Development and Method Validation of RP-HPLC For Simultaneous Determination of Pregabalin and Methylcobalamin in Pure and Pharmaceutical Dosage Form

 

Sreekanth.D¹, P. Ramya¹, Vishwanadham.Y²,Vanitha. R³

¹Department of Pharmaceutical Analysis, Palamuru University, Mahaboobnagar, TS, India.

²Department of Pharmaceutical Chemistry, Vishnu institute of Pharmaceutical education &Research(VIPER), Narsapur, Medak-TS India.

³Department of Pharmaceutics, TRR college of Pharmacy, Hyderabad.

*Corresponding Author E-mail: vishwanadham.y@gmail.com

To develop a simple, sensitive, precise and accurate RP-HPLC method was developed for the quantitative estimation of Pregabalin and Methylcobalamin in bulk drug and the most common from delivery of drugs oral, sublingual, injection pharmaceutical dosage forms. This method was Pregabalin and Methylcobalamin was freely soluble in ethanol, methanol and sparingly soluble in water. The %RSD values were within 2 and the method was found to be precise. The results expressed in Tables for RP-HPLC method was promising. The RP-HPLC method is more sensitive, accurate and precise compared to the Spectrophotometric methods. This method can be used for the routine determination of Pregabalin and Methylcobalamin in bulk drug and in Pharmaceutical dosage forms.

 

 

 

INTRODUCTION:

Pregabalin, marketed under the brand name Lyrica among others, is a medication used to treat epilepsy, neuropathic pain, fibromyalgia, and generalized anxiety disorder. Its use for epilepsy is as an add-on therapy for partial seizures with or without secondary generalization in adults. Some off-label uses of pregabalin include restless leg syndrome, prevention of migraines, social anxiety disorder, and alcohol withdrawal. When used before surgery it does not appear to affect pain after surgery but may decrease the use of opioids^[1].

 

Pregabalin chemically known as (3S)-3-(aminomethyl)-5-methylhexanoic acid is an antiepileptic, anticonvulsant and neurotransmitter. This drug produces its actions by binding to the alpha2-delta (α2δ) subunit of the voltage-gated calcium channels. It is freely soluble in water both in acid and basic aqueous solution. It is well absorbed after oral administration and largely excreted by renal excretion ^[2]. Pregabalin is the 3-isobutylsubstituted analogue of _-amino butyric acid (GABA) but is inactive at GABA receptors ^[3]. The pharmacological activities of pregabalin result from its binding to the alpha-2-delta-2 protein, an auxiliary protein associated with voltage-gated calcium channels in the central nervous system ^[4]. It is considered to have a low potential for abuse, and a limited dependence liability if misused, and is thus classified as a Schedule V drug in the U.S^[5]. Although various bio-analytical methods for estimation of pregabalin in human serum and spectrophotometric methods for estimation of pregabalin in dosage form^{[6, 7]}. And HPLC methods for development and validation of pregabalin in capsules have been reported in the literature. ^[8,9]. The results of analysis were validated using International Conference on Harmonization (ICH) guidelines. The present work describes a simple, precise and accurate RP HPLC method for estimation of pregabalin in commercial dosage form. The main objective of this study was to develop a fast, sensitive, robust and cheap method to validate pregabalin using isotropically labeled internal standard (ISTD), in order to extend its application to assess the bioequivalence of two lamotrigine 50 mg tablet formulations in healthy volunteers. Methylcobalamin is a form of vitamin B12 used in the treatment of trigeminal neuralgia, megaloplastic anemia, diabetic neuropathy and facial paralysis in Bell’s pasly syndrome. It is chemically Coα-[α-(5,6-dimethylbenz-1H-imidazolyl)]-Coβmethylcobamide^[10-15].

 

PREGABALIN

Drug :  Pregabalin

Synonym:(S)-3-Isobutyl gaba, CI-1008, Lyrica

Drug category: Analgesics, Anticonvulsant

Structure  :

 

IUPAC Name: (S)-(+)-4-Amino-3-(2-methylpropyl) butanoic acid

Molecular Formula:C₈H₁₇NO₂

Molecular Weight: 159.23 g.mol−1

 

PHYSICOCHEMICAL PROPERTIES:

Description (Physical State): white to off-white, crystalline solid

Solubility: freely soluble in water and both basic and acidic aqueous solutions    

Storage Conditions: Store at 25°C (77°F)

 

METHYLCOBALAMIN

Drug :  Methylcobalamin

Synonym: Mecobalamin

Structure:

 

Molecular Formula: C₆₃H₉₁CoN₁₃O₁₄P

Molecular Weight: 1344.4gm/mole.

