Development And Validation Of RP-HPLC Methods For Estimation of Pregabalin and Pyridoxine Hydrochloride in Synthetic Mixture

Pregabalin and Pyridoxine Hydrochloride are both pharmacologically significant agents used in the management of neurological conditions, especially those involving nerve damage or dysfunction. Their combination in a combination dosage form is designed to address the multifactorial nature of certain neurological disorders, providing a comprehensive treatment approach. The development of an analytical method to estimate these drugs simultaneously is crucial for ensuring the safety, efficacy, and quality of such complex formulations.

Pregabalin:

Pregabalin is a gamma-aminobutyric acid (GABA) analog that acts on the central nervous system. Pregabalin is an anti-convulsants drugs. It is primarily used for the treatment of various neuropathic conditions. Neuropathic pain, which results from nerve injury or damage, is one of the most common conditions treated with Pregabalin. The chemical name of Pregabalin is (S)-3-(aminomethyl)-5-methylhexanoic acid. It has a molecular formula of C₈H₁₇NO_2. Molecular weight 159.23 g/mol.

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Fig 1: Structure of Pregabalin

Pyridoxine Hydrochloride (Vitamin B6):

Pyridoxine Hydrochloride is often used alongside other therapeutic agents to enhance nerve function and alleviate symptoms. It also acts as a cofactor in the synthesis of neurotransmitters like serotonin, dopamine, and gamma- aminobutyric acid (GABA), which are crucial for nerve healthThe chemical name of Pyridoxine Hydrochloride is 4,5-bis(hydroxymethyl)-2-methylpyridin-3-ol;hydrochloride. It has a molecular formula of C8H11NO3. Molecular weight 205.64 g/mol.

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Fig 2: Structure of Pyridoxine Hydrochloride

MATERIALS AND METHODS:

Table:1 Instrument specification for High Performance LC

Table:2 Instrument specifications for weighing balance

Table:3 Instrument specification for melting point apparatus

Table:4 Instrument Specification for UV double beam Spectrophotometer

Table.5: Procurement of pure APIs

Identification of Drugs:

Solution Stability

The solubility of Pregabalin & Pyridoxine Hydrochloride practically determined separately by taking 100 mg of both the drugs in 100 ml volumetric flasks, adding required quantity of solvent at room temperature and shaken for few minutes. Solubility data for each study was observed and recorded in Table 9.

Table.6: Solubility Table

Table.7: Solubility Data for Pregabalin Derivative Benzyl Chloride & Pyridoxine Hydrochloride

Identification by Melting Point Determination

Melting point of Pregabalin & Pyridoxine Hydrochloride hydrochloride has been determined. The melting points of the compounds were taken by open capillary method.

Table.8: Melting Point of Drugs

IR Spectra for Identification of drugs:

IR Spectra of pregabalin:

Table.9: IR Interpretation of Pregabalin

IR Spectra of Pyridoxine Hydrochloride:

Table.10: IR Interpretation of Pyridoxine Hydrochloride

Development and Optimization of HPLC Method:

Selection of Wavelength

To determine wavelength for measurement, standard spectra of Pregabalin Derivative Benzyl Chloride & Pyridoxine Hydrochloride were scanned between 200-400 nm against diluents. Absorbance maxima of Pregabalin Derivative Benzyl Chloride & Pyridoxine Hydrochloride detected at 240 nm & 263 nm. Chromatogram was taken at 250 nm, both drugs give good peak height and shape. So, 250nm was selected for Simultaneous estimation of Pregabalin Derivative Benzyl Chloride & Pyridoxine Hydrochloride their formulation. The UV overlain spectrum for Pregabalin & Pyridoxine Hydrochloride shown in fig No.5.

Selection of Chromatographic Conditions

Proper selection of the HPLC method depends upon the nature of the sample (ionic or ionisable or neutral molecule), its molecular weight, pKa and solubility. HPLC was selected for the initial separation based on literature survey and its simplicity and suitability. To optimize the chromatographic conditions the effect of chromatographic variables such as mobile phase, flow rate and solvent ratio were studied. Finally, the chromatographic condition was chosen that give the best resolution, symmetry and capacity factor for estimation of both drugs.

Selection of Column

For HPLC Method, various columns are available but based on literature survey C-18 (id 4.6 x 150 mm, 5 µm) was selected over the other columns.

Preparation of Solution

Preparation of Mobile Phase

HPLC method was followed by isocratic elution technique. Mobile phase comprised of 1% v/v orthophosphoric acid and acetonitrile in a ratio of 55:45% v/v. pH Adjustment to 3.0 using triethylamine as it elutes both drugs peak efficiently in short time with satisfactory resolution, tailing factor and theoretical plates. The mobile phase is ideal for promoting good separation and accurate quantification of the analytes in a controlled and reproducible manner. The acidic nature of the orthophosphoric acid helps with the ionization of the compounds, while the acetonitrile provides the necessary polarity to enhance resolution and shorten analysis times.

