INTRODUCTION:

Pregabalin (PRG), (S)-3-(amino methyl)-5-methylhexanoic acid (Figure 1), It is a white crystalline solid, which is soluble in water and in both basic and acidic aqueous solutions and used as anticonvulsant, analgesic medication and neurotransmitter. It is an antiepileptic and structurally related to the inhibitory neurotransmitter aminobutyric acid (GABA)^[1]. It was recently approved for adjunctive treatment of partial seizures in adults in United States and Europe and for the treatment of neuropathic pain from post therapeutic neuralgia and diabetic neuropathy.

Recent studies have shown that pregabalin is effective at treating chronic pain in disorders such as fibromyalgia and spinal card injury. It is considered to have a low potential for abuse, and a limited dependence liability if missed, and is thus classified as a schedule V drug in the U.S. For analysis of pregabalin various methods like HPLC, LC-MS and UV spectrophotometric methods have been reported so far for determination of pregabalin alone and its combination with other drugs^[2]. However, HPLC and LC-MS equipment are expensive. Moreover, these techniques may suffer from some other disadvantages such as high maintenance and acquisition cost, time-consuming analysis, requirement for sample pre-treatment, and in some cases low sensitivity. That is why these methods are time consuming and tedious. Further, most of the reported spectrometric methods do not involve the derivatizing agent.^{[3],[4],[5],[6]}Therefore, in present work a new simple spectrophotometric method was proposed by using derivatizing agent for rapid analysis of pregabalin.

Figure 1: Chemical structure of pregabalin

MATERIALS AND METHODS:

Instruments:

All UV spectrophotometric measurements were carried out using Shimadzu 1600 UV spectrophotometer (Japan) equipped with deuterium and tungsten lamp, photomultiplier tube detector and 1cm quartz cell. Slit width for both monochromators were set at 1.5nm. The spectrometer is connected to a PC computer loaded with the UV Probe software.

Reagents and Chemicals:

All the solvents were of analytical-reagent grade, and purified whenever necessary. Pregabalin was generously supplied by Sun Pharmaceuticals, Mumbai. Tablets containing pregabalin was purchased from the local market.

Preparation of Standard Solutions:

Accurately weighed 15 mg of powered drug in 100 ml of volumetric flask. Add 17 mg of derivatizing agent to it. Further add 50 ml of Methanol. Heat the solution in water bath at 40° C for 10 min. Finally, volume in the flask was made up to the mark by using Methanol.^[7]

Preparation of Sample Solution:

Twenty tablets were weighed, finely powdered and mixed thoroughly. An accurately weighed amount of the powder equivalent to 10 mg of drug was transferred into a 100 mL volumetric flask, dissolved in about 10 mL methanol and resultant mixture was allowed to sonication for 15 minutes, diluted to the mark with methanol. Mixed well and filtered; the first portion of the filtrate was rejected. Further dilutions with the same solvent were made to obtain sample solution containing the specified concentration for each drug as mentioned under the preparation of standard solutions.

General Analytical Procedure:

Absorbance of all solutions prepared is recorded in following manner. About 4 mL of each solution was transferred to quart cell and absorbance of each solution was recorded at 226nm.

Linearity:

To assess linearity of analytical method, series of standard solutions of drug exhibiting concentration 2-20 µgmL^-1 were prepared in separate 10 mL volumetric flask by diluting suitable volume of stock solution using methanol as solvent. Absorbance of all solutions was recorded at wavelength of maximum absorbance 226 using methanol as blank. (Table 1)

Table 1: Linearity data for the developed method

Sr.	Concentration (µg mL^-1)	Absorbance
1	1	0.529
2	2	0.530
3	3	0.789
4	4	0.870
5	5	1.12
6	6	1.38
7	7	1.56
8	8	1.75
9	9	1.99
10	10	2.299
11	11	2.232
12	12	2.368
13	13	2.568
14	14	2.750
15	15	2.87
16	16	3.12
17	17	3.450
18	18	3.60
19	19	3.75
20	20	3.87

Accuracy:

Accuracy of method was done by spiking known amount of drug in to sample solutions and recovery study was done. To ensure the accuracy, known amounts of pure drug (80%, 100%, and 120%) were added to the sample solution and these samples were reanalyzed by the proposed method and also % recovery was determined. (Table 2)

Table 2: Results of accuracy data obtained from recovery study

% Recovery Level	Conc. Added (µg mL^-1)	Conc. Found (µg mL^-1)	Recovery (%)	RSD (%)*
80% of test conc.	1.36	3.06	75.72	0.559
100% of test conc.	1.70	3.4	95.75	0.472
120% of test conc.	2.04	3.7	106.41	0.336

Table 3: The intra-day and inter-day precision study of the developed method confirmed adequate sample stability and method reliability where all the Relative Standard Deviation were below 2%

Sr. No	Intra-day precision		Inter-day precision
Sr. No	Concentration (µg mL^-1)	Absorbance	Concentration (µg mL¹)	Absorbance
1	3.06	1.401	3.06	1.400
2	3.06	1.400	3.06	1.402
3	3.06	1.407	3.06	1.407
4	306	1.418	3.06	1.400
5	3.06	1.402	3.06	1.415
6	3.06	1.418	3.06	1.400
7	3.06	1.405	3.06	1.407
8	Mean	1.407	Mean	1.404
9	SD	0.50428	SD	0.3673
10	%RSD	0.640	%RSD	0.468