Functions: This vitamer is one of two active coenzymes used by vitamin B12-dependent enzymes and is the specific vitamin B12 form used by 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), also known as methionine synthase.

 

EXPERIMENTAL WORK:

HPLC METHOD DEVELOPMENT:

Preparation of standard solution:

Accurately weigh and transfer 10 mg of Pregabalin and Methylcobalamin working standard into a 10ml of clean dry volumetric flasks add about 7ml of Methanol and sonicate to dissolve and removal of air completely and make volume up to the mark with the same Methanol. Further pipette 0.15ml of the above Pregabalin and 1.5ml of Methylcobalamin stock solutions into a 10ml volumetric flask and dilute up to the mark with Methanol.

 

Procedure:

Inject the samples by changing the chromatographic conditions and record the chromatograms, note the conditions of proper peak elution for performing validation parameters as per ICH guidelines.

 

Mobile Phase Optimization:

Initially the mobile phase tried was Methanol: Water and Water: Acetonitrile and Methanol: TEA Buffer: ACN with varying proportions. Finally, the mobile phase was optimized to Methanol: TEA Buffer: CAN in proportion 65:15:20 v/v respectively. 

 

Optimization of Column:

The method was performed with various columns like C18 column, Symmetry and Zodiac column. X-Terra C18 (4.6×150mm, 5µ) was found to be ideal as it gave good peak shape and resolution at 1ml/min flow.

 

OPTIMIZED CHROMATOGRAPHIC CONDITIONS:

Instrument used   : Waters HPLC with auto sampler and PDA Detector 996 model.

Temperature:         Ambient

Column: X-Terra C18 (4.6×150mm, 5µ)

Buffer:    Dissolve 1.5ml of triethyl amine in 250 ml HPLC water and adjust the pH 4.5. Filter and sonicate the solution by vacuum filtration and ultra-sonication.

pH: 4.5

Mobile phase: Methanol: TEA buffer: ACN (65:15:20 v/v)

Flow rate: 1ml/min

Wavelength: 212 nm

Injection volume  : 10 ml

Run time: 10 min

 

VALIDATION

PREPARATION OF BUFFER AND MOBILE PHASE:

Preparation of Triethylamine (TEA) buffer (pH-4.5):

Dissolve 1.5ml of Triethylamine in 250 ml HPLC water and adjust the p^H 4.5. Filter and sonicate the solution by vacuum filtration and ultra-sonication.

 

Preparation of mobile phase:

Accurately measured 650 ml (65%) of Methanol, 150 ml of Triethylamine buffer (15%) and 200 ml of Acetonitrile (20%) were mixed and degassed in digital ultrasonicater for 10 minutes and then filtered through 0.45 µ filter under vacuum filtration.

 

Diluent Preparation:

The Mobile phase was used as the diluent.

 

VALIDATION PARAMETERS SYSTEM SUITABILITY:

Accurately weigh and transfer 10 mg of Pregabalin and 10mg of Methylcobalamin working standard into a 10ml of clean dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution) Further pipette 0.15ml of the above Pregabalin and 1.5ml of Methylcobalamin stock solutions into a 10ml volumetric flask and dilute up to the mark with Diluent.

 

Procedure:

The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits.

 

SPECIFICITY STUDY OF DRUG:

Preparation of Standard Solution:

Accurately weigh and transfer 10 mg of Pregabalin and 10mg of Methylcobalamin working standard into a 10ml of clean dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solutions) further pipette 0.15ml of the above Pregabalin and 1.5ml of Methylcobalamin stock solutions into a 10ml volumetric flask and dilute up to the mark with Diluent.

 

Preparation of Sample Solution:

Take average weight of one Tablet and crush in a mortar by using pestle and weight 10 mg equivalent weight of Pregabalin and Methylcobalamin sample into a 10mL clean dry volumetric flask and add about 7mL of Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent.

Further pipette 1.5ml of above stock solution into a 10ml volumetric flask and dilute up to the mark with Diluent.

 

Procedure:

Inject the three replicate injections of standard and sample solutions and calculate the assay by using formula:

 

%ASSAY =

 

Sample area        Weight of standard       Dilution of sample      Purity        Weight of tablet

 ___________ ×   ________________ × _______________×_______×______________×100

  Standard area      Dilution of standard     Weight of sample        100            Label claim

 

 

PREPARATION OF DRUG SOLUTIONS FOR LINEARITY:

Accurately weigh and transfer 10 mg of Pregabalinand 10mg of Methylcobalamin working standard into a 10ml of clean dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

 

PRECISION REPEATABILITY

Preparation of Pregabalin and Methylcobalamin Product Solution for Precision:

Accurately weigh and transfer 10 mg of Pregabalinand 10mg of Methylcobalamin working standard into a 10ml of clean dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)

 

Further pipette 0.15ml of the above Pregabalin and 1.5ml of Methylcobalamin stock solutions into a 10ml volumetric flask and dilute up to the mark with Diluent.