Preparation of Standard Stock Solution A: (Pregabalin Stock Solution)

Accurately weighed quantity of 100 mg of Pregabalin in 1ml of (DCM). Add 0.1ml of Benzyl Chloride to pregabalin solution. Add 0.1ml of 1M NaOH. Transferred into 100 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 1000 μg/mL. Withdraw 7.5 ml from Stock Solution and make up to 10 ml to get 750 μg/mL.

Preparation of Standard Stock Solution B: (Pyridoxine Hydrochloride stock solution)

Accurately weighed quantity of Pyridoxine Hydrochloride 10 mg was transferred into 100 mL volumetric flask, dissolved in methanol and diluted up to mark with methanol. This will give a stock solution having strength of 100 μg/mL.Withdraw 1.5 ml from Stock Solution and make up to 10 ml with to get 15 μg/mL.

Chromatographic condition

The chromatographic separation of Pregabalin and Pyridoxine Hydrochloride were achieved on C-18 (id 4.6 x 150 mm, 5 µm) by using mobile phase composed of 1% v/v orthophosphoric acid and acetonitrile in a ratio of 55:45% v/v, at flow rate 1.0 ml/min with run time of 30 minutes. Detection of both drugs was carried out at 254 nm by using diluent as mobile phase.

Method of validation

As per ICH guideline (Q2R1), the method validation parameters studied were specificity, linearity, accuracy, precision, limit of detection, limit of quantitation and robustness.

Specificity

The analytical method for specificity was evaluated by injecting the following solutions. Diluent was prepared and inject into the HPLC system in triplicate. Sample solution was prepared with appropriate levels of excipients as a placebo sample and inject into the HPLC system in triplicate for all the dosage strengths. Placebo was prepared by mixing all excipients without active ingredients. Standard and sample solutions were prepared for assay (100% Conc.) and inject into the HPLC system in triplicate.

Linearity and Range

Preparation of Solution for linearity studies: For the purpose of linearity, accurately weighed amount of Pregabalin Derivative Benzyl Chloride (75 mg), and Pyridoxine Hydrochloride (1.5 mg) was taken into the volumetric flask (10 ml) and volume of the flask was raised to 10 ml with methyl alcohol to give stock solution containing 750µg/ml of Pregabalin, and 15 µg/ml of Pyridoxine Hydrochloride. Various aliquots from this stock solution were transferred to another 10 ml volumetric flask and volume was raised to the mark with mobile phase to give final solutions containing 50+1, 75+1.5, 100+2, 125+2.5 and 150+3µg/ml of Pregabalin and Pyridoxine Hydrochloride respectively.

Precision

Repeatability

Prepared standard working solution of mixtures having concentration of Pregabalin Derivative Benzyl Chloride (750 μg/ml) and Pyridoxine Hydrochloride (15 μg/ml) were injected at volume of 20 μL into column by employing optimized chromatographic conditions. Each standard mixture was injected 5 time and peak area was monitored. Each concentration was monitored for repeatability by RSD.

Intra-day and Inter-day Precision

• Method precision was determined by performing intraday and inter day precision.
• Mixture that represents overall range (Pregabalin Derivative Benzyl Chloride +Pyridoxine Hydrochloride = 50+1, 100+2 and 150+3 µg/ml) were analyzed on same day at different time interval for intraday precision.
• Mixture that represents overall range (Pregabalin Derivative Benzyl Chloride +Pyridoxine Hydrochloride = 50+1, 100+2 and 150+3 µg/ml)were analyzed on different days for inter-day precision.

System Suitability Parameters

Solution of Pregabalin + Pyridoxine Hydrochloride (100+100 μg.ml-1) was injected 3 times for determination of System suitability parameters which includes Retention time (Rt), Tailing factor (Tf), Resolution (Rs) and number of theoretical plates. System suitability parameters   for selected concentration were determined by C.V.

Accuracy

Accuracy of the analytical method has been performed by spiking of sample with the standard. Spiking of the placebo was performed at 50,100 and 150 % of the target concentration

Limit of detection and Limit of Quantification

The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the standard deviation of y-intercept of calibration curve. The limit of detection (LOD) and the limit of quantification (LOQ):

LOQ = 10 σ/s and LOD = 3.3 σ/s

Where, σ = the standard deviation of the response.

S = the slope of the calibration curve

Robustness

Following parameters were altered one by one for determination of robustness of the method and their effect was observed by comparing with the standard preparation. Mobile phase flowrate (± 0.1 mL/min), optimized flowrate was 1.0 mL/min. Mobile phase composition (± 2 mL), in optimized ratio 2 determinations of Pregabalin Derivative Benzyl Chloride + Pyridoxine Hydrochloride for each alteration were carried out and RSD was measured.

