Report

RESEARCH PRACTICE

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ASSIGNMENT

A Report

On

CHROMATOGRAPHIC ANALYTICAL METHOD DEVLOPMENT AND

PREPERATION, OPTIMIZATION OF MUCOADHESIVE BUCCAL

FORMULATION FOR SUITABLE DRUG CANDIDATE DRUG

By

PRAVIN G.LONDHE

ID NO: 2011H108172P

Submitted in partial fulfillment of the course

BITS G540: Research Practice

Submitted to

Mr. Jaipal A.

(Instructor In-charge)

BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE, PILANI,

RAJASTHAN – 333 031

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AIM: Chromatographic analytical method development and preparation and optimization

of Mucoadhesive buccal formulations for a suitable drug candidate.

INTRODUCTION:

Oral drug is considered as a one of the best patient compliant route for the delivery of drug molecules

and is economical. Since, some drugs either degrade in the stomach due to harsh acidic pH conditions

in the stomach or metabolize due to extensive first-pass metabolism. Parenteral route is also better

option for delivery of drugs that has gastric degradation and have extensive first pass metabolism, but

parenteral route is not patient compliant, non-economical and requires skilled professional to

administer the drug. So, there is a need to develop an alternate dosage form for the drugs that cannot

withstand harsh pH conditions and high metabolism. Buccal delivery is one of the options with an

advantage of delivering drugs to systemic circulation without hepatc firstpass metabolism and for a

prolonged period of time due to mucoadhesion nature of buccal mucosa.

Buspirone :

Mechanism of Action:

Buspirone binds to 5-HT type 1A serotonin receptors on presynaptic neurons in the dorsal raphe and

on postsynaptic neurons in the hippocampus, thus inhibiting the firing rate of 5-HT-containing

neurons in the dorsal raphe. Buspirone also binds at dopamine type 2 (DA2) receptors, blocking

presynaptic dopamine receptors. Buspirone increases firing in the locus ceruleus, an area of brain

where norepinephrine cell bodies are found in high concentration. The net result of buspirone actions

is that serotonergic activity is suppressed while noradrenergic and dopaminergic cell firing is

enhanced

Pharmacokinetic Properties:

Biotransformation:Metabolized hepatically, primarily by oxidation by cytochrome P450 3A4

producing several hydroxylated derivatives and a pharmacologically active metabolite, 1-

pyrimidinylpiperazine (1-PP)

Protein Binding: 95% (approximately 70% bound to albumin, 30% bound to alpha 1 -acid

glycoprotein)

Absorption:Rapidly absorbed in man. Bioavailability is low and variable (approximately 5%) due to

extensive first pass metabolism.

Half-Life: 2-3 hours (although the action of a single dose is much longer than the short halflife

indicates).

Toxicity: Oral, rat LD 50 = 136 mg/kg. Symptoms of overdose include dizziness, drowsiness, nausea

or vomiting, severe stomach upset, and unusually small pupils.

Interactions: Inhibitors and Inducers of Cytochrome P450 3A4 (CYP3A4): Buspirone has been shown

in vitro to be metabolized by CYP3A4. This finding is consistent with the in vivo interactions

observed between Buspirone and the following

Itraconazole: Increased plasma level of buspirone.

Nefazodone: Increased plasma levels of buspirone.

Rifampicin: Decreased plasma levels of buspirone.

Carbamazepine: Reduced plasma levels of buspirone.

Haloperidol: Increased plasma levels of haloperidol.

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Grapefruit or grapefruit juice: Significantly increases the plasma levels of buspirone. The probable

mechanism of this interaction caused by grapefruit juice is delayed gastric emptying and inhibition of

the cytochrome P450 3A4-mediated first-pass metabolism of buspirone.

