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Research Scholar
Miss. Tejaswini K. Mankar

Research Guide
Dr. U. N. Mahajan

Dadasaheb Balpande College of Pharmacy,

Rashtrasant Tukadoji Maharaj, Nagpur University,
Nagpur-440037 (MS)

2018-2019
CONTENTS

INTRODUCTION
LITERATURE REVIEW
AIM, OBJECTIVE AND NEED OF RESEARCH WORK
PLAN OF RESEARCH WORK
REFERENCES
1. INTRODUCTION
Pharmaceutical analytical chemistry is an important part in monitoring the quality of
pharmaceutical products for safety and efficacy. With the advancement in synthetic organic
chemistry and other branches of chemistry including bioanalytical sciences and biotechnology,
the scope of analytical chemistry The choice of analytical methodology is based on many
considerations, such as: chemical properties of the analyte and its concentration sample matrix,
the speed and cost of the analysis, type of measurements i.e., quantitative or qualitative and the
number of samples. A qualitative method yields information of the chemical identity of the
species in the sample. A quantitative method provides numerical information regarding the
relative amounts of one or more of the analytes in the sample.has enhanced too, much higher
levels.
Analytical chemistry deals with methods for identification, separation, and quantification of the
chemical components of natural and artificial materials.[1]
Analytical methods development and validation play important roles in the discovery,
development, and manufacture of pharmaceuticals. Pharmaceutical products formulated with
more than one drug, typically referred to as combination products are intended to meet
previously unmet patients need by combining the therapeutic effects of two or more drugs in one
product. These combination products can present daunting challenges to the analytical chemist
responsible for the development and validation of analytical method. Analytic methods are
intended to establish the identity, purity, physical characteristics and potency of the drugs that we
use. Methods are developed to support drug testing against specifications during manufacturing
and quality release operations, as well as during long-term stability studies. Methods may also
support safety and characterization studies or evaluations of drug performance. According to the
International Conference on Harmonization (ICH). [2] the most common types of analytic
procedures are:
1. Identification tests
2. Quantitative tests of the active moiety in samples of API or drug product or other selected
component(s) in the drug product
3. Limits tests for the control of impurities.
Analytical methods development and validation play important roles in the discovery,
development, and manufacture of pharmaceuticals.

1.1 RP-HPLC
Reversed-phase high-performance liquid chromatography (RP-HPLC) involves the
separation of molecules on the basis of hydrophobicity. The separation depends on the
hydrophobic binding of the solute molecule from the mobile phase to the immobilized
hydrophobic ligands attached to the stationary phase, i.e., the sorbent. The solute mixture is
initially applied to the sorbent in the presence of aqueous buffers, and the solutes are eluted by
the addition of organic solvent to the mobile phase. Elution can proceed either by isocratic
conditions where the concentration of organic solvent is constant, or by gradient elution
whereby the amount of organic solvent is increased over a period of time. The solutes are
eluted in order of increasing molecular hydrophobicity.[3]

1.2 ANALYTICAL METHOD VALIDATION:-

Analytical method validation according to USP is performed to ensure that an analytical
methodology is accurate, specific, reproducible, precise and rugged over the specified range that
an analyte will be analysed. Validation of method is the process by which a method is tested by a
developer or user for reliability, accuracy and preciseneness of it intended purpose.
The international conference on harmonization (ICH) of technical requirement for the
registration of pharmaceutical for human use. It has developed suitable text on the validation of
analytical procedure. The document included definitions for eight validation parameter. The
United State Food and Drug Administration (US-FDA) have proposed guideline on submitting
sample and analytical data for method validation. The United State Pharmacopoeia (USP) has
published specific guidelines for method validation for compound evaluation.[4]
Advantages of analytical method validation:-

 The biggest advantage of method validation is that builds a degree of confidence, not
only for the developer but also to the user.

 Although the validation exercise may appear costly and time consuming, it results
inexpensive, eliminates frustrating repetitions and leads to better time management in the
end.