The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits.

 

INTERMEDIATE PRECISION:

To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision was performed on different days by maintaining same conditions. 

 

Procedure:

DAY 1:

The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six replicate injections was found to be within the specified limits.

 

DAY 2:

The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six replicate injections was found to be within the specified limits.

 

ACCURACY:

For preparation of 50% Standard stock solution:

Accurately weigh and transfer 10 mg of Pregabalinand 10mg of Methylcobalamin working standard into a 10ml of clean dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)Further pipette 0.075ml of the above Pregabalin and 0.75ml of Methylcobalamin stock solutions into a 10ml volumetric flask and dilute up to the mark with Diluent.

 

For preparation of 100% Standard stock solution:

Accurately weigh and transfer 10 mg of Pregabalinand 10mg of Methylcobalamin working standard into a 10ml of clean dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)Further pipette 0.15ml of the above Pregabalin and 1.5ml of Methylcobalamin stock solutions into a 10ml volumetric flask and dilute up to the mark with Diluent.

 

For preparation of 150% Standard stock solution:

Accurately weigh and transfer 10 mg of Pregabalinand 10mg of Methylcobalamin working standard into a 10ml of clean dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)Further pipette 0.225ml of the above Pregabalin and 2.25ml of Methylcobalamin stock solutions into a 10ml volumetric flask and dilute up to the mark with Diluent.

 

Procedure:

Inject the Three replicate injections of individual concentrations (50%, 100%, 150%) were made under the optimized conditions. Recorded the chromatograms and measured the peak responses. Calculate the Amount found and Amount added for Pregabalinand Methylcobalaminand calculate the individual recovery and mean recovery values.

 

ROBUSTNESS:

The analysis was performed in different conditions to find the variability of test results. The following conditions are checked for variation of results.

 

For preparation of Standard solution:

Accurately weigh and transfer 10 mg of Pregabalinand 10mg of Methylcobalamin working standard into a 10ml of clean dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution)Further pipette 0.15ml of the above Pregabalin and 1.5ml of Methylcobalamin stock solutions into a 10ml volumetric flask and dilute up to the mark with Diluent.

 

Effect of Variation of flow conditions:

The sample was analyzed at 0.9 ml/min and 1.1 ml/min instead of 1ml/min, remaining conditions are same. 10µl of the above sample was injected and chromatograms were recorded

 

Effect of Variation of mobile phase organic composition:

The sample was analyzed by variation of mobile phase i.e. Methanol: TEA Buffer: Acetonitrilewas taken in the ratio and 70:5:25, 60:30:10 instead (65:15:20), remaining conditions are same. 10µl of the above sample was injected and chromatograms were recorded.

 

RESULTS AND DISCUSSION:

Optimized Chromatogram (Standard)

Mobile phase: Methanol: TEA Buffer pH 4.5: Acetonitrile (65:15:20)                                  

Column: X-Terra C18 (4.6×150mm, 5.0 µm)

Flow rate : 1 ml/min

Wavelength: 212 nm

Column temp: Ambient

Injection Volume: 10 µl

Run time: 10 minutes

 

Figure No. 1:Optimized Chromatogram

 

Observation: From the above chromatogram it was observed that the Pregabalin and Methylcobalamin peaks are well separated and they shows proper retention time, resolution, peak tail and plate count. So it’s optimized trial.

 

 

 

 

Table No.1: - Peak results for optimized

 

 

Optimized Chromatogram (Sample)

 

 

Figure No.2: Optimized Chromatogram (Sample)

 

Table No.2: Optimized Chromatogram (Sample)

 

Acceptance criteria:

·       Resolution between two drugs must be not less than 2

·       Theoretical plates must be not less than 2000

·       Tailing factor must be not less than 0.9 and not more than 2.

·       It was found from above data that all the system suitability parameters for developed method were within the limit.

 

SPECIFICITY

The ICH documents define specificity as the ability to assess unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products, and matrix components.

 

Analytical method was tested for specificity to measure accurately quantitate Pregabalin and Methylcobalamin in drug product. 