Polymers for buccal tablet:

o Carbopols (CP934, and CP940) – swellable with water

o polycarbophil (PC) - swells in water to a range of volume depending primarily on pH,

insol. In water , in dilute acids, in dilute alkali, and in common organic solvents.

o Sodium carboxymethyl cellulose (Scmc) - Easy Solubility in cold and hot water, Yield

viscous colloidal solution with water; insoluble in ethanol, anionic water soluble

polymer.

o Pectin representing the anionic type - Partially soluble in cold water. Soluble in 20

parts of water. Dissolves more readily in water if it is first moistened with alcohol

glycerol or sugar syrup, or if first mixed with 3 or more pars of sucrose. Insoluble in

alcohol, dilute alcohol, organic solvents.

o White chitosan (Ch) as cationic polymer - o Hydroxypropylmethyl cellulose (HPMC) as a non-ionic polymer - soluble in cold

water, glacial acetic acid, ethanol, methanol and propylene glycol, slightly soluble in

acetone depending on the degree of substitutions, practically insoluble in hot water,

ethylene glycol and toluene. HPMC is dissolved in a mixture of 10% methanol and 90%

methylene chloride to form colloidal solutions.

o Xantham gum- Exhibits pseudoplastic behavior in solution (i.e. high viscosity at rest,

viscosity decreases with increasing shear rates, and instantly returns as shear is reduced

or removed). Higher degree of pseudoplasticity than other commercial hydrocolloids.

Viscosity is maintained over a wide pH range (pH 2 to 12). Compatible with most

common acids, and most basic compounds. pH increases in the presence of soluble

multivalent cations, which can cause cross-linking to an insoluble gel. Viscosity increases

in the presence of a salt, including divalent metal salts such as calcium and magnesium.

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Review of literature for the method development and validation

1. Ramesh Gannu et al had developed RP-HPLC method for buspirone HCL in rabbit serum , they

have used the c8 column for the pharmacokinetic study .the elutent was monitored bu using UV

at 235 nm using isocratic mode of elution , the linearity range was found to be 1-3000 ng ml -1

,

the method was found to be precise accurate and sensitive during the study.

2. M. V. Basaveswara Rao et al had developed RP-HPLC method Validated RP-HPLCMethod for the

Determination of Buspiron in Pharmaceutical Formulations, they have used isocratic elution with flow

rate of 1 ml/min and used the c18 column , the mobile phase consisted of the water :ACN:methanol

45:35:20 (v/v ) the UV detection wavelength was 210 nm and 20 ul samples were injected

3. A. azeem et al had done development and validation of the stability indicating LC-UV method

for rapid analysis of buspirone in pharmaceutical dosage form they have used RP c 18 column

with the methanol and 0.01 M sodium dihydrogen phosphate buffer ( pH 3.5) at flow rate of the

0.8ml/min UV detection was at 244 nm , responce was linear over the range of the 0.05-20 ug/ml

-1 ( r= 0.9998) ,the method was validated according to the ICH guidelines ,and was found to be

accurate, precise , and sensitive .

Most literature methods for analysis of buspirone are concerned with biological fluids. Buspirone

and its metabolites have been quantified in plasma by use of HPLC coupled with UV , electrochemical ,

and coulometric detection. LC–MS and LC–MS–MS methods have also been introduced recently.

Buspirone had been quantified in se-rum by solid-phase extraction and two-dimensional HPLC with UV

detec-tion . Pharmacokinetic analysis of the drug in rat plasma had been per-formed by means of brain

microdialysis coupled to HPLC with electro-chemical detection . The structure profile of buspirone

metabolites has also been assessed by use of LC–MS

Buspirone had also been analyzed by capillary zone electrophore-sis , voltammetry , HPLC , TLC ,

potentiometry , and differential pulse polarography . Four impurities of buspirone have been fractionated

by use of liquid chromatography with electrospray ioni-zation mass spectrometry [27]. Colorimetric

methods, with laborious deri-vatization, have been reported for analysis of buspirone in tablets.

The objective of the present study was to develop a simple, reliable, rapid and sensitive analytical

method with better detection range, with improved retention time for the estimation of buspirone

hydrochloride in bulk, pharmaceutical formulation

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Material and method

1. Drug profile ( buspirone hydrochloride) :

Physiochemical properties of the drug :

IUPAC Name: 8-[4-(4-pyrimidin-2-ylpiperazin-1-yl)butyl]-8-azaspiro[4.5]decane-

7,9-dione

Structure

Log P 1.95, 1.78

Refractivity 108.89

pKa 1.22, 7.32

Melting point 201.5-202.5oC

Water solubility 1g/ml

BCS class Class-1 (high solubility and high permeability)

Tablet marketed 5 mg / tablet, 7.5 mg / tablet 10 mg / tablet, 15 mg / tablet, 30 mg /

tablet.