 Minor changes in the conditions such as reagent supplier or grade, analytical setup are
unavoidable due to obvious reasons but the method validation absorbs the shock of such
condition and pays for more than invested on the process.

1.4 STABILITY INDICATING ASSAY METHOD:-

The main contemporary goal of stability indicating methods (SIM) is to provide
information about condition for stress testing so as to establish the stability of drug
substances and product. SIMs are used to differentiate the API from its potential
decomposition product. Force degradation is required to demonstrate the specificity when
developing SIMs and for this reason, it should be perform prior to implementing the
stability studies. Force degradation of drug standard and excipients is carried out under
different conditions to determine whether the analytical method is stability indicating.[3]
Chemical stability of pharmaceutical molecules is a matter of great concern as it
affects the safety and efficacy of the drug product. The FDA and ICH guidance states the
requirement of stability testing data to understand how the quality of a drug substance
and drug product changes with time under the influence of various environmental factors.
Knowledge of the stability of molecule helps in selecting proper formulation and package
as well as providing proper storage conditions and shelf life, which is essential for
regulatory documentation. Forced degradation is a process that involves degradation of
drug products and drug substances at conditions more severe than accelerated conditions
and thus generates degradation products that can be studied to determine the stability of
the molecule.[5,6]
1.5 KINETIC DEGRADATION:-

Chemical kinetics is forms, the study of rate of chemical changes taking place during
chemical reactions. As applied to pharmaceutical formulation , this includes a study of the
physical and chemical reactions in drug and dosage. Chemical kinetics is forms, factors
influencing the rate of these chemical reactions, accelerated stability testing and prediction of
shelf life of formulation. The kinetic parameters like rate constant (K), half life (t 1/2), shelf life
are calculated and order of reaction are determined. [7]

1.6 PEAK PURITY:-

Peak-purity is of vital importance for documenting the safety and the efficacy of a
pharmaceutical drug. The improvement of the chemical synthesis and the pharmaceutical
processes requires knowledge about purity. The peak purity is determined by the purity
angle value and peak threshold value. If the purity angle is less than threshold, the
method will be specific for the determination of drug.[8]

1.7 MASS BALANCE:-

The process of adding together the assay value and levels of degradation products
to see how closely these add up to 100% of the initial value, with due consideration of the
margin of analytical error.It is critical to investigate the mass balance of a method if it
needs to be stability indicating. To meet the commitments of ongoing stability testing
these methods are required for all products.For convenience, the same methods are often
used for release an stability testing. Whether the assessment of mass balance is performed
during method development or during method validation often depends on the type of
drug being manufactured.[9]
1.8 DRUG PROFILE:-
Tenofovir:-
Tenofovir is a class of antiretroviral drugs known as nucleotide analogue reverse transcriptase
inhibitors (NtRTIs), which block reverse transcriptase, an enzyme crucial to viral production in
HIV-infected people.[10]

Chemical Name: (R)-(1-(6-amino-9H-purin-9-yl)propan-2-yloxy)methylphosphonic acid

Drug Class: Nucleoside Reverse Transcriptase Inhibitors

Solubility:- slightly soluble in water, soluble in methanol

Molecular Formula: C9 H14 N5 O4 P

Chemical Class: Purine Nucleotides

Molecular Weight: 287.261 g/mol

Melting point:- 276 – 280 0C

pKa:- 7.91
EMITRICITABINE:-
Emtricitabine (EMT):- EMT is a nucleoside reverse transcriptase inhibitor (NRTI) for the
treatment of HIV infection in adults. EMT is an analogue of cytidine. The drug works by
inhibiting reverse transcriptase, the enzyme that copies HIV RNA into new viral DNA. [11]
Description :- white to off white powder

