analysis.

Ruggedness of the method:

Method ruggedness was evaluated by performing the analysis following the recommended procedures by three different analysts. From the %RSD values presented, one can conclude that the proposed method is rugged.

Limit of detection (LOD) and Limit of quantification (LOQ):

LOD and LOQ values were calculated to check the detection limit and quantification limit of the method.

RESULT:

Wavelength Selection:

Pregabalin standard solutions were prepared separately and followed the method developed, noted the maximum absorbance obtained at 592nm for Pregabalin. The absorption spectrum of Pregabalin is given in Figure 2.

Estimation of formulation:

Estimation of Pregabalin in Capsule dosage form by UV- Visible spectroscopy was carried out using optimized condition. The assay procedure was repeated for three times and the absorbance was recorded. The results of analysis of marketed formulation are given in the Table 1.

Figure 2: Absorption spectrum of Pregabalin

Figure 3: Visible spectrum of Pregabalin Capsule Formulation

Table 1: Results of marketed formulation by Colorimetry

Marketed formulation

Drug

Label claim (mg)

Estimated amount (mg)

% purity

% RSD

Pregabalin capsule

PGB

50.6

50.9

100

101.2

101.8

0.97

Method validation:

Linearity:

Pregabalin is found to be in the linear in the concentration range 50-90μg/ml. The calibration graph (Figure 4) plotted by using absorbance Vs concentration (Table 2).

Table 2: Linearity of Pregabalin

Concentration (μg/ml)	Absorbance
50	0.097
60	0.126
70	0.153
80	0.178
90	0.198

Figure 4: Calibration curve of Pregabalin

Accuracy:

The accuracy of the method was determined at three percentage level 50%, 100% and 150% levels. The recovery studies were carried out three times and the percentage recovery and percentage relative standard deviation was found to be less than 2 and given in Table 3.

Table 3: Accuracy studies of Pregabalin

Drug	Theoretical % Target level	Amount added (µg/ml)	Amount recovered (mg)	% Recovery	% RSD
Pregabalin	50	35	50.9	101.86	1.74
			50.32	100.64
			49.2	98.4
	100	70	49.5	99	0.50
			49.8	99.6
			50	100
	150	105	49.9	99.8	0.70
			50.38	100.6
			49.6	99.2

Precision:

To determine the precision of the proposed method, pure drug solutions (PGB) at a concentration within the working range were prepared and analyzed in three replicates during the same day and on three consecutive days and the results are presented in Table 4. Percentage relative standard deviation (%RSD) for intra-day was 0.97 for PGB and for inter-day %RSD was 0.99 for PGB indicating repeatability.

Table 4: Precision studies of Pregabalin

Drug	Amount (µg/ml)	Intra-day		Inter-day
		% content	% RSD	% content*	% RSD
Pregabalin	70	100.6	1.03	101.53	1.204
		98.6		101.73
		99.2		99.53

Ruggedness:

Method ruggedness was evaluated by performing the analysis following the recommended procedures by three different analysts. From the %RSD values obtained in (Table 5) it concluded that the proposed method was rugged.

Table 5: Ruggedness results

Drug	Analyst	Amount taken (mg)	Amount Found (mg)	% Content	% RSD
PGB	Analyst I	50	50.6	100.2	0.592
	Analyst II		50.9	101.8
	Analyst III		50.3	100.6

Limit of detection (LOD) and Limit of quantification (LOQ):

LOD and LOQ values were calculated to check the sensitivity of the method. The values are concluded in Table 6.

Table 6: LOD and LOQ results

LOD (µg/ml)	LOQ (µg/ml)
10.91	33.06

Analytical data:

The linear regression equations were obtained by the method of least squares and the Beer's law range, detection wavelength, correlation coefficient, slope and intercept of methods are summarized in Table 7.

Table7: Analytical Data Parameters

Parameter	Pregabalin
Detection wavelength	592 nm
Beer’s law Limit	50-90 µg/ml
Regression equation	y=0.002x - 0.027
Correlation coefficient	0.995
Slope	0.002
Intercept	0.027
LOD	10.91µg/ml
LOQ	33.06µg/ml

DISCUSSION:

Literature survey reveals that UV- Visible spectrophotometric and HPLC method were done for the analysis of estimation of Pregabalin in its dosage form. No other UV-Visible studies using Bromophenol Blue were reported for the estimation of Pregabalin in its pure and dosage form. Hence an attempt was made to develop a validated simple, accurate and precise method for the determination of Pregabalin using Bromophenol Blue.

The developed method was validated as per ICH guideline in terms of linearity, accuracy, precision, ruggedness, LOD and LOQ. In this method the solvent used for the preparation of Pregabalin was distilled water. The method is based on the formation of brown colored ion-pair complex of Pregabalin with Bromophenol Blue reagent. The ion pair complex was produced in presence of Acid Phthalate Buffer of pH 3. The developed brown colour was stable at room temperature. The developed brown coloured complex showed maximum absorbance at 592nm. Linearity was found in the concentration range of 50, 60, 70, 80 and 90 µg/ml with regression coefficient value 0.995. Precision studies for intra-day and inter-day shows that percentage relative standard deviation was within range of acceptable limit. The recovery studies were carried out at 50%, 100% and 150% levels. The result shows that the method was found to be accurate. The limit of detection (LOD) and limit of quantification (LOQ) was found at 10.91µg/ml and 33.06µg/ml respectively. A ruggedness study was carried out using different analysts; assure that the method was rugged. Hence the method can be applied for routine quality control of the drug.

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Received on 10.05.2022 Modified on 15.07.2022

Asian J. Research Chem. 2022; 15(6):417-421.

DOI: 10.52711/0974-4150.2022.00073