Colour

White

Melting point 201.5.5-202.5oC

Mechanism of action :

o Buspirone is the partial agonist at the 5-HT 1A receptor and is used to treat various anxiety

disorder , it also binds to the dopamine receptors it also inhibit the activity of the NA in locus

coerulus .thus useful in the anxiety

Side effects :

o Its main side effects are dizziness , nausea , hedache and restlessness which genrally seems to be

less troublesome. \

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2.GELLAN GUM:

CAS Number: 71010-52-1

Trade Names: Kelcogel®

Gelrite

Phytagel

Gel-Gro

Gellan gum is an anionic, high molecular weight, deacetylated exocellular polysaccharide gum

produced as a fermentative final product by a pure culture of Pseudomonas elodea. The

production organism is an aerobic, well characterized, non-pathogenic, gram-negative bacterium.

Gellan gum has the characteristic property of temperature-dependent and cation-induced

gelation. This gelation involves the formation of double helical junction zones followed by a

Gellan gumregation of the double helical segments to form a three-dimensional network by

complexation with cations and hydrogen bonding with water.

Gellan gum can be used to produce easy-to-swallow solid dosage forms, such as gels beads and

coated tablets, and also to modify the release rates of active ingredients from tablets and capsules

It is also conveniently used for controlled or sustained release of various drugs by the preparation

of various types of rate controlling dosage form.

CHEMICAL CHARACTERISTICS:

There are two chemical forms of gellan gum; these are native or natural form, which has high

acyl contents, and low or deacetylated form. Both forms have a similar linear structure. The

natural form of gellan is composed of the linear structure of a repeating tetrasaccharide unit of

glucose, glucuronic acid and rhamnose in a molar ratio of 2:1:1. It is partially acetylated with

acetyl and L-glyceryl groups located on the glucose residues. The native form contains two acyl

substituents, namely acetate and glycerate, both being located on the same glucose residue and on

the average, there is one glycerate and a half acetate group per every tetrasaccharide repeating

unit. This difference in substitutions leads to a difference in the gelling potential. However, the

presence of acetyl groups interferes in ion bonding ability. The native polysaccharide is partially

esterified with L-glycerate and acetate but the commercial product Gelrite® has been completely

de-esterified by alkali treatment.

The exact molecular formula of gellan gum may vary slightly (e.g., depending on the degree to

which the glucuronic acid is neutralized with various salts). Because of the presence of free

carboxylate groups in gellan gum, it becomes anionic in nature and thus, would possess the

characteristic property of undergoing ionic gelation in the presence of mono- and divalent cations.

However, the affinity for divalent cations is much stronger than the monovalent cations.

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Repeating units of Chemical structure of (a) native and (b) Deacetylated gellan gum.

PHYSICAL CHARACTERISTICS:

Properties Values

Nature Off white powder

Solubility Water soluble, forms viscous gels but insoluble

in ethanol

Stability Stable in normal room temperature

Bulk density Near about 836 kg/m3

Moisture content 98.6%wb or 67.6% db

Loss on drying Not more than 15% (105°, 2½h)

Specific gravity Less than 1 (<1)

Gel strength 550-850 (gm/cm).

pH Neutral (7).

Molecular mass Approximately 5, 00,000

Lead content Not more than 2 mg/kg

Nitrogen content Not more than 3 %

Isopropyl alcohol content Not more than 750 mg/kg

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APPROVED LEGAL USES OF THE SUBSTANCE:

According to EPA, gellan gum is exempt from the requirement for a pesticide tolerance when used as an

inert ingredient in pesticide formulations (EPA, 2004). Approval for the use of gellan gum as an inert

ingredient in pesticide formulations is promulgated at 40 CFR 180.950.

According to FDA, gellan gum may be safely used as a direct food additive for human consumption as

long as its use is in accordance with 21 CFR 172.665. According to 21 CFR 172.665, gellan gum is

produced from P. elodea (now known as S. elodea) by a pure culture fermentation process and purified by

recovery with isopropyl alcohol. Residual isopropyl alcohol in the gellan gum must not exceed 0.075

percent. Additionally, it is exempt from the threshold of regulation (21 CFR 170.39) for its use as a

coating or sizing agent on food contact articles.

