A novel method for spectrophotometric determination of pregabalin in pure form and in capsules

Method

In our efforts to design a novel spectrophotometric method for quantification of pregabalin, we investigated its derivatization with ninhydrin ((triketohydrindene hydrate) for generation of a chromophoric product. Figure 3 shows the UV/visible spectrum of the chromophoric derivative. The procedure involves formation of purple colored product by reaction of pregabalin with ninhydrin by heating at a temperature of 70-75°C for 20 minutes. Reaction of ninhydrin 1 with amines, alpha amino acids, peptides, and proteins yields an aldehyde with one carbon atom less than the alpha-amino acid; and carbon dioxide in stoichlometric amounts and varying amounts of ammonia, hydrindantin and a chromophoric compound known as Ruhemann's Purple (2-(3-hydroxy-1-oxo-1H-inden-2-ylimino)-2H-indene-1,3-dione). This pigment serves as the basis of detection and quantitative estimation of alpha-amino acids.¹³ Mechanism proposed (Figure 4) for the reaction involves removal of a water molecule from ninhydrin hydrate 1 to generate 1,2,3-indantrione 2 in the first step, which then, forms a Schiff's base with the amino group of pregabalin resulting in the ketimine 3. Removal of the aldehyde RCHO generates an intermediate amine 4 (2-amino-l,3-indandione). Condensation of this intermediate amine with another molecule of ninhydrin follows to form the expected chromophore 5 (Ruhemann's Purple). The rate-determining step in the entire sequence of the ninhydrin reaction is the nucleophilic-type displacement of a hydroxy group of ninhydrin hydrate by a non-protonated amino group.

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

Linearity and range

The regression plots showed compliance with Beer Lambert's law (linearity) in the concentration range of 50 μg/mL-1000 μg/mL with a correlation coefficient (r²) of 0.992. The standard plot is given in Figure 5. Table 1 summarizes the performance data and statistical parameters for the proposed method including concentration ranges, linear regression equation, correlation coefficient, molar absorptivity, Sandell sensitivity limit and these indicate a good linearity over the working concentration ranges.

Precision

Accuracy

Where

C_t is the total drug concentration measured after standard addition;

C_i drug concentration in the formulation sample;

C_a, drug concentration added.

The percentage relative error was calculated as: E_r % = [(found - added)/added] × 100

Interference

Satisfactory values of the mean recovery values ± SD, RSD % and E_r % 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)

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.

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.

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 λ_max of the drug (402.6 nm) against corresponding concentration values and compliance with Beer Lambert's law was assessed.

Analysis of pharmaceutical formulation