217 CHAPTER- 5 2.5 DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLCMETHOD FOR THE DETERMINATON OF PREGABALIN IN ITS CAPSULES DOSAGE FORMS CONTENTS 1. Drug profile 2. Review of the past work on the analytical methods for Pregabalin. 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
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217
CHAPTER- 5
2.5 DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLCMETHOD FOR THE DETERMINATON OF PREGABALIN IN ITS CAPSULES DOSAGE FORMS
CONTENTS
1. Drug profile
2. Review of the past work on the analytical methods for Pregabalin.
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
218
1. DRUG PROFILE- PREGABALIN
Pregabalin1-6 is an anticonvulsant drug for neuropathic pain. It is also an adjunct therapy
for partial seizures, and it is reported as being used for general anxiety disorder and for
treatment of epilepsy. neuropathic pain. It was designed as a more potent successor to a
related drug, gabapentin. Pregabalin binds to the alpha2-delta subunit of the voltage-
gated calcium channel in the central nervous system. While pregabalin is a structural
derivative of the inhibitory neurotransmitter gamma- aminobutyric acid (GABA), it
does not bind directly to GABAA, GABAB, or benzodiazepine receptors, does not
augment GABAA responses in cultured neurons, does not alter rat brain GABA
concentration or have acute effects on GABA uptake or degradation. However, in
cultured neurons prolonged application of pregabalin increases the density of GABA
transporter protein and increases the rate of functional GABA transport. Pregabalin does
not block sodium channels, is not active at opiate receptors, and does not alter
cyclooxygenase enzyme activity. It is inactive at serotonin and dopamine receptors and
does not inhibit dopamine, serotonin, or noradrenaline reuptake
Mechanism of Action
Pregabalin binds with high affinity to the alpha2-delta site (an auxiliary subunit of
voltage-gated calcium channels) in central nervous system tissues. Although the
mechanism of action of pregabalin is unknown, results with genetically modified mice
and with compounds structurally related to pregabalin (such as gabapentin) suggest that
binding to the alpha2-delta subunit may be involved in pregabalinís antinociceptive and
antiseizure effects in animal models. In vitro, pregabalin reduces the calcium-dependent
release of several neurotransmitters, possibly by modulation of calcium channel
function.
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Chemical Structure:
Chemical name (IUPAC ) : (S)-3-(aminomethyl)-5-methylhexanoic
Chemical formula : C8H17 NO2
Molecular weight : 159.23
Physical state : A white crystalline powder
Melting point : 186-1880 C
Solubility : Slightly solublw in Ethanol, DMSO and Soluble
in Phosphate buffer
Official Status of the drug :The drug is official in Merck Index
Table 2.5.1 Important brand names of Pregabalin formulations
S.No
Brand
Name Company Composition Packing
1 Gabanext ABBOTT HC Pregabalin 75mg 10 SG-acp
Pregabalin 150mg 10 SG-acp
2 Galinerve SUN (ARIAN) Pregabalin 75mg 10 cap
Pregabalin 150mg 10 cap
3
Nervup-
PG
ABBOTT HC
Each hard gelatin cap
cont:Pregabalin 75mg,
Methylcobalamin 750 mcg,
Alpha lipoic acid100mg 10 cap
(as 2 film coated tablets each
contain 50 mg of Alpha lipoic
acid. Colour:Quinoline yellow
4 Nuramed ZYDUS (CND) Pregabalin 75mg 10 cap
Pregabalin 150mg 10 cap
5 Pregamet ABBOTT HC
Pregabalin 75mg,
Methylcobalamin 750 mcg 10 cap
6 Pregatar LUPIN(PINNACLE)
Pregabalin 75mg 10 cap
Pregabalin 150mg 10 cap
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2. Review of the past work on the analytical methods for Pregabalin
T.A.C. Vermeij et al 7 , proposed a HPLC method for simultaneous determination of
the γ-amino-n-butyric acid (GABA) derivatives pregabalin (PGB), gabapentin (GBP)
and vigabatrin (VGB) in human serum. Separation is achieved on a Alltima 3C18
column using isocratic elution; the drugs are monitored using fluorescence detection.
Norvaline is used as an internal standard. Within-day precision (COV; n = 10) is 1.2%
for PGB (serum concentration 10.0 mg/l), 1.1% for GBP (serum concentration 15.8
mg/l) and 0.3% for VGB (serum concentration 15.5 mg/l). The method is linear up to at
least 63 mg/l for PGB, 40 mg/l for GBP and 62 mg/l for VGB. Lower limits of
quantitation (LOQ) are 0.13 mg/l for PGB, 0.53 mg/l for GBP and 0.06 mg/l for VGB.
Berry et al 8, proposed a HPLC method for determination of pregabalin in
serum/plasma. Using C8 column The assay is calibrated over the range 0.5 mg/L to 8
mg/L. concentration measurements in predose samples from a group of patients with
dose escalated to 600 mg/d pregabalin are presented. The drug concentrations measured
were in the range 2.8-8.2 mg/L at steady state.
A. S. Jadhav et al 9, proposed a HPLC method for the determination of pregabalin in
bulk drugs using reversed-phase ODS column with a 60:40 (v/v) mixture of aqueous
0.2% triethylamine (pH adjusted to 3.5 with dilute orthophosphoric acid) and
acetonitrile as mobile phase. Concentration over the range 750 (LOQ) to
7,500 ng L−1 for the Renantiomer. The limits of detection and quantification of
the R enantiomer were 250 and 750 ng L−1, respectively, for an injection volume of
10 µL. Recovery of the R enantiomer from bulk drug samples of pregabalin ranged from
97.5 to 101.76%. Solutions of pregabalin in water and in the mobile phase were found
to be stable for at least 48 h.
Yizhong Zhang et al 10, proposed a method for direct chiral separation of pregabalin
from its R-enantiomer and HPLC/MS/MS assays have been validated to support isolated
perfused rat kidney studies. The separation was developed through serial coupling of
various macrocyclic glycopeptide stationary phases until partial separation of the
enantiomers was achieved. Identification of the resolving stationary phase followed by
221
optimization of the mobile phase enabled the baseline resolution of the enantiomers
using mass spectrometry compatible solvents and modifiers.
Önal Armağan et al 11, proposed a spectrophotometric method for determination of
pregabalin (Pgb) in pharmaceutical preparations. The method is based on the reaction of
Pgb as n-electron donors with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and
7,7,8,8-tetracyanoquinodimethane (TCNQ) as π-acceptors to give highly colored
complex species. The colored products were quantitated spectrophotometrically at 494
and 841 nm for DDQ and TCNQ, respectively. Optimization of the different
experimental conditions was conducted. Beer's law was obeyed in the concentration
ranges 2.0–30.0 and 1.5–10 g·mL−1 for DDQ and TCNQ methods, respectively. The
third method is based on the interaction of ninhydrin (NN) with primary amine present
in the pregabaline. This reaction produces a blue coloured product in N,N-
dimethylformamide (DMF) medium, which absorbs maximally at 573 nm. Beer's law
was found in the concentration range 40.0–180.0 µg·mL−1.
Ramakrishna Nirogi et al 12, proposed a HPLC positive ion atmospheric pressure
chemical ionization tandem mass spectrometry method for the quantification of
pregabalin in human plasma. Following liquid–liquid extraction, the analyte was
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 160–142 for pregabalin and m/z 482–258 for the internal standard.
The assay exhibited a linear dynamic range of 1–10,000 ng/mL for pregabalin in human
plasma. The lower limit of quantification was 1 ng/mL with a relative standard
deviation of less than 11.4%.
M. N. Farooqui et al 13, proposed a HPLC for the determination of pregabalin in
capsule dosage form. Using Hypersil BDS, C8, 150×4.6 mm, 5 µm column,
photodiode array detector. The mobile phase consisting of phosphate buffer pH 6.9 and
acetonitrile in the ratio of 95:05 with flow rate of 1 ml/min. Lower limit of
quantification is 0.6 mg/l. The sample solution was stable at room temperature for about
26 h.
M. I. Walash et al 14 , proposed a spectrofluorimetric method for the determination of
pregabalin (PG) in capsules. The method is based on the reaction between pregabalin
222
and fluorescamine in borate buffer solution of pH 10 to give a highly fluorescent
derivative that is measured at 487 nm after excitation at 390 nm. The fluorescence
intensity concentration plot was rectilinear over the range of 0.01–0.3µg/mL−1 with a
lower detection limit of 0.0017µg/mL−1 and limit of quantitation of 0.005µg/mL−1. The
developed method was successfully applied to the analysis of the drug in its commercial
capsules. The mean percentage recovery of PG in its capsule was 99.93±1.24 (n = 3).
Ashu M et al 15, proposed a RP-HPLC method for the determination of pregabalin in
the capsule dosage form. Stationary phase as waters spherisorb 5µ ODS 24.6mm x
250mm column using a mobile phase of acetonitrile:buffer (30:70 V/V) at a flow rate of
1ml/min with detection of analyte at 210 nm. The retention time for pregabalini is
3.1±0.3 min. Peak width 5.26s and SD 1.3152 for the sample peak. Linearity in the
range of 200-800 µg/ml. The intra and inter day R.S.D ranged from 0.79-1.85%. The
recovery (mean ±S.D.) of low, middle and high concentrations were 100.02± 0.80,
100.05 ± 0.42, 100.03 ± 0.35 respectively.
The present investigation by the author describes the development of a rapid, accurate
and precise RP-HPLC method for the determination of Pregabalin in capsule dosage
forms
223
3) EXPERIMENTAL AND RESULTS
a) MATERIALS AND METHODS Instrumentation
The author had attempted to develop a liquid chromatographic method for
simultaneous estimation of Pregabalin. The separation of the analyte was done by using
an isocratic Agilent HPLC instrument, on a Grace Kromasil C8 column (150 x 4.6mm;
5µ).The instrument was equipped with a pump (G1311A), injector, DAD (G13158)
Detector and column oven. Data acquisition was done by using Agilent 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 Pregabalin was gifted by M/s LUPIN Ltd. The samples of
branded formulations of pregabalin (Pregastar capsules of Lupin) were procured from
the local market.
Reagents
Potassium dihydrogen phosphate : GR grade
Potassium hydroxide pellets : GR grade
Acetonitrile : HPLC grade
Water : Milli-Q / HPLC grade
Preparation of 5N Potassium hydroxide solution
28g of Potassium hydroxide pellets was dissolved in 100 mL of water.
Preparation of Buffer
1.2g of Potassium dihydrogen phosphate was dissolved in 1000 mL of water. The pH
was adjusted to 6.7 ± 0.05 with 5N Potassium hydroxide solution. This solution was
filtered through a 0.45µm membrane filter.
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Preparation of Mobile phase:
The above buffer solution and acetonitrile were mixed in the ratio of 970: 30 (v/v),
filtered and degassed.
Diluent:
The above buffer used as a diluent also in preparing drug solutions.
Preparation of working standard solution:
About 50mg of Pregabalin standard was accurately weighed and transferred into a 50
mL volumetric flask, about 30 mL of the diluent was added and sonicated to dissolve,
dilute to volume with the diluent. The working standard solution containing 1000
mcg/mL. The solution was filtered through a 0.45µm Nylon membrane filter.
Preparation of formulation sample solution
Determined 20 capsules average fill weight of the Pregabalin Capsules (Pregastar of
Lupin Pharmaceutical Ltd.).A quantity of equivalent to 100 mg of Pregabalin was
transferred into a 100 mL volumetric flask, about 60 mL of the diluent was added and
the contents were sonicated for about 30 min with intermittent shaking. The flask was
cooled to room temperature and the solution was made up to the volume with the
diluent and mixed well. The solution was filtered through a 0.45µ Millipore nylon
membrane filter.
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.
The mobile phase and the flow rate
In order to get sharp peak 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.
225
To find out the most suitable mobile phase to effect ideal separation of the drug
under isocratic 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. 1.2 g of potassium dihydrogen phospahate
was dissolved into 1000 mL of water and the pH was adjusted to 6.7 (±0.05) with 5N
potassium hydroxide. Buffer and acetonitrile in a ratio of 97:3 v/v was proved to be the
most suitable of all the combinations since the chromatographic peaks obtained were
better defined and resolved and almost free from tailing.
A mobile phase flow rate of 1.0 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 200 nm. Hence detection of the drug was made at 200 nm.
Retention time of Pregabalin
A model chromatogram showing the separation of Pregabalin is presented in Fig
2.5.1. Under the above optimized conditions retention time of Pregabalin was obtained
at about 4.75 min.
After a thorough study of the various parameters the following optimized
conditions mentioned in Table 2.5.2 were followed for the determination of Pregabalin
bulk samples and pharmaceutical formulations.
226
Fig 2.5.1 A Model Chromatogram showing the separation of Pregabalin peak
Table 2.5.2 Optimized Chromatographic Conditions
Parameter Value
Column Grace Kromasil C8 (150 x 4.6mm;
5µ)
Mobile Phase Buffer(pH 6.7):acetonitrile
(97:03)
Flow Rate 1.0 mL/min
Run Time 8 min
Column Temperature 30±1 ˚C
Volume Of Injection 20 µL
Detection Wave Length 200 nm
Retention Time 4.75 min
227
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,