RILPIVIRINE UV-VISIBLE SPECTROSCOPY HPLC

1. METHOD DEVELOPMENT AND VALIDATIONS OF
RILPIVIRINE BY UV AND HPLC METHODS

2. INTRODUCTION
Pharmaceutical 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.

3. DIFFERENT ANALYTICAL METHODS
•Qualitative analysis
•Quantitative analysis
•Dual wavelength spectrophotometry
•Derivative spectra

4. 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.

5. 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

10. THEORY INVOLVED
Molecular absorption & fluorescence

11. INSTRUMENTATION & WORKING
Types of Uv- vis
spectrophotometer
Single Beam Double Beam

12. SINGLE 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.

13. SINGLE BEAM

14. DOUBLE 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.

15. DOUBLE BEAM

16. 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 .

17. 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

19. INSTRUMENTATION
 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.

20. AIM OF THE PROJECT
 We are going to develop a new analytical
technique by using UV and HPLC methods for
the following drug.

21. RILPIVIRINE
Rilpivirine 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-

22. IUPAC NAME-
4-{[4-({4-[(E)-2-
cyanovinyl]-2,6-
dimethylphenyl}amino)
pyrimidin-2-
yl]amino}benzonitrile
MOLECULAR
FORMULA-C
22H18N6
BRAND NAME-Edurant
MOLECULAR
WEIGHT- 402.88
MOLECULAR
MASS-
366.42 g/mol
NATURE-Rilpivirine
hydrochloride is a
white to almost white
powder.

23. 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.

24. 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.

25. CONTD.
Ch Venkata Reddiah et al have developed effective estimation of
Rilpivirine by HPLC method in tablet dosage forms and its invitro
dissolution 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.

26. 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.

27. PLAN OF WORK
Method Development of Rilpivirine by Using UV Spectroscopy
• 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.

28. Method
Development of
Rilpivirine by Using
HPLC :-

29. 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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