Precision:

Precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision was assessed inter and intra-day by recording absorbance of six different drug solutions of concentration 20 µgmL^-1. (Table 3)

Ruggedness:

Analysis of an homogeneous sample in different laboratories, by different analysts, under prevalent environmental conditions using the specified parameters. (Table 4)

Table 4: Ruggedness data for the develop method

Sr. No	Concentration (µg mL^-1)	Absorbance
1	3.4	0.900	0.908
2	3.4	0.908	0.915
3	3.4	0.902	0.905
4	3.4	0.905	0.908
5	3.4	0.900	0.915
6	3.4	0.905	0.910
7	3.4	0.910	0.903
8	Mean	0.9042	0.909
9	SD	0.6584	0.6814
10	%RSD	0.67	0.69

Table 5: Robustness data for the develop method

Sr No	Concentration (µg mL^-1)	Absorbance
1	3.7	1.780	1.782
2	3.7	1.782	1.784
3	3.7	1.785	1.787
4	3.7	1.780	1.781
5	3.7	1.782	1.783
6	3.7	1.781	1.785
7	3.7	1.783	1.786
8	Mean	1.781	1.784
9	SD	0.118659	0.1618
10	%RSD	0.11	0.15

Robustness:

The ability to remain unaffected by small but deliberate variations in method parameters- evaluates reliability. Provides an indication of the reliability of the method during normal usage. Analysis of an homogeneous sample in different laboratories, by different analysts, under prevalent environmental conditions using the specified parameters. In order to check ruggedness of the method six different standard solutions of analyte were prepared of concentration of 20 µgmL^-1followed by recording of absorbance by two different analyst. SD and RSD were determined.^[8](Table 5)

RESULTS AND DISCUSSION:

A novel UV spectrophotometric method for analysis of Pregabalin is developed by using least harmful solvent Methanol for routine analysis. The given method is found to be linear in the concentration range of 2-20 µgmL^-1. Standard calibration curve was obtained by plotting the absorbance against its concentration measured at wavelength 226 nm. The regression coefficient was found to be 0.9963 and slope was found to be 0.1847 (Table1).

Accuracy was done by recovery method at 80, 100 and 120 per- cent level. It has been observed that recoveries of 99.36, 99.208 and 100.23% were observed at 80, 100 and 120% level respectively. Furthermore % RSD of 0.559,0.472 and 0.336 which are less than 2.

CONCLUSION:

Pregabalin is antiepileptic drug of class gabapentenoid class. As drug has shown very less absorbance at its reported wavelength 210nm by UV spectroscopy. Hence in the present work derivatising agent was used to increase the absorbance for the development of UV spectrophotometric method for determination of pregabalin. The developed method is validated according ICH guidelines, method is fast, accurate, precise and robust. Present method can be therefore used for routine analysis of pregabalin. Method involves no tedious sample preparation requirements and hence it is simple and fast.

ACKNOWLEDGEMENTS:

Authors acknowledge sincere thanks to management Mumbai Educational Trust’s Institute of Pharmacy, Bhujbal Knowledge City, Nashik (MS) India for making available required facilities to complete present work. Authors are thankful to Sun Pharmaceuticals, Mumbai for providing pregabalin gift sample. Tablets containing pregabalin was purchased from the local market.

REFERENCE:

1. Indian Pharmacopoeia, 2010, volume3, published by Indian Pharmacopoeia Commission, Ghaziabad, Government of India Ministry of Health and Family Welfare, pg. no 2242.

2. Jampala B, Bandi R and Naidu NVS. Analytical RP-HPLC method for development and validation of pregabalin in bulk and the determination of pregabalin in capsule dosage form. International Journal of Innovative Research in Science (Engineering and Technology, 3(4); 2014:11094-11098.

3. Pawar PY, Zanje LS, Tambe SS, Nandgaonkar AU, Funde PV and Vyavhare AA. Simultaneous estimation of pregabalin and paracetamol by UV spectroscopic method in bulk and tablet formulation. World Journal of Pharmacy and Pharmaceutical Sciences,3(4);2014:743-750.

4. Bhatt KK, Patelia EM and Mori A. Simultaneous estimation of pregabalin and methylcobalamine in pharmaceutical formulation by RP-HPLC method. Journal of Analytical and Bioanalytical Techniques.4(1);2013:1-4.

5. Karavadi TM, Challa BR. Bioanalytical method development and validation of pregabalin in rat plasma by solid phase extraction with HPLC-MS/MS: application to a pharmacokinetic study. Journal of Liquid Chromatography and Related Technologies.37; 2014:130-144.

6. Sowjanya P. RP HPLC method development of pregabalin in bulk, dosage form and validation parameters. Journal of Hospital and Clinical Pharmacy. 2(4); 2016:61-75.

7. Vogels. Textbook of Practical Organic Chemistry. Furniss et al, India, 2006.

8. ICH Harmonized Tripartite Guidelines, Validation of analytical procedure: Text and Methodology Q2(R1), 4th version, Parent Guidelines dated 27 oct 1994.

Received on 10.01.2019 Accepted on 20.02.2019

Asian J. Pharm. Ana. 2019; 9(1):15-18.

DOI: 10.5958/2231-5675.2019.00005.X