Development and validation of colorimetric method for the determination of pregabalin in bulk and pharmaceutical formulations

Areej Abdelmonim Mohamed*1,2, Tilal Elsaman3, Mohammed E Adam1, Elrasheed A Gadkariem1, Shaza W Shantier1 1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Khartoum, Khartoum, Sudan 2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Sudan University of Science and Technology, Khartoum, Sudan 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Omdurman Islamic University, Omdurman, Sudan


PGN (
) is an antiepileptic amino acid used for treatment of partial seizure and neuropathic pain. It is a GABA (gamma amino butyric acid) analog, works by binding to the voltage-gated Ca 2+ channels (Katzung et al., 2012). It has a simple aliphatic structure with no chromophore, hence exhibits weak UV absorbance making spectrophotometric determination of the drug somehow dif icult. Many spectrophotometric (Shep and Lahoti, 2013), (Patel et al., 2016) spectro luorimetric (Derayea et al., 2018), luorometric (Yoshikawa et al., 2016) and chromatographic (HPLC) (Akther et al., 2015), (A and P, 2016), GC-MS (Tafesse et al., 2018), UPLC (Sravanthi and Madhavi, 2019) methods were reported for determination of PGN, alone and in combination with other drugs. Ascorbic acid (vitamin C) is a naturally occurring antioxidant. It has been widely used as an analytical reagent in pharmaceutical analysis. It is known to have a selective reaction with ammonia and primary aliphatic amines of the type R-CH 2 -NH 2 (λ max 390 and 530 nm) (Pesez and Bartos, 1974). This reagent was reported to be used in the determination of various drugs including lisinopril (Rahman et al., 2005), paromomycin (Adam et al., 2016b), gabapentin (Adam et al., 2016a), and tranexamic acid (Gadkariem et al., 2013). Most of the reported methods for PGN analysis need highly trained personnel, expensive or unavailable equipment or reagents. Based on these reports, simple, precise, accurate and inexpensive colorimetric method was developed for the determination of PGN in bulk and capsule forms using ascorbic acid as a chromogen.

MATERIALS AND METHODS
All materials and reagents used were of analytical grade. PGN authentic standard (M.wt 159.2 and 99% claimed purity) was kindly provided by Pharmaland Pharmaceuticals, Sudan. Sample Z of PGN capsules (150mg) was obtained from the local market. Ascorbic acid, Dimethylsulfoxide (DMSO) and Dimethylformamide (DMF) were purchased from S.D Fine chem. Limited, India. Spectrophotometric studies were carried out on Shimadzu UV-1800ENG240V, Kyoto, Japan.

Preparation of stock solutions
Standard stock solution PGN standard (100mg) was accurately weighed and transferred into a 10 ml volumetric lask. 7 ml of distilled water were added and the solution was sonicated for 30 min. to ensure dissolution. The volume was then completed to the mark with distilled water (Solution A; 1%w/v; 10mg/ml).

Sample stock solution
Out of 20 capsules of sample Z, an amount equivalent to 100mg of PGN was accurately weighed and treated as PGN standard (Solutions B; 1%w/v; 10mg/ml).

Ascorbic acid solution
Fifty milligrams of ascorbic acid was accurately weighed and transferred into 25 ml volumetric lask. About 20 ml of DMSO were added and the solution was shaken for 5 minutes to ensure dissolution. The volume was completed to the mark with DMSO and mixed well (0.2%w/v).

Reagent blank
About 100 µl of distilled water were transferred into a 10 ml volumetric lask. 1 ml of freshly prepared 2% w/v ascorbic acid solution was added and the volume was made up to the mark with DMSO.

Procedure Optimization
To determine the optimum solvent, one ml of ascorbic acid solution was transferred into a 10 ml volumetric lask containing 40 µl of solution A. the volume was made up to the mark with DMSO and heated for 30 min. then cooled and scanned against blank solutions. (The same experiment was carried out with DMF) In order to get the best heating time, one ml of ascorbic acid solution was added to a set of volumetric lasks containing 60 µl of solution A. The solutions were treated as above and heated in a boiling water bath for 10, 20, 30 & 40 min., respectively. After cooling the solutions were scanned against blank solutions.

Linearity
Serial volumes of solution A (5-30 µl) were transferred into a set of 10 ml volumetric lasks. Distilled water was added to each lask to obtain 100 µl. 1 ml of freshly prepared ascorbic acid solution (2%) solution was added to each lask and the volume was made up to the mark with DMSO. The solutions were transferred into stoppered glass test tubes and heated for 30 min. in a boiling water bath. After cooling to room temperature, the absorbance of each solution was measured against reagent blank at 390nm and 532nm. Absorbance values obtained were plotted against PGN concentration values and the calibration curves at 390nm and 532nm were constructed. Linearity was con irmed using Minitab 14 software to produce standardized residual versus itted values plot.

