Method development and validation of pregabalin in bulk and tablet dosage forms by UV spectroscopy

Introduction

Pregabalin (PGB) is chemically as (S)-3-amino methyl-5-methyl hexanoic acid and is structurally related to the naturally occurring amino acids L-leucine and gammaaminobutyric acid (GABA). It is a white to off-white crystalline, non-hygroscopic and water soluble (Freely soluble below pH-3.7) powder. It contains one chiral center but is synthesized as the single enantiomer S. PGB exists as a single anhydrous and not solvated crystal form. PGB is an anti-convulsant and analgesic medication that was recently approved for adjunctive treatment of partial seizures in adults in both the United States and Europe and for the treatment of neuropathic pain from postherpetic neuralgia and diabetic neuropathy [1][2][3] . The mechanism of action is still unclear, pregabalin decreases central neuronal excitability by binding to an auxiliary subunit (α-δ protein) of a voltage-gated calcium channel on neurons in the central nervous system. The methods adapted to the analysis of PGB include High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Mass Spectrophotometry (LC-MS) and spectrofluorimetry. In addition, these methods require long and tedious pretreatment of the samples and laborious clean-up procedures prior to analysis. An official monograph of PGB does not exist in any pharmacopoeia and the determination of PGB in bulk and pharmaceutical formulations has not been yet described. Therefore, it is very imperative to develop a simple and suitable analytical method for the determination of PGB in bulk and pharmaceutical formulations [6][7][8][9] . PGB reduces the release of several neurotransmitters include glutamate, norepinephrine, and calcitonin gene related peptide from certain brain tissues and also reduce calcium influx in synaptosomes [4,5] . Pregabalin undergoes minimal metabolism in human with unchanged parent representing the majority (≥90%) of drugderived material. This contrasts with gabapentin, which is absorbed via a capacity-limited L-amino acid transport system from the proximal small bowel into the bloodstream. The therapeutic importance of pregabalin was behind the development of numerous methods for its determination. UV-visible spectrophotometry is the technique of choice in research laboratories, hospitals and pharmaceutical industries due to its low cost and inherent simplicity. This paper reports a simple, sensitive and accurate spectrophotometric method for the determination of PGB.

The method is based on the direct measurement of the native absorbance of the drug at 210 nm against the reagent blank.

The proposed method was extended to the determination of PGB in bulk, pharmaceutical formulations.

Materials and Methods

Instruments used

UV-visible spectrophotometer with UV Win software.

Weighing balances and matching quartz cells with a 1 cm cell path length were utilized along with the mentioned equipment, which had an automatic wavelength accuracy of 0.1 nm.

Chemicals and reagents

Pharmaceutical grade pregabalin (API) was procured as gift sample from Hetero Drugs Ltd., Hyderabad, Telangana, India. The marketed pharmaceutical dosage form of pregabalin sitagliptin tablets (Preganerve 100 mg) was purchased from a local Pharmacy, in Hyderabad, Telangana, India. All chemicals and reagents were of analytical grade. Solvent selection: A number of trails were done to find out the ideal solvent for dissolving the drug. The solvents such as double distilled water, methanol and HCl buffer were tried based on the solubility of the drug.

Selection of detection wavelength

Appropriate volume 1 ml of standard stock solution of pregabalin was transferred into a 10 ml volumetric flask, diluted to a mark with HCl buffer to give concentration of 10 μg/ml. The resulting solution was scanned in the UV range (200-400 nm).

Preparation of stock solution

A precisely weighed, 10 mg of pregabalin was transferred to 10 ml volumetric flask (Clean and dry). Then few ml of HCl buffer was added and dissolved the drug by vigorous shaking. The volume was then made up to the mark with HCl buffer to obtain the stock solution of 1000 µg/ml.

Preparation of working standard solution

From stock solution 1 ml was pipetted out and further diluted to 10 ml with HCl buffer to get the solution having a concentration of 100 μg/ml.

Preparation of calibration curve

From the working standard solution, pipetted out 0.2ml, 0.4 ml, and 0.6 ml, 0.8 ml,1ml and diluted to 10 ml using HCl buffer to produce, 2, 4, 6, 8, 10 μg/ml, solutions, respectively. The absorbance of the solutions at the λ max of 210 nm using HCl buffer as blank was measured. The calibration curve was plotted by taking concentration on Xaxis and absorbance on the Y-axis. The curve shows linearity in the concentration range of 2 to 10 μg/ml. The correlation coefficient (r²) was found to be 0.9998.