 

Assay (Standard):               

 

Figure No. 3: Chromatogram showing assay of standard injection -1

 

Figure No..4: Chromatogram showing assay of standard injection -2

 

Figure No.5: Chromatogram showing assay of standard injection -3

 

Table No.3: Peak results for assay standard

 

 

Assay (Sample):

 

Figure No.6: Chromatogram showing assay of sample injection-1

 

Figure No..7. Chromatogram showing assay of sample injection-2

 

 

Figure No.8: Chromatogram showing assay of sample injection-3

 

Peak results for Assay sample Table No.4: Peak results for Assay sample

 

%ASSAY =

  Sample area        Weight of standard     Dilution of sample     Purity      Weight of tablet

 ___________ ×   ________________ × _______________×_______×______________×100

  Standard area      Dilution of standard    Weight of sample       100          Label claim

 

=355917/352888.7×10/150×150/0.0149×99.7/100×1.2360/825×100

=100.5%

The % purity of Pregabalin and Methylcobalamin in pharmaceutical dosage form was found to be100.5%.

 

CHROMATOGRAPHIC DATA FOR LINEARITY STUDY: 

Results of linearity Pregabalin:     

Table No.5: Results of linearity Pregabalin:

Figure No.  9: calibration graph for Pregabalin

 

LINEARITY PLOT:

The plot of Concentration (x) versus the Average Peak Area (y) data of Pregabalin is a straight line.

Y = mx + c

Slope (m) = 24679

Intercept (c) = 3242

Correlation Coefficient (r)   =   0.999

 

VALIDATION CRITERIA:

The response linearity is verified if the Correlation Coefficient is 0.99 or greater.

 

CONCLUSION:

Correlation Coefficient (r) is 0.99, and the intercept is 3242. These   values meet the validation criteria.

 

Results of linearity Methylcobalamin

Table No.6:Results of linearity Methylcobalamin

 

Figure No. 10: calibration graph for Methylcobalamin

 

ACCURACY:

Accuracy at different concentrations (50%, 100%, and 150%) were prepared and the % recovery was calculated.

 

Table No.7: The accuracy results for Pregabalin

%Concentration (at specification Level)	Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	192446.6	7.5	7.5	100	99.7%
100%	374222	15	14.98	99.8
150%	555891.3	22.5	22.49	99.5

 

Table No.8: The accuracy results for Methylcobalamin

%Concentration (at specification Level)	Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	2001752	75	74.87	99.8	99.9%
100%	3927797	150	149.9	99.9
150%	5858665	225	224.9	99.9

 

Acceptance Criteria:

·                     The percentage recovery was found to be within the limit (98-102%).

·                     The results obtained for recovery at 50%, 100%, 150% are within the limits. Hence method is accurate.

 

LIMIT OF DETECTION:

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

                    LOD= 3.3 × σ / s

Where 

σ = Standard deviation of the response   

S = Slope of the calibration curve

 

Result:

Pregabalin:

=3.3 × 5088.675424/24679

=0.68µg/ml

 

Methylcobalamin:

=3.3 × 84406.48871/25807

=10.7µg/ml

 

LIMIT OF QUANTITATION

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

 

                                            LOQ=10×σ/S

Where 

σ = Standard deviation of the response   

S = Slope of the calibration curve

Result:

Pregabalin:

=10×5088.675424/24679

= 1.9µg/ml

Methylcobalamin:

=10 × 84406.48871/25807

= 32.7µg/ml

 

Robustness

The robustness was performed for the flow rate variations from 0.9 ml/min to 1.1ml/min and mobile phase ratio variation from more organic phase to less organic phase ratio for Pregabalin and Methylcobalamin. The method is robust only in less flow condition and the method is robust even by change in the Mobile phase ±5%. The standard and samples of Pregabalin and Methylcobalamin were injected by changing the conditions of chromatography. There was no significant change in the parameters like resolution, tailing factor, asymmetric factor, and plate count.

 

 

Results for Robustness Pregabalin:

Table No.9: Results for Robustness Pregabalin

 

The tailing factor should be less than 2.0 and the number of theoretical plates (N) should be more than 2000.

 

Results for Robustness Methylcobalamin

 

Table.No.10: Results for Robustness Methylcobalamin

 

 

Acceptance criteria:

The tailing factor should be less than 2.0 and the number of theoretical plates (N) should be more than 2000.

 

The analytical method was developed by studying different parameters.First of all, maximum absorbance was found to be at 212 nm and the peak purity was excellent. Injection volume was selected to be 10µl which gave a good peak area. The column used for study was X-Terra C₁₈because it was giving good peak.Ambient temperature was found to be suitable for the nature of drug solution. The flow rate was fixed at 1.0ml/min because of good peak area and satisfactory retention time. Mobile phase is Methanol: TEA Buffer pH 4.5: Acetonitrile (65:15:20) was fixed due to good symmetrical peak. So this mobile phase was used for the proposed study.