Chemical name:- 2',3'-dideoxy-5-fluoro-3'-thiacytidine[

Molecular formula :-C8H10FN3O3S

Molecular weight 247.247

Melting point 136-140 °C

Solubility:- Freely soluble in methanol and water, practically

insoluble in methylene chloride

pKa:- 2.65
2. LITERATURE REVIEW

 Bhushan p. badgujar et.al (2017) had developed and validated RP-HPLC method for
simultaneous estimation of Emtricitabine and Tenofovir alafenamide in bulk and tablet
dosage formulation on C18 (4.6X250 mm, 5µ) column with a mobile phase composed of
Methanol: Distill water (60:40 v/v) at a flow rate of 1 ml/min. The pH of mobile was
adjusted by 0.05% ortho phosphoric acid (pH-3). The retention times of Emtricitabine
and Tenofovir were 3.10 min and 7.38 min respectively. Total run time emtricitabine and
tenofovir alefenamide was 10 min.

 N. MD. Akram et.al (2017) had developed and validated RP-HPLC method for
simultaneous estimation of Emtricitabine and Tenofovir alafenamide in bulk and tablet
dosage formulation. On C18 (4.6×250mm) 5µm) column with mobile phase composed of
(30:70v/v) Mixed phosphate buffer (KH2PO4 and K2HPO4) pH 3 (pH was adjusted with
orthophosphoric acid) ACN, the flow rate of 1ml/min. detection wave length was selected
273 nm used was WATERS HPLC.

 Bala Rami Reddy Yenumula et.al (2015) had developed and validated RP-HPLC
method for simultaneous estimation of Emtricitabine and Tenofovir disoproxil fumarate
in tablet dosage form. On phenomenex - luna C 18 (25cm x 4.60mm)particle size 5µm)
column with mobile phase composed of mixture of 10mm phosphate buffer (pH 6.8):
Acetonitrile: 40:60(v/v) at flow rate 1ml/min. The retension time of Emtricitabine and
Tenofovir were 2.81 min and 7.42 min respectively. Total run time emitricitabine and
tenofovir disoproxil fumarate was 10 min.
 Parthiben C. et.al (2011) had developed and validated RP-HPLC method for
simultaneous estimation of tenofovir disoproxil fumarate and emtricitabine in tablet
dasage form. On thermoscientific C18 (4.6 x 250 mm), particle size (5µm) column with
mobile phase acetonitrile : water (70:30 v/v). flow rate of 1.5 ml/min. the retension time
of emtricitabine and tenofovir disproxil fumarate were 2.82 and 4.38 min respectively.

 Rajesh Sharma et.al (2009) had developed and validated RP-HPLC method for
simultaneous estimation of tenofovir disoproxil fumarate and emtricitabine in tablet
dosage form on Luna C18 (25cm x 4.60 mm, particle size 5µm) column with a mixture
of acetonitrile: potassium dihydrogen phosphate buffer (pH 3.0 ± 0.05 adjusted with
orthophosphoric acid): triethylamine in the ratio of 70:30:0.5(v/v) as mobile phase. UV
detection was performed at 260 nm. The retention time was 1.78 and 2.27 min. for
emtricitabine and tenofovir disoproxil fumarate respectively and total run time was 4
min. at a flow rate of 1.5 mLmin-1.
 1. AIM, OBJECTIVE AND NEED OF RESEARCH

1.1 AIM:-
Development and validation of RP-HPLC method for simultaneous estimation of
tenofovir alafenamide and emtricitabine drug in their combined solid dasage form.

1.2 OBJECTIVE:-
The main objectives of the study are:-

 To develop new, simple, accurate, rapid and sensitive, robust analytical method for the
determination of tenofovir alafenamide and emitricitabine by RP-HPLC.
 To validate the method in accordance with USP and ICH guidelines for the intended
analytical application i.e., to apply the method for analysis of the drug in its solid dosage
form.
 To study the interference of major impurities of tenofovir alafenamide amd emtricitabine
in an optimizes method.
 To develop stability indicating assay method for the simultaneous determination of
selected drug along with its degradent product.