Assay of pharmaceutical formulation

20 Tablets of pregabalin marketed formulations were weighed and powdered. A quantity of tablet powder equivalent to 50 mg of pregabalin was transferred to 100 ml volumetric flask and volume was made up to the mark with HCl buffer. The absorbance of the resulting solution was measured at 210 nm and the amount of pregabalin was computed from its calibration plot.

Method development and validation [10][11][12][13][14] These current validation characteristics describe the validation parameters stated by the International Conference on Harmonization [ICH] guidelines.

Linearity

The linearity of an analytical procedure is its ability to obtain test results, which are directly proportional to the concentration of analytic in the sample. Linearity can be assessed by performing single measurements at several analyte concentrations. A linearity correlation coefficient above 0.998 is acceptable for most methods, especially for major components in assay methods. The range of an analytical procedure is the interval between the upper and lower concentration of analyte in the sample.

Precision

The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogenous sample under prescribed conditions. Precision was determined by intra-day and inter-day study. The repeatability of the method was evaluated by carrying out the assay 3 times on the same day and intermediate precision was evaluated by carrying out the assay on 3 consecutive days for the sample solution. The percent relative standard deviation (% RSD) was calculated.

Accuracy (Recovery studies)

The accuracy of analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted true value. Accuracy studies were performed at three different levels (80%, 100% and 120%) by standard addition method and the samples were analyzed in triplicate by the proposed method. Known amount of standard sitagliptin at 80%, 100% and 120% of predetermined sample was added to a pre-quantified tablet sample.

Ruggedness

Method ruggedness is defined as the reproducibility of results when the method is performed under actual use conditions. This includes different analysts, laboratories, columns, instruments, sources of reagents, chemicals, solvents and so on. Method ruggedness may not be known when a method is first developed, but insight is obtained during subsequent use of that method.

Robustness

The concept of robustness of an analytical procedure has been defined by the ICH as "a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters". The most important aspect of robustness is to develop methods that allow for expected variations in the separation parameters. For the determination of a method's robustness, parameters such as variation in detector wavelength are varied within a realistic range and the quantitative influence of the variables is determined. If the influence of the parameter is within a previously specified tolerance, the parameter is said to be within the method's robustness range. The absorbance was measured and the assay was calculated for six times.

LOD and LOQ

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantified as an exact value.

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

Where, σ = Standard deviation of the response, and S = Slope of the calibration curve.

Results and Discussion

Determination of absorbance maxima

From the above working standard solution, 1 ml was pipetted out into a 10 ml volumetric flask and the volume was made up to the mark with HCl buffer to prepare a concentration of 10 μg/ml. The sample was then scanned in UV/Vis-spectrophotometer in the range 200-400 nm using HCl buffer as blank and the wavelength corresponding to maximum absorbance was found to be 210 nm. It was observed that the drug showed maximum absorbance at 210 nm which was selected as the wavelength for detection. The absorption maxima curve was shown in Figure 2. New spectrophotometric methods were developed for the determination of pregabalin in bulk and pharmaceutical dosage forms. The absorption spectra were recorded in the wavelength region of 200-400 nm by UV. The proposed method obeyed Beer's law in the concentration range of 2-10 μg/ml with a good correlation coefficient of r 2 =0.997.

Calibration data is represented in Table 1. Beer's law range was confirmed by the linearity of the calibration curve of pregabalin as shown in Figure 3. The precision of the method was reported in terms of relative standard deviation and it should be evaluated by using a minimum of 3 determinations over which shows % RSD less than 2 indicates that the method was precise and the results are presented in Table 2. Recovery studies were carried out for the developed method by addition of known amount of standard drug solution of Pregabalin to pre-analyzed tablet sample solution at three different concentration levels. The resulting solutions were analyzed by the proposed methods. The recovery (Table 3) was in the range of 98.12-101.17%. The results were reported to be within the limits. In fact, there was no difference in mean assay results of the method obtained from two instruments of different manufacturers.

Conclusion

For the determination of a method's robustness, parameters such as variation in detector wavelength are varied within a realistic range and the quantitative influences of the variables were determined. The results are within the specified limits which states that this method is robust. The developed method was applied to the analysis of tablet formulations of pregabalin found to be within the proposed limits. The developed method has good linearity, accuracy and precision results indicates that the high quality of the method. The developed method is validated according to ICH guidelines, a method is fast, accurate, precise and robust. The present method can be therefore used for routine analysis of pregabalin. The result shows the developed method of pregabalin is yet another suitable method for assay which can help in the analysis of pregabalin in different formulations.

Acknowledgement

AcknowledgementWe express our indebtedness and sense of gratitude the management of CMR College of Pharmacy, Kandlakoya, Medical, Hyderabad, Telangana, India for providing the necessary equipment for research, facilities and support.

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