- Research article
- Open Access
A novel method for spectrophotometric determination of pregabalin in pure form and in capsules
- Alka Bali1Email author and
- Prateek Gaur1
- Received: 19 July 2011
- Accepted: 7 October 2011
- Published: 7 October 2011
Abstract
Background
Pregabalin, a γ-amino-n-butyric acid derivative, is an antiepileptic drug not yet official in any pharmacopeia and development of analytical procedures for this drug in bulk/formulation forms is a necessity. We herein, report a new, simple, extraction free, cost effective, sensitive and reproducible spectrophotometric method for the determination of the pregabalin.
Results
Pregabalin, as a primary amine was reacted with ninhydrin in phosphate buffer pH 7.4 to form blue violet colored chromogen which could be measured spectrophotometrically at λmax 402.6 nm. The method was validated with respect to linearity, accuracy, precision and robustness. The method showed linearity in a wide concentration range of 50-1000 μg mL-1 with good correlation coefficient (0.992). The limits of assays detection was found to be 6.0 μg mL-1 and quantitation limit was 20.0 μg mL-1. The suggested method was applied to the determination of the drug in capsules. No interference could be observed from the additives in the capsules. The percentage recovery was found to be 100.43 ± 1.24.
Conclusion
The developed method was successfully validated and applied to the determination of pregabalin in bulk and pharmaceutical formulations without any interference from common excipients. Hence, this method can be potentially useful for routine laboratory analysis of pregabalin.
Keywords
- Pregabalin
- Ninhydrin
- Pure Drug
- Percentage Relative Error
- Lactose Monohydrate
Background
Chemical structure of pregabalin.
UV/Visible scan of pregabalin without derivatization (λ max 196.2 nm).
Results and discussion
Method
UV/Visible scan of the chromophoric product of pregabalin (λ max 402.6 nm).
Mechanism of generation of chromophore (Ruhemann's Purple) by reaction of pregabalin with ninhydrin.
Effect of pH
Of the buffers investigated (acetate buffer, phosphate buffer), colour development was noted in case of phosphate buffer. The optimum buffer pH was found to be 7.4 and lower pH ranges resulted in an insufficient colour development.
Effect of reagent concentration
The addition of 1.0 mL of ninhydrin solution (0.2% w/v) was sufficient to obtain the maximum and reproducible absorbance values for the various concentration ranges of PGB. Smaller amounts resulted in incomplete reaction. Further increase in the concentration had no significant effect on complex formation, although absorbance increased slightly owing to the reagent background used.
Effects of temperature and heating time
The effects of temperature and heating time on the formation of the coloured complex were also optimized. At room temperature, the addition of ninhydrin did not lead to the formation of any coloured product and higher temperatures were required to accelerate the reaction. The colour intensity increased with increasing temperature and maximum absorbance was obtained following heating on a water bath at a temperature of 70-75°C for 20 minutes. Further heating caused no appreciable change in the colour. The complex obtained was highly stable for more than 6 h.
Validation
Validation data for determination of pregabalin by proposed method
Validation parameter | Results |
---|---|
Absorption maximum | 402.6 nm |
Regression equation | y = 1.908x - 0.056 |
Slope | 1.908 |
Intercept | 0.056 |
Beer's law limit (μg mL-1) | 50-1000 |
Molar Absorptivity (L/mol-1/cm-1) | 1198.4 |
Sandell's sensitivity (μg/cm2/0.001 A.U) | 0.1328 |
Coefficient of correlation(r) | 0.992 |
Limit of detection (LOD) | 0.006 mg/mL |
Limit of quantification (LOQ) | 0.02 mg/mL |
Accuracy | 100.43 ± 1.24 |
Precision | Intra-day = 0.62 |
Inter-day = 0.61 | |
Robustness | Less than 1% |
% Purity | 99.8% |
Linearity and range
Standard plot of pregabalin by ninhydrin method.
Precision
Precision of the proposed methods for the analysis of pregabalin
Pregabalin (mg/ml) | Within day, n = 3 | Between day, n = 3 | ||
---|---|---|---|---|
Mean ± SD | RSD (%) | Mean ± SD | RSD (%) | |
0.2 | 0.288 ± 0.14 | 0.49 | 0.287 ± 0.0005 | 0.0020 |
0.4 | 0.551 ± 0.28 | 0.51 | 0.551 ± 0.0004 | 0.0007 |
0.6 | 0.890 ± 0.42 | 0.48 | 0.890 ± 0.0003 | 0.0004 |
0.8 | 1.451 ± 0.56 | 0.39 | 1.452 ± 0.0005 | 0.0004 |
1.0 | 1.989 ± 0.71 | 0.36 | 1.990 ± 0.0003 | 0.0002 |
Accuracy
Where
Ct is the total drug concentration measured after standard addition;
Ci drug concentration in the formulation sample;
Ca, drug concentration added.