 

Run time was selected to be 10 min because analyze gave peak around 2.090, 5.289 ±0.02min respectively and also to reduce the total run time.The percent recovery was found to be 98.0-102 was linear and precise over the same range. Both system and method precision was found to be accurate and well within range. The analytical method was found linearity over the range 5-25ppm of Pregabalin and 50-250ppm of Methylcobalamin of the target concentration. The analytical passed both robustness and ruggedness tests. On both cases, relative standard deviation was well satisfactory.

 

CONCLUSION:

In the present investigation, a simple, sensitive, precise and accurate RP-HPLC method was developed for the quantitative estimation of Pregabalin and Methylcobalamin in bulk drug and pharmaceutical dosage forms.

This method was simple, since diluted samples are directly used without any preliminary chemical derivatisation or purification steps.

 

Pregabalin and Methylcobalamin was freely soluble in ethanol, methanol and sparingly soluble in water.

 

Methanol: TEA Buffer pH 4.5: Acetonitrile (65:15:20) was chosen as the mobile phase. The solvent system used in this method was economical. The %RSD values were within 2 and the method was found to be precise. The results expressed inTablesfor RP-HPLC method was promising. The RP-HPLC method is more sensitive, accurate and precise compared to the Spectrophotometric methods.

 

This method can be used for the routine determination of Pregabalin and Methylcobalamin in bulk drug and in Pharmaceutical dosage forms.

 

REFERENCES:

1.   Vishwanadham Yerragunta, and Gali, Haritha. "Development and validation of RP-HPLC method for simultaneous estimation of naproxen and esomeprazole in pharmaceutical dosage form." Asian Journal of Research in Chemistry 9.8 (2016): 366-368.

2.   Indian Pharmacopeia, vol III, six ed., Ghaziabad, 2010.

3.   Yerragunta, Vishwanadham, et al. "Pyrimidine and Its Biological Activity: A Review."  Pharma Tutor 1.2 (2013): 39-44.

4.   Sandhya Mandadi, Vishwanadham Yerragunta, formulation and evaluation of atorvastatin calcium sustained release tablets., Int J Pharm 2016; 6(3): 124-130.

5.   Vishwanadham Yerragunta, Divakar Kanakagiri , A. Hari Prakash ,  formulation and   evaluation of sustained release dosage form of anti - diabetic drug: glimepiride., WJPRT 2016; 4(5):11-18

6.   Gujral RS., Manirul Haque SK and Shanker P, et al. A Sensitive Spectrophotometric Method for the Determination of Pregabalin in Bulk, Pharmaceutical Formulations and in Human Urine Samples, Int J Biomed Sci., 2009, 5(4): 421-427.

7.   Bali A, Gaur P. A novel method for spectrophotometric determination of pregabalin in pure form and in capsules. Chemistry Central Journal, 2011, 5: 1-7.

8.   Kasawar GB, Farooqui MN, et al. Development and Validation of HPLC Method for the Determination of Pregabalin in Capsules. Indian J Pharm Sci. 2010, 72(4):517–519.

9.   Thacker PS, Patel D. RP-HPLC method development and validation for eslicarbazepine  acetate in api. IJARPB, 2012; 1 (2) : 84-94.

10. Saravanan J, Shajan A, Joshi NH, Varatharajan R, Valliappan K. A simple and validated RP-HPLC method for the estimation of Methylcobalamin in bulk and capsule dosage form.  International Journal of Chemical and Pharmaceutical Sciences 2010;1(2):13-16.

11. Beckette AH, Stenlake JB. Practical Pharmaceutical Chemistry. 4thEdn.New Delhi: CBS Publishers; 1997, 275.

12 .          British Pharmacopeia. British Pharmacopeial Commission. Vol III, London: 2004, 2334.

13. Bentley AO, Driver JE. Textbook of Pharmaceutical Chemistry.

14. Sethi PD. HPLC -Quantitative Analysis of Pharmaceutical formulations 1stEdn. New Delhi: CBS Publishers and Distributors;1985, 212 and 879.

15. HiguchiT. Pharmaceutical Analysis. 1sted. New Delhi: CBS Publishers and Distributors; 2004, 671.

16.           Vanitha Rapaka, G. Haritha, D. Sreekanth, Y. Vishwanadham, Int J Pharm 2017; 7(2):138-144.

 

 

 

 

 

Received on 01.06.2017         Modified on 20.06.2017

Accepted on 11.07.2017         © AJRC All right reserved