Assay
Serial dilutions of solutions B were treated as under calibration curve. The content of capsules was determined by direct sample/standard comparison.

Added recovery
Ten microlitre of solution A and B were transferred into two separate 10 ml volumetric lasks. Ten microlitre of solutions A and B were mixed in a third one and all the three were treated as under calibration curve (100% level). The process was repeated at 50% and 150% levels. Percent added recovery was calculated as follows:

Standard addition
Zero, 10, 20 and 30 µl of solution A were transferred into separate 10 ml volumetric lasks. 10 µl of solution B was added to each and treated as under calibration curve. The concentration of the irst solution (X) is then calculated by taking Y = zero in the calibration curve equation and represented as a percent of the added amount.
The molar ratio of the reaction was obtained from a plot of concentration ratio ([PGN]/ [ascorbic acid]) versus absorbance values.

RESULTS AND DISCUSSION
PGN is a simple aliphatic amino acid with weak UV absorbance (210nm) (Shep and Lahoti, 2013). This absorbance is highly susceptible to overlapping with most solvents as they absorb at the same UV region. Reaction with a suitable chromogen can produce a chromophore enabling the analysis of PGN by spectrophotometry. Ascorbic acid was found to react with PGN in DMSO, giving a purple-coloured complex with two λ max (390nm and 532nm) suggesting two transitions. Experimental factors affecting the colour development, intensity and stability were studied and optimized to produce the spectrum presented in Figure 2. DMSO was found to give better      peak shapes and more intense absorbance than DMF (Table 1).
Heating to 100 • C was found necessary for colour development. The most available maximum of absorbance was reached with heating for 30 minutes ( Table 2).
The highest absorbance levels were observed with 1 ml of 0.2% ascorbic acid solution (Table 3). The developed colour was found stable for more than 180 minutes after which the absorbance intensity decreased very slightly with passage of time.

Linearity
The constructed calibration curves (Figure 3) obeyed Beer's law over the concentration range 5−30 µg/ml. The linearity data was calculated at 95% con idence limit and summarized in Table 4. The two plots in Figure 4 shows a narrow range of magnitude difference between the standardized residuals and the itted values of data points. This re lects the closeness of actual points to the best-itted line and con irms the linearity of the regression curve. Additionally, the normal scattering of data points above and below the zero line means there is no systematic error.

Accuracy and precision
Accuracy of the developed method was tested by recovery percent and standard addition methods. The obtained results of the percent recovery of sample Z are summarized in Table 5. Standard addition test results were (93% and 96%; at 390nm and 532nm, respectively. The obtained results re lected the freedom from any interference.
The developed method was then applied for the assay of pharmaceutical formulation. Results are summarized in Table 6. Statistical comparison was conducted using the following formula: where x is the calculated mean, µ is the true mean and s is the standard deviation.
It was found that there are no signi icant differences as the calculated t value was less than the tabulated one (4.30, n=3).
The precision of the developed method was evaluated by three concentrations of PGN within the linearity range. The obtained RSD% values for the within-day and between-days determination were within the range of 0.82-2.04% and 0.14-1.77%; n=3, at 390nm and 532nm respectively. These low values (less than or equal to 2%) re lected the precision of the developed method.

Proposed reaction mechanism
Ascorbic acid is oxidized by heat into dehydroascorbic acid (DHA). This step is essential for the reaction to take place (Pesez and Bartos, 1974).
As proposed in Scheme 1, the reaction proceeds by nucleophilic addition. The electron lone pair of the amino group in PGN attacks the most electrophilic carbonyl carbon in ascorbic acid to give an imine which undergoes hydrolysis into an amine. The amine then couples with another molecule of dehydroascorbic acid to give the coloured complex.
This proposed mechanism is con irmed by the molar ratio results [drug: reagent] to be 1:2 ( Figure 5).

CONCLUSION
The developed method was proven to be simple, accurate and precise for the determination of PGN in bulk and dosage forms. Ascorbic acid is considered a suitable, cheap and available reagent for the analysis of PGN. The developed method can be used for the routine analysis of PGN. Comparison of the developed method with the of icial methods is highly recommended to further prove the accuracy. Veri ication of the chemical structures of the formed complexes may contribute to better understanding of the reaction conditions. It is recommended to modify the developed methods in order to apply them to analyze PGN in biological samples.