The percentage relative error was calculated as: Er % = [(found - added)/added] × 100
Results of recovery studies with pure drug pregabalin by standard addition method
Initial Conc. (mg/mL) | Volume (mL) | Amount added (μg) | Amount recovered (μg) | Recovery (%) ± SDa | RSD (%) |
---|---|---|---|---|---|
0.2 | 5.0 | 100.0 | 98.7 | 98.70 ± 0.890 | 0.91 |
0.4 | 5.0 | 100.0 | 96.5 | 96.50 ± 0.923 | 0.95 |
0.6 | 5.0 | 100.0 | 99.7 | 99.70 ± 0.624 | 0.63 |
0.8 | 5.0 | 100.0 | 100.3 | 100.3 ± 1.600 | 1.57 |
1.0 | 5.0 | 100.0 | 98.1 | 98.10 ± 1.718 | 1.72 |
Recovery studies with pregabalin capsules by standard addition method
Initial Conc. (mg/mL) | Volume (mL) | Amount of PGB added (μg) | Amount recovered (μg) | Recovery (%) ± SDa | Er% | RSD (%) |
---|---|---|---|---|---|---|
0.05 | 5.0 | 50.0 | 49.5 | 98.85 ± 0.145 | -1.14 | 0.14 |
0.05 | 5.0 | 100.0 | 99.5 | 99.09 ± 0.370 | -0.91 | 0.37 |
0.05 | 5.0 | 200.0 | 199.1 | 99.45 ± 0.124 | -0.550 | 0.13 |
0.1 | 5.0 | 50.0 | 49.1 | 98.71 ± 0.471 | -1.29 | 0.47 |
0.1 | 5.0 | 100.0 | 98.1 | 98.16 ± 0.271 | -1.83 | 0.28 |
0.1 | 5.0 | 200.0 | 98.1 | 98.16 ± 0.271 | -1.83 | 0.28 |
0.2 | 5.0 | 50.0 | 49.0 | 98.44 ± 0.426 | -1.55 | 0.43 |
0.2 | 5.0 | 100.0 | 99.7 | 99.67 ± 0.186 | -0.32 | 0.19 |
0.2 | 5.0 | 200.0 | 199.0 | 99.49 ± 0.165 | -0.510 | 0.17 |
0.4 | 5.0 | 50.0 | 49.7 | 99.27 ± 0.251 | -0.73 | 0.25 |
0.4 | 5.0 | 100.0 | 97.6 | 97.54 ± 0.196 | -2.45 | 0.20 |
0.4 | 5.0 | 200.0 | 199.7 | 99.74 ± 0.123 | -0.253 | 0.12 |
0.6 | 5.0 | 50.0 | 49.8 | 99.07 ± 0.483 | -0.92 | 0.48 |
0.6 | 5.0 | 100.0 | 98.2 | 98.29 ± 0.134 | -1.70 | 0.14 |
0.6 | 5.0 | 200.0 | 198.9 | 98.71 ± 0.646 | -1.287 | 0.65 |
0.8 | 5.0 | 50.0 | 49.1 | 98.62 ± 0.479 | -1.38 | 0.48 |
0.8 | 5.0 | 100.0 | 99.2 | 99.25 ± 0.098 | -0.74 | 0.10 |
0.8 | 5.0 | 200.0 | 198.2 | 99.22 ± 0.136 | -0.777 | 0.14 |
1.0 | 5.0 | 50.0 | 50.0 | 99.49 ± 0.495 | -0.51 | 0.49 |
1.0 | 5.0 | 100.0 | 97.0 | 97.09 ± 0.147 | -2.91 | 0.15 |
1.0 | 5.0 | 200.0 | 199.5 | 99.66 ± 0.198 | -0.333 | 0.20 |
Interference
Satisfactory values of the mean recovery values ± SD, RSD % and Er % in recovery studies in drug formulation (Table 4) revealed that there is no potential interference of the excipients listed by the manufacturer, i.e., talc, lactose monohydrate and maize starch. This may be attributed to the dependence of the reaction in the proposed method on the presence of a primary aliphatic amino group in the drug molecule which is not present in any of these excepients.
Limit of detection (LOD) and limit of quantitation (LOQ)
LOD and LOQ of the method were established using calibration standards (Table 1). LOD and LOQ were calculated as 3.3 σ/s and 10 σ/s, respectively, as per ICH definitions, where, σ is the mean standard deviation of replicate determination values under the same conditions as the sample analysis in the absence of the analyte (blank determination), and s is the sensitivity, namely the slope of the calibration graphs.