Assay

Procedure of for derivatization of Pregabalin

Structure of Pregabalin and its suitability for derivatization.

Pregabalin chemically (S)-3-(aminomethyl)-5-methylhexanoic acid. The molecule is highly statured, aliphatic chain with single bonds except for C=O bond. Such bonding chemistry of Pregabalin, makes it poor candidate for UV light absorption and hence making it almost ineligible for analysis by techniques which explore phenomenon UV radiation such as HPLC-UV or UV-Spectrophotometry. Although presence of COOH group makes its estimation pretty easy by titration from bulk API lots but is unsuitable for analysis pregabalin in the dosage forms which contain interfering excipients.  The structure as shown in the Figure-1, has two functional groups, carboxylic acid (COOH) and primary amine (Nh2), both groups amenable for derivitization. The derivatization of drugs for the purpose of analysis, needs to be simple, cost effective, safe and environment friendly. Most importantly, the dervitizing reactions essentially be short, single step and can be carried out at general laboratory conditions. The presence of both primary amine and carboxylic acid groups in the structure makes molecule more explicable for modication into UV into absorbing species, however considering structural constrains, it appears that exploring amine groups appears to less complicated.

Benzoylation of Pregabalin

The benzoylation generally reaction of primary amine with or OH group with benzoyl chloride resulting in the formation of an amide or ester. The benzoylation of phenolic OH requires drastic conditions of 0 high temperatures of 125-175 C with addition of benzoyl chloride. On other hand, benzoylation at primary amine was reported to be relatively less complex.  Benzoylation of amines involves, reaction acyl chloride with an amine so that an amide is formed, together with a proton and a chloride ion. Addition of a base is required to neutralize this acidic proton, otherwise the reaction will not proceed. Generally the aqueous solution of a base is slowly added to the reaction mixture to neutralize incoming proton. For zwitterion molecules such as amino acids, base also keeps COOH group ionized and prevents amine group being protonated. General scheme for benzoylation of amines is given below.

RESULT AND DISCUSSION:

Selection of Wavelength

To determine wavelength for measurement, standard spectra of Pregabalin Derivative Benzyl Chloride & Pyridoxine Hydrochloride were scanned between 200-400 nm against diluents. Absorbance maxima of Pregabalin Derivative Benzyl Chloride & Pyridoxine Hydrochloride detected at 240 nm & 263 nm. Chromatogram was taken at 250 nm, both drugs give good peak height and shape. So, 250nm was selected for Simultaneous estimation of Pregabalin Derivative Benzyl Chloride & Pyridoxine Hydrochloride their formulation. The UV overlain spectrum for Pregabalin & Pyridoxine Hydrochloride in their formulation.

Selection of Mobile phase

Trail 1

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Acetonitrile:Water(30:9.0v/v)

Detection: 250 nm

Flow rate:1 ml/min

Run Time: 10 minutes

Observations: No peak detected.

Trail 2

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Acetonitrile:Water(50:50v/v)

Detection: 250 nm

Flow rate:1 ml/min

Run Time: 10 minutes

Observations: Only one Peak detected but broad peaks observe.

Trail 3

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Acetonitrile:Water (80:20v/v)

Detection: 250 nm

Flow rate:1 ml/min

Run Time: 10 minutes

Observations: Only one Peak detected but broad peaks observe.

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Ethanol: Phosphate Buffer (60:40v/v)

Detection: 250 nm

Flow rate:1 ml/min

Run Time: 10minutes

Observations: only one peak detected.

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Fig.10 Trial 4: Chromatogram of PGB + Pyri HCL Ethanol: Phosphate Buffer (60:40v/v)

Trail 5

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Chloroform: Acetonitrile (9.0:30v/v)

Detection: 250 nm

Flow rate:1 ml/min

Run Time: 10minutes

Observations: only one peak detected.

Trail 6

Column: C-18 (id 4.6 x 250 mm, 5 µm)

Mobile Phase: Chloroform: Acetonitrile (50:50v/v)

Detection: 250 nm

Flow rate:1 ml/min

Run Time: 10minutes

Observations: Peaks detected and separated, but broad peaks observe.

Trail 7

Column: C-18 (id 4.6 x 150 mm, 5 µm)

Mobile Phase: 1% v/v orthophosphoric acid and acetonitrile in a ratio of (30:70v/v)

Detection: 250 nm

Flow rate:1 ml/min

Run Time: 30 minutes

Observations: No peak detected.

Trail 8

Column: C-18 (id 4.6 x 150 mm, 5 µm)

Mobile Phase: 1% v/v orthophosphoric acid and acetonitrile in a ratio of (50:50v/v)

Detection: 250 nm

Flow rate:1 ml/min

Run Time: 30minutes

Observations: only one peak detected.