1.3 NEED OF RESEARCH:-

 It literature survey reveals that their were are not any method documented based on
stability indicating analytical method of the selected drugs.
 It has been also found that the peak observed in the develope method were sluggish,
assymetric and appeared at very lower retension time.
 The method has not analysed by PDA detector hence the develope method did not
determine peak purity and peak threshold.
 This promoted us to develop and validate new stability indicating assay method for the
estimation of antiretroviral drug.
3.PLAN OF WORK:-
1. Literature Survey.
2. Procurement of drug sample and other chemicals.
3. Identification of drugs by UV Aand FTIR Spectroscopy.
4. Method development by RP-HPLC.
 Selection of preliminary HPLC conditions.
 Selection of mobile phase
 Selection of column
 Selection of wavelength
5. Validation of proposed methd
a) Specificity
b) Accuracy
c) Precision
i) Repeatability precision
ii) Intermediate precision
iii)Reproducibility
d) Linearity
e) Limit of Detection
f) Limit of Quantification
g) Selectivity / specificity
h) Robustness
i) Ruggedness
6. Relative Humidity
7. Stability indicating parameter
8. Complation of data
4. REFERENCES:
1. Skoog DA, West DM, Holler FJ (1996) Fundamentals of analytical chemistry.
(8thEdn), Fort Worth: Saunders College Pub.

2. Song HH, Choi KS, Kim CW, Kwon YE (2009) Pharmacokinetic Profiles of Two
Branded Formulations of Piroxicam 20mg in Healthy Korean Volunteers by a Rapid
Isocratic HPLC Method. J Bioequiv Availab 1: 074-079.

3. ICH guideline, Q2 (R1) step 4, Validation of Analytical Procedures: Text and

Methodology (2005).
4. Aguilar, M. I. and Hearn, M. T. W. (1996) High resolution reversed phase high
performance liquid chromatography of peptides and proteins. Meth. Enzymol. 270, 3–
26.PubMedCrossRefGoogle Scholar
5. Mant, C. T and Hodges, R. S. (1996) Analysis of peptides by high performance liquid
chromatography. Meth. Enzymol. 271, 3–50.PubMedCrossRefGoogle
6. ICH guideline, Q2 (R1) step 4, Validation of Analytical Procedures: Text and
Methodology (2005).

7. Scholarhttps://www.slideshare.net/bknanjwade/kinetics-and-drug-stability-52487252
5:30@22/7/18
8. https://slideplayer.com/slide/7483505/ 5:58@22/7/18

9. https://www.linkedin.com/pulse/mass-balance-analytical-method-critical-oona-
mcpolin5:40@22/7/18

10. file:///C:/Users/Admin/Desktop/tenofovir/ANA%20MD%202.htm11:06@21/7/18.
11. Bhushan P. Badgujar*, Moreshwar P.Mahajan, Sanjay the Determination of
Emtricitabine and Tenofovir AF in its Bulk and Pharmaceutical Dosage Forms. Journal
of Chemical and Pharmaceutical Sciences. 2015

12. Bala Rami Reddy.Yenumula1*#, Mutta Reddy.Singampalli2 and Bala Sekhara

Reddy.Challa3#. Simultaneous Estimation of Emtricitabine and Tenofovir Disoproxil D.
Sawant. Development and Validation of RP-HPLC Method for the Simultaneous
Estimation of Tenofovir Alafenamide and Emtricitabine in Bulk and Tablet Dosage
Form. International Journal of ChemTech Research vol.10 No 5. 2017,731-739.

13. N.MD. Akram*1, M. Umamahesh2 A New. Validated RP-HPLC Method for tenofovir
Fumarate in Tablet Dosage Form by Reverse Phase High-performance Liquid
Chromatography. International Journal of Analytical Techniques.2015.

14. Parthiban C1, Bhagavan Raju M2, sudhakar M1. Simple RP-HPLC method for
simultaneous estimation of Tenofovir Disoproxil fumarate and Emtricitabine in tablet
dasage form. IRJP.2011.

15. Rajesh sharma 1B, Pooja Gupta1. A Validated RP - HPLC Method for Simulataneous
Estimation of Emtricitabine and Tenofovir Disoproxil Fumarate in a Tablet Dosage form.
Eurasian J Anal Chem 2009;4(3):276–284 .