APPLICATION OF GELLAN GUM IN DRUG DELIVERY SYSTEM:

For the delivery of the drug product in the specific area gellan gum is used as a very good carrier by the

preparation of different types of attractive formulations. The application of gellan gum is based on the

following features-

1. It controls the delivery of the drug product to the specific pharmacological target.

2. It acts as a permeation enhancer and increases drug products permeability across bio

membranes, such as the small intestinal membrane.

3. In the formulations it also used to increases the drug products stability.

4. It increases the drug products solubility in biofluids such as intestinal fluid.

5. It decrease drug candidate metabolism, and/or decrease its elimination.

One of the most remarkable and useful features of gellan gum is its swelling ability as its swelling can be

triggered by a change in the environment surrounding to the delivery system. Depending upon the

polymer, the environmental change can involve pH, temperature or ionic strength and the system can

either shrink or swell upon a change in any of these environmental factors of these sensitive systems.

Drug release is accomplished only when the polymer swells and because gellan gum is potentially most

useful pH sensitive polymer that swells at high pH values and collapse at low pH values, triggered drug

delivery occurs upon an increase in the pH of the environment. That’s why gellan gum is very ideal for

systems such as oral delivery, in which the drug is not released at low pH values in the stomach, but

rather at high pH values in the upper small intestine.

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Observation and results:

1.Optimization of the formula for Buccal Tablets:

Batch I

S. NO. INGREDIENTS RATIONALE FOR 1 TABLET

(mg)

FOR 20

TABLETS

(mg)

1. Buspirone Drug 2.5 50

2. Gellan gum Polymer 30 600

3. Micro crystalline

cellulose

Diluent (Directly

compressible)

22.5 450

4. Mannitol Channeling and

Cooling agent

11 220

5. Magnesium

stearate

Lubricant 1.5 30

6. Kollidon SR Dry binder 7.5 150

Batch II


(mg)

FOR 20

TABLETS

(mg)


2. Gellan gum Polymer 41.25 825

3. Micro crystalline

cellulose

Diluent (Directly

compressible)

11.25 222.5


Cooling agent

7.5 150

5. Magnesium

stearate

Lubricant 1.5 30

6. Kollidon SR Dry binder 10.25 210

Batch III


(mg)

FOR 20

TABLETS

(mg)




Cooling agent

8 160

4. Magnesium

stearate

Lubricant 1.5 30

5. Kollidon SR Dry binder 15 300

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Batch IV


(mg)

FOR 20

TABLETS

(mg)




Cooling agent

26 529

4. Magnesium

stearate

Lubricant 1.5 30

Problem faced: Disintegration of tablet when added to water.

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2.Analytical Method Development by High Performance Liquid Chromatography

Every day many chromatographers face the need to develop a high-performanceliquid chromatography

(HPLC) separation. Where as individual approachesmav exhibit considerable diversity. method

development often follows theseries of steps summarized in Fig. we review the importance of each of

these steps, in preparation for a more detailed examination in Our philosophy of method development is

based on several considerations. There exists today a good practical understanding of chromatographic

separation and how it varies with the sample and with experimental conditions. Any systematic approach

to HPLC method development should be based on this knowledge of the chromatographic process. In

most cases, a desired separation can be achieved easily with only a few experiments. In other cases, a

considerahle amount experimentation is needed .the good method devloment stratergies strategy should

require only as many experimental runs as are necessary to

achieve the desired final result.

Steps in method development :

Validation of the method for routine analysis.

A. recover purified material

B.quantititive calibration C qualitative method

Check for problem or requirement of special condition

Optimise the separation conditions

Choose LC method preliminary run; estimate best separation conditions

Choose detector and detector settings

Need of special procedure , sample pretreatment

Information of the sample and separation goals

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Instrumental parameters: The optimised separation conditions were found to be

Instrument: Shimadzu HPLC

Software: LC Solution

Column used: C-8 (250mm*25mm; particle size = 5 microns)

Column Temperature : 25˚C

Mobile phase: 25mM Phosphate Buffer pH 3.0 : Methanol (50:50)

Flow rate:

Pump A = 0.5 ml/min (166-175 kgf/c)

Pump B = 0.5 ml/min (166-175 kgf/c)

Runtime: 10 minutes

λ max of Buspirone: 238 nm

Detector : UV Detector

Injection volume : 50 microlitres

The preliminary trials were done as following :

500 ng/ml concentration

Mobile phase: 25mM Phosphate Buffer pH 3.0 : Methanol (50:50)

Retention time = 7.022 min

Area under peak = 88500

Tailing factor = 1.14

Problem faced: ‘Ghost Peak’ in Blank sample.

