Impurities profiling of ATZ

GTU/PHD/NIRAV SONI/189999901009 1

Doctoral Progress Review Comment-2

Justification of DPC-2
 Extensive Literature survey has done.
 The title is finalized.
 Writing references done accordingly GTU guidelines.
 Method development of Atazanavir sulfate (ATZ) is done.

Doctoral Progress Review Comment-3

Justification of DPC-3
 I have done optimized mobile phase and UV spectra (Overlapping) graph ATZ and
its impurities and wavelength finalized as per comment sheet.
 Method validation and forced degradation stability done.
 Also, I have done Estimation of marketed dosage form.
 Writing references done accordingly GTU guidelines.

Contents
1. Introduction
1.1 Drugs
1.2 Anti-HIV drugs
1.3 HIV Life cycle
1.4 Classification of anti-HIV drugs
1.5 Analytical methods
1.6 Modes of separation
1.6.1 RP-HPLC
1.7 Parameters that are affected by chromatographic conditions

1.8 Analytical method validation
1.9 Impurities profile
1.10 Sources of impurities
1.11 Classification of impurities
1.11.1 Designation of impurities
1.12 Identification of impurities
1.13 Quantitation of impurities
2.0 Literature review
2.1 Official method reported in pharmacopoeia
2.2 Reported method for selected anti-HIV Drugs
2.3 Reported method for other class of anti-HIV Drugs

3. Rationale.
4. Aim and objectives.
5. Proposed plan of work.
6. Introduction of drug profiles.
6.1 Drug profile of Atazanavir sulphate
6.2 List of marketed formulation of atazanavir and its combination or combined dosage
forms.
6.2.1 List of single component marketed formulations of Atazanavir Sulfate.
6.2.2 List of combination formulations of Atazanavir Sulfate.
6.3 List of known impurities profile.
6.3.1 Impurities used in this project

7. Materials and Instrumentals Specification
7.1 Apparatus and Instruments.
7.2 Reagents and Materials.
8. Identification of Drugs.
8.1 Identification by Melting Point.
8.2 Identification by Solubility.
8.3 Identification by IR Spectra.
9.0 Selection of wavelength.
10. Method developments (MD).
10.1 Development and optimization of the RP-HPLC method.
10.2 Selection of buffer strength and pH.
10.3 Evaluation of stationary phase

10.4 Optimization of gradient program
10.5 Gradient program .
10.6 Evaluation of flow rate and column oven temperature.
10.7 Selection of detector wavelength.
10.8 Selection of sample concentration and injection volume.
10.9 Optimized chromatographic conditions for proposed method.
10.10. Selection of Mobile phase.
10.11 System Suitability Test (SST).
11. Experimental works.
11.1 Preparation of stock solution.
11.2 Preparation of standard working solution.
11.3 Preparation of mobile phase.

1.1 Introduction of drugs
• A drug may be defined as a substance meant for diagnosis, cure, mitigation and
prevention, treatment of diseases in human beings or animals, for altering in
structure or function of the body of human beings or animals1. Pharmaceutical
chemistry is a science that makes use of general laws of chemistry to study drugs
i.e. their preparation, chemical nature, composition, structure, influence on an
organism, the methods of quality control and the conditions of their storage etc.2-6

1.2 Introduction of Anti-HIV Drugs.
• Introduction of Anti-HIV Drug:- HIV-the Human Immunodeficiency Virus is the
retrovirus that causes AIDS. HIV belongs to the retrovirus subfamily lentivirus.
HIV attaches to cells with CD4 receptors (T4 cells and macrophages). AIDS
remains a serious threat because of the expense and inaccessibility of antiretroviral
agents in the developing countries in which the disease is most prevalent. In
addition, the effectiveness of antiretroviral drugs has been diminished by the
emergence of multidrug- resistant virus,1,5,7

1.3 HIV Life Cycle1,2

1.4 Classification of Anti-HIV Drugs 3,4
Sr.No Class Drugs
1. Nucleoside reverse
transcriptase inhibitors
(NRTIs)
Zidovudine, Stavudine, Lamivudine, Abacavir,
Zalcitabine, Emtricitabine, Didanosine,
Tenofovir disoproxil, Tenofovir alafenamide.
2. Non nucleoside reverse
(NNRTIs)
Efavirenz, Nevirapine, Delaviridine,
Rilpivirine, Etravirine Doravirine

