22

2.1 Introduction

This section deals with the development of a simple, sensitive, specific,

spectrophotometric, HPLC and HPTLC methods for the determination of pregabalin in

bulk and pharmaceutical formulation. An extensive literature survey shows that there are

few reports on determining pregabalin contents in pharmaceuticals have been published,

involving spectrophotometric - spectrofluorimetric methods1

and pre column

derivatization method using internal standard 2, 3

. Synthesis and characterization of

pregabalin lactose degradation product was also reported 4. Literature survey further

revealed the availability of the UV method (bulk, formulation, human urine sample) 5

,

Colorimetric methods6, HPLC methods

7-11 and analysis of pregabalin in Human

12-15 and

rat plasma 16

. The development of a gradient RP-HPLC method and validation for the

determination of Pregabalin and its related substances in bulk drug as well as its

pharmaceutical formulations was also addressed in this section. Pregabalin is official in

Pharmacopoeia 17

. However the Pharmacopoeial method and the reported HPLC methods

was not able to well resolve all potential impurities that may form during the synthetic

process (Figure 2.2) or carried over from the starting material, so it is felt necessary to

develop a suitable stability-indicating LC method with an objective to resolve all the

potential impurities. A simple and sensitive thin-layer chromatographic method for the

analysis of pregabalin in bulk and its pharmaceutical dosage form was also carried upon.

Since there are no reported HPTLC method found through computer assisted literature

survey, the developed planar- chromatographic method (HPTLC) would serve as a rapid

and reliable method for the determination of pregabalin in bulk and its pharmaceutical

dosage form.

23

2.2. Experimental

2.2.1. UV Method

2.2.1.1. Materials

Pregabalin was obtained from Aurabindo Pharmaceuticals Ltd (Hyderabad, India). It was

used without further purification. Its Pharmaceutical preparation, Lyrica Capsule

(Manufacturer- Pfizer) was purchased from a local drug store; it contained 25mg active

material.

2.2.1.2. Preparation of the standard stock solution

A standard drug solution of pregabalin was prepared by dissolving 100 mg of pregabalin

in 10 mL of double distilled water and this was transferred into a 100 mL volumetric

flask to obtain a stock solution of 1000 µg/mL.

2.2.1.3. Preparation of the working solution

From the above stock solution, 10 mL of the sample was transferred into a 100 mL

volumetric flask and the volume was made up to the mark with distilled water to prepare

a concentration of 100 µg/mL. The solution was further diluted and scanned in the range

of 200-400 nm using distilled water as blank. The wavelength corresponding to the

maximum absorbance (λmax) was found to be 210 nm. This was further utilized to obtain

a calibration curve.

2.2.1.4. Preparation of sample solution

The proposed method was applied to analyze the commercially available pregabalin

capsules. Twenty capsules were weighed and amount of capsule powder equivalent to 10

mg of pregabalin was weighed accurately and transferred to a 100 mL volumetric flask

dissolved in distilled water, then the volume was brought up to 100 mL by using the same

24

solvent. Filtered through 0.45µ whattmann filter paper. The solution was diluted suitably

with distilled water to get a final concentration of 10 µg/mL. This was subsequently

analyzed using a double beam UV-VIS spectrophotometer against distilled water as a

blank. The drug content of the sample was calculatedby using regression analysis.

2.2.1.5. Method Validation

The method was validated for different parameters like Linearity, Accuracy, Precision,

Specificity, Limit of Detection (LOD) and Limit of Quantification (LOQ).

2.2.1.5.1. Linearity

Various aliquots were prepared from the stock solution (100 µg/mL) ranging from 2.5 –

12.5 µg/mL. The samples were analyzed with the help of a UV-VIS Spectrophotometer

using distilled water as the blank.

2.2.1.5.2. Accuracy

The accuracy of the method was determined by preparing solutions of different

concentrations, i.e , 80, 100, 120% in which the amount of marketed formulation was

kept constant(10mg) and the amount of pure drug was varied, that is 8 mg, 10 mg, 12 mg

for 80,100, 120%, respectively. The solutions were prepared in triplicate and the accuracy

was indicated by % recovery.

2.2.1.5.3. Precision

The precision of the method was demonstrated by intra-day and inter-day variation

studies. In the inter-day variation study, 7.5 µg/mL of solution was prepared and

analyzed thrice, for three consecutive days, and the absorbance was recorded. In the intra-

day variation study, six different solutions of the same concentration (7.5 µg/mL) was

25

prepared and analyzed thrice a day (morning, afternoon, and evening). The results were

indicated by % RSD.

2.2.1.5.4. Limit of Detection & Limit of quantification

The LOD & LOQ were calculated using the formula involving the standard deviation of

response and the slope of the calibration curve. LOD = Cd × Syx / b and LOQ = Cq ×

Syx / b. Where Cd and Cq are the coefficients for LOD and LOQ. Syx is the residual

Variance of the Regression, and b is the Slope. Calculation was performed by using

values of Cd and Cq of 3.3 and 10.

2.2.2. HPLC Method

2.2.2.1. Materials

Reference standard of Pregabalin and its related impurities (Figure 2.3) were procured

from Aurabindo Pharmaceuticals (Hyderabad, India). The potential impurities in

pregabalin are 3-isobutylglutaric acid (Impurity A), (R)-(-)-3-Carbamoylmethyl-

5methylhexanoic acid (Impurity- B), and 4-isobutylpyrrolidin-2-one (Impurity C). The

HPLC grade acetonitrile was purchased from Merck, India. Analytical grade potassium

dihydrogen phosphate, potassium hydroxide and methanol were obtained from S.D. Fine

Chemicals Ltd. (Mumbai, India). High purity water was prepared by using Millipore

Milli-Q plus purification system (Millipore, Bedford, USA).

2.2.2.2. Instrumentation

Waters HPLC equipped with quaternary pump and PDA detector was used. The output

signal was monitored and integrated by waters empower 2 software.

26

2.2.2.3. Chromatographic conditions

The chromatographic column used was Inertsil ODS 3V, 250 x 4.6 mm, 5 µm particles.

Mobile phase consisted of A and B. A was prepared by dissolving 2.72g of potassium

dihydrogen orthophosphate in 1000 mL of water, adjusted the pH of the solution to 5.9 ±

0.05 with dilute potassium hydroxide solution and filtered by using 0.45 µm nylon filter

and prepare a degassed mixture of buffer and methanol in the ratio of 90:10. This is

mobile phase A. Mobile phase B was pure acetonitile. Mobile phase flow rate was set at

1.0 mL min-1

. The applied gradient program is shown in Table 2.6. The temperature of

the column was maintained at 35oC. A wavelength of 210nm was used for the detection

and the injection volume used was 20µL.

2.2.2.4. Standard preparation

Weigh accurately about 25mg of reference standard of pregabalin in 25 mL volumetric

flask. Add about 10 mL of the diluent with intermittent shaking and made up to the mark

by using diluent (water is used as diluent). Pipette 2 mL of this solution in to a 25 mL

volumetric flask and dilute to volume with diluent and mix well. Filter about 2 mL

through 0.45 µm pall pharma lab nylon 66 membrane filters or 0.45 µm Dura pore PVDF

hydrophilic membrane filter.

2.2.2.5. Sample preparation

To determine the pregabalin content in capsules, (Lyrica- Brand Name) which contained

25 mg of pregabalin per capsule was selected. Capsule powder equivalent to 25 mg was

weighed accurately and transferred in to 25 mL standard flask, add 17 mL of diluent,

sonicate for 30 min, then dilute to volume and mix well. Further dilutions were carried

out to obtain same concentration as standard.

27

2.2.2.6. System suitability solution

A study to establish the interference of impurities was conducted. Prepared the impurity

solutions in the concentration of 0.3% of all impurities with respect to standard

preparation. Spiked the standard preparation with impurity blend solution and injected in

to HPLC system.

2.2.2.7. System suitability parameters

Resolution between Impurity -A and pregabalin obtained from the chromatogram from

the system suitability solution should not be less than 2.0. The % RSD for the peak areas

of six replicate injections for pregabalin is not more than 5.0%. Theoretical plates for

pregabalin and its three main impurities should be more than 2000 and the tailing factor

should not be more than 2.0, respectively.

2.2.2.8. Method Validation

2.2.2.8.1. Precision

The precision was carried out by six independent assay determination of pregabalin

against a reference standard and the % R.S.D of the assay was calculated. In the case of

related substances, the precision was checked by spiking the known impurities to

pregabalin reference standard and calculating the % R.S.D of area for each impurity.

Intermediate precision was conducted by different analyst in different days in accordance

with ICH guidelines.

2.2.2.8.2. Limit of detection (LOD) and limit of quantification (LOQ)

The LOD and LOQ for the impurities A, B and C were estimated at a signal-to-noise

ratio of 3:1 and 10:1, respectively. Precision was also carried out at LOQ level by

28

injecting the individual preparations of all the impurities six times and calculating the %

R.S.D of the peak area.

2.2.2.8.3. Linearity

Linearity test solutions for both drug and its related impurities were prepared over the

concentration range of 0.5 µg/mL-1.50µg/mL. The above test was carried for 3

consecutive days. The % R.S.D. value for the slope and the Y-intercept of the calibration

curve was calculated.

2.2.2.8.4. Accuracy

The accuracy test was carried out by spiking experiment. Impurities A, B, and C were

spiked into the drug sample at 50%, 75%, 100%, 125% and 150% of the specified limit

concentration. The experiment was performed in triplicate.

2.2.2.8.5. Specificity

The specificity of the method for pregabalin was determined in presence of its impurities

and its degradation products. Intentional degradation was performed on pregabalin to

provide an indication of the stability indicating property and specificity of the method.

Stress condition study included acid, alkali, oxidative, photolytic and thermal

degradation. Forced degradation studies were performed at an initial concentration of 100

µg/mL. Pregabalin was refluxed with 0.01N HCl at 70oC for 3 hours on mantel followed

by neutralization and adjusting the pH to 7 with 0.01N NaOH for acid degradation. Alkali

degradation was carried out by dissolving pregabalin in 0.01N NaOH on bench top for 3

hours followed by neutralization by adjusting pH to 7 with 0.01N HCl. For oxidative

degradation, pregabalin was refluxed with 1% H2O2 by heating on water bath at 40oC for

1 hour. (2600 Lux/119h & 40 min) was the condition used under photolytic stress. For

29

thermal degradation, samples were exposed to temperature at 105oC for 14 hours. Further

dilution of the above mentioned stress conditioned samples were done to required

concentration with diluent.

2.2.2.8.6. Robustness

Robustness of the method was carried out by deliberately altering the experimental

conditions. The flow rate of the mobile phase was changed by 0.2 units from 1.0 to 0.8

and 1.2 mL min-1

. The effect of column temperature was also studied at 30 and 40oC

instead of 35oC. The effect of pH on resolution of the impurities was studied by varying

the buffer pH by ± 0.1 units from 5.9 to 5.8 and 6.0. The effect of use of a column from a

different batch was also investigated. In all these studies mobile phase composition was

held constant.

2.2.2.9. Solution stability

Solution stability was checked by keeping the test solution spiked with impurities in

tightly capped volumetric flask at temperature 25o ± 2

oC on a laboratory bench for 24

hours. Content of impurities was checked for every 6 hours interval and compared with

freshly prepared solution. Mobile phase stability was also checked out by determining the

content of impurities in sample solution spiked with all the related impurities, which were

prepared freshly at every 6h up to 24h.

2.2.3. HPTLC Method

2.2.3.1. Materials and Reagents

The chemicals and reagents used for the present research work were of AR grade and

procured from S.D. Fine-Chem. (New Delhi, India).

30

2.2.3.2. HPTLC Instrumentation

Chromatography was performed on 10 cm x 10 cm aluminium foil plates precoated with

0.2-mm layers of silica gel 60F254 (E. Merck, Germany). Before use the plates were

prewashed by development with methanol then dried in the current of dry air and

activated at 600C for 5 min. Samples were applied as bands 6 mm wide, 15 mm apart, by

use of a Camag (Switzerland) Linomat 5 equipped with a microlitre syringe. A constant

application rate of 150 nL s-1

was used. Chloroform: methanol (6:6 v/v) used as mobile

phase. Linear ascending development was performed in a twin-trough glass chamber and

saturated with iodine vapour for 30 min at room temperature (RT, 25± 20C) and relative

humidity 60 ± 5%. The development distance was approximately 80 mm. After

development the plates were dried in current of air by use of an air dryer. Densitometric

scanning was performed with camag TLC scanner III in the absorbance reflectance mode

at 290 nm and operated by WINCATS software (V 1.4.3camag) resident in the system.

The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum

between 200-400 nm and concentrations of the compound chromatographed were

determined from the intensity of diffusely reflected light.

2.2.3.3. Calibration Plots of pregabalin

A stock solution containing 1 mg/mL of pregabalin was prepared by dissolving an

accurately weighed 100 mg portion of the drug in methanol in 100 mL volumetric flask

(1000 ng/μL) Different volumes of stock solution (1, 2, 3, 4, 5, 6, 7, 8 and 9 µL) were

spotted on an HPTLC plate in triplicate to obtain concentrations of 1, 2, 3, 4, 5, 6, 7, 8

and 9 μg/spot of pregabalin, respectively. The data of peak area versus drug concentration

were treated by linear least-squares regression to obtain the calibration graphs.

31

2.2.3.4. Method Validation

2.2.3.4.1. Precision

The precision of the method was verified by repeatability and intermediate precision

studies. Repeatability studies were performed by analyzing three different concentrations

(2, 4. 6 (μg/spot) of the drug by six times on the same day. The intermediate precision of

the method was checked by repeating on three different days.

2.2.3.4.2. Robustness

The analytical conditions were deliberately changed, by introducing small changes in

mobile phase composition (±0.1mL), mobile phase volume (±2%), chamber saturation

period (±10%), development distance (±10%), and time from application to development

(0, 10, 15, 20min), time from development to scanning (0, 30, 60, 90 min) were carried

out.

2.2.3.4.3. Limit of detection and Limit of quantification

The method was used to determine the limit of detection (LOD) and limit of

quantification (LOQ). Blank methanol was spotted six times, and the SD (Sb) of the peak

area of the blanks was calculated. The limits were determined from the slope (S) of the

calibration plot and the SD of the response for the blank sample (Sb) by use of the

formula:

LOD = 3.3 x Sb / S and LOQ = 10 x Sb / S

2.2.3.4.4. Accuracy

To check the degree of accuracy of the method, recovery studies were performed in

triplicate by standard addition method at 50, 100 and 150%. Known amount of standard

32

pregabalin was added to pre-analysed samples and were subjected to the proposed

HPTLC method. Six determinations were performed at each level of recovery.

2.2.3.4.5. Specificity

The specificity of the method was determined by comparing the results for the standard

drug and the sample. The peak purity of the sample was assessed by comparing the

spectra at peak start, peak apex, and peak end positions of the spot.

2.2.3.4.6. Forced Degradation studies

Forced degradation studies were performed to prove the stability-indicating property of

the drug to the various stressed conditions. The drug was exposed to different stress

condition similar to the procedure adopted in HPLC method and 5 µL of the sample was

applied on an HPTLC plate and the procedure was carried out as discussed in 3.9.2.

section.

2.2.3.5. Analysis of pregabalin in capsules

To determine the pregabalin content in capsules, twenty capsules were weighed. Capsule

powder equivalent to 100 mg of pregabalin (about 454 mg of pregabalin capsules

powder) was weighed accurately and transferred in to a 100mL volumetric flask,

extracted with methanol, sonicated for 30 min, and diluted to volume with same solvent.

The resulting solution was filtered through a 0.45 µm filter (Millifilter; Milford, MA;

USA). The solution (5 µL, 5 μg/spot of pregabalin) was applied in triplicate on an

HPTLC plate for quantification using the proposed method. The possibility of excipient

interference with the analysis was examined.

33

2.3. Results and Discussions

2.3.1. UV Method

The aqueous pregabalin solution using distilled water as the solvent obeyed beer’s law in

the concentration range of 2.5-12.5 µg/mL (Figure 2.1). The optical characteristics and

the data concerning the proposed method were represented in Table 2.1. The recovery

studies were carried out for the developed method by the addition of standard drug

solution of pregabalin to pre-analyzed solution. The recovery studies were satisfactory

and the percentage of drug recovered (Table 2.2) was in the range of 100.4-101.6%,

which showed that there was no interference from excipients. The precision of the

method expressed as % RSD of intraday and interday validation is given in Table no.2.3,

2.4 & 2.5, respectively. The sensitivity was estimated in terms of limit of quantification

(LOQ), The smallest amounts detected were estimated in terms of limit of detection

(LOD) The results were also shown in Table 2.1. It was successfully applied for

determination of drugs in their formulated pharmaceutical formulations. The drug

content in the capsule formulation was found to be 98.97%, (n=6).

2.3.2. HPLC Method

2.3.2.1. Optimization of chromatographic condition

The objective of the present work is to develop a Liquid chromatographic method for the

determination of related substances in pregabalin API as well as its pharmaceutical

formulations. Different reverse phase stationary phase were employed during method

development and different kind of mobile phase were studied with combination of

acetonitrile. The resolution between impurity B and C was critical also the tailing of the

peak was observed high. To reduce the run time and to bring about the good resolution

34

between the main component and all the potential impurities, several preliminary

chromatographic runs were performed to investigate the suitability for drug content

estimation and cost because of the increasing importance of rapid economic analysis in

pharmaceutical analysis to increase the throughput. Optimization of the gradient program

was done in order to elute all the three impurities and the degradation products. The

separation of all the three impurities from the main peak of the drug was achieved

successfully by following the gradient program shown in Table 2.6. Chromatograms of

blank interference as well as placebo interference were also checked for the developed

method and it is depicted in Figure 2.4 and 2.5. The typical retention times (RT) of

pregabalin, impurity A, B, and C are about 5.48, 3.33, 7.32 and 8.42 min, respectively

(Figure 2.6a). The relative retention time (RRT), tailing factor and theoretical plates of

pregabalin and its three main impurities are shown in Table 2. 7.

2.3.2.2. Forced degradation studies

Stress degradation studies in acid, alkali, H2O2, photolytic and thermal conditions were

conducted to demonstrate the effective separation of degradants from pregabalin peak.

The drug underwent mild degradation in all the conditions and the degraded peaks

obtained under the forced degradation studies were well resolved from pregabalin peak.

The peak purity of the pregabalin stressed sample chromatograms were evaluated by

Empowers software. In all stress conditions, pregabalin peak purity angle was less than

purity threshold. The results are given under Table 2.8 and Figure 2.7, respectively.

2.3.2.3. Precision

The % RSD of retention time for the drug was observed to be 0.01%, which was well

within the acceptance (of NMT 0.50%) and the % RSD of area of the impurities A, B

35

and C was observed to be 0.217%, 0.342%, and 0.367% which is well within the

acceptance criteria of not more than 10.0%. The % R.S.D. of the assay results obtained in

the intermediate precision was less than 5% which confirms the good precision of the

method.

2.3.2.4. LOD and LOQ

The limit of detection (LOD) and limit of quantification (LOQ) for all impurities are

tabulated in Table 2.9. The precision at LOQ concentrations for all the impurities was

found to be less than 5%.

2.3.2.5. Linearity

Linearity regression analysis demonstrated acceptability of the method. The relation

between impurity concentration (x) and its corresponding peak area rates (Y) was

expressed by the regression equation Y = mx + b. The values of slope and intercept

obtained from calibration curves of Impurity A, B and C are 467.07 x (slope) - 700.5

(intercept), 854.19 x + 999.5 and 547.3 x – 1000, respectively. The correlation coefficient

determined from linear calibration plot for all the three impurities were found to be

greater than 0.999.

2.3.2.6. Accuracy

Accuracy of the method was demonstrated by recovery studies at three different

concentration levels in triplicate. The analysis was carried out at 50%, 100% and 150% of

specification limit. The mean recoveries of all the impurities were found to be within the

acceptance criteria of 97-103%. The results of the recovery studies are summarized in

Table 2.10.

36

2.3.2.7. Robustness

Close study of the analytical results obtained after deliberate alteration of the

chromatographic conditions (mobile phase flow rate, pH, and column temperature)

revealed the robustness of the method. In all the above mentioned conditions the

resolution between the impurities and the resolution between drug and impurities were

greater than 2.

2.3.2.8. Solution stability

After 24 hours on bench top at room temperature, no significant change or degradation

was observed in % of impurities of pregabalin.

2.3.2.9. Applicability of the proposed method for the estimation of pregabalin in

marketed formulation

The peak at tR 5.448 min for pregabalin was observed in the chromatogram of the drug

samples extracted from capsules (Figure 2.6 b) Experimental results of the amount of

pregabalin in capsules (Brand name: Lyrica, label claim: 25 mg per capsules), expressed

as percentage of label claim were in good agreement with the label claims thereby

suggesting that there is no interference from any excipients, which are normally present

in capsules. The drug content was found to be 99.84% ± 1.65 (% R.S.D. of 1.03) for

pregabalin.

2.3.3. HPTLC Method

2.3.3.1. Optimization of the Mobile Phase

Several solvent mixtures in different ratios were tested to obtain a compact band of

pregabalin. Chloroform: methanol (6:6 v/v) was found to give a compact band for

pregabalin with an Rf value of 0.3±0.02 (Figure 2.8a). The selected mobile phase

37

composition gave a sharp and symmetrical peak which is clearly visible under UV light

saturated with Iodine vapour (Figure 2.11). Thirty minutes was found to be sufficient for

saturation of the development chamber. A 20 mL aliquot of mobile phase was used for a

20 min development over a distance of 80 mm.

2.3.3.2. Calibration Plots of pregabalin

The linear regression analysis data for the calibration plots showed a good linear

relationship (r2 = 0.99808±0.000108) with respect to peak area (Figure 2.9) The mean

values of the slope and intercept were 920.94±2.24 and 362.83±10.48, for densitometric

analysis at 290nm (Table 2.11). The 3D pictorial representation of pregabalin of various

concentrations is depicted in the Figure 2.10.

2.3.3.3. Method Validation18

2.3.3.3.1. Precision

The results of the repeatability and intermediate precision experiments are shown in

(Table 2.12). The developed method was found to be precise as the RSD values for

repeatability and intermediate precision studies were <2%, respectively as recommended

by ICH guideline.

2.3.3.3.2. Robustness

The low values of RSD obtained after introducing small, deliberate changes in the mobile

phase composition, mobile phase volume, chamber saturation time, time from application

to development and time from development to scanning in the developed HPTLC method

indicated the robustness of the method (Table 2.14).

38

2.3.3.3.3. LOD and LOQ

LOD and LOQ were determined by the SD method and were found to be 0.38 and 1.15

μg/spot, respectively. The low values of LOD and LOQ indicate the good sensitivity of

the proposed method (Table 2.11).

2.3.3.3.4. Accuracy

Accuracy of the method was obtained by recovery after spiking with 50, 100 and 150%

of additional drug. Recovery of pregabalin in samples was 99.6 - 101.2 % (Table 2.13).

2.3.3.3.5. Specificity

The Rf value (0.30±0.02) of the sample and standard was almost identical, and spectra of

the sample and the standard were superimposable. These results indicated the specificity

of the method (Figure 2.12)

2.3.3.3.6. Forced Degradation studies

The drug was found to undergo mild degradation in all the stressed condition and the

degraded peak was found to be well separated from the main peak thus proving the

method as the stability – indicating.

2.3.3.4. Analysis of pregabalin in Capsules

A single spot at Rf – 0.30 was observed in the densitogram of the drug samples extracted

from capsules (Figure 2.8b). There was no interference from the excipients commonly

present in the capsules. The drug content was found to be 98.95% ± 0.31 with a RSD of

0.73 % (n=6). It may therefore be inferred that pregabalin had no quantifiable additional

impurities in the marketed formulation analysed by use of the proposed method.

39

Table 2.1: Validation parameters of pregabalin by UV method

Parameter Result

Absorption maxima λmax (nm) 210 nm

Beer’s law limits (µg/mL) 2.5-12.5 µg/ mL

Standard regression equation Y=bC+a Y=0.0756x-0.0173

Correlation coefficient 0.9971

Sandell’s sensitivity

(mg/cm2/0.001 absorbance unit) 0.013908

Molar absorptivity

(lit.mol-1

cm-1

) 0.312421×105

LOD 0.81 µg/ mL

LOQ 2.4 µg/ mL

Assay(n=6) 98.97

*Y= bC+ a where C is the concentration of pregabalin in µg/mL and Y is the absorbance

at the respective λmax

Table 2.2: Determination of accuracy by the percentage recovery (n=3)

Drug Sample Level of Amount Drug %

concentration addition Added(µg) found Recovery

(µg/mL) (%) (µg/mL) ±SD

10 80 8 18.4682 101.6

± 1.15

Pregabalin 10 100 10 20.271 101.35

± 0.1

10 120 12 22.0888 100.4

± 1.05

40

Table 2.3: Precision results showing repeatability of pregabalin by UV method

Concentration (µg/mL) Absorbance Mean ± SD

7.5 0.5496

7.5 0.5527

7.5 0.5516 0.551 ± 0.0016406

7.5 0.5489

7.5 0.5535

7.5 0.5504

Table 2.4: Intra-Day assay study (n=3) of pregabalin by UV method

Concentration Average

(µg/mL) %RSD %RSD

Absorbance 1 Absorbance 2 Absorbance3

7.5 0.29 0.32 0.38 0.33

Table 2.5: Inter-Day assay study (n=3) of pregabalin by UV method

Concentration Average

(µg/mL) %RSD %RSD

Absorbance 1 Absorbance 2 Absorbance3

7.5 0.30 0.38 0.35 0.34

41

Table 2.6: Gradient Program for HPLC

Time (min) Buffer pH: Methanol Acetonitrile

5.9 (% v/v) (% v/v)

0.01 100 0

05 70 30

10 45 55

15 100 0

20 100 0

Table 2.7: System suitability parameters of pregabalin and its impurities

Name Theoretical plates Tailing factor RRT

Impurity-A 6607 1.10 0.61

Impurity-B 6843 1.2 1.34

Impurity-C 6201 0.9 1.54

Pregabalin 98479 1.0 ----

42

Table 2.8: Results for specificity of the method by HPLC.

Mode of Condition % Assay % Purity Purity Purity

degradation degradation angle threshold flag

w.r.t. control

Acid

degradation 70oC/3h 98.47 0.70 0.031 0.230 NO

Base

degradation 70oC/3h 99.00 0.17 0.035 0.231 NO

Oxidative

degradation 40oC/1h 98.85 0.32 0.031 0.228 NO

Photolytic 2600Lux/

degradation 119h & 98.85 0.32 0.042 0.228 NO

40 min

Thermal

degradation 105oC/14h 98.76 0.41 0.037 0.230 NO

Table 2.9: LODs and LOQs of impurities by signal to noise ratio method.

Impurity Concentration with respect to Concentration with respect

sample at LOD, % to sample at LOQ, %

A 0.006 0.020

B 0.008 0.021

C 0.007 0.020

43

Table 2.10: Parameters of recovery of impurities of pregabalin by HPLC method

Sample spike level mg added mg found % recovery mean % recovery

no.

1 50.0% 10 10.10 100.77

2 50.0% 10 10.10 101.22 101.0

3 50.0% 10 10.10 100.96

1 75.0% 15 15.40 102.48

2 75.0% 15 15.40 102.41 102.4

3 75.0% 15 15.30 102.16

1 100.0% 20 20.60 103.21

2 100.0% 20 20.60 102.79 103.0

3 100.0% 20 20.60 102.88

1 125.0% 25 25.30 101.30

2 125.0% 25 25.30 101.18 101.4

3 125.0% 25 25.40 101.61

1 150.0% 30 30.90 102.81

2 150.0% 30 30.80 102.41 102.5

3 150.0% 30 30.70 102.21

Table 2.11: Linear regression data for the calibration curves (n=6) HPTLC method

Parameters TLC densitometry

Linear range 1-9 μg/spot

Correlation coefficient (r) ±SD 0.99808±0.000108

Slope±SD 920.94±2.24

Intercept±SD

362.83±10.48

LOD (μg/spot) 0.38

LOQ (μg/spot) 1.15

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Table 2.12: Intra and inter-day precision by Proposed HPTLC method (n=6)

Amount Repeatability Intermediate precision

(μg/spot) Mean Area (AU) ±SD %RSD Mean Area (AU) ±SD %RSD

2 2297±11.25 0.48 2312±15.32 0.66

4 4193 ±16.85 0.40 4224±19.6 0.46

6 5823±21.12 0.36 5883±25.4 0.43

Table 2.13: Recovery studies of pregabalin (n=6) by the proposed HPTLC method

Excess of drug added Amount of Recovery % R.S.D

to the analyte (mcg/ spot) drug found (ng) (%)

1 0.996 99.66 1.07

2 2.023 101.2 0.37

3 3.030 101.0 0.49

Matrix containing 2 μg of drug.

Table 2.14: Robustness of the method (n=6) by HPTLC

Parameters TLC densitometry± SD

% Change in mobile phase 0.72± 0.01

Chamber saturation time 0.74± 0.02

Development distance 0.76± 0.01

Time from application to development 0.75± 0.02

Time from development to scanning 0.74± 0.01

45

Figure 2.1: Calibration curve of pregabalin

46

COOH

COOH

OO O

CONH2

COOH

3-CABAMOYL METHYL-5-METHYL HEXANOIC ACID

(R)-PHENYL ETHYLAMINE

Aq.HCL

CONH2

COOH

R-(-)-3-CARBAMOYL METHYL HEXANOIC ACID

Br2

NAOH

ISO BUTANOL

ISO PROPANOL

NH2

COOH

PREGABALIN

3-ISO BUTYL GLUTARIC ACID

Aq.NH3

AC2O

Figure 2.2: Synthetic scheme of pregabalin

47

Figure 2.3: Chemical structure of pregabalin and its process - related impurities

48

Figure 2.4: Chromatogram of blank interference

Figure 2.5: Chromatogram of Placebo interference

49

Figure 2.6 (a): HPLC Chromatogram of standard pregabalin spiked with 0.3% of its

known impurities.

Figure 2.6 (b): HPLC Chromatogram of pregabalin extracted from the capsule

formulation.

50

Typical chromatogram of Acid stressed sample

Chromatogram of Base stressed sample

Chromatogram of Peroxide stressed sample

Chromatogram of Sunlight stressed sample

Chromatogram of Thermal degradation

Figure 2.7: Typical chromatograms of pregabalin under various stressed condition

51

Figure 2.8 (a): A typical densitogram of standard pregabalin (Rf -0.30±0.02) at 290nm

using chloroform: methanol (6:6 v/v).

Figure 2.8 (b): A typical densitogram of pregabalin extracted from sample ( Rf –

0.30±0.02) at 290nm using chloroform: methanol (6:6 v/v).

52

Figure 2.9: Calibration curve of standard pregabalin with respect to the area under curve

at various concentrations.

Figure 2.10: 3D Densitogram of standard pregabalin at different concentrations from 1-9

μg/spot

53

Figure 2.11: Video image of standard pregabalin saturated with iodine vapour at 290nm.

Figure 2.12: Insitu UV spectra of pregabalin standard and sample.

54

2.4. Correlation of the Results of Pregabalin by Statistical Analysis (Anova Test)

This write up brings about the correlation of the results which was applied for the

estimation of pregabalin by UV, HPLC as well as HPTLC methods. Anova-test was

applied to compare the results for the developed Spectrophotometric and

Chromatographic techniques.

Six different samples taken during in process control of capsule manufacturing were

determined by UV, HPLC and HPTLC. To test differences between the proposed

analytical methods, statistical tests were performed for the level of confidence 95% (p =

0.05). The obtained value of F stat is lower than two tail F crit, which leads to the

conclusion that there is no significant difference between the means. The results of

ANOVA test are given in Tables 2.15.

55

Table 2.15: Comparative results of pregabalin by the developed UV, HPLC and HPTLC

methods by ANOVA-test.

S.No. UV method HPLC method HPTLC method

1 99.23 98.23 98.53

2 99.73 98.27 98.29

3 98.92 97.99 99.98

4 100.12 98.99 100.29

5 99.23 99.21 99.88

6 99.23 98.29 99.77

Groups Count Sum Average Variance

Column 1 6 591.98 98.66333 0.294827

Column 2 6 590.98 99.49667 0.234827

Column 3 6 596.74 99.45667 0.693107

ANOVA

Source of variation SS dF MS Fstat P-value Fcrit

Between groups 3.157511 2 1.578756 3.68232 0.044033 3.873423

Within groups 6.1138 15 0.407587

Total 9.271311 17

Fstat < Fcrit

From the Table 2.15 it was concluded that F-stat < F-critical (i.e F stat value is less than

F critical value) which leads to the conclusion that there is no significant difference

between the proposed methods.

2.5. Conclusions

UV Spectrophotometric method developed for the estimation of pregabalin was found to

be accurate and simple. The developed Liquid chromatographic method for the

determination of related substances in pregabalin was stability indicating. The method

was fully validated according to ICH guidelines. This method can be adopted for the

56

routine quality control department for both the assay and the related substances

determination and also for the stability studies. As there are no reported methods for the

estimation of pregabalin by HPTLC, so it was felt necessary by the author to develop a

simple and precise method for the quantitative analysis of pregabalin in bulk and its

formulation. Thus the proposed UV, HPLC and HPTLC methods are accurate and

reproducible for quantitative analysis of pregabalin in bulk as well as its dosage form.

Statistical tests indicate that the proposed methods reduce the duration of analysis and

appear to be equally suitable for routine determination of pregabalin in pharmaceutical

formulation in quality control laboratories, where economy and time are essential.

57

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