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Possible cause for getting the ghost peak.

1.Contamination in the injector or column

2. Late eluting peak usually broad present in the sample.

Remedy we have used for removing the ghost peak

Flushing of the injector between analysis we have runned strong solvent through the column here

we have used the ACN as the solvent but we were still facing the same problem

We can include final wash step so as to remove the strongally retained compound from the

column

The third possible cause was sample preparation which we have checked many times but we

werw still facing the same problem.

.

Trials for removing the ghost peak…

Multiple Column washings given.

Ratio of Aqueous to Organic phase (of Mobile phase) varied.

After all this trial we were getting the same problem so we thought to change the mobile phase

Change of Mobile phase composition(organic phase) : Use of Acetonitrile

Mobile phase = 25mM Phosphate Buffer pH 3.0 : ACN (65:35)

Other conditions kept same.

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Problems :

1. Greater baseline noise

2. Appearance of Ghost peaks

Retention time = 7.323


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Reverse flushing of column was done; trials were done but Ghost peaks persisted.

Use of Another C-8 column:

Mobile phase: 25mM Phosphate Buffer pH 3.0 : ACN (70:30)

Multiple column washings given…

Still the same problem is coming ghost peak is persisting…


Area = 88605


Problems:

1. Tailing factor greater than 1.2 - not acceptable.

2. Bulge observed in the peak.

3. Persistent Ghost peak.

So we thought it is the problem with the column so now we are shifting to the c18 column.

Use of C-18 column and Acetate buffer in Mobile phase

Mobile phase = Methanol : Acetate buffer pH 4.0 (70:30)


Area = 64722


Problem:

1. T.F. > 1.2

2. Noise and bigger peaks at the other retention times

3. Ghost peak

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The same problem we are facing in the C18 column also but we are thinking we will do troubleshooting

with the C18 column..

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REFERENCES:

1. Shojaei A. H., Buccal mucosa as a route for systemic drug delivery: a review. Journal of

Pharmacy and Pharmaceutical Science. 1998, 1: 15-30.

2. Aulton M E., The design of dosage forms. In: Pharmacutics-The science of dosage form design.

(Peter York., Eds), 3rd ed. Churchill Livingston, Oxford, UK.2001, 1-12.

3. Ikinci G., senel S., Akıncıbay H., Kas S., Ercis S., Wilson C G., Hıncal A A., Effect of chitosan

on a periodontal pathogen Porphyromonas gingivalis. International Journal of Pharmaceutics.

2002, 235: 121-127

4. Voorspoels J., Remon J P., Eechaute W., and De Sy W., Buccal absorption of testosterone and its

esters using a bioadhesive tablet in dogs. Pharmaceutical Research. 1996, 13: 1228-1232.

5. Eunsook C., Hyesun G., Inkoo C., Formulation and evaluation of ondansetron nasal delivery

systems. International Journal of Pharmaceutics. 2008, 349: 101–107.

6. Ramesh Gannu, Shravan Kumar Yamsani, Chinna Reddy Palem, Development of high

performance liquid chromatography method for buspirone in rabbit serum: Application to

pharmacokinetic study, Analytica Chimica Acta 647 (2009) 226–230

7. M. V. Basaveswara Rao, A. V. D. Nagendrakumar, SushantaMaiti, and Guttikonda Raja Validated RP-HPLCMethod for the Determination of Buspirone in Pharmaceutical Formulations,

Chromatography Research International Volume 2011, Article ID 232505, 3 pages

8. A. AZEEM. RIZWAN, F.J.AHMAD, Z IQBAL, R.K.KHAR, M. AQIL,S.TALEGAONKAR

Development and Validation of a Stability- Indicating LC–UV Method for Rapid Analysis of

Buspirone in Pharmaceutical Dosage Forms Acta Chromatographica 21(2009)2, 283–297

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