3. Nucleotide reverse
(NTRTIs)
Tenofovir
4. Protease inhibitors (PIs) Saquinavir, Indinavir, Nelfinavir,
Amprenavir, Fosamprenavir, Ritonavir,
Lopinavir, Atazanavir.
5. Entry/Fusion inhibitors Enfuvirtide, Zidovudine, Stavudine,
Lamivudine, Abacavir, Zalcitabine,
Emtricitabine, Didanosine, Maraviroc

6. Integrase inhibitors Bictegravir, Dolutegravir, Elvitegravir,
Raltegravir
7. Booster drugs Cobicistat,Ritonavir

1.5 Introduction to analytical method 5-8
• High-performance liquid chromatography (HPLC), sometimes called high-pressure
liquid chromatography, is a separation technique based on a solid stationary phase
and a liquid mobile phase. The work out flow about High Performance Liquid
Chromatography (HPLC) is depicted in given figure.

1.6 Modes of separation in HPLC 5,14,15,17
• Normal phase mode.
• Reversed phase mode.
• Ion exchange chromatography.
• Reverse phase ion pair chromatography.
• Affinity chromatography and
• Size exclusion chromatography.

1.6.1 RP- HPLC
 Reversed phase mode is the most popular mode for analytical and preparative
separations of compound of interest in chemical, biological, pharmaceutical, food and
biomedical sciences. In this mode, the stationary phase is non polar hydrophobic
packing with octyl or octadecyl functional group bonded to silica gel and the mobile
phase is polar solvent.
 An aqueous mobile phase allows the use of secondary solute chemical equilibrium
(such as ionization control, ion suppression, ion pairing and complexation) to control
retention and selectivity. The polar compound gets eluted first in this mode and non
polar compounds are retained for longer time.

 As most of the drugs and pharmaceuticals are polar in nature, they are not retained
for longer times and hence elute faster. The different columns used are
octadecylsilane (ODS) or C18, C8, C4, etc., (in the order of increasing polarity of the
stationary phase).

1.7 Parameters that are affected by
chromatographic conditions 5-8,14,15,17
• Resolution (Rs).
• Column efficiency/ Number of theoretical Plates (N)
• Capacity factor (k').
• Peak asymmetry factor (As).

1.8 Analytical Method Validation 5,7
 Method validation is the process used to confirm that the analytical procedure
employed for a specific test is suitable for its intended use. Results from method
validation can be used to judge the quality, reliability and consistency of analytical
results; it is an integral part of any good analytical practice.

1.9 Introduction to impurity profiles 7-10
 Definition:- An impurity is any constituent present in excipient, drug substance
(Active Pharmaceutical Ingredient or bulk material) or drug product (Dosage
form or Finished product) that is not an excipient, active drug substance,
formulated drug product.
 This definition of impurity is broad enough to include degradation products as
impurities. The term degradation product (DP) is defined in ICH as follows:
(www.ich.org, ICH guidelines, 2012).
 Degradation Products (DP):-“A molecule resultant from a modification in the
active drug substance or formulated drug product brought over time”.

1.10 Source of impurities
 Bulk drug Substance process development and formulated drug Product
formulation development are two main areas of the pharmaceutical drug
development process. Impurities can be produced in either of the processes.
 During the Drug Substance synthesis development process, impurities can be
generated from the synthetic process or as a result of degradation. In drug product
formulation development, impurities can also be generated as the degradation
products, as a product of drug excipient interaction, or external contamination or
from packaging components.

1.11 Classification of impurities 9,14-22
 Theoretically possible (potential) impurities are classified (Figure ) as following
types (Guidance for industry, 1998).

 Based on the source of impurities they are classified (Figure) as
follows:

1.11.1 Designation of impurities 9,14-25
 Impurities have been titled differently by various groups of scientists who deal with
them. Commonly used terms are displayed in (Figure) and those terms have been
found acceptable by ICH and various regulatory bodies.