Robustness
Repeatability is based on the results of the method operating over short interval of time under same conditions. Robustness was examined by evaluating the small variations in different experimental conditions such as heating temperatures (± 2° C), working wavelengths, volume and concentration of reagents. Three replicate determinations at six different concentration levels of the drugs were carried out. The within-day RSD values were found to be less than 0.6% indicating that the proposed method has reasonable robustness.
Stability
The stability of the final sample solutions was examined by their absorbance values and responses were found to be stable for at least 6 hours at room temperature.
Analysis of marketed formulation (Pregabalin capsules)
Assay results for pregabalin determination in capsule formulation
Formulation | Label claim (mg) | Mean Recovery (mg) ± SDa | Mean % Recoverya± SDa |
---|---|---|---|
Pregabalin 75 (Torrent Pharmaceuticals) | 75 | 74.61 ± 0.55 | 99.48 ± 0.73 |
Analytical applications
The results with the proposed method for the determination of pregabalin in its pharmaceutical formulation (Pregabalin75 capsules) suggest satisfactory recovery. Further, standard addition technique followed to check the validity of the method have given good recoveries of the drug in presence of formulation suggesting a noninterference from formulation excepients. Hence, this method can be recommended for adoption in routine analysis of pregabalin in in quality control laboratories.
Conclusion
The method proposed is simple, rapid, inexpensive and sensitive for the determination of pregabalin in bulk as well as in marketed form (capsules). There is no requirement of any sophisticated apparatus as in chromatographic methods. Omission of an extraction step with organic solvents is an added advantage. The method has been validated in terms of its sensitivity, simplicity, reproducibility, precision, accuracy and stability of the coloured species for ≥ 6 h suggesting its suitablility for the routine analysis of PGB in pure form (in bulk analysis) as well as pharmaceutical formulations without interference from excipients.
Experimental
Apparatus
All absorption spectra were recorded using a Perkin Elmer lambda 15 UV-Visible spectrophotometer (German) with a scanning speed of 60 nm/min and a band width of 2.0 nm, equipped with 10 mm matched quartz cells. A CyberScan pH 510 (Eutech instruments) pH meter was used for checking the pH of buffer solutions.
Materials and reagents
All chemicals and materials were of analytical grade and were purchased from Qualigens fine chemicals, Mumbai, India. All solutions were freshly prepared in double distilled water.
Pure samples
Pregabalin (PGB) pure grade was graciously provided as a gift samples by Vardhman Chemtech limited, Derabassi, Punjab, India.
Market samples
Pregabalin75 capsules (label amount 75 mg PGB/Capsule) Torrent Pharmaceuticals were purchased from the market.
Preparation of phosphate buffer pH 7.4
Phosphate buffer pH 7.4 was prepared by mixing 250 mL of 0.2 M potassium dihydrogen phosphate with 195.5 ml of 0.2 M NaOH and making up the volume to 1000 ml with distilled water. The pH of the buffer was adjusted to 7.4 using a precalibrated pH meter.
Standard Stock solutions
Stock solution of pregabalin (2 mg/ml) was prepared by dissolving 200 mg of pregabalin in 100 mL of phosphate buffer (pH 7.4).
Preparation of ninhydrin solution
The 0.2% solution of ninhydrin was prepared by dissolving 200 mg of ninhydrin in 100 ml of ethanol and was kept in an amber colored bottle.
Method
Standard plot
Different aliquots were taken from the stock solution (2 mg/ml) and diluted with phosphate buffer pH 7.4 to prepare a series of concentrations ranging from 50 to 1000 μg/mL of pregabalin. To 5.0 mL of these aliquots taken in stoppered tubes, 1.0 mL of ninhydrin solution (0.2% w/v) was added and heated on a water bath at a temperature of 70-75°C for 20 minutes. The tubes were kept covered to avoid the loss of solvent due to evaporation. After cooling the solution to room temperature, the absorbance values were measured in triplicate at 402.6 nm against mixture of 5.0 mL phosphate buffer (pH 7.4) and 1.0 mL 0.2% ninhydrin as reagent blank. The calibration graph was obtained by plotting the absorbance values at the λmax of the drug (402.6 nm) against corresponding concentration values and compliance with Beer Lambert's law was assessed.
Analysis of pharmaceutical formulation
Preparation of capsule sample solution
The contents of twenty capsules were mixed and weighed accurately. Separate quantities of the powder equivalent to 30 mg, 60 mg and 90 mg of PRG were transferred into a 100 mL volumetric flasks, dissolved in water, and sonicated for 5 min., the volume was then completed with water, shaken well for 5 min. and filtered into a dry flask. To 5.0 mL aliquots of the filtrate taken in stoppered tubes, 1.0 mL of ninhydrin solution (2.0% w/v) was added and solution heated on a water bath at a temperature of 70-75°C for 20 minutes. Solutions were cooled to room temperature and the absorbance values noted in triplicate at 402.6 nm against reagent blank.
Declarations
Authors’ Affiliations
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