Trial 9

Column: C-18 (id 4.6 x 150 mm, 5 µm)

Mobile Phase: 1% v/v orthophosphoric acid and acetonitrile in a ratio of (20:80v/v)

Detection: 250 nm

Flow rate:1 ml/min

Run Time: 30minutes

Observations: Peaks detected and separated, but broad peaks observe.

Column: C-18 (id 4.6 x 150 mm, 5 µm)

Mobile Phase: 1% v/v orthophosphoric acid and acetonitrile in a ratio of 55:45%

Detection: 250 nm

Flow rate:1 ml/min

Run Time: 30 minutes

Observations: Good peaks with Adequate solution was observed.

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Fig.16 Trial 4: Chromatogram of PGB + Pyri HCL with 1% v/v OPA acid and ACN in a ratio of (55:45v/v)

 7.5.3 Chromatographic conditions for optimized mobile phase trial

Stationary phase: C-18 (id 4.6 x 150 mm, 5 µm)

Mobile Phase: 1% v/v orthophosphoric acid and acetonitrile in a ratio of 55:45%

Detection: 250 nm

Flow rate:1 ml/min

Run Time: 30 minutes

Detector: UV detector

Injection volume: 20 μl

Column Temperature: 40ºC

Mode: Isocratic

Method Validation

Linearity

For the purpose of linearity, accurately weighed amount of Pregabalin Derivative Benzyl Chloride (10 mg), and Pyridoxine Hydrochloride (10 mg) was taken into the volumetric flask (10 ml) and volume of the flask was raised to 10 ml with methyl alcohol to give stock solution containing 100 µg/ml of Pregabalin, and 100 µg/ml of Pyridoxine Hydrochloride. Various aliquots from this stock solution were transferred to another 10 ml volumetric flask and volume was raised to the mark with mobile phase to give final solutions containing 50+1, 75+1.5, 100+2, 125+2.5 and 150+3 µg/ml of Pregabalin and Pyridoxine Hydrochloride respectively.

Table.11: Linearity data for Pregabalin Derivative Benzyl Chloride and Pyridoxine Hydrochloride

Table.12: Linearity results for Pregabalin Derivative Benzyl Chloride and Pyridoxine Hydrochloride

Precision

Repeatability

The data for repeatability for Pregabalin Derivative Benzyl Chloride and Pyridoxine Hydrochloride is shown in table 13. The % R.S.D For Repeatability data was found to be 0.48 % for Pregabalin derivative benzyl chloride and 0.49 % for Pyridoxine hydrochloride.

Table.13: Repeatability data for Pregabalin and Pyridoxine Hydrochloride

Inter-day precision  

The data for interday precision for Pregabalin Derivative Benzyl Chloride and Pyridoxine Hydrochloride is shown in table 14. The % R.S.D for intraday precision was found to be 0.19-0.58 % for Pregabalin and 0.10-0.43 % for Pyridoxine Hydrochloride.

Table.14: Inter-day precision data for estimation of Pregabalin Derivative Benzyl Chloride and Pyridoxine Hydrochloride

Intra-day precision

The data for intra-day precision for Pregabalin Derivative Benzyl Chloride and Pyridoxine Hydrochloride is shown in table 15. The % R.S.D for intraday precision was found to be 0.06-0.13 % for Pregabalin and 0.09 -0.17 % for Pyridoxine Hydrochloride.

Table.15: Intra-day precision data for estimation of Pregabalin Derivative Benzyl Chloride and Pyridoxine Hydrochloride

Accuracy

Accuracy of the method was confirmed by recovery study from synthetic mixture at three level standard additions. Percentage recovery for Pregabalin Derivative Benzyl Chloride & Pyridoxine Hydrochloride was found to be 99.48- 99.78% and 99.33-100.59 % respectively. The results are shown in table.16-17.

Table.16: Recovery data for Pregabalin Derivative Benzyl Chloride

Table.17: Recovery data for Pyridoxine Hydrochloride

LOD and LOQ

The limit of detection (LOD) and Limit of Quantification (LOQ) was found to be as per below:

Table.18: LOD and LOQ Limit for Pregabalin Derivative Benzyl Chloride & Pyridoxine Hydrochloride

Selectivity

There is no interference in the mixture.

Robustness

The method is found to be robust as the results were not significantly affected by slight variation in Mobile Phase Composition and flow rate of mobile phase. The results are shown in table 19. Variation seen was within the acceptable range respect to peak asymmetry and theoretical plates, so the method was found to be robust.

Table.19: Robustness data for Pregabalin Derivative Benzyl Chloride & Pyridoxine Hydrochloride

Analysis of marketed product

The proposed method was successfully applied to analysis of the commercially available tablet formulation. The % drugs were found satisfactory, which is comparable with the corresponding label claim.

Summary Of Method Validation:

Table.21: Summary of validation parameter of RP-HPLC method