 Identification and characterization of impurities is an analytical activity aiming to
elucidate the chemical structures and the possible mechanisms of formation of
unknown impurities. Because of the complexity and diversity of the impurities in
both their origins and properties, the identification strategies are determined by the
specific situations. A general strategy can be set for the identification and
characterization of the impurity of bulk drug substances by the rational use of
analytical techniques. The schematic use of the methods for impurity profiling of
drug substances is shown in below figure.
1.12 Identification of impurities 9

 The following techniques are being regularly used for the
quantitation of impurities and degradation products:
1) High Performance Liquid Chromatography (HPLC),
2) Gas Chromatography (GC),
3) Thin Layer Chromatography (TLC),
4) High Performance Thin Layer Chromatography (HPTLC),
5) Capillary Electrophoresis (CE),
1.13 Quantitation of Impurities 5,7,9

6) Supercritical Fluid Chromatography (SFC),
7) Gel Permeation Chromatography (GPC).
8) Ultra Performance Liquid Chromatography (UPLC)
Since, I have used only HPLC in our present work.

 I have found stability method, Chemical synthesis and
characterization, method development (MD) and validation (MV)
for selected anti-HIV class of drugs. Also, Simultaneous
estimation of analytical method and validation of anti-HIV class.
 There is a very few method reported for impurity profiling and
quantification of selected anti-HIV drugs by chromatography
technique.
2.0 Literature review 26-56

Sr.No Drugs Types of research Methods
1. Atazanavir
suplhate
Related substances
Mode:- Gradient
Column :- Intersil ODS-3 (5µm, 25 cm × 4.6mm)
Mobile phase:- Sodium dihydrogen
orthophosphate monohydrate (pH 2.5) and
Acetonitrile (ACN)
Flow rate :- 1.0 ml/min
Volume of injection :- 20 μl
Wavelength:- 210 nm.
Column temperature:-45 0C
HPLC 26
2.1 Official method reported in pharmacopoeia 26

2.2 Reported method for selected anti-HIV Drugs27-29
1. Atazanavir
suplhate
Mode:- Gradient
Column :- Ascentis, Express C8,
(150 mm × 4.6 mm, 2.7 μm)
Mobile phase:- potassium dihydrogen
phosphate (pH 3.5, 0.02 M) and ACN
Injection volume:- 20 µl
Column temperature:-300C
Detector:- PDA (Photo Diode Array)
RP-HPLC27

2. Atazanavir
suplhate
Mode:- Gradient
Column :- Express C8 (150 mm*4.6
mm , 2.7 µm)
Mobile phase:- Potassium dihydrogen
phosphate (pH 3.5, 0.02M) and ACN
RP-HPLC28

3. Atazanavir suplhate
+ Ritonavir
Mode:- Gradient
Column :- Acquity BEH C18 (100mm
× 2.1mm), 1.7 μ
Mobile phase: 0.01M
monobasic potassium hydrogen
phosphate adjusted the pH to 3.6 and
ACN
Run time:- 18 min
RP-UPLC29

1. Lopinavir +
Ritonavir
Bulk drug and pharmaceutical formulation
with its impurities
UPLC30
2. Emtricitabine Related degradation substances HPLC 31
3. Darunavir Unknown impurities RP-HPLC 32
4. Zidovudine: A stability-indicating method for
identification, characterization and toxicity
prediction of two major acid degradation
products
LC-MS/MS33
2.3 Reported method for other class of anti-HIV Drugs
30-57

5. Doravirine Characterization of
impurities of
UHPLC-high
resolution MS and
tandem MS
analysis34
6. Liponavir Impurities HPLC35
7. Tenofovir disoproxil
fumarate
Synthesis and
characterization
LC-MS and NMR
Spectroscopy36
8. Atazanavir sulfate Method development and
validation
RP-HPLC37

9. Atazanavir sulfate Method development and
validation
RP-HPLC 38
10. Atazanavir +
Cobicistat
Stability indicating methods (SIM) RP-HPLC 39
11. Raltegravir Method development and
validation
RP-HPLC 40
12. Raltegravir Stability indicating methods (SIM) RP-HPLC 41
13. Darunavir +
Raltegravir
Simultaneous determination UV-HPLC
method 42

14. Raltegravir Method developement and
validation for the quantification
Liquid chromatography–
tandem mass spectrometry
in the negative ionization
mode 43
15. Raltegravir potassium
+ Rilpivirine HCl
Method developement and
validation
HPLC and HPTLC 44
16. Raltegravir Forced degradation studies RP-HPLC and
Characterization of
Degradants by LC-MS/MS
45
17. Maraviroc +
Raltegravir
Simultaneous determination in
human plasma
HPLC‐UV46
18. Lamivudine and
Raltegravir
Simultaneous estimation binary
mixture by using design of
experiment
RP-HPLC 47

validation in blood plasma
HPLC48
20. Darunavir
Ethanolate
Stability indicating methods
(sim)
HPLC Method 49
21. Darunavir
Ethanolate
(sim)
HPLC50
22. Darunavir Simultaneous determination of
six process related impurities
UPLC-MS/MS51
23. Ritonavir Determination of three phenol
impurities
UPLC-MS/MS 52

validation in blood plasma
HPLC48
20. Darunavir
Ethanolate
(sim)
HPLC Method 49
21. Darunavir
Ethanolate
(sim)
HPLC50
22. Darunavir Simultaneous determination of
six process related impurities
UPLC-MS/MS51
23. Ritonavir Determination of three phenol
impurities
UPLC-MS/MS 52

24. Etravirine Stability indicating methods (sim) UPLC 53
25. Efavirenz Synthesis, isolation and characterization
and in process impurities in the presence
of tetrahydrofuran (THF) solvent
RP-HPLC54
26. Cobicistat +
Atazanavir
sulphate
Method development for the assay and
degradation study
RP-HPLC55
27. Atazanavir +
cobicistat
Stability indicating simultaneous
estimation
RP-HPLC56
28. Darunavir Method development and validation RP-HPLC57

CHAPTER-3

 Atazanavir (ATZ) is indicated for the prevention of Antiretroviral, HIV Protease
Inhibitors (PI).
 There is a very few method reported for impurity profiling and quantification of
selected anti-HIV drugs by chromatography technique. Therefore, it is worthwhile to
develop and validate the impurity profiling of separation, identification and
quantification for selected anti-HIV drugs.

CHAPTER-4

4.1 Aims
 Extensive literature survey reveals that a very few chromatographic methods for the
determination of atazanavir in combination with other anti-retroviral drugs/agents in
biological fluids and two assay with separation, identification and quantification of
impurities method in active pharmaceutical ingredient (API).The present research aims
at reporting simple, rapid, precise and simultaneous method to efficiently separate
the impurities present in the ATZ in their fixed dose. Since the product is a single
molecules, impurities having structural similarity will present in the drug product.
 The criticality of this method involves in separating active ingredients along with their
specified and unspecified impurities with satisfactory resolution and at shorter runtime.
The sensitivity of developed method should be sufficient enough to quantify the lower
levels of impurities to ensure safety and efficacy of drug product. The method
development was carried out to achieve these specified goals.

4.2 Objectives
 The main objective of the present research work is outlined as follows :
To develop and validate RP-HPLC method for separation, identification of
known impurities and quantification of unknown impurities of atazanavir
sulfate in marketed formulation.
 The primary objective of the present method is to develop a simple, rapid and
simultaneous method to efficiently separate the impurities present in Atazanvir
sulfate in their dosage form. Since the product is a single molecule, impurities
having structural similarity will present in the drug product. The criticality of
this method involves in separating both active ingredients along with their
specified and unspecified impurities with satisfactory resolution and at shorter
runtime.
 The sensitivity of developed method should be sufficient enough to quantify
the lower levels of impurities to ensure safety and efficacy of drug products.

CHAPTER-5
Proposed plan
of work

Selection of drug
Review of literature
Synopsis preparation & submission
Study of physicochemical characteristics of drug
Study of impurities profile of selected anti-HIV drugs
Method development and validation of selected anti-HIV drugs
Validation of developed method
Preparation of Final Report, Discussion and Thesis writing
Submission of Thesis

Introduction to drug profile
CHAPTER-6

6.1 Drug profile of Atazanavir sulphate 58-68
Physicochemical Properties
Name
Atazanavir sulphate
Official in
IP,BