258

CHAPTER- 6 2.6 DEVELOPMENT AND VALIDATION OF STABILITY INDICAT ING RP-HPLC METHOD FOR THE DETERMINATON OF PRAMIPEXOLE DIHYDROCHLORIDE MONOHYDRATE IN PRAMIPEXOLE DIHYDROCHLORIDE EXTENDED RELEASE TABLETS

CONTENTS

1. Drug profile

2. Review of the past work on the analytical methods for Pramipexole dihydrochloride monohydrate.

3. Experimental and results

a) Material and methods

b) Optimization of chromatographic conditions and method development

c) Validation of the proposed method

4. Summary of the results and Conclusion

5. References

259

1. DRUG PROFILE-PRAMIPEXOLE DIHYDROCHLORIDE

Pramipexole dihydrochloride tablets contain pramipexole1-6, a nonergot dopamine

agonist. The chemical name of pramipexole dihydrochloride is (S)-2-amino-4, 5, 6, 7-

tetrahydro-6-(propylamino) benzothiazole dihydrochloride monohydrate. Its molecular

formula is C10 H17 N3 S · 2HCl · H2O, and its molecular weight is 302.27.

Mechanism of Action

Pramipexole is a nonergot dopamine agonist with high relative in vitro specificity and

full intrinsic activity at the D2 subfamily of dopamine receptors, binding with higher

affinity to D3 than to D2 or D4 receptor subtypes.

Parkinson’s disease

The precise mechanism of action of pramipexole as a treatment for Parkinson’s disease

is unknown, although it is believed to be related to its ability to stimulate dopamine

receptors in the striatum. This conclusion is supported by electrophysiologic studies in

animals that have demonstrated that pramipexole influences striatal neuronal firing rates

via activation of dopamine receptors in the striatum and the substantia nigra, the site of

neurons that send projections to the striatum. The relevance of D3 receptor binding in

Parkinson’s disease is unknown.

Pharmacokinetics

Pramipexole displays linear pharmacokinetics over the clinical dosage range. Its

terminal half-life is about 8 hours in young healthy volunteers and about 12 hours in

elderly volunteers (see CLINICAL PHARMACOLOGY, Pharmacokinetics in

Special Populations). Steady-state concentrations are achieved within 2 days of

dosing.

260

Chemical Structure:

Chemical name (IUPAC ) : (S)-2-amino-4, 5, 6, 7-tetrahydro-6-propylamino)

benzothiazole dihydrochloride monohydrate

Chemical formula : C10H17 N3S-2HCl-H2 O

Molecular weight : 302.27

Physical state : A white to off white powder

Melting point : 296-3010 C

Solubility : Freely Soluble in Methanol,

Official Status of the drug : The drug is official in Merck Index

Table 2.6.1 Important brand names of Pramepexole dihydrochloride monohydrate

Brand Name Formulation Dosage Manufacturer

Mirapex Tablets 0.375,0.75,1.5,3.0 and 4.0 mg

Boehringer Ingelheim International GmbH

261

2. Review of the past work on the analytical methods for Pramipexole dihydrochloridemonohydrate

Panchal JG, et al 7, proposed a GC/MS method for the determination of pramipexole in

rat plasma. Analytes were determined using electron impact ionization in a single

quadrupole mass spectrometer. GC/MS was performed in the selected ion monitoring

mode using target ions at m/z 211, 212 and 152 for pramipexole and m/z 194 and 165

for caffeine as internal standard. A linear calibration curve was plotted over the range of

20-1000�pg/mL for pramipexole (r(2) > 0.996). The LLOQ was 20.0 pg/mL,

respectively, which offered high sensitivity and selectivity enough for bioanalytical

investigation. Inter- and intraday precisions ranged from 0.3 to 8.8% and from 0.9 to

11.33%, respectively.The recovery of pramipexole from plasma ranged from 82.4 ± 7.1

to 87.8 ± 5.7%.

G. Srinubabu et al 8, proposed a RP-HPLC method with UV detection for routine

control of pramipexole in tablets was developed. Chromatography was performed with

mobile phase containing a mixture of acetonitrile/phosphate buffer (60/40; v/v) with a

flow rate of 0.8 mL min−1. Quantitation was accomplished with the internal standard

method; the procedure was validated by linearity (correlation coefficient = 0.99892),

accuracy, robustness and intermediate precision. Limit of quantitation and limit of

detection were found to be 4.5 µg and 1.4 µg respectively.

D. Vijaya Bharathi 9, proposed a LC-MS/MS method for the estimation of pramipexole

(PPX) with 500 µL human plasma using memantine as an internal standard (IS). The

resolution of peaks was achieved with 0.01 M ammonium acetate buffer (pH

4.4):acetonitrile (30:70, v/v) on a Discovery CN column. The total chromatographic run

time was 3.0 min and the elution of PPX and IS occurred at approximately 2.32 and

2.52, respectively. The MS/MS ion transitions monitored were 212.10 → 153.10 for

PPX and 180.20 → 107.30 for IS. The method was proved to be accurate and precise at

linearity range of 20–3540 pg/mL with a correlation coefficient (r) of ≥0.999.

Ramakrishna V. S. Nirogi et al 10, proposed a high-performance liquid

chromatography/electrospray ionization tandem mass spectrometry method for the

quantification of pramipexole in human plasma. Following liquid–liquid extraction, the

262

analytes were separated using an isocratic mobile phase on a reverse-phase column and

analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M +

H]+ions, m/z 212/152 for pramipexole and m/z 409/228 for the IS. The method exhibited

a linear dynamic range of 200–8000 pg/mL for pramipexole in human plasma. The

lower limit of quantification was 200 pg/mL with a relative standard deviation of less

than 8%.

D.B. Pathare et al 11 , proposed a chiral liquid chromatographic method for the

enantiomeric resolution of Pramipexole dihydrochloride monohydrate, (S)-2-amino-

4,5,6,7-tetra-hydro-6-(propylamino) benzothiazole dihydrochloride monohydrate, a

dopamine agonist in bulk drugs. The enantiomers of Pramipexole dihydrochloride

monohydrate were resolved on a Chiralpak AD (250 mm × 4.6 mm, 10 µm) column

using a mobile phase system containing n-hexane:ethanol:diethylamine (70:30:0.1,

v/v/v). The resolution between the enantiomers was found not less than eight. The limit

of detection and limit of quantification of (R)-enantiomer are 300 and 900 ng/ml,

respectively for 20 µl injection volume. The percentage recovery of (R)-enantiomer was

ranged from 97.3 to 102.0 in bulk drug samples of Pramipexole dihydrochloride

monohydrate.

Yadav Manish 12, proposed a UPLC-MS-MS method for the determination of

pramipexole, a dopamine agonist, in human plasma. The chromatographic separation is

achieved on a Waters Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 µm) analytical

column using an isocratic mobile phase, consisting of 10 mM ammonium formate (pH

7.50)-acetonitrile (15:85, v/v), at a flow-rate of 0.5 mL/min. The precursor → product

ion transition for pramipexole (m/z 212.1 → 153.0) and IS (m/z 315.0 → 176.1) were

monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction

monitoring (MRM) and positive ion mode.

B.M. Gurupadayya et al 13, proposed two methods A and B for the quantitative

estimation of pramipexole dihydrochloride drug and its formulations. Method A is

based on the diazotization of primary amine group of pramipexole with sodium nitrate

and hydrochloric acid followed by coupling with N-(1-napthyl) ethylene diamine

hydrochloride (BM Reagent) to form a colored chromogen with a characteristic

absorption maximum at 616 nm. Method B is based on the reaction of the drug in

263

methanolic solution with paradimethylaminobenzaldehyde (PDAB) in acidic condition

producing Schiff’s base having a 8 at 474.5nm. Beer’s law is obeyed in concentrations

ranging from 4-20 µg/ml for method max A and 50-150 µg/ml for method B.

Petikam Lavudu1 et al 14, proposed three vis-spectrophotometric methods for the

determination of pramipexole dihydrochloride in bulk and tablet dosage forms. The

methods (A & B) are based on the oxidation of pramipexole dihydrochloride with

iron(III) and the subsequent formation of an intensive orange red complex between the

liberated iron(II) and 1,10- phenanthroline (method A) or 2,2'-bipyridyl (method B)

reagents. Method C is based on the fact that iron(III) is reduced to iron(II) by

pramipexole dihydrochloride, then the in situ formed iron(II) reacts with potassium

ferricyanide to give the soluble Prussian blue in acidic conditions. By measuring the

absorbance of iron(II)-1,10-phenanthroline complex, iron(II)-2,2'-bipyridyl and soluble

Prussian blue at the absorption maximum of 530, 530 and 735 nm, respectively, the

indirect determination of pramipexole dihydrochloride can be obtained. A good linear

relationship of the concentration of pramipexole dihydrochloride versus absorbance is

observed with a linear range of 4-40, 3-30 and 2-20 µg mL-1 for methods A, B and C,

respectively.

C.vinodhini et al 15, proposed two spectrophotometric methods in ultraviolet and

visible region for the estimation of Pramipexole dihydrochloride monohydrate in

pharmaceutical dosage forms. Method I was based on Pramipexole dihydrochloride

monohydrate showing absorption maximum at 265nm in methanol. Method II was

based on reaction of Pramipexole dihydrochloride monohydrate with ferric nitrate under

acidic condition to yield a yellowish green color. This color has a characteristics light

absorption in the visible region with the absorption maximum at 435nm. Method I and

II obeyed Beer’s law in the concentration range of 10 - 80µg/ml and 5 – 25µg/ml

respectively.

The present investigation by the author describes the development of a rapid, accurate

and precise RP-HPLC method for the determination of Pramipexole dihydrochloride

monohydrate in tablet dosage forms.

264

3) EXPERIMENTAL AND RESULTS a) MATERIALS AND METHODS Instrumentation

The author had attempted to develop a liquid chromatographic method for

simultaneous estimation of Pramipexole dihydrochloride. The separation of the analyte

was done by using a Gradient Waters Allaince HPLC instrument, on a Inertsil C8

column (250 x 4.6mm; 5µm). The instrument was equipped with a pump (2695),

injector, PDA Detector (2996) and column oven. Data acquisition was done by using

Empower software

Degassing of the mobile phase was done by using a Spectra lab model DGA

20A3 ultrasonic bath sonicator. A Sartorious electronic balance was used for weighing

the materials. Class ‘A’ Borosil glassware was employed for volumetric and general

purpose in the study.

Drugs

The reference sample of Pramipexole dihydrochloride was gifted by M/s

Wockhardt Pharmaceutical limited. The Branded formulations of Pramipexole

dihydrochloride (Mirapex tablets of Boehringer Ingelheim International GmbH) were

procured from the local market.

Reagents

Potassium dihydrogen phosphate : AR/GR grade

Octane-1 sulphonic acid sodium salt : AR/GR grade

Orthophosphoric acid (min 88%) : AR/GR grade

Concentrated hydrochloric acid (min. 35%) : AR/GR grade

Acetonitrile : HPLC grade

Methanol : HPLC grade

Water : Milli-Q / HPLC grade

Preparation of mobile phase A

9.1 g of potassium dihydrogen phosphate and about 5.0 g of Octane-1 sulphonic acid

sodium salt was dissolved in 1000 mL of water. The pH was adjusted to 3.0 (+0.05)

with orthophosphoric acid. This solution was filtered through a 0.45µm membrane

filter.

265

Preparation of mobile phase B

Acetonitrile used as a mobile phase B

Preparation of diluent-1

8.5 mL concentrated hydrochloric acid was diluted to 1000 mL with methanol and

mixed well.

Preparation of diluent-2

8.5 mL concentrated hydrochloric acid was diluted to 1000 mL with water and mixed

well.

Chromatographic conditions

Column : Inertsil C 8-3 (250 x 4.6mm; 5µm)

Flow rate : 1.5 mL/min

Wavelength : 264 nm

Injection volume : 50µL

Column Oven Temperature : 40°C

Runtime : 20 min

Gradient Program

Time (min) % Mobile phase A % Mobile phase B

0 80 20

8 67 33

11 67 33

12 80 20

20 80 20

Preparation of Blank

5.0 mL of diluent-1 was taken into 20 mL volumetric flask; volume made up to the

mark with diluent -2 and mixed well. The solution was filtered through a 0.45µ PTFE

membrane filter by discarding 5 mL of filtrate.

266

Preparation of working standard solution

About 60 mg of Pramipexole dihydrochloride monohydrate standard was accurately

weighed and transferred into a 100 mL volumetric flask, about 60 mL of the diluent-1

was added and the contents were sonicated to dissolve and further diluted to volume

with the diluent-1 and mixed well. 5.0 mL of this solution was diluted into a 50 mL

volumetric flask and volume made up to the mark with the the diluent-1 and mixed well.

Further 5.0 mL of this solution was diluted into a 20 mL volumetric flask and volume

made up with the diluent-2 and mixed well. The solution was filtered through a 0.45µ

PTFE membrane filter by discarding 5 mL of filtrate.

Preparation of Formulation sample solution

16 intact tablets of Pramipexole dihydrochloride monohydrate were taken into (Mirapex

tablets of Boehringer Ingelheim International GmbH) a 200 mL dry volumetric flask,

about 100.0 mL of the diluent-1was added by using 50 mL pipette and the contents were

stirred on magnetic stirrer for about 30 min. The solution was sonicated for 10 min with

intermittent vigorous shaking. The flask was cooled to room temperature. A portion of

this solution was centrifuged at about 5000 rpm for 5 min in stoppered centrifuge tube.

5.0 mL of this supernatant solution was diluted into a 20 mL volumetric flask with the

diluent-2 and mixed well. The solution was filtered through a 0.45µ PTFE membrane

filter by discarding 5 mL of filtrate.

b) OPTIMIZATION OF THE CHROMATOGRAPHIC

CONDITIONS AND METHOD DEVELOPMENT

For developing the HPLC method, a systematic study of the effect of various

factors for ideal separation of the drugs was undertaken. This was done by varying one

parameter at a time and keeping all other conditions constant. The following studies

were conducted for this purpose. A non-polar C8 column was chosen as the stationary

phase for this study.

267

The mobile phase and the flow rate

In order to get sharp peaks and good base line separation of the components,

the author carried out a number of experiments by varying the commonly used solvents,

their compositions and flow rate.

To find out the most suitable mobile phase to effect ideal separation of the drugs under

Gradient conditions, mixtures of commonly used solvents like water, methanol and

acetonitrile with or without different buffers in different combinations were tested as

mobile phases on a C8 stationary phase.

Mobile phase A: 9.1 g of potassium dihydrogen phosphate and about 5.0 g of Octane-1

sulphonic acid sodium salt was dissolved in 1000 mL of water. The pH of this solution

was adjusted to 3.0 (+0.05) with orthophosphoric acid. This solution was filtered

through a 0.45µm (or fine porosity) membrane filters and degassed.

Mobile phase B: Acetonitrile was used as a mobile phase B

A mobile phase flow rate of 1.5 mL/min was found to be suitable in the study

range of 0.5 -2.0 mL/min.

Detection wave length

The UV absorption spectrum of the drug was taken in methanol and the λ max

found to be at 264 nm. Hence, detection of the drug was made at 264nm.

Retention time of Pramipexole

A model chromatogram showing the separation of Pramipexole is presented in

Fig 2.6.1 Under the above optimized conditions a retention time of Pramipexole about

8.53 min.

After a thorough study of the various parameters the following optimized

conditions mentioned in Table 2.6.2 were followed for the determination of

Pramipexole bulk samples and Pharmaceutical formulations.

268

Fig 2.6.1 A Model Chromatogram showing the separation of Pramipexole

Table 2.6.2 Optimized Chromatographic Conditions

Parameter Value

Column Inertsil C8, (250 x 4.6mm; 5µm)

Mobile Phase Mobile phase A and Mobile phase B

(Gradient)

Flow Rate 1.5 mL/min

Run Time 20 min

Column Temperature 30±1 ˚C

Volume Of Injection 20 µL

Detection Wave Length 264 nm

Retention Time 8.53 min

269

c) VALIDATION OF THE PROPOSED METHOD The method was validated in compliance with ICH guidelines16-19. The

parameters determined for validation were specificity, precision, accuracy, robustness,

Linearity, Forced Degradation, Limit of Quantification and Limit of Detection, system

suitability and stability of analytical solution.

1. Specificity

The method specificity was assessed by comparing the chromatograms obtained from a

placebo solution containing a mixture of most commonly used excipients without the

drug and another solution containing the excpeints with the drug. These solutions were

prepared in the diluent. The drug to excipient ratio used was similar to that in the

commercial formulation. The commonly used excipients in formulations like lactose,

starch, microcrystalline cellulose, ethyl cellulose, hydroxyl propyl methylcellulose,

magnesium stearate and colloidal silicon dioxide were taken up for the study. The

mixtures were filtered through 0.45µ membrane filter before injection. The placebo

solution and the sample solution (placebo and the drug) were injected into HPLC

system separately in triplicate and the relevant chromatograms observed. There was no

interference from blank and placebo at the retention time of analyte peak. The absence

of additional peaks in the chromatogram indicates non interference of the commonly

used excipients in the tablets and hence the method is specific. The relevant

chromatograms are given in Fig 2.6.2, 2.6.3 and 2.6.4 for chromatograms of blank,

placebo and placebo with the drug sample solutions respectively.

Acceptance criteriaon

a) No interfering peak should appear at the retention time of Pramipexole peak from

blank and placebo. Peak purity for analyte peak should pass.

Conclusion

The proposed method is specific for determination of Pramipexole dihydrochloride

monohydrate in its formulation tablet dosage forms as method meets acceptance

criterion.

270

2. Forced degradation study

Forced degradation study was carried out by exposing the placebo and the formulation

sample of to the following conditions.

1. Treatment with hydrochloric acid.

2. Treatment with sodium hydroxide.

3. Treatment with hydrogen peroxide.

4. Thermal exposure.

5. Photolytic exposure.

6. Exposure to humidity

a) Acid degradation

Transferred 16 tablets of sample and Placebo powder equivalent to 6 mg into two

separate 200 mL volumetric flasks. 96 mL of diluent-1was added to each flask and

stir on magnetic stirrer for about 30 min, this solution was sonicated for 10 min with

intermittent shaking. To each flask added 2.0 mL of 10N hydrochloric acid and kept

at room temperature for about 3 hrs. After 3 hrs the resulting solution were

neutralized using 2.0 mL of 10N sodium hydroxide and mixed well. Centrifuged the

portion of solution at 5000 rpm for 5 min. 5.0 mL of this supernant solution was

diluted into a 20 mL with diluent-2 and mixed well. Filtered the solutions through a

0.45µ PTFE filter by discarding 5.0 mL of the filtrate.

b) Alkali degradation

Transferred 16 tablets of sample and Placebo powder equivalent to 6 mg into two

separate 200 mL volumetric flasks. 94 mL of diluent-1was added to each flask and stir

on magnetic stirrer for about 30 min, this solution was sonicated for 10 min with

intermittent shaking. To each flask added 3.0 mL of 10 N Sodium hydroxide solution

and kept at room temperature for about 3 hrs. After 3 hrs the resulting solution was

neutralized using 3.0 mL of 10N hydrochloric acid and mixed well. Centrifuged the

portion of solution at 5000 rpm for 5 min. 5.0 mL of this supernant solution was diluted

into a 20 mL with diluent-2 and mixed well. Filtered the solutions through a 0.45µ

PTFE filter by discarding 5.0 mL of the filtrate.

c) Peroxide degradation

Transferred 16 tablets of sample and Placebo powder equivalent to 6 mg in to two

separate 200 mL volumetric flasks. 99 mL of diluent-1 1was added to each flask and

stir on magnetic stirrer for about 30 min. This solution was sonicated for 10 min with

intermittent shaking. To each flask added 1.0 mL of 30% w/v Hydrogen peroxide

271

solution and mixed well. Immediately centrifuged the portion of solution at 5000 rpm

for 5 min. 5.0 mL of this supernant solution was diluted into a 20 mL with diluent-2

and mixed well. Filtered the solutions through a 0.45µ PTFE filter by discarding first

5.0 mL of the filtrate.

d) Thermal degradation

Transferred 16 tablets of sample and Placebo powder equivalent to 6 mg in to two

separate 200 mL volumetric flasks. Placebo and sample were exposed to heat at 80°C

for about 24 hr. 100 mL of diluent-1 1was added to each flask and stir on magnetic

stirrer for about 30 min.This solution was sonicated for 10 min with intermittent

shaking. Centrifuged the portion of solution at 5000 rpm for 5 min. 5.0 mL of this

supernant solution to 20 mL with diluent-2 and mixed well. Filtered the solutions

through 0.45µ PTFE filter by discarding 5.0 mL of the filtrate.

e) Photolytic degradation

Transferred 16 tablets of sample and Placebo powder equivalent to 6 mg into two

separate 200 mL volumetric flasks. Placebo and sample were exposed to photolytic

treatment for about 22 hrs (1.2 million lux hrs).100 mL of diluent-1 1was added to each

flask and stir on magnetic stirrer for about 30 min.This solution was sonicated for 10

min with intermittent shaking. Centrifuged the portion of solution at 5000 rpm for 5

min. 5.0 mL of this supernant solution was diluted into a 20 mL with diluent-2 and

mixed well. Filtered the solutions through a 0.45µ PTFE filter by discarding 5.0 mL of

the filtrate.

f) Humidity degradation

Transferred 16 tablets of sample and Placebo powder equivalent to 6 mg in to two

separate 200 mL volumetric flask. Placebo and sample were exposed to humidity at

40°C/75%RH for about 86 hrs 100 mL of diluent-1 1was added to each flask and stir on

magnetic stirrer for about 30 min, this solution was sonicated for 10 min with

intermittent shaking. Centrifuged the portion of solution at 5000 rpm for 5 min. 5.0 mL

of this supernant solution was diluted into 20 mL with diluent-2 and mixed well.

Filtered the solutions through a 0.45µ PTFE filter by discarding 5.0 mL of the filtrate.

The % Assay values with respect to untreated sample and peak purity data of

Pramipexole at each condition are tabulated in Table 2.6.3. Refer Fig 2.6.5 for

chromatograms and purity plots of untreated sample. Refer in Fig

2.6.6,2.6.7,2.6.8,2.6.9,2.6.10 and 2.6.11 for chromatograms and purity plots of Acid,

Alkali, Peroxide, Thermal, Photolytic and humidity treated sample solutions.

272

Acceptance criterion

Peak purity of analyte peak should pass.

Table 2.6.3 Forced degradation data

Sr. No.

Sample condition

%

Assay

% Degradation

w.r.t. Untreated

sample

Peak purity data for Pramipexole peak

Purity Angle

Purity Threshold

Purity Flag

1. Untreated sample* 103.60 - 0.077 0.283 No

2. Acid Treated

Treated with 2.0 mL of 10N HCl at room temperature for 3 hr.

102.38 1.18 0.262 0.678 No

3. Alkali Treated

Treated with 3.0 mL of 10N NaOH at room temperature for 3 hr.

104.06 0.00 0.260 0.668 No

4. Peroxide Treated

Treated with 1.0mL of 30% w/v hydrogen peroxide solution and immediately prepared

94.94 8.36 0.211 0.509 No

5. Thermal Treated

Exposed in oven at 80°C for 24 hr.

101.95 1.59 0.239 0.534 No

6. Photolytic Treated

Exposed to photolytic treatment for 22 hr. (1.2 million lux hr.)

101.61 1.92 0.268 0.605 No

7. Humidity Treated

Exposed at 40°C/75%RH for about 86 hr.

104.22 0.00 0.327 0.675 No

*Untreated sample data taken from method precision experiment. Conclusion

As the method meets acceptance criteria, it is concluded that the method is stability

indicating for determination of Pramipexole dihydrochloride monohydrate in

Pramipexole dihydrochloride extended release tablets.

273

3. Precision

3.1 System precision

Six replicate injections of standard solution were injected into HPLC system. Mean, SD

and % RSD were calculated for peak area counts of Pramipexole. The results are

tabulated in Table 2.6.4.

Acceptance criterion

% RSD should not be more than 2.0 for Pramipexole peak area counts.

Table 2.6.4 System precision data

.

Sr. No. Pramipexole peak area

counts (µV*sec)

1. 752200

2. 751520

3. 752795

4. 752618

5. 751847

6. 752348

Mean 752221

SD 476.4

% RSD 0.06

274

3.2 Method precision

Six sample preparations were made from a single batch and analyzed as per the

proposed method. % Assay of Pramipexole dihydrochloride monohydrate for six

samples was calculated. The results are tabulated in Table 2.6.5.

Acceptance criterion

% RSD for % Assay of six preparations should not be more than 2.0

Table 2.6.5 Method precision data

Sr. No.

Assay of Pramipexole

dihydrochloride monohydrate

1. 102.52

2. 103.07

3. 104.29

4. 103.74

5. 103.68

6. 104.30

Mean 103.60

SD 0.698

% RSD 0.67

275

3.3 Intermediate precision (Ruggedness)

Ruggedness of the method was verified by analyzing six sample preparations of same

batch used under method precision as per proposed method by different analysts using

different instrument and different column on different day.

The amount of Pramipexole dihydrochloride monohydrate for six samples was

determined. The %RSD for Assay and overall %RSD for above results of the method

precision were calculated. The results are tabulated in Table 2.6.6.

Acceptance criteria

% RSD for % Assay of six preparations should not be more than 2.0 and overall %RSD

should not be more than 2.0

Table 2.6.6 Ruggedness data

Sr. No.

Assay of Pramipexole dihydrochloride monohydrate

Method precision

Ruggedness

1. 102.52 101.87

2. 103.07 102.85

3. 104.29 103.67

4. 103.74 101.43

5. 103.68 102.43

6. 104.30 104.20

Mean 103.60 102.74

SD 0.698 1.056

% RSD 0.67 1.03

Overall Mean 103.17

Overall SD 0.964

Overall %RSD

0.93

Conclusion:

The proposed analytical method meets the acceptance criteria for precision. Hence, the

method is precise.

276

4. Accuracy

The placebo was spiked with known amounts of Pramipexole dihydrochloride

monohydrate API at about 50%, 100% and 150% of test concentration in triplicate at

each level. Amount of Pramipexole dihydrochloride monohydrate was quantified and %

recovery was calculated from amount found and actual amount added. Data is tabulated

in Table 2.6.7.

Acceptance criteria

% Recovery at each level should be between 98.0 to 102.0 and %RSD should not be

more than 2.0

Overall %recovery should be between 98.0 to 102.0 and %RSD should not be more than

2.0

Table 2.6.7 Accuracy data

Conclusion

The proposed analytical method meets acceptance criteria for recovery study. Hence,

the method is accurate and precise.

Spike level (%)

Actual Amount of Pramipexole

dihydrochloride monohydrate added (mg)

Amount of Pramipexole

dihydrochloride monohydrate found (mg)

% Recovery

Mean

SD

% RSD

50

3.0203 3.0230 100.09

100.05 0.038 0.04 3.0203 3.0208 100.02

3.0203 3.0213 100.03

100

6.0406 6.0330 99.87

100.31 0.398 0.40 6.0406 6.0800 100.65

6.0406 6.0645 100.40

150

9.0609 8.9983 99.31

99.67 0.338 0.34 9.0609 9.0355 99.72

9.0609 9.0590 99.98

Overall mean 100.01

Overall SD 0.381

Overall % RSD 0.38

277

5. Linearity

Linearity of response for Pramipexole dihydrochloride monohydrate was performed

using the standard solution in a range of 7.705 mcg/mL to 23.115 mcg/mL[about 50%

to 150% of the test concentration]. Results are tabulated in Table 2.6.8 and represented

graphically in Fig 2.6.12.

Acceptance criterion

Correlation coefficient (r) value should not be less than 0.99

Table 2.6.8 Linearity data

Conclusion

Response of Pramipexole dihydrochloride monohydrate was found linear in mentioned

range of 7.705 to 23.115 mcg/mL.

Linearity level (%)

Concentration in mcg/mL

Pramipexole average peak area counts

(µV*sec)

50 7.705 395416

60 9.246 471957

70 10.787 545932

80 12.328 632844

90 13.869 708402

100 15.410 787282

110 16.951 865870

120 18.492 941393

130 20.033 1024589

140 21.574 1099292

150 23.115 1180302

Slope 50996

Intercept 816

CC 0.99996

278

6. Stability in analytical solution

Stability of Pramipexole dihydrochloride monohydrate in analytical solution was

verified by analyzing sample solution initially and also at different time intervals up to

25 hrs and 46 min by storing sample solution at room temperature. Cumulative % RSD

for Pramipexole peak area counts was calculated. The results are tabulated in Table

2.6.9.

Acceptance criteria

Cumulative %RSD should not be more than 2.0 for Pramipexole peak area counts at

each time interval.

Table 2.6.9 Stability in analytical solution data

Time Pramipexole peak

area counts (µV*sec) Cumulative

% RSD

Initial 780458 -

22 Min 780744 0.03

41 Min 780736 0.02

02 Hr 06 Min 780756 0.02

03 Hr 52 Min 780594 0.02

07 Hr 24 Min 781658 0.05

11 Hr 38 Min 781822 0.07

15 Hr 52 Min 782302 0.09

21 Hr 49 Min 782257 0.10

25 Hr 46 Min 781627 0.09

Conclusion

It was found that the solution is stable up to 25 hrs at room temperature and hence, it is

concluded that the proposed analytical method meets the pre-established acceptance

criteria,

279

7. Robustness

To evaluate its robustness, following small deliberate variations were made in the

method. The samples were analyzed in triplicate for each parameter.

1. Flow rate (±10%)

2. Organic content variation in mobile phase (±2% absolute by gradient change)

3. Column oven Temperature (± 5°C)

4. Wavelength (± 5 nm)

5. pH of mobile phase (± 0.1 unit)

System suitability was evaluated in each condition and results were compared with

method precision results. Results are tabulated in Table 2.6.10.

Acceptance criterion

Overall %RSD should not be more than 2.0 for individual experiment.

Table 2.6.10 Robustness data

Sr.No. M.P. -Flow +Flow -Temp +Temp - nm + nm - pH + pH -Org +Org

1 102.52 102.71 102.66 103.15 103.10 102.47 102.43 101.75 102.10 102.19 102.12

2 103.07 101.93 101.89 102.34 102.24 103.04 103.04 103.31 103.73 103.26 103.33

3 104.29 102.75 102.76 103.08 103.07 104.26 104.27 102.17 102.99 103.52 103.71

4 103.74 - - - - - - - - - -

5 103.68 - - - - - - - - - -

6 104.30 - - - - - - - - - -

Overall mean 103.22 103.21 103.35 103.33 103.49 103.48 103.20 103.38 103.40 103.42

Overall SD 0.825 0.837 0.702 0.723 0.737 0.745 0.907 0.762 0.723 0.743

Overall % RSD

0.80 0.81 0.68 0.70 0.71 0.72 0.88 0.74 0.70 0.72

280

M.P. Method precision data

-Flow Flow rate (1.35 mL/minute)

+Flow Flow rate (1.65 mL/minute)

-Temp Column oven temperature (35°C)

+Temp Column oven temperature (45°C)

-nm Wavelength (259 nm)

+nm Wavelength (269 nm)

-Org Organic content variation in mobile phase (- 2% absolute by gradient change)

+Org Organic content variation in mobile phase (+ 2% absolute by gradient change)

- pH pH of buffer in mobile phase (2.90)

+ pH pH of buffer in mobile phase (3.10)

Conclusion

As method meets acceptance criteria, the method considered to be robust for changes in

flow rate, column oven temperature, wavelength, pH of buffer in mobile phase and

organic content variation in mobile phase.

8. Limit of Detection and Limit of Quantification

Limit of detection (LOD) is defined as the lowest concentration of analyte that gives a

measurable response. LOD is determined based on signal to noise ratio (S/N) of three

times typically for HPLC methods.The limit of quantification (LOQ) is defined as the

lowest concentration that can be quantified reliably with a specified level of accuracy

and precision. It is the lowest concentration at which the precision expressed by an RSD

of less than 2%. In this study the analyte response is 10 times greater than the noise

response. For this study six replicates of the analyte at lowest concentration in the

calibration range were measured and quantified. The LOD and LOQ of Pramipexole

obtained by the proposed method were 0.035 and 0.122 µg/mL respectively

281

9. Summary of system suitability

System suitability was evaluated by injecting standard solution during different days of

validationand monitoring tailing factor and theoretical plates for different parameters.

The % Relative standard deviation for the peak area counts of Pramipexole from five

replicate injections of standard solution were verified during every experiment. The

results are tabulated in Table 2.6.11.

Acceptance criteria

1. Column efficiency determined for the Pramipexole peak in standard solution should

be not less than 15000 theoretical plates and tailing factor for the same peak should be

not more than 2.0

2. Percentage relative standard deviation for peak area counts of Pramipexole from

five replicate injection of standard solution should be not more than 2.0

Table 2.6.11 Summary of system suitability data

Sr. No.

Name of experiment

Theoretical Plate counts of Pramipexole

peak

Tailing factor of Pramipexole

peak % RSD

1 System precision, Method precision

58292 1.0 0.07

2 Robustness (- Wavelength) 58262 1.0 0.09

3 Robustness (+ Wavelength) 58310 1.0 0.07

4 Solution stability 58979 1.0 0.04

5 Linearity 80377 1.0 0.32

6 Specificity 55419 1.0 0.34

7 Recovery 78255 1.0 0.07

8 Robustness (- Flow Rate) 63765 1.0 0.10

9 Robustness (+ Flow Rate) 53835 1.0 0.03

10 Robustness (- Temperature) 56499 1.0 0.05

11 Robustness (+ Temperature) 59090 1.0 0.06

12 Ruggedness 72940 1.0 0.10

13 Robustness (- pH) 55263 1.0 0.06

14 Robustness (+ pH) 55358 1.0 0.10

15 Robustness (-Organic) 94705 1.0 0.04

16 Robustness (+Organic) 53807 1.0 0.03

17 Forced Degradation-1 55590 1.0 0.15

18 Forced Degradation-2 54932 1.0 0.19

282

4) SUMMARY OF THE RESULTS AND CONCLUSION

The present study was aimed at developing a simple, precise and accurate HPLC

method for the analysis of pramipexole dihydrochloride monohydrate from tablet

dosage forms. A non-polar C8 analytical chromatographic column was chosen as the

stationary phase for the separation and determination of Pregabalin. For the selection of

the mobile phase a number of eluting systems were examined. Mixtures of commonly

used solvents like water, and acetonitrile with or without different buffers in different

combinations were tested as mobile phases on a C8 stationary phase. The choice of the

optimum composition is based on the chromatographic response factor, a good peak

shape with minimum tailing. A buffer mixture containing 9.1 g of potassium dihydrogen

phosphate and about 5.0 g of Octane-1 sulphonic acid sodium salt was dissolved in

1000 mL of water. The pH was adjusted to 3.0 (+0.05) with orthophosphoric acid and

acetonitrile with the gradient program was proved to be the most suitable of all the

combinations since the chromatographic peak obtained was better defined and resolved

and almost free from tailing. The retention time of the drug was found at 8.53 min.

Summary of Validation:

Specificity

No interfering peak was observed at the retention time of Pramipexole dihydrochloride

monohydrate from blank and placebo samples. Thus, the Peak purity for the analyte

passed.

Hence, it is concluded that method is specific for determination of Pramipexole

dihydrochloride monohydrate in Pramipexole dihydrochloride extended release tablets.

Forced Degradation

Forced degradation study was carried out by subjecting the placebo and sample to the

following conditions.

1. Treatment with hydrochloric acid.

2. Treatment with sodium hydroxide.

3. Treatment with hydrogen peroxide.

4. Thermal exposure.

5. Photolytic exposure.

6. Exposure to humidity

283

In all the above conditions, met the acceptance criterion for peak purity. Hence, the

method can be considered as stability indicating for determination of Pramipexole

dihydrochloride monohydrate in Pramipexole dihydrochloride extended release tablets.

Observation: During Force Degradation study it was observed that Pramipexole

dihydrochloride monohydrate is sensitive for Peroxide degradation.

System Precision

The % RSD of Pramipexole peak area from six replicate injections of Standard solution

was less than 2.0 and it meets the acceptance criterion.

Method Precision

Six samples from a single batch were prepared and analyzed as per test method. % RSD

for % assay of Pramipexole dihydrochloride monohydrate was calculated for six

preparations. The % RSD for % assay of Pramipexole dihydrochloride monohydrate

was less than 2.0 and meets the acceptance criteria.

Ruggedness

Six samples from a single batch (same batch used under Method precision) were

prepared and analysed as per test method by different analyst by using different column,

different HPLC system on different day.

% RSD for % assay of Pramipexole dihydrochloride monohydrate was calculated for six

preparations.

% RSD for % assay of Pramipexole dihydrochloride monohydrate was less than 2.0 and

meets the acceptance criteria.

Overall % RSD for % assay of Pramipexole dihydrochloride monohydrate obtained

from ruggedness and method was less than 2.0 and meets the acceptance criteria.

The proposed analytical method meets acceptance criteria for precision. Hence the

method is precise.

Accuracy (Recovery)

The sample solutions were prepared at each level by spiking the placebo with

Pramipexole dihydrochloride monohydrate API with at about 50%, 100 % and 150 % of

test concentration. The % Recovery at each level was calculated.

284

Analytical method meets acceptance criteria for recovery study. Hence, the method is

accurate and precise.

Linearity

Linearity range for Pramipexole dihydrochloride monohydrate was determined using

solutions containing about 50% to 150% of test concentration. It was found that

response for Pramipexole dihydrochloride monohydrate was linear in the range of 7.705

mcg/mL to 23.115 mcg/mL and the relevant correlation coefficient value is more than

0.99.

Stability in analytical solution

By analyzing the sample solution, at different time intervals, stability of the drug in

analytical solution was carried out. It was found that sample solution was stable up to

25hrs at room temperature.

Robustness

Robustness of analytical method was carried out by deliberately varying optimized

chromatographic conditions of flow rate (+10%), column oven temperature (±5°C),

wavelength (±5nm) and organic content of mobile phase (+2% absolute by gradient

change), change in pH of buffer in mobile phase (± 0.1unit). The method is robust for

change in organic content in mobile phase, change in flow rate, change in column oven

temperature, change in pH of mobile phase and change in wavelength, as method meets

acceptance criteria

Limit of detection and Limit of Quantification

The lowest values of LOD and LOQ as obtained by the proposed method indicate the

method is sensitive.

Conclusion

The validation data proves that the proposed method for determination of Pramipexole

dihydrochloride monohydrate in Pramipexole dihydrochloride extended release tablets

is specific, precise, accurate, robust and linear under the given conditions of

methodology and is suitable for use.

285

Fig 2.6.2: HPLC Chromatogram of blank

286

Fig 2.6.3: HPLC Chromatogram of placebo solution

287

Fig 2.6.4: HPLC Chromatogram and purity plot of placebo with drug sample

solution

288

Fig 2.6.5: HPLC Chromatogram and purity plot of untreated sample

289

Fig 2.6.6: HPLC Chromatogram and purity plot of acid treated sample

290

Fig 2.6.7: HPLC Chromatogram and purity plot of alkali treated sample

291

Fig 2.6.8: HPLC Chromatogram and purity plot of peroxide treated sample

292

Fig 2.6.9: HPLC Chromatogram and purity plot of thermal treated sample

293

Fig 2.6.10: HPLC Chromatogram and purity plot of photolytic treated sample

294

Fig 2.611: HPLC Chromatogram and purity plot of humidity treated sample

295

Fig 2.6.12: Linearity plot for Pramipexole dihydrochloride monohydrate

Linearity level (%)

Concentration (mcg/mL)

Pramipexole average peak area counts (µV*sec)

50 7.705 395416

60 9.246 471957

70 10.787 545932

80 12.328 632844

90 13.869 708402

100 15.410 787282

110 16.951 865870

120 18.492 941393

130 20.033 1024589

140 21.574 1099292

150 23.115 1180302

Slope 50996

Intercept 816

CC 0.99996

296

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