METHOD DEVELOPMENT AND VALIDATIONS OF

RILPIVIRINE BY UV AND HPLC METHODS

INTRODUCTIONPharmaceutical analysis is a branch of

practical chemistry that involves a series of

process for identification, determination,

quantification and purification of a substance,

separation of the

components of a solution or mixture, or

determination of structure of chemical

compounds.

DIFFERENT ANALYTICAL METHODS

•Qualitative analysis

•Quantitative analysis

•Dual wavelength spectrophotometry

•Derivative spectra

ULTRA VIOLET-VISIBLE SPECTROSCOPY When a beam of light is passed through a

spectrum, then It gets dispersed in to seven

colors i.e. VIBGYOR, and these set of colors

or bands known as SPECTRUM.

The arrangement obtained by arranging

these bands or waves called

electromagnetic waves in the order of their

increased wave length or decreased

frequencies are known as

ELECTROMAGNETIC RADIATIONS.

So, the study of the relationship between the

electromagnetic radiations with matter as

the function of wavelength is known as

SPECTROSCOPY.

PRINCIPLE

UV visible spectroscopy measures the response of a sample to ultra violet and visible range of electromagnetic radiation.Molecules have either of electrons. These electrons absorb uv radiation & undergoes transitions from ground state to excited state

THEORY INVOLVEDMolecular absorption & fluorescence

INSTRUMENTATION & WORKING

Types of Uv- visspectrophotometer

Single Beam Double Beam

Earliest design.

The light beam do not split. All of it

passes through the sample or reference.

First a reference is used to calibrate the

instrument.

Then sample is measured, replacing the

reference.

SINGLE BEAM

SINGLE BEAM

The light beam is split into 2; one passes through sample and

other through reference.

They consists of rotating discs called Beam choppers or

Chopper mirrors.

Some instruments contain only a single detector and the

measurement is done alternatively.

Some instruments contain 2 detectors and both the sample

and reference are measured simultaneously.

DOUBLE BEAM

DOUBLE BEAM

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Chromatography is a physical process whereby

components ( solutes ) of a sample mixture are

separated by their differential distribution

between stationary & mobile phases .

Planar & column are two basic forms of

chromatography .

High performance liquid chromatography is a

form of column chromatography .

PRINCIPLE INVOLVED In analytical liquid chromatography the mobile phase or eluent , exits

from the column & passes through a detector or a series of detectors that produce a series of electronic signals that are plotted as a function of time distance or volume , the resulting graph is a chromatogram .

The retention time ( tR ) is the time taken for each analyte peak to emerge from the column .

• Under defined chromatographic conditions tR is a charcteristic of the

analyte .

• The volume of the mobile phase required to elute the analyte under

defined chromatographic conditions is referred to as retention ( or )

elution volume ( VR ) .

VR = tR Fc

The increased resolution achieved in HPLC compared to classical chromatography is primarily the result of adsorbents of very small particle size ( less then 20µm )& large surface areas .

The smallest gel beads used in gel exclusion chromatography are superfine grade with diameters of 20-50µm .

A combination of high pressure & adsorbents of smaller size leads to high resolution power & short analysis time in HPLC.

INSTRUMENTATION

AIM OF THE PROJECT

We are going to develop a new analytical

technique by using UV and HPLC methods for

the following drug.

RILPIVIRINERilpivirine is the second generation of

non-nucleoside reverse transcriptase

inhibitors (NNRTIS) recently marketed

for the treatment of HIV infection. It is

superior to the first generation

NNRTI(4-6) in that it is active against

NNRTI resistant HIV-I.

STRUCTURE-

IUPAC NAME-4-{[4-({4-[(E)-2-cyanovinyl]-2,6-dimethylphenyl}amino)pyrimidin-2-yl]amino}benzonitrile

MOLECULAR FORMULA-C22H18N6

BRAND NAME-Edurant

MOLECULAR WEIGHT- 402.88

MOLECULAR MASS-366.42 g/mol

NATURE-Rilpivirinehydrochloride is a white to almost white powder.

REVIEW OF LITERATURE Mohanreddy Chilukuri et al have developed Degradation pathway for Rilpivirine

hydrochloride by validated stability indicating UP-LC method. Rilpivirine is subjected

to stress conditions of acid, base, oxidation, thermal and photolysis and then six

impurities are studied and major degradant is identified by LC-MS and spectral

analysis. Chromatographic separation is achieved on a Shimpack XR-ODS-11

stationary phase with simple mobile phase combination and quantification is carried at

295nm with flow rate of 1.0ml/min. This developed LC method is validated with respect

to specificity, linearity and range, accuracy, precision, and robustness for impurities and

degradant determination.

CONTD.

D. Pranitha et al have published an research article on simultaneous

estimation of emtricitabine, tenofovir, disoproxil fumerate and

rilpivirine in bulk form by RP-HPLC method. The method employs

Thermo Hypersil ODS C-18 column and flow rate of 1ml/min with load

of 20μl. Acetonitrile and phosphate buffer (60:40) pH 3 was used as

mobile phase. Detection carried out at 260nm and percentage recovery

was found be 98-102%. This newly developed method was successfully

utilized for the quantitative estimation of drugs in bulk form.

CONTD.

Ch Venkata Reddiah et al have developed effective estimation of Rilpivirine by HPLC method in tablet dosage forms and its invitrodissolution assessment(8). Solvents used was acetonitril : buffer (55 : 45) %v/v and absorption maxima of drug was found to be 280nm. Linear response was observed in range of 5.5 –41.25μg/ml with R equal to 0.99. This method was successfully employed for quality control assay of compound simultaneously and dissolution data helpful in generating the further information regarding invivo absorption rate in tablet dosage form.

CONTD.

T.Sudha et al have developed a reverse phase high performance and HPTLC methods

for the determination of Rilpivirine bulk and in tablet dosage form. This method

depends on RP-HPLC, the mobile phase used consists of mixed phosphate buffer :

acetonitrile (60 : 40% v/v) with pH 6.8 and flow rate of 1.0ml/min in isocratic mode.

Separation was carried out by UV – detector at 272nm. Percentage recovery was

found as 100.53%. Second method depends on HPTLC and mobile phase used is ethyl

acetate : methanol : chloroform (8:1:1% v/v/v). Densiometric analysis was carried out

at 254nm and percentage recovery was found as 100.17%. This proposed method was

validated statistically and recovery study for the determination of Rilpivirine in bulk

and in tablet dosage form was performed.

PLAN OF WORK

• Solubility:- Rilpivirine is practically insoluble in water over a

wide pH range.

• Selection of Wavelength:- The sample of rilpivirine was prepared in

concentration of 10µ g/ml. This sample was scanned in the range of

the 200-400nm using UV-visible spectrophotometer.

Method Development of Rilpivirine by Using UV Spectroscopy

Method Development of Rilpivirine by Using HPLC :-

REFERENCES Laurence L, John S, Keith L. The Pharmacological Basis of Therapeutics. 2006.

Scientific Discussion.

Barry CJ, Gary TP, Ganesha R, Disha P, Joseph DB, Heather LB, et al. A comparison of

the ability of rilpivirine (TMC278) and selected analogues to inhibit clinically relevant

HIV-1 reverse transcriptase mutants. BioMed Central. [Research]. 2012(10.1186/1742-

4690-9-99).

Calvin JC, Jaime A-V, Bonaventura C, Jan F, Margaret AJ, Kiat R, et al. Rilpivirine

versus efavirenz with two background nucleoside or nucleotide reverse transcriptase

inhibitors in treatment-naive adults infected with HIV-1 (THRIVE): a phase 3,

randomised, non-inferiority trial. 2011 16;378(9787):229 - 37.

Cohen C, Molina J, Cahn P, Clotet B, Fourie J, Grinsztejn B, et al. Efficacy and safety of

rilpivirine (TMC278) versus efavirenz at 48 weeks in treatment-naive HIV-1-infected

patients: pooled results from the phase 3 double-blind randomized ECHO and THRIVE

Trials. Journal Acquir Immune Deficiency Syndrome. 2012;1;60(1):33-42.

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