Studies - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/23805/8/08_chapter 2.pdf · Preformulation Page 46 Introduction Preformulation testing is the first step in the rational

Chapter 2

Preformulation

Studies

Chapter 2

Preformulation Page 46

Introduction

Preformulation testing is the first step in the rational development of dosage forms of

a drug substance. In preformulation physical and chemical properties of a drug

substance alone and when combined with excipients are investigated. These studies

commence when a newly synthesized drug molecule shows sufficient

pharmacological promise in animal models to warrant evaluation in man.The overall

objective of preformulation testing is to generate information useful to the formulator

in developing stable and bioavailable dosage forms which can be mass-produced.

During the early development of a new drug substance, the synthetic chemist, alone or

in cooperation with specialists in other disciplines, may record some data which can

be appropriately considered as preformulation data (Lakings et al., 1995).

Active drug Gabapentin and all excipients were standardized for their

physicochemical properties alone and in mixture

2.1 DRUG PROFILE

Generic name: Gabapentin

Brand names: Gralise, Horizant, Neurontin, Gabarone

Chemical Abstracts Registry No.: 60142-96-3

IUPAC Name: 2-[1-(aminomethyl)cyclohexyl]acetic acid

Molecular Formula: C9 H17NO2

Molecular weight: 171.24.

2.1.1 PHYSICOCHEMICAL PROPERTIES OF GABAPENTIN

Appearance: White to off-white crystalline solid

Dissociation constant: pKa1- 3.7, pKa2- 10.7

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

Partition coefficient (Log P): (n-octanol/0.05M phosphate buffer) at pH 7.4 is -1.25

http://www.chemindustry.com/chemicals/554179.html

Chapter 2


2.1.2 PHARMACOLOGY

2.1.2.1 Indications and Usage

Gabapentin is used primarily for the treatment of seizures, neuropathic pain, and hot

flashes. There are, however, concerns regarding the quality of the research on its use

to treat migraines, bipolar disorders, and pain (Vedula et al., 2009).

Pain

Gabapentin provides significant pain relief in about a third of people who take it for

fibromyalgia or chronic neuropathic pain (Moore et al., 2011). It is also effective in

reducing narcotic usage post operatively (Ho et al., 2006) and is helpful in

neuropathic pain due to cancer (Bar et al., 2010). It has not been shown to be useful

for HIV associated sensory neuropathy (Philips et al., 2010). When used for

neuropathic pain it does not appear superior to carbamazepine (Wiffen et al., 2005).

Further evidence is needed to determine if it is effective for migraine prevention

(Mulleners et al., 2008). It appears to be equally effective as pregabalin and is of

lower cost (Finnerup et al., 2010).

Seizures

Gabapentin is approved for treatment of focal seizures in a number of countries

(Johannessen et al., 2006) and evidence supports its use for treating partial and

mixed seizure disorders with and without secondary generalization in patients over 12

years of age with epilepsy however there is insufficient evidence for its use in

generalized epilepsy (French et al., 2004). It is also indicated as adjunctive therapy in

the treatment of partial seizures in pediatric patients age 3 ¾ 12 years.

Other

There is some evidence of benefit in acquired pendular nystagmus and infantile

nystagmus but not in periodic alternating nystagmus (McLean et al., 2009; Strupp,

et al., 2009). Gabapentin may help with menopausal symptoms (Cheema et al.,

2007). It may be effective in reducing pain and spasticity in multiple sclerosis

(Mack et al., 2003) Gabapentin is not supported for alcohol withdrawal, (Prince et

al., 2008) and treatment of smoking cessation have had mixed results (Sood et al.,

2007; Sood et al., 2010). Gabapentin helps with itching due to renal failure, known as

uremic pruritus. (Berger et al., 2011)

Chapter 2


2.1.2.2 Mechanism of action

Gabapentin is a gamma-aminobutyric acid analogue, is structurally related to the

neurotransmitter GABA (gamma-aminobutyric acid) but it does not modify GABA or

GABAB radioligand binding, it is not converted metabolically into GABA or a

GABA agonist, and it is not an inhibitor of GABA uptake or degradation.

Its site of action is alpha2-delta (α2-δ) protein, an auxiliary subunit of voltage-gated

calcium channels. It reduces the synaptic release of several neurotransmitters,

apparently by binding to (α2-δ) subunits, and possibly accounting for its actions in

vivo to reduce neuronal excitability and seizures.

2.1.2.3 Dosage and Administration

Gabapentin is given orally with or without food

Postherpeutic Neuralgia: In adults with postherpeutic neuralgia, gabapentin

therapy may be initiated as a single 300-mg dose on Day 1, 600 mg/day on Day 2

(divided BID), and 900 mg/day on Day 3 (divided TID). The dose can

subsequently be titrated up as needed for pain relief to a daily dose of 1800 mg

(divided TID). In clinical studies, efficacy was demonstrated over a range of

doses from 1800 mg/day to 3600 mg/day with comparable effects across the dose

range. Additional benefit of using doses greater than 1800 mg/day was not

demonstrated.

Epilepsy: Gabapentin is recommended for add-on therapy in patients 3 years of

age and older. Effectiveness in pediatric patients below the age of 3 years has not

been established.

Patients >12 years of age: The effective dose of gabapentin is 900 to 1800 mg/day and

given in divided doses (three times a day) using 300 or 400 mg capsules, or 600 or

800 mg tablets. The starting dose is 300 mg three times a day. If necessary, the dose

may be increased using 300 or 400 mg capsules, or 600 or 800 mg tablets three times

a day up to 1800 mg/day. Dosages up to 2400 mg/day have been well tolerated in

long-term clinical studies. Daily maintenance doses should be given in 3 equally

divided doses, and the maximum time between doses in a 3 times daily schedule

should not exceed 12 hours.

Chapter 2


2.1.2.4 Pharmacokinetics

Pharmacokinetic parameters of GBP are as stated below:

Volume of distribution 1.0 (L/kg)

Half-life 5–7 (hrs)

Plasma clearance 0.120-0.130 (l/kg/hr)

tmax 2 to 4 (hrs)

Cmax 4 (mcg/ml)

AUC 30 (mcg.hr/ml)

% Protein binding < 3

% Excreted unchanged in urine ≈100

Absorption: Absorption is rapid. Gabapentin is absorbed in part by the L-amino acid

transport system, which is a carrier-mediated, saturable transport system; as the dose

increases, bioavailability decreases. Gabapentin bioavailability is not dose dependent.

Bioavailability of gabapentin is approximately 60%, 47%, 34%, 33%, and 27%

following 900, 1200, 2400, 3600, and 4800 mg/day given in 3 divided doses,

respectively. Plasma gabapentin concentrations are dose-proportional at doses of 300

to 400 mg q8h, ranging between 1 µg/mL and 10 µg/mL, but are less than dose-

proportional above the clinical range (>600 mg q8h). There is no correlation between

plasma levels and efficacy. Gabapentin pharmacokinetics is not affected by repeated

administration, and steady-state plasma concentrations are predictable from single-

dose data.

2.1.2.5 Adverse actions

The most commonly observed adverse events associated with the use of gabapentin

were somnolence, dizziness, ataxia, fatigue, nystagmus and tremor.

2.2 Standardization of Drug: Gabapentin

Gabapentin was purchased from Sashun chemicals Ltd., Cuddalore. The drug was

screened and tested for the following parameters as per monographic specifications

and Certificates of Analysis. Tables 2.1 illustrate various tests, observations and

specifications for drugs. It was standardized as per USP and purity and identity were

checked with Certificate of Analysis provided by supplier. Gabapentin was tested for

the following.

Chapter 2


Appearance: Color of drug was observed visually.

Solubility: Solubility was checked in alcohol, methanol, and phosphate buffer of

different pH.

Identification tests: Infrared spectrum of drugs was investigated using FTIR

Infrared Spectrophotometer using potassium disk method. Spectrum was scanned

over the wave number range 4000-400 cm-1

.

Loss on drying: Drug (1gm) was weighed and dried in an oven at 100°C- 105°C

to constant weight for 4 hours. The weight was again recorded.

Melting point: This was determined using melting point testing apparatus.

UV spectrum: Ultraviolet spectrum of drug was taken using JASCO Ultraviolet

Spectrophotometer in phosphate buffer pH 6.8, as a solvent. Spectrum was

scanned over wavelength range of 200-400 nm.

Assay: Percent drug content was considered as mentioned in Certificate of

Analysis of drug obtained from the suppliers and confirmed by the analytical

method described in later section.

The results of the tests performed are mentioned in Table 2.1.1

Table 2.1.1: Monographic evaluation of Gabapentin

Tests Specifications Results

Appearance White crystalline powder Conforms

Identification By IR, To match with

working standard

The IR spectrum of gabapentin

have absorption peaks at (cm-1

)

1615,1546,1476,1420,1300,1165,

1091, 1080, 1064, 976,928, 922,

890, 749 and 709 (Fig 2.1.1)

Solubility Soluble in water,

sparingly soluble in

methanol

Complies

Residual

solvents

Meets the requirements Complies

Melting point 163°C 163-165°C

Assay 98.5-101.0% 99.17%

Lactam

impurity

0.03% (w/w) Not more than 0.5%w/w

Chapter 2


Fig 2.1.1: Certificate of Analysis (Shasun Chemicals and Drugs Ltd.)

Chapter 2


Fig 2.1.2: Infrared spectrum of gabapentin

2.2 Standardization of Polymers

The choice of the excipients depends on several factors; namely, the drug used, the

process involved, the formulator and the cost of excipients. All the excipients used in

the formulation development were procured from authentic vendors and Certificates

of Analysis were obtained for the same. Some of the important tests were performed

as per monographs and Certificate of Analysis to confirm the quality of the excipients.

Majority of the excipients used for the formulation development work are as listed in

Table 2.1.2-2.1.4

Chapter 2


Table 2.1.2: List of Excipients used in Formulation Development

Name of the Excipient Source Use Sucralose Gangwal Chemicals Sweetener Xylitol Lactitol Mannitol Glyceryl Monostearate Fine organics Ltd.,

Mumbai, India Lipid carrier for melt

granulation Cetyl Alcohol Loba Chemicals, Mumbai Lipid carrier for melt

granulation Polyox WSR 10 & 90 Dow Chemical Company Binder Polyox WSR 90 Microcrystalline cellulose

101,102,105 & 113 Signet Chemical

Corporation Pvt. Ltd. Diluent

Kollidon® 30 BASF Binder Polyvinyl Alcohol S. D. Fine Chemicals Viscosity builder Triehyl citrate Sigma Aldrich Solvent Propylene Carbonate Solvent Propylene Glycol Solvent Arlasolve Croda Chemicals

(India)Private Limited Solvent

Glycerine S.D. Fine Chemicals Solvent Tetraglycol Croda Chemicals (India)

Private Limited Cosolvent

Polyethylene glycol 400 S.D. Fine Chemicals Solubiliser HPMC E15 Colorcon Asia Pvt. Ltd Binder Carbopol 974P NF Lubrizol, Mumbai Gelling agent, polymer Carbopol 971 P Lubrizol, Mumbai Gelling agent, polymer Eudragit L100/55 Evonik Laboratory Anionic polymer Viscarin GP109 Signet Chemical

Corporation Pvt. Ltd. As Film forming Polymer

Blanose 7 LF Signet Chemical

Corporation Pvt. Ltd. As Film forming Polymer

Aerosil R972 Evonik Laboratory Glidant for solid dosage

form Compritol ATO 888 Gattefosse Lubricant Pharmaspheres 200µ Signet Chemical

Corporation Pvt. Ltd Neutral pellets for

adsorption of drug Pearlitol DC 500 Signet Chemical

Corporation Pvt. Ltd. Base for loading of drug

Medium Chain Triglycerides Croda Chemicals (India)

Private Limited Vehicle

Indion 234 Ion Exchange (India) Ltd Taste masking aid Indion 244 Ion Exchange (India) Ltd Taste masking aid Indion 464 Ion Exchange (India) Ltd Taste masking aid Indion 294 Ion Exchange (India) Ltd Taste masking aid Indion 414 Ion Exchange (India) Ltd Taste masking aid Indion 454 Ion Exchange (India) Ltd Taste masking aid Indion 214 Ion Exchange (India) Ltd Taste masking aid Indion 204 Ion Exchange (India) Ltd Taste masking aid

Chapter 2


Table 2.1.3: List of Excipients used in Formulation Development

1. Sucralose

Sucralose is used as a sweetening agent in beverages, foods, and pharmaceutical

applications. It has a sweetening power approximately 300–1000 times that of sucrose

and has no aftertaste. It has no nutritional value, is noncariogenic, and produces no

glycemic response.

Table 2.1.4: Monographic evaluation of Sucralose


Appearance

White to Off-white crystalline powder Complies

Solubility Freely soluble in methanol, ethanol and water Complies

Refractive Index 1.36 Complies

Optical rotation +850 Complies

Bulk Density 0.36gm/cm3 Complies

2. Xylitol

As xylitol has an equal sweetness intensity to sucrose, combined with a distinct

cooling effect upon dissolution of the crystal, it is highly effective in enhancing the

Name of the Excipient Source Use

Beta-cyclodextrin Cerestar Taste masking aid

Eudragit EPO Evonik Laboratory

Insulating coatings

having applications

Taste masking and

Moisture protection

Sodium Bicarbonate S.D. Fine Chemicals

Salt Cinnamic acid Acid Hydroxypropyl

methylcellulose (HPMC)

K15 M Colorcon Asia Pvt. Ltd

Release retardant HPMC K100M HPMC K100LV Sodium Alginate S.D. Fine Chemicals Release retardant

Blanose 7 HF Signet Chemical Corporation

Pvt. Ltd. Release retardant

Klucel HXF Signet Chemical Corporation

Pvt. Ltd. Release retardant

Polyox 301 & 303 Dow Chemical Company Release retardant Natrosol 250 HX & 250

HHX Hercules International Ltd. Release retardant

Chapter 2


flavor of tablets and syrups and masking the unpleasant or bitter flavors associated

with some pharmaceutical actives and excipients (Soderling et al., 1997).

Table 2.1.5: Monographic evaluation of Xylitol


Appearance

Homogeneous white to off-white crystalline

powder

Complies

Solubility Freely soluble in water, slightly soluble in

ethanol

Complies

Odour Odourless Complies

Taste Intensely sweet, no off test Complies

3. Lactitol Monohydrate

Lactitol is used as a noncariogenic replacement for sucrose. It is also used as a diluent

in solid dosage forms (Allen et al., 2000). A direct-compression form is available,

(Muzikova et al., 2003) as is a direct-compression blend of lactose and lactitol.

Table 2.1.6: Monographic evaluation of Lactitol Monohydrate


Appearance

White orthorhombic crystals Complies

Solubility Soluble in water, slightly soluble in ethanol Complies


Taste Sweet taste imparting a cooling sensation Complies

Specific optical

rotation

13.50 to 15.5

0 Complies

4. Mannitol

Mannitol is widely used in pharmaceutical formulations and food products. In

pharmaceutical preparations it is primarily used as a diluent (10–90% w/w) in tablet

formulations, where it is of particular value since it is not hygroscopic and may thus

be used with moisture-sensitive active ingredients (Allen et al.,2000; Yoshinari et

al., 2003). Mannitol may be used in direct-compression tablet applications, (Kanig et

al., 1964; Ward et al., 1969; Ghanem et al., 1986) for which the granular and spray-

dried forms are available, or in wet granulations (Mendes et al., 1978). Granulations

containing mannitol have the advantage of being dried easily.

Chapter 2


Table 2.1.7: Monographic evaluation of Mannitol


Appearance

White crystalline powder Complies

Solubility Freely soluble in water Complies


Taste Sweet taste imparting a cooling sensation Complies

Specific optical rotation +23° to +25° +24°

5. Glyceryl Monostearate

The many varieties of glyceryl monostearate are used as nonionic emulsifiers,

stabilizers, emollients, and plasticizers in a variety of food, pharmaceutical, and

cosmetic applications. Glyceryl monostearate has also been used in a novel fluidized

hot-melt granulation technique for the production of granules and tablets

(Kidokoro et al., 2002). Glyceryl monostearate is a lubricant for tablet manufacturing

and may be used to form sustained-release matrices for solid dosage forms

(Peh et al., 1995; Peh et al., 2000).

Table 2.1.8: Monographic evaluation of Glyceryl Monostearate


Appearance

Cream-colored, wax like solid in the

form of beads

Complies

Solubility Soluble in hot ethanol, ether,

chloroform, Practically insoluble in

water

Complies

Odour Slight fatty odor Complies

Melting Point 55–60°C

58°C

Saponification value 157–170 164

Acid value ≤3.0 2.5

HLB value

3.8 3.2

6. Cetyl Alcohol

Cetyl alcohol is widely used in cosmetics and pharmaceutical formulations such as

suppositories, modified-release solid dosage forms, emulsions, lotions, creams, and

ointments. Cetyl Alcohol has been used in a novel hot-melt granulation technique for

the production of granules and tablets (Peterson et al., 2010).

Chapter 2


Table 2.1.9: Monographic evaluation of Cetyl Alcohol


Appearance waxy and white flakes Complies

Solubility freely soluble in ethanol (95%) and

ether, solubility increasing with

increasing temperature; practically

insoluble in water.

Complies

Odour faint characteristic odor Complies

Taste Bland

Melting Point 45–52°C

48°C

Acid value ≤1.0 0.8

7. Polyox WSR Polymers

POLYOX Water-Soluble Resins are nonionic, high molecular weight water-soluble

poly (ethylene oxide) polymers. They are hydrophilic powders supplied in a wide

variety of molecular weight grades, ranging from one hundred thousand to eight

million. Lower molecular weight grades polyethylene oxide can be used as a tablet

binder at concentrations of 5–85%. The higher molecular weight grades provide

delayed drug release via the hydrophilic matrix approach. They have a long history of

successful applications in pharmaceutical products, in uses such as controlled release

solid dose matrix systems (Choi et al., 2003; Hardy et al., 2008), in direct

compression tablet binding, in melt extrusion and in gastro-retentive dosage forms

transdermal drug delivery systems, and mucosal bioadhesives. (Dhawan et al., 2005)

It was obtained from Dow Chemical Company.

Table 2.1.10: Number of repeat units and molecular weight as a function of

polymer grade for polyethylene oxide

Polyox grade Approximate number of

repeating units

Approximate molecular

weight

WSR N-10 2 275 100 000

WSR N-80 4 500 200 000

WSR 301 90 000 4 000 000

WSR 303 159 000 7 000 000

Chapter 2


Fig 2.1.3: Certificate of Analysis of Polyox Fig 2.1.4: Certificate of Analysis of Polyox

WSR N-301 WSR N-303

8. Microcrystalline Cellulose

Avicel PH, FMC’s innovator brand of microcrystalline cellulose, is a purified,

partially depolymerized alpha-cellulose made by acid hydrolysis of specialty wood

pulp and the process of polymerization involves high levels of quality and

stringency. Microcrystalline cellulose is widely used in pharmaceuticals, primarily as

a binder/diluent in oral tablet and capsule formulations where it is used in both wet-

granulation and direct-compression processes (Lamberson et al., 1976; Chilamkurti

et al., 1982; Wallace et al., 1983). In addition to its use as a binder/diluent,

microcrystalline cellulose also has some lubricant (Omray et al., 1986) and

disintegrant properties that make it useful in tableting. Microcrystalline cellulose is

also used in cosmetics and food products Avicel PH’s unique properties – superior

compactibility, drug carrying capacity and rapid disintegration - make it the excipient

of choice in direct compression applications.

Chapter 2


Avicel PH 112 & 113 grade have very low moisture with fine particle size. It

improves product stability by extending the shelf life particularly of moisture

sensitive actives.

Table 2.1.11: Properties of selected commercially available grades of

microcrystalline cellulose

Grade Nominal mean

particle size (μm)

Particle size analysis Moisture content

(%) Mesh

size

Amount retained

(%)

Avicel PH-101

50 60 ≤1.0 ≤5.0

200 ≤30.0

Avicel PH-102

100 60 ≤8.0 ≤5.0

200 ≥45.0

Avicel PH-105 20 400 ≤1.0 ≤5.0

Avicel PH-112 100 60 ≤8.0 ≤1.5

Avicel PH-113 50 60 ≤1.0 ≤1.5

Fig 2.1.5: Certificate of Analysis of Avicel PH 101 Fig 2.1.6: Certificate of Analysis of Avicel

PH 112

Chapter 2


Fig 2.1.7: Certificate of Analysis of Avicel PH 113 Fig 2.1.8: Certificate of Analysis of Avicel

PH 105

Standardization was carried out as per certificate of analysis provided by Signet

Chemical Corporation.

Table 2.1.12:Physicochemical evaluation of Avicel PH 113.

Sr.

No.

Test Specification Results

1 Characteristics White, free-flowing

odourless powder

Confirms

2 Bulk Density(g/cc) 0.27-0.34 0.31

3 Moisture content (%) NMT2% 1.5%

4 pH 5.2 5-7

5 Nominal Particle Size (µm) 50 Pass

Chapter 2


9. Kollidon® 30 (Polyvinylpyrrolidone)

Polyvinylpyrrolidone (PVP), also commonly called Polyvidone or Povidone, is a

water-soluble polymer made from the monomer N-vinylpyrrolidone. Although

povidone is used in a variety of pharmaceutical formulations, it is primarily used in

solid-dosage forms. In tableting, povidone solutions are used as binders in wet-

granulation processes (Becker et al., 1997; Stubberud et al., 1996).

Fig 2.1.9: Certificate of Analysis of Kollidon® 30 (Polyvinylpyrrolidone)

10. Polyvinyl alcohol

It is a hydrophilic, semicrystalline polymer. It has been used as viscosity builder in

ophthalmic solutions. It was standardized as per the monograph given in USP. (US

Pharmacopoeia., 1990) The tests and results are recorded in Table 2.1.14

Chapter 2


Table 2.1.13: Characterization of PVA

Sr.

no

Tests Specification Result

1. Appearance White colored

granular and

odorless

powder

Complies

2. Solubility Soluble in hot

water

Complies

3. Viscosity at

200C

85-115% of

label claim of

25 cps

95%

4. pH 5.0-8.0 5.5

5. Melting point 2280C 228

0C

6. Loss on drying < 5.0% 1.5%

11. Propylene glycol

It is widely used as water miscible co-solvent, extractant, preservative and antiseptic

in a variety of pharmaceutical formulations.

Table 2.1.14: Monographic evaluation of Propylene glycol


Description Clear, colorless, viscous, practically

odorless liquid with a sweet, slightly acrid

taste resembling that of glycerin.

Complies

Identification tests According to reference standards Complies

Residue on ignition ≤3.5 mg 2.9 mg

Specific gravity 1.035–1.037 g/mL 1.035 g/mL

12. Carbopol

Carbopol polymer is high molecular weight crosslinked polymer of acrylic acid,

which confirms to USP/INF specifications. It was obtained from Lubrizol Pvt. Ltd.

Carbomer having low residuals only of ethyl acetate, such as carbomer 971P or 974P,

may be used in oral preparations, in suspensions, tablets, or sustained release tablet

formulations. (Singla et al., 2000)

Chapter 2


Fig 2.1.10: Certificate of Analysis of Carbopol 971 P

Fig 2.1.11: Certificate of Analysis of Carbopol 974 P

Chapter 2


13. Eudragit EPO

EUDRAGIT® E PO is a cationic copolymer based on dimethylaminoethyl

methacrylate, butyl methacrylate, and methyl methacrylate. Eudragit EPO is known to

have taste masking properties (Alpana et al., 2012).

Fig 2.1.12: Certificate of Analysis of Eudragit EPO

Chapter 2


14. Ion Exchange resins

Ion exchange Resins are solid and suitably insoluble high molecular weight poly-

electrolytes that can exchange their mobile ions of equal charge with the

surrounding medium. (Suhagiya et al., 2010) Both cation exchange and anion

exchange resins has been utilized in present research work for taste masking aid.

Fig 2.1.13: Certificate of Analysis of Indion 414 Fig 2.1.14: Certificate of Analysis of Indion 464

Fig 2.1.15: Certificate of Analysis of Indion 234 Fig 2.1.16: Certificate of Analysis of Indion 244

Chapter 2


Fig 2.1.17: Certificate of Analysis of Indion 294

16. Hydroxyehylcellulose (HEC)

Natrosol, Hydroxyethyl cellulose is a nonionic, water-soluble polymer widely used in

pharmaceutical formulations. It is primarily used as a thickening agent in ophthalmic

(Grove et al., 1990) and topical formulations (Gauger et al., 1984), although it is also

used as a binder (Delonca et al., 1978) and film-coating agent for tablets (Kovacs et

al., 1990). High viscosity grades of HEC viz. Natrosol 250 HX & 250 HHX are used

in modified release tablets as release retardant. (Shyr-Yi Lina., 2008)

Standardization was carried out as per certificate of analysis provided by Hercules

International Ltd.

Table 2.1.15: Physicochemical evaluation of Natrosol 250 HHX PHARM

Sr.

No.

Test Specification Results

1 Characteristics A white to light tan,

free-flowing powder

Confirms

2 Solubility Soluble in cold and hot

water

Confirms

3 Apparent viscosity cps 3500-5500 5000

4 Moisture content (%) less than 5% 4

5 pH in water 6-8.5 7.1

Chapter 2


Fig 2.1.18: Certificate of Analysis of Natrosol 250HHX

17. Hydroxypropylcellulose (HPC)

Hydroxypropylcellulose (HPC) is nonionic water-soluble cellulose ether with a

versatile combination of properties. It combines dual solubility in aqueous and polar

organic solvents, thermoplasticity, and surface activity with the thickening and

stabilizing properties of other water soluble cellulose polymers. In oral products,

hydroxypropyl cellulose is primarily used in tableting as a binder (Skinner et al.,

1999), film-coating, and extended-release-matrix former (Alderman et al., 1987;

Lee Dy et al., 2000) Standardization was carried out as per certificate of analysis

provided by Hercules International Ltd.

Chapter 2


Table 2.1.16: Physicochemical evaluation of KLUCEL HXF PHARM

Sr.

No.

Test Specification Result

1 Characteristics Off-white tasteless powder Confirms

2 Solubility Soluble in water and polar

solvents

Confirms

3 Apparent viscosity (cps) 1500-3000 2300

4 Moisture content (%) less than 5% 2.2

5 pH in water 5-7.5 7.1

Fig 2.1.19: Certificate of Analysis of Klucel HXF

Chapter 2


18. Hydroxypropylmethylcellulose (HPMC)

METHOCEL™ Cellulose Ethers are water-soluble polymers derived from cellulose,

the most abundant polymer in nature. In addition Methocel™ Cellulose Ethers are the

first choice for the formulation of hydrophilic matrix systems, providing a robust

mechanism for the slow release of drugs from oral solid dosage forms. With a choice

of viscosity grades, Methocel™ provides a simple solution to meet a range of drug

solubility needs. It is widely used in oral, ophthalmic and topical pharmaceutical

formulations. In oral products, is primarily used as a tablet binder (Chowhan et al.,

1980), in film-coating, (Alderman et al.,1989; Patell et al., 1990) and as a matrix for

use in extended-release tablet formulations (Shah et al., 1989; Wilson HC et al.,

1989; Dahl TC et al., 1990).

Standardization was carried out as per certificate of analysis provided by Colorcon

Asia Private Limited.

Table 2.1.17: Physicochemical evaluation of METHOCEL K100 CR

Sr.

No.


1 Characteristics Fine white powder Confirms

2 Solubility Soluble in cold water

Confirms

3 Viscosity (cps)

75000-14,0000 93404

4 Moisture content (%) less than 5% 2

5 pH in water 5-8 6.7

19. BLANOSE CMC 7HF PHARM

BLANOSE CMC 7HF PHARM is an anionic water-soluble polymer derived from

cellulose. It acts as a thickener, binder, stabilizer, protective colloid, suspending

agent, and rheology, or flow control agent.

Carboxymethylcellulose sodium is widely used in oral and topical pharmaceutical

formulations, primarily for its viscosity-increasing properties. Viscous aqueous

solutions are used to suspend powders intended for either topical application or oral

and parenteral administration (Chang et al., 2004). Carboxymethylcellulose sodium

may also be used as a tablet binder and disintegrant, (Dabbagh et al., 1999) and to

stabilize emulsions (Adeyeye et al., 2002).

Standardization was carried out as per certificate of analysis provided by Hercules

International Ltd.

Chapter 2


Table 2.1.18: Physicochemical evaluation of BLANOSE CMC 7HF PHARM

Fig 2.1.20: Certificate of Analysis of Blanose 7HF

20. Glyceryl Behanate

Compritol 888 ATO (USP, 2007) is a fine white powder of well-controlled particle

size distribution with an indicative particle size of 50µm.It is a mixture of

Sr.

No.

Test Observation Specification and inference

1 Characteristics Fine white powder Confirms

2 Solubility Soluble in water

Confirms

3 Viscosity (mPa.s))

1850 Pass (1500-2500)

4 Moisture content (%) 6 Pass (less than 8%)

5 pH in water 8 Pass (6.5-8)

Chapter 2


approximately 15 % mono-, 50% di- and 35% triglycerides of behenic acid (C22)

while other fatty acids than behenic acid account for less than 20 %.

It is chemically inert and highly compatible with other ingredients. Pharmaceutically

it is used as lubricant for tablets and capsules, as a binding agent and lipophilic matrix

for sustained-release tablets and capsules.

Table 2.1.19: Physicochemical evaluation of Glyceryl Dibehenate.

Sr.

No.


1 Characteristics White-yellow powder with light

odor

Confirms

2 Solubility Insoluble in water soluble in

chloroform and dichloromethane

Confirms

3 Melting point 710C Confirms

4 Moisture content

(%)

less than 0.1 0.04

5 Saponification

value mgKOH/g

145-165 Pass

6 Iodine value

g I2/100g

<3 0.3

21. Aerosil ® R 972

AEROSIL® R 972 Pharma is a high purity, amorphous, anhydrous, hydrophobic

colloidal silica for use in pharmaceutical products.

Colloidal silicon dioxide is widely used in pharmaceuticals, cosmetics, and food

products. Its small particle size and large specific surface area give it desirable flow

characteristics that are exploited to improve the flow properties of dry powders in a

number of processes such as tableting (Lerk et al., 1977). AEROSIL® R 972

Pharma is a colloidal silicon dioxide that has been chemically treated to render its

surface hydrophobic. AEROSIL® R 972 Pharma is less agglomerated than other

AEROSIL® types, and is an excellent glidant with extremely low moisture

absorption.

Chapter 2


2.3 Drug and Excipient Interaction Studies

Excipients are usually biologically inactive; the same cannot be said from

a chemical perspective. Excipients, and any impurities present, can stabilise or

destabilise drug products. The objective of drug/excipient compatibility

considerations and practical studies is to delineate, as quickly as possible, possible

interactions between potential formulation excipients and the API. This is an

important risk reduction exercise early in formulation development. In the present

research work, the effect of some commonly used excipients in the manufacturing of

tablets on the API, Gabapentin was studied under isothermal stress conditions of

40°±2°C/ 75%±5% RH. Isothermal stress testing involves challenging drug-excipient

mixtures in presence of moisture to degradation. The inactive ingredients elected for

the study included different hydrocolloid polymers, diluents, and lubricants. The

Gabapentin: Excipient blends were taken in ratio of 1:1, except for glidants and

lubricants, where the ratio was 9:0.1.Aliquots of these mixtures and the drug alone

were kept in open 5mL glass vials, exposed to 40°C and 75% relative humidity

conditions for one month. At intervals of 2 weeks and 4 weeks, the samples were

withdrawn to make physical observations and analyzed for related substances formed

after the exposure of the drug and excipient. Initial assay of each blend was

determined and considered as 100%. The assay of these blends determined at the end

of study was expressed as percentage of the initial assay. Results of drug and

excipient Interaction studies are given in Tables 2.1.20 and 2.1.21.

Table 2.1.20: Physical observations in Drug excipient compatibility studies Sr.

no.

Ingredients Ratio Initial 40°C/ 75% RH

for 2 weeks

40°C/ 75% RH

for 2 weeks

1 Drug - White crystalline powder No change No change

2 HPMC K100 M 1:1 White crystalline powder No change No change

3 Klucel HXF 1:1 Off white crystalline

powder

No change No change

4 Natrosol

1:1

Off white crystalline

powder

No change No change

5 Carbopol 971P 5:1 White crystalline powder Yellowish

lumpy

agglomerate

Yellowish lumpy

agglomerate

6 Poyox 301 1:1 White crystalline powder No change No change

7 Polyox 303 1:1 White crystalline powder No change No change

8 Blanose 7 HF 1:1 White crystalline powder No change No change

9 Viscarin GP 109 1:1 Light brown crystalline

powder

No change No change

Chapter 2


Table 2.1.21: Results of Drug excipient compatibility studies

Sr.

no.

Ingredients

Initial total

impurity

%w/w

40°C/ 75% RH

for 2 weeks

Impurity %

w/w

40°C/ 75% RH

for 4 weeks Impurity %

w/w

Remarks

1 Drug 0.01 0.06 0.06 Compatible

2 HPMC K100 M 0.04 0.08 0.09 Compatible

3 Klucel HXF 0.05 0.08 0.08 Compatible

4 Natrosol 0.04 0.09 0.08 Compatible

5 Carbopol 971P 0.08 0.2 0.2 Incompatible

6 Poyox 301 0.05 0.085 0.085 Compatible

7 Polyox 303 0.05 0.086 0.086 Compatible

8 Blanose 7 HF 0.03 0.087 0.087 Compatible

9 Viscarin GP 109 0.04 0.09 0.09 Compatible

2.4 DSC thermograms for drug and sustained release polymers and other

exicipients used in the formulation

To investigate any drastic changes with thermal behavior of either drug or excipients,

the interaction studies were carried out using differential scanning calorimetry.

Differential scanning calorimetry or DSC is a thermoanalytical technique in which the

difference in the amount of heat required to increase the temperature of a sample and

a reference are measured as a function of temperature. Both the sample and reference

are maintained nearly at the same temperature throughout the experiment. The

temperature programme for DSC analysis is designed such that the sample holder

temperature increases linearly as a function of time. The reference sample should

have a well-defined heat capacity over the range of temperatures to be scanned.

The basic principle underlying DSC technique is that when the sample undergoes

physical transformation such as phase transitions, more or less heat will need to flow

to it than the reference to maintain both at the same temperature. Whether more or

less heat must flow to the sample depends on whether the process is exothermic or

endothermic. For example, as a solid sample melts to a liquid it requires more heat

flowing to the sample to increase its temperature at the same rate as the reference.

This is due to the absorption of heat by the sample as it undergoes the endothermic

phase transition from solid to liquid. Likewise, as the sample undergoes exothermic

processes (such as crystallization) less heat is required to raise the sample

temperature. By observing the difference in heat flow between the sample and

Chapter 2


reference, differential scanning calorimeters are able to measure the amount of heat

absorbed or released during such transitions. DSC may also be used to observe more

subtle phase changes, such as glass transitions. DSC is widely used in industrial

settings as a quality control instrument due to its applicability in evaluating sample

purity and for studying polymer curing. Here drug interaction studies were

investigated by using this technique.

Thermal analysis of Gabapentin and other excipients used in the optimised

formulation was carried out employing Differential Scanning Calorimeter (Mettler

Toledo DSC). Samples (approximately 5-7 mg) were accurately weighed into

aluminum pans and sealed. The temperature was gradually increased by 10oC from

room temperature to 400oC and thermograms were obtained as shown in Figures

2.1.21-2.1.25.

Fig 2.1.21.: DSC thermogram of pure gabapentin

Fig 2.1.22. : DSC thermogram of Klucel HXF

Chapter 2


Fig 2.1.23 : DSC Thermogram of preformulation mix with Klucel HXF

Fig 2.1.24: DSC thermogram of Polyox WSR N-303

Fig 2.1.25: DSC thermogram of preformulation mix with Polyox WSR N-303

Chapter 2


2.5 Results and Discussion

2.5.1 Standardization of drug and excipients

The standardization of drugs and excipients is an integral part of any research work

and ensures quality of the research outcomes.

Gabapentin was standardized as per the specifications given in the monograph

in British Pharmacopoeia 2012. Table 2.1.1 enlists the various tests,

observations and specifications. The drug passed all the tests for identity and

was well within pharmacopoeial limits. The FT-IR spectra is shown in

Fig 2.1.2. The COA provided by the Sashun chemicals Ltd.is also attached.

Sweeteners such as sucralose, xylitol, lactitol and mannitol were standardized

as per the specifications of IP’2007. Table 2.1.4- 2.1.7 enlists the various

tests, observations and specifications. The sweeteners passed all the tests for

identity and were well within pharmacopoeial limits.

Lipid carriers like glyceryl monostearate and cetyl alcohol were standardized

as per the specifications of IP’2007. Table 2.1.8 -2.1.9 enlists the various

tests, observations and specifications. The lipid carriers passed all the tests for

identity and were well within pharmacopoeial limits.

Polymers such as Hydroxyethyl cellulose, Hydroxypropyl cellulose,

Hydroxypropylmethyl cellulose and Sodium carboxy methyl Cellulose, were

standardized as per the specifications. Table 2.1.15, 2.1.16, 2.1.17, 2.1.18,

enlists the various tests, observations and specifications. The polymer passed

all the tests for identity and was well within pharmacopoeial limits.

2.5.2 Drug-Excipient interaction studies

No color change was observed in any excipient and all the combinations except

Carbopol 971P exhibited impurity profiles within the reference limits (the total

impurity should not be more than 0.4%). Based on these results all the hydrocolloid

polymers screened at different concentrations for development of the matrix

bioadhesive tablet complied with the tests for quality and purity. The selected

excipients were found to be compatible with gabapentin displaying no obvious signs

of degradation on visual observation and from HPLC impurity analysis studies. Hence

further experimentation for formulation development using these excipients could be

initiated.

Chapter 2


2.5.3 DSC thermograms for drug and sustained release polymers and other

excipients used in the formulation

It is indicated from the thermogram in Fig 2.1.23 and Fig 2.1.25 that well

characterized and recognizable endotherm of drug appeared at the temperature near

177.7± 10C.

Endothermic peak for Klucel HXF and Polyox 303, were found to be at 75.640C and

55.570C respectively. From Fig 2.1.23 and 2.1.25 it is evident there is no overlapping

of peaks which confirms that there is no interaction between drug and other excipients

used. Hence the excipients can be safely used in the formulation.

Chapter 2


2B Analytical Method Development and Validation

2B.1 Introduction

GBP [1–(amino methyl)–cyclohexaneaceticacid] is a cyclic GABA [gamma – amino

butyric acid] analogue. Although, it is structurally related to GABA, GBP has no

direct GABA mimetic effect. GBP with a trade name of Neurontin® is extensively

used for the treatment of convulsive-type cerebral disorders, such as epilepsy,

hypokinesia and cranial trachoma (McLean et al., 2003).

GBP was originally approved in the U.S. by the Food and Drug Administration

(FDA) in 1994 for use as an adjunctive medication to control partial seizures

(effective when added to other antseizures drugs). There is a wide individual variation

in the rate of clearance of these drugs and there is always a need to perform

compliance testing, ascertain toxicity, and elucidate possible clinical interactions.

Accordingly, the therapeutic monitoring of GBP is highly desirable (Patsalos et al,

1996). GBP is highly water soluble, with an octanol/ buffer (pH 7.4) log P value of -

1.10, and is zwitterionic at physiological pH (pKa value of 3.68 and 10.7)

(McLean.,et al., 1995). Absolute bioavailability of GBP is dose dependent. The

literature reveals that numerous analytical methods have been reported for the

determination of GBP in pharmaceutical preparations and human serum. These

methods are based on spectrophotometry (Hisham et al., 2003; Patel et al, 2011)

HPTLC (Sane et al., 2003), Gas chromatography (Wolf et al., 1996; Wolf et al.,

1999) Gas chromatography – Mass spectrophotometry (Borrey et al., 2005), HPLC

(George et al., 2000 ;Zhu et al.,2002; Cetin et al, 2004; Vermeij et al., 2004),

Capillary electrophoresis (Garcia et al., 1994; Lin et al., 2004) and fluorometry

(Zehouri et al., 2004; Olgun et al., 2002; Belal et al 2002). For the present work

colorimetric and HPLC methods were developed for analysis of GBP.

UV spectrophotometric and HPLC methods were developed for quantification of GBP

in various media as well as plasma for in vivo studies.

Following methods were tried out for the analysis of GBP.

UV-Visible Spectroscopy for routine analysis

Colorimetric method for estimation of GBP

HPTLC method for estimation of the GBP

HPLC method for estimation of GBP

Chapter 2


Bioanalytical method development of GBP using HPLC with

spectroflourimetric detection for estimation in the plasma

HPLC method with spectroflourimetric detection for estimation GABA in the

brain tissue

2B.2 Analytical Method Validation

Validation of analytical methodologies is widely accepted as pivotal before they are

put into routine use. A method must be tested for effectiveness and must be

appropriate for the particular analysis to be undertaken. Method validation is defined

as the process of proving, through scientific studies, that an analytical method is

acceptable for its intended use and it instills confidence that the method can generate

test data of acceptable quality.

Recent guidelines for methods development and validation for new non-compendial

test methods are provided by the FDA draft document, ‘Analytical Procedures and

Method Validation: Chemistry, Manufacturing and Controls Documentation’. In

recent years, a great deal of effort has been devoted to the harmonization of

pharmaceutical regulatory requirements in the United States, Europe and Japan. As

part of this initiative, the International Conference on Harmonization (ICH) has issued

guidelines for analytical method validation. The recent FDA method validation draft

guidance document as well as the United States Pharmacopoeia (USP) both refers to

the ICH guidelines.

Linearity and range

Linearity defines the analytical response as a function of solute concentration and

range prescribes a region over which acceptable linearity, precision and accuracy are

achieved. Linearity is generally reported as the variance of the slope of the regression

line. Range is the interval between the upper and lower concentrations of solute that

have been demonstrated to be determined with precision, accuracy and linearity using

the method. The ICH guidelines specify a minimum of five concentrations, along with

certain minimum specified ranges

Accuracy and bias

Accuracy is the measure of exactness of an analytical method, or the closeness of

agreement between the measured value and the value that is accepted either as a

Chapter 2


conventional, true value or an accepted reference value. Bias assesses the influence of

the analyst on the performance of the method.

Precision

Precision quantifies the variability of an analytical result as a function of operator,

method manipulations and day-to-day environment. It is also the measure of the

degree of repeatability of an analytical method under normal operation and is

expressed as the percent RSD for a statistically significant number of samples.

Precision experiments give a good indication of the performance of the method and

should be repeated regularly. Generally, any increase of the RSD above 2.0% should

be investigated. According to ICH, three types of precision can be defined and should

all be assessed as described below.

Repeatability

Repeatability refers to the results of the method operating over a short time interval

under the same conditions (inter-assay precision). It expresses the degree of variation

arising during replicate assays performed consecutively and non-consecutively but on

the same day. Repeatability should be determined from a minimum of nine

determinations covering the specified concentration range of the procedure.

Intermediate precision (ruggedness)

Intermediate precision refers to the results from laboratory variations due to random

events such as differences in experimental periods, analysts and equipment.

Reproducibility

Reproducibility is an indication of the ability of the method to be transferred from one

laboratory to another.

Specificity and selectivity

A method is specific if it produces a response for only one single solute. Since it is

almost impossible to develop a chromatographic assay for a drug in a matrix that will

respond to only the compound of interest, the term selectivity is more appropriate.

Selectivity describes the ability of an analytical method to differentiate various

substances in the sample and is applicable to methods in which two or more

components are separated and quantitated in a complex matrix. It is a measure of

degree of interference from such things as other active ingredients, excipients,

impurities and degradation products, ensuring that a peak response is due only to a

single component, i.e., that no co-elution exists.

Chapter 2


Limit of detection and Limit of Quantitation

The USP requires that the limit of detection (LOD) and the limit of quantitation

(LOQ) be determined for studies that involve the detection and quantitation of

components at or near trace levels. Such studies include purity testing of active

pharmaceutical ingredients, stability testing of dosage forms and the analysis of

manufacturing equipment cleaning validation samples. For many pharmaceutical

applications the LOQ is generally a more useful parameter than the LOD. The LOD is

defined as the lowest concentration of a solute in a sample that can be detected,

though not necessarily quantitated and the LOQ is defined as the lowest concentration

of a solute in a sample that can be determined with acceptable precision and accuracy

under the stated operational conditions of the method.

The drug passed the tests for identity, purity and the results were found to comply

with the pharmacopoeial standards and hence were used for further incorporation in

the formulation of controlled drug delivery systems.

2B.3 UV method for determination of GBP

Initially the standard curve was prepared in both methanol and 0.1 N HCl for the

estimation of drug content and analysis of dissolution samples.

2B.3.1 Instrumentation

All spectral measurements were made by using Jasco UV visible spectrophotometer.

2B.3.2 Preparation of standard solution in methanol

GBP (500 mg) was accurately weighed and transferred to 100 ml volumetric flask. It

was dissolved properly and diluted up to the mark with methanol to obtain

concentration of 5 mg/ml. This solution was used as working standard solution. The

absorbance of the solution containing GBP at 5mg/ml was determined in the UV

range 200-400nm against methanol as blank. The λmax was found to be 215nm. The

absorbance of standard solution was determined on U.V. Spectrophotometer, Jasco V-

530 at 215 nm. The UV scan for the GBP is depicted in Fig 2B.1.1

Chapter 2


Fig 2B.1.1: UV Scan for GBP at 215 nm in methanol

2B.3.3 Preparation of calibration curve of GBP in methanol

Accurately weighed 500mg of GBP was dissolved in sufficient amount of methanol in

a 50 ml volumetric flask and diluted to volume with methanol. From the above

solution, samples of 1, 2, 3, 4, 5 and 6ml was withdrawn in 10 ml volumetric flasks

and diluted to volume with methanol so as to obtain standard solutions of

concentrations 500, 1000, 1500, 2000, 2500 and 3000 µg/ml respectively. A standard

plot of absorbance verses concentration of GBP was obtained. Absorbance and

concentration was subjected to least square line regression analysis to calculate the

calibration equation and coefficient of correlation (r2). The results for the same are

given in Table 2B.1.1 and Fig 2B.1.2. The regression coefficient for the calibration

curve was found to be 0.9904 and linear concentration was found to be in the range of

500µg/ml to 3000 µg/ml.

Table 2B.1.1: Calibration curve for GBP by UV at 215nm

Sr.

no.

Concentration

(ppm)

Absorbance

1 500 0.1657

2 1000 0.2886

3 1500 0.4449

4 2000 0.5653

5 2500 0.6935

6 3000 0.8005

Chapter 2


Fig 2B.1.2: Standard curve of GBP by UV spectrophotometry

2B.3.4 Preparation of standard solution in 0.1N HCl

An accurately weighed sample (500mg) of GBP reference standard was transferred to

100 ml volumetric flask and dissolved in 0.1 N HCl to make solution of 5 mg/ml. UV

scan was taken from 200-400 nm. A sharp peak of drug was obtained at 212 nm but

the drug was less sensitive to UV range.

Fig 2B.1.3: UV Scan for GBP at 212 nm in 0.1N HCl

2B.3.5 Preparation of calibration curve of GBP in 0.1N HCl

Accurately weighed 500mg of GBP was dissolved in sufficient amount of 0.1N HCl

in a 50 ml volumetric flask and diluted to volume with 0.1N HCl. From the above

solution, samples of 2, 3, 4, 5, 6, 7 and 7.5ml was withdrawn in 10 ml volumetric

flasks and diluted to volume with 0.1N HCl so as to obtain standard solutions of

concentrations 500, 1000, 1500, 2000, 2500, 3000, 3500 and 4000 µg/ml respectively.

The absorbance of standard solution was determined at 212 nm. A standard plot of

absorbance verses concentration of GBP was obtained. Absorbance and concentration

y = 0.0003x

R2 = 0.9904

0

0.2

0.4

0.6

0.8

1

0 500 1000 1500 2000 2500 3000 3500

abso

rban

ce

concentration(ppm)

Absorbance vs Concentration

absobance

Chapter 2


was subjected to least square line regression analysis to calculate the calibration

equation and coefficient of correlation (r2). The results for the same are given in Table

2B.1.2 and Fig 2B.1.4. The regression coefficient for the calibration curve was found

to be 0.994 and linear concentration was found to be in the range of 500 µg/ml to

4000 µg/ml.

Table 2B.1.2: Calibration curve for GBP by UV at 212nm in 0.1 N HCl

Fig 2B.1.4: Standard curve of GBP in 0.1N HCl by UV spectrophotometry

2B.4 Colorimetric method for determination of GBP

This method is based on the reaction of the primary amino group of GBP with

ninhydrin reagent in N,N1-dimethylformamide (DMF) medium producing a colored

product which absorbs maximally at 572 nm.

2B.4.1 Reagents

Ninhydrin reagent and N,N1-Dimethyl formamide. All other solvents were used are

of analytical grade.

y = 0.0003x R² = 0.994

0

0.2

0.4

0.6

0.8

1

1.2

0 1000 2000 3000 4000 5000

Ab

sorb

ance

Concentration in ppm

Series1

Linear (Series1)

Sr.

no

Concentration

(ppm)

Absorbance

1 500 0.1652

2 1000 0.2841

3 1500 0.4180

4 2000 0.5884

5 2500 0.6999

6 3000 0.8531

7 3500 0.9376

8 4000 1.0499

Chapter 2



All spectral measurements were made by using Jasco UV visible spectrophotometer.

2B.4.3 Preparation of Calibration Curve of GBP

Into 10 ml measuring flasks, different aliquots of drug solution were transferred to

provide final concentration range 40-280 mg/ ml. To each flask, 2 ml of ninhydrin

reagent in N,N1-DMF was added. The volume was made up to the mark with distilled

water and the flask was heated on a waterbath at 90±5oC for 10 min. After the flask

had been cooled to room temperature and the solution was made up to the mark with

water. The absorbance of the solution was measured against a reagent as blank at 572

nm. The calibration graph was prepared by plotting absorbance vs. concentration of

GBP. Absorbance and concentration was subjected to least square line regression

analysis to calculate the calibration equation and coefficient of correlation (r2). The

results for the same are given in Table 2B.1.3 and Fig 2B.1.6.The regression

coefficient for the calibration curve was found to be 0.9909 and linear concentration

was found to be in the range of 40 µg/ml to 240 µg/ml.

Fig 2B.1.5: UV Scan for GBP at 572 nm

Table 2B.1.3: Calibration curve for GBP by colorimetric at 572nm

Sr.

No.

Concentration

(ppm)

Absorbance

1 40 0.0154

2 80 0.0413

3 120 0.0694

4 160 0.104

5 200 0.1337

6 240 0.1664

Chapter 2


Fig 2B.1.6: Standard curve of GBP by colorimetry at 572 nm

2B.5 HPTLC method for determination of GBP

HPTLC method was tried and developed by using the method reported in literature

(Saner et al., 2003) so that it can be alternative to HPLC for day-to-day analysis of

dissolution and drug content samples.

2B.5.1 Reagents

0.2% alcoholic ninhydrin solution, analytical grade glacial acetic acid and n-butanol


A Camag, Linomat sampler applicator was used for spotting of plates, Camag twin

trough glass chamber (10 x 10), Camag TLC scanner, Spectral range 190 – 800nm,

Camag UV cabinet with dual wavelength UV lamp: Dual wavelength 254 / 366nm,

Stationary Phase: Silica gel G60 F254 coated on aluminum sheet, Hamilton 100µl

HPTLC syringe.

.

2B.5.3 Preparation of Calibration Curve of GBP

Initially weighed amount of drug was dissolved in methanol to give the concentration

of 100mcg/ml i.e. 100ng/mcl. In this method the mobile phase of n-butanol: water:

Glacial acetic acid (2.4:1.2:0.6) was used. The plate was then sprayed with 0.2%

alcoholic ninhydrin solution and dried at 1050C for 10 min. Detection and

quantification of GBP was performed by densitometry at λ, 490nm. The calibration

graph was prepared by plotting absorbance vs. concentration of GBP. Area and

concentration was subjected to least square line regression analysis to calculate the

calibration equation and coefficient of correlation (r2).

Abs. vs Conc.

y = 0.0007x - 0.0098

R2 = 0.9909

-0.05

0

0.05

0.1

0.15

0.2

0 50 100 150 200 250 300

conc(ppm)

abs

abs

Linear (abs)

Chapter 2


Fig 2B.1.7: HPTLC Chromatogram of GBP 490 nm

Table 2B.1.4: Calibration curve for GBP by HPTLC at 490nm

Fig 2B.1.8: Standard curve of GBP by HPTLC at 490nm

2B.6 Development of HPLC method for determination of GBP from dissolution

samples

HPLC is a well-documented analytical technique having greater accuracy and

precision as compared to UV spectrophotometry. GBP is a weak ultraviolet (UV)

absorber requiring spectral analysis at short UV wavelengths. At 210 nm GBP

absorbance is approximately one order of magnitude less than the lactam. Thus,

traditional dissolution analysis by direct UV was not used since low levels of the

y = 12.615x + 5802.4 R² = 0.9916

0

5000

10000

15000

20000

25000

0 500 1000 1500

area

Linear (area)

Sr.

no

Concentration

(ppm)

Absorbance

1 200 7811

2 400 10982

3 600 13825

4 800 16076.9

5 1000 18728.3

6 1200 20373.7

Chapter 2


lactam may interfere with GBP quantitation. For accurate quantification of GBP and

the lactam impurity, HPLC method reported in USP was used.

2B.6.1 Reagents: GBP, HPLC grade Acetonitrile and methanol, Monobasic

Ammonium Phosphate Buffer pH 1.8. All the solvents used were filtered through

0.45-µm-membrane filter and degassed using sonicator.

2B.6.2 Instrumentation: Tosoh HPLC, Japan

2B.6.3 Chromatographic conditions:

Instrument: Tosoh HPLC, Japan

Column: Perfectsil C18 column (46mm x 250 mm) 2.5 µm

Mobile phase: Monobasic Ammonium Phosphate Buffer pH 1.8: Acetonitrile

(76:24)

Flow rate: 1 ml/min

λmax: 215 nm

Loop capacity: 20µl

2B.6.4 Preparation of stock solution

Accurately weighed 100mg of GBP and 10 mg of lactam were taken in 100 ml

volumetric flask and diluted to volume with monobasic ammonium phosphate buffer

pH 2 and used as stock solutions in preparation of calibration curves. The reference

solution was diluted with diluent to obtain concentration ranges of GBP (50-500 ppm)

and lactam (5-50 ppm). Area and concentration was subjected to least square line

regression analysis to calculate the calibration equation and coefficient of correlation

(r2). The calibration curves were plotted as peak area vs. concentration as depicted in

Fig 2B.1.10 and 2B.1.11. The regression coefficient for the calibration curve of GBP

was found to be 0.9991 and linear concentration was found to be in the range of 50

µg/ml to 500µg/ml. The regression coefficient for the calibration curve of GBP

lactam was found to be 0.9998 and linear concentration was found to be in the range

of 10µg/ml to 50µg/ml.

Chapter 2


Fig 2B.1.9: HPLC Chromatogram showing peak of GBP and lactam

Table 2B.1.5: Calibration curve of GBP Table 2B.1.6: Calibration curve of Lactam

Fig 2B.1.10: Standard curve of GBP Fig 2B.1.11: Standard curve of Lactam

Standard curve of gabapentin

y = 247.97x

R2 = 0.9991

0

40000

80000

120000

160000

0 200 400 600

conc (ppm)

are

a

Standard curve of lactum y = 4120.9x

R2 = 0.9998

0

50000

100000

150000

200000

250000

0 20 40 60

conc (ppm)

are

a

Sr.

no

Concentration

(ppm)

Absorbance

1 0 0 2 50 11026 3 100 24705

4 200 47380

5 300 76804

6 400 100409

7 500 121431

8 600 149904

Sr.

no

Concentration

(ppm)

Absorbance

1 0 0

2 10 126032

3 20 242056

4 30 357238

5 40 461256

6 50 582576

Chapter 2


2B.6.5 Method validation

The method validation was performed according to the United States Pharmacopoeia

and results are tabulated. The following validation characteristics were studied:

linearity, range, accuracy, and precision, limit of quantitation and limit of detection.

1. System precision (injection repeatability): It was determined by performing six

repeated analysis of working standard solution.

2. Linearity: It was determined by building calibration curves. For the construction of

calibration curve six calibration standard solutions were prepared at concentrations

ranging from 50 to 500µg/ml of GBP and 5-50 µg/ml of lactam. Each standard

solution was injected once. Calibration curves of standard GBP were generated by

plotting analyte peak area vs. concentration of the drugs.

3. Limit of detection: It is the lowest concentration of an analyte that the procedure

can reliably differentiate from background noise. It was determined by injecting the

mobile phase three times into the system and the value with the highest peak area in

the range of the retention time was determined. The concentration corresponding to

three times the value of noise peak gave estimate of limit of detection. (Table 2B.1.7)

4. Limit of Quantification: It was determined as the lowest concentration that can

be established with acceptable accuracy and precision. The noise of the instrument

was determined as above. The concentration corresponding to 10 times the area of

noise peak gave an estimate of limit of quantification. (Table 2B.1.7)

5. Intermediate precision: It was assessed by analyzing three replicate injections of

reference solution at three levels on three different days (inter-day), and results were

reported in Table 2B.1.8. Precision was expressed by the % R.S.D. of the analyte

peaks.

6. Accuracy (Recovery)

Accuracy was established by evaluating the amount determined from the quality

control standards and the lactam and comparing to the respective nominal value

expressed as percent recovery. Results were reported on Table 2B.1.9.

Chapter 2


Table 2B.1.7: Method validation data for GBP and lactam

Analytical parameters GBP Lactam impurity

Retention time 6.8 ±0.3 26.2 ±0.3

LOQ (µg/ml) 20 2

LOD (µg/ml) 5 0.5

Linearity

Range (µg/ml) 50-500 5-50

Slopea ± % RSD 247.55 ± 0.715 4120.9± 1.26

System Precision

Amount taken 300 20

Amount detectedb(µg/ml) 296.83 19.55

% RSD 0.752 1.76

R2 0.9991 0.9998

aMean (n=4), bMean (n=6)

Table 2B.1.8: Interday-intermediate precision using proposed method(n=3)

GBP Lactam

Level

Amount

taken

(µg)

Amount

detected

(µg)

% RSD

Amount

taken

(µg)

Amount

detected

(µg)

% RSD

1 50 50.33 1.51 10 10.1 0.99

2 100 99.86 1.10 20 20.26 1.24

3 200 199.2 0.77 30 30.13 1.68

Table 2B.1.9: Accuracy experiment using proposed method

GBP Lactam

Level Amount

taken

(µg)

Amount

detected

(µg)

%

recovery

Amount

taken

(µg)

Amount

detected

(µg)

%

recovery

1 50 50.33 100.6 10 10.1 100.16

2 100 99.86 99.8 20 20.26 100.3

3 200 199.6 99.6 30 30.13 100.7

Mean % recovery (n= 9) 100.4 Mean % recovery (n= 9) 100.4

%RSD 1.16 %RSD 1.27

Chapter 2


2B.7 Development of HPLC method with Spectrofluorimetric detection for

estimation of gammaaminobutyric acid (GABA) in the brain

GABA is an amino acid and important inhibitory neurotransmitter which has been

implicated in CNS diseases, ranging from dementia to anxiety and mood disorders.

The selection of molecules that influence GABA release or uptake requires both a

viable animal model and a reliable analytical method. Brain tissue homogenates

primarily reflect intracellular GABA which is present at a considerably higher

concentration. GBP, marketed for the treatment of seizures and neuropathic pain, has

been shown to increase in vivo GABA concentration in the brain of both rodents and

humans (Loscher et al., 1991; Petroff et al., 2000). In the present research work

developed mucoadhesive tablet formulations were also evaluated for their in vivo

efficacy. The pentylenetetrazole induced seizure activity model was used for

evaluating anticonvulsant potential of developed mucoadhesive formulation. Brain

tissue levels of neurotransmitter, Gammaaminobutyric acid (GABA) was analysed in

both treated animals and untreated animals after inducing seizure activity. For

analysis of GABA in brain samples more sensitive HPLC method was developed with

spectrofluorimetric detection.

Most of the amino acids, upon reacting with o-phthaldehyde (OPA) at an alkaline pH

give rise to fluorescent derivatives which could be separated on a C18 column by

reverse phase high pressure liquid chromatography. Precolumn derivatization of

amino acid by OPA reagent forms highly fluorescent 1- alkylthio-2-alkylisoindole

derivatives at room temperature which are less polar than their respective amino acids

and can be separated on C-18 reversed phase HPLC. Reverse phase separations are

routinely carried out in the pH range of 6.0 to 7.5 for optimal fluorescence.

2B.7.1 Reagents: Gammaaminobutyricacid, o-phthaldehyde, 2-mercaptoethanol,

0.1M sodium tetraborate solution, HPLC grade Acetonitrile and ethanol. All the

solvents used were filtered through 0.45-µm-membrane filter and degassed using

sonicator.

OPA-BME reagent for derivatisation: Accurately weighed 30 mg of o-

phthaldehyde was dissolved in 2 ml ethanol and 20 µl of 2-mercaptoethanol and 10

ml of 0.1M sodium tetraborate was added to the solution. 2 ml of this solution was

diluted to 10 ml with 0.1M sodium tetraborate. It was keep it on ice and used within 5

hours.

Chapter 2


2B.7.2 Preparation of standard solutions

10 mM GABA solution was prepared by dissolving 10.3 mg of GABA in 10 ml

distilled water. The reference solution was diluted with diluent to obtain different

concentration GABA (10, 30, 50, 70, 80 and 100ng/ml). 20 µl of these solutions were

mixed with 50 µl of the OPA-BME reagent for derivatisation. Derivatised sample was

shaken for 80 seconds and immediately injected. Area and concentration was

subjected to least square line regression analysis to calculate the calibration equation

and coefficient of correlation (r2). The regression coefficient for the calibration curve

of GABA was found to be 0.9991 and linear concentration was found to be in the

range of 10ng/ml to 100ng/ml.

2B.7.3 Instrumentation: All the chromatographic measurements were carried out by

using a HPLC Merck Hitachi Model comprising of La Chrom L-7100 pump,

Fluorimetric detector and L-7200 auto sampler.

2B.7.4 Chromatographic conditions

Column : C18, 150 m X 4.6 mm (MKR 44)

Flow rate : 1 ml/min

Column temp : 37 0C

Mobile Phase : Buffer: Acetonitrile (77:23)

Buffer : 80 mM NaOH: Dissolve 3.2 g of sodium hydroxide in 1000 ml of

water. Adjust to pH 5.4 with glacial acetic acid

Detector : FL Excitation at 330 nm and emission at 450 nm

Inj. Vol. : 20 µl

Run time : 15 minutes

2B.7.5 Test solution: The brains were rapidly removed from decapitated animals and

then they were rapidly dissected and immediately homogenized in 2 ml of 80 %

ethanol for 2 minutes in ice cold conditions. 0.1 M perchloric acid was then added to

the homogenized tissue for deproteinisation and centrifuged for 30 minutes at 5000

rpm at 4° C. Supernatant was separated and filtered through Millipore cellulose

acetate membrane and stored in deep freezer for further use. 20 µl of this solution was

mixed with 50 µl of the OPA-BME reagent for derivatisation. Derivatised sample was

shaken for 80 seconds and immediately injected.

Chapter 2


Fig 2B.1.12: HPLC Chromatogram showing peak of GABA in brain homogenate by

spectrofluorimetry

Fig 2B.1.13: Standard Curve of GABA

2B.7.6 Validation of analytical method for estimating GABA content in brain

tissue

In order to confirm the reliability, accuracy and precision of the method, several

parameters were investigated according to ICH guidelines for analyzing GABA in

brain homogenate.

Linearity

Linearity curves to assay GABA in were constructed by assaying GABA derivatives

prepared with OPA-BME reagent in the concentration range of 10ng to 100ng/ml.

The linearity was repeated on three different days. The regression coefficient was

calculated for each day.

Precision

Intra-day precision

y = 3367.9x R² = 0.9991

0

50000

100000

150000

200000

250000

300000

350000

400000

0 50 100 150

Are

a

Concentration (ng/ml)

Area

Linear (Area)

Chapter 2


The intra-day precision was determined by sequential analysis at three concentration

levels of GABA (10, 50 and 100 ng/ml). Each sample was analyzed at three different

times a day, totaling to nine analyses in a day.

Inter-day precision

Three different concentrations 10, 50 and 100 ng/ml of GABA were analyzed on three

different days. Three replicates per concentration were injected in to the HPLC. SD

and standard error of mean (SE) were compared as a measure of scatter of method

precision.

Repeatability

System repeatability was determined by injecting 6 replicates of standard 50 ng/ml

GABA solution after appropriate derivitisation. The entire procedure was repeated on

three different days and the relative standard deviation (% RSD) was calculated to

determine the deviation of each measurement from mean value for each day.

Recovery

High recovery of the drug from the homogenate matrix is a desirable outcome of

sample preparation and is therefore an important characteristic of the extraction

procedure. The absolute recovery was determined as the ratio of response measured

for the spiked sample (in homogenate matrix) treated according to the extraction

procedure to that of a nonbiological sample spiked with the same quantity of the drug

and directly injected into the chromatographic system. Three different concentration

levels (10, 50 and 100 ng/ml) were investigated for determination of extraction

recovery.

Table 2B.1.10: Method validation data for GABA

Analytical parameters GABA

Retention time 18.36 ±0.5

LOQ (ng/ml) 10

LOD (ng/ml) 5

Linearity

Range (ng/ml) 10-100

Slopea ± % RSD 3367.9 ± 0.852

System precision

Amount taken 50

Amount detectedb(ng/ml) 49.3

% RSD 0.98

R2 0.9991


Chapter 2


Table 2B.1.11: Interday-intermediate precision using proposed method(n=3) for

GABA

GABA

Level

Amount

taken

(ng)

Amount

detected

(ng)

% RSD

1 10 9.7 1.6

2 50 49.7 1.2

3 100 99.8 1.89

Table 2B.1.12: Validation of developed analytical method for accuracy

GABA

Level Amount

taken

(ng)

Amount

detected

(ng)

%

recovery

1 10 9.8 98

2 50 48.32 96.6

3 100 98.1 98.1 Mean % recovery (n= 9) 97.56

%RSD 0.678

2B.8 Analytical method development for estimation of GBP in plasma by HPLC

In vivo efficacy of the developed mucoadhesive formulations were assessed by

pharmacokinetic studies in Wistar rats. Measurement of drug concentration in plasma

is essential to estimate the pharmacokinetic parameters. A sensitive and reliable

bioanalytical HPLC method was developed for the estimation of GBP in rat plasma

during pharmacokinetic studies. For determination of concentration of GBP plasma,

HPLC was performed using mobile phases as reported in the literature (Forrest et al.,

1996).

Chapter 2


2B.8.1Chromatographic conditions

Column : C18, 150 m X 4.6 mm (MKR 44)

Flow rate : 2 ml/min

Column temp : 37 0C

Mobile Phase : 0.33M acetate buffer (Containing 100mg/ml EDTA): methanol:

Acetonitrile (40:30:30, v/v)

Acetate buffer : Acetate buffer was prepared by diluting 7.5 ml glacial acetic acid

(approximately 17.4 M) to 400 ml with water, Adding 40mg of

EDTA and adjusting the pH to 3.7 with 3 M NaOH. Adjust to

pH 5.4 with glacial acetic acid

Detector : FL Excitation at 330 nm and emission at 450 nm

Inj. Vol. : 20 µl

Run time : 15 minutes

2B.8.2 Extraction of GBP

The blood samples (750µl) were withdrawn in a centrifuge tube containing disodium

EDTA (400µl of 4% Solution) as an anticoagulant. The blood samples were

centrifuged at 4000 rpm at 40C for 20mins for plasma separation. Separated plasma

was treated with 0.2 M perchloric acid for precipitation of the plasma proteins to

avoid the interference from it during HPLC studies. Again the solution was subjected

to centrifugation and the supernatant was separated. The known amount of GBP was

added to the supernatant which was previously separated and derivitised in similar

manner as GABA samples. GBP standards were prepared by addition of appropriate

working standard (1-10 ppm) to blank plasma, without drug treated in a similar

manner.

Fig 2B.1.14: Standard curve for GBP in plasma by Spectrofluorimetry

y = 39861x + 2631.9

R² = 0.9995

0

100000

200000

300000

400000

500000

0 5 10 15

A

r

e

a

Concentration (ppm)

Standard curve of Gabapentin by

spectrofluorimetric detection

Area

Chapter 2


Fig 2B.1.15: HPLC Chromatogram showing peak of GBP spiked in plasma by

spectrofluorimetry

2B.8.3 Validation of analytical method for estimating GBP in Plasma

A. Specificity

The specificity of the method was verified by evaluating the interference of plasma

components on the assay of active, GBP. To determine the method specificity, the

control brain samples were treated with the same procedure as that to extract actives

and the spectra obtained from these injections were compared to the separated plasma

spiked with GBP.

B. Linearity

Linearity curves to assay GBP in plasma samples were constructed by assaying

plasma samples spiked with GBP the concentration range of 1 to 10 µg/ml. The

samples were injected into the HPLC and chromatograms were recorded at FL

Excitation of 330 nm and emission of 450 nm. An appropriate concentration of

internal standard was selected for spiking the plasma based on preliminary studies.

The final solutions were injected in triplicate. The calibration curve was obtained by

plotting peak area ratio of GBP v/s concentration. The linearity was repeated on three

different days. The regression coefficient was calculated for each day.

C. Precision

Intra-day precision

The intra-day precision was determined by sequential analysis at three concentration

levels of GBP (1.0, 5, 10 µg/mL). Each sample was analyzed at three different times a

day, totaling to nine analyses in a day.

Inter-day precision

Three different concentrations 1, 5, 10 µg/mL of GBP were analyzed on three

different days. Three replicates per concentration were injected in to the HPLC. SD

Chapter 2


and standard error of mean (SE) were compared as a measure of scatter of method

precision.

Repeatability

System repeatability was determined by injecting 6 replicates of 4.0 µg/mL of GBP

solution respectively which was prepared by spiking the appropriate concentrations of

drug and internal standard into the separated plasma followed by extraction. The

entire procedure was repeated on three different days and the relative standard

deviation (% RSD) was calculated to determine the deviation of each measurement

from mean value for each day.

Recovery

High recovery of the drug from the plasma sample is a desirable outcome of sample

preparation and is therefore an important characteristic of the extraction procedure.

The absolute recovery was determined as the ratio of response measured for the

spiked sample (in separated plasma) treated according to the extraction procedure to

that of a nonbiological sample spiked with the same quantity of the drug and directly

injected into the chromatographic system. Three different concentration levels (2.0,

5.0 and 10.0 µg/mL) were investigated for determination of extraction recovery.

Table 2B.1.13: Method validation data for GBP

Analytical parameters GBP

Retention time 8.13 ±0.5

LOQ (µg/ml) 0.5

LOD (µg/ml) 1

Linearity

Range (µg/ml) 1-10

Slopea ± % RSD 39861 ± 0.152

System precision

Amount taken 4

Amount detectedb(µg/ml) 3.98

% RSD 0.752

R2 0.9995


Chapter 2


Table 2B.1.14: Interday-intermediate precision using proposed method(n=3) for

GBP

GBP

Level

Amount

taken

(µg)

Amount

detected

(µg)

% RSD

1 1 9.74 1.6

2 5 4.94 1.2

3 10 9.89 1.89

Table 2B.1.15: Validation of developed analytical method for Accuracy

GBP

Level Amount

taken

(µg)

Amount

detected

(µg)

%

recovery

1 1 9.78 97.82

2 5 4.9 97.3

3 10 98.1 98.1 Mean % recovery (n= 9) 97.56

%RSD 0.412

Results and Discussion:

2B.9.1 UV method for determination of GBP

In the present work, U.V spectrophotometric method for the quantitation of GBP in

oral dosage form was developed for routine analysis. The method was developed

using 0.01N HCL solution methanol as solvent systems as the drug showed good

solubility in both the solvent systems. In proposed method, absorption maxima was

obtained at 215 nm for GBP in solvent system methanol and 212 nm for GBP in

0.01N HCl and the calibration curve obeyed Beer’s Lamberts law in the concentration

range of 500-3000 μg/ml with correlation coefficient (r²) of 0.9904 (Fig 2B.1.2) and

0.994 in methanol and 0.1 N HCl (Fig 2B.1.4) respectively. Since the UV method

was not sensitive enough to estimate the drug content and for monitoring of the drug

release during dissolution studies, this method was not found to be useful for the

routine analysis of drug.

2B.9.2 Colorimetric method for determination of GBP

This method was found to be more sensitive than the UV method i.e. in the range of

40-240 ppm (Fig 2B.1.6). But since the GBP react with ninhydrin at the neutral pH

only, it was difficult to estimate the drug content in acidic and basic vehicles. In

Chapter 2


dissolution studies of the GBP tablets the dissolution media for mucoadhesive tablet

was 0.1N HCl. When the samples were subjected to colorimetric analysis the

solutions were not giving purple color indicating that the reaction has not occurred.

Also the qualitative and quantitative estimation of the toxic lactam impurity was not

possible with this method which is commonly exhibited by the drug during the

process of formulation.

2B.9.3 HPTLC method for determination of GBP

HPTLC method for the quantitation of GBP in oral dosage form was developed for

routine analysis. To increase sensitivity, in proposed method GBP was mixed with

ninhydrin reagent and analyzed for GBP. The results for the same are given in Table

2B.1.4 and Fig 2B.1.8. The regression coefficient for the calibration curve was found

to be 0.9916 and linear concentration was found to be in the range of 200ng/ml to

1200ng/ml. This procedure was too lengthy and tedious for day-to-day routine

analysis of the dissolution samples so this method was discontinued. Also the

quantitation of lactam was difficult with HPTLC. So HPLC method was further

investigated for analysis of both GBP and lactam in dissolution samples.

2B.9.4 Development of HPLC method for determination of GBP from dissolution

samples

The linear regression analysis of calibration curve of GBP and lactam showed

excellent correlation coefficients as indicated by the linearity result reported in Table

2B.7. The calibration curves were linear in the range of 50-500 ppm for GBP (R2=

0.9991) and 5-50 ppm for lactam (R2=0.9998). The system precision and intermediate

precision were expressed in terms of relative standard deviation. Excellent system

precision was evident with % RSD of 0.752 and 1.76 for GBP and lactam respectively

that indicates repeatability of the method. The intermediate precision of reference

solutions at three concentration levels (n=3) on three different days was also evident

with a low percent RSD providing the ruggedness of the method (Table 2B.8).

Accuracy of the method was determined by performing the recovery experiment.

Three replicate samples at each concentration level were prepared and the % recovery

at each level (n = 3) and mean % recovery (n = 9) were determined (Table 2B.9). The

mean recovery was found to be 100.04 and 100.4 for GBP and lactam respectively.

Thus, the developed method was found to be accurate and precise. This method was

found to be useful for the estimation of drug content and analysis dissolution samples.

Chapter 2


2B.9.5 Development of HPLC method with Spectrofluorimetric detection for

estimation of gammaaminobutyric acid (GABA) in the brain

Linearity: Calibration curves constructed for GABA by plotting the graph of

concentration versus GABA area was found to be linear in the range of 10 to 100

ng/ml as shown in the Fig 2B.1.13. The analytical method showed a regression

coefficient greater than 0.9905 on all the three days. Thus the linear regression

analysis demonstrated acceptability of the method for quantitative analysis of GABA.

Intra-day and Inter-day Precision: The observed lower values of relative standard

deviation, lower % RSD values < 2, at both, intra-day and inter-day analysis indicated

the method to be precise. It showed the acceptability of the method with adequate

intra-day and inter-day precision (Table 2B.1.11).

Repeatability: % RSD low variance for three separate days for GABA as shown in the

Table 2B.1.11. This demonstrates the method to be repeatable for the analysis of

GABA.

Recovery: The recovery was calculated from the GABA concentration with standard

sample and compared with the spiked brain homogenate. The mean recovery of

GABA was 98.0%, 96.6% and 98.1% at concentrations of 10, 50 and 100 µg/mL

respectively (Table 2B.1.12). The average recovery over the entire analytical range

was 97.56%. From the recovery rates, it can be concluded that the extraction

procedure provided a reliable quantitative determination of the GABA from brain

homogenate.

2B.9.6 Bioanalytical method development for estimation of GBP in plasma by

HPLC:

GBP could be easily extracted from the blood plasma using extraction procedure

mentioned in section 2B.8.2. The suitability of the method was further verified by

performing the detailed validation of the method as per ICH guidelines.

Specificity: The control plasma sample HPLC spectra were compared with HPLC

spectra from GBP spiked plasma sample. The retention time of the drug was recorded

at 8.1 min. In the chromatogram of the control plasma, plasma components did not

interfere with the peak of interest. Since no interference between the drug and plasma

components were observed in the HPLC spectra, the method was proved to be

selective and specific.

Chapter 2


Linearity: Calibration curves constructed for GBP by plotting the graph of

concentration versus GBP area was found to be linear in the range of 1.0 to 10µg/ml

as shown in the Fig 2B.1.14. The analytical method showed a regression coefficient

greater than 0.9995 on all the three days. Thus the linear regression analysis

demonstrated acceptability of the method for quantitative analysis of GBP in the

plasma samples.

Intra-day and Inter-day Precision: The observed lower values of relative standard

deviation, lower % RSD values <2, at both, intra-day and inter-day analysis indicated

the method to be precise. It showed the acceptability of the method with adequate

intra-day and inter-day precision (Table 2B.1.14).

Repeatability: % RSD displayed low variance for three separate days for GBP as

shown in the Table 2B.1.13. This demonstrates the method to be repeatable for the

analysis of GBP from the plasma sample.

Recovery: The recovery was calculated from the GBP concentration with the

nonbiological sample and compared with the sample spiked with plasma. The mean

recovery of GBP was 97.82%, 97.3% and 98.1% at concentrations of 1.0, 5.0 and 10.0

µg/mL respectively (Table 2B.1.15). The average recovery over the entire analytical

range was 97.56%. From the recovery rates, it can be concluded that the extraction

procedure provided a reliable quantitative determination of the drug in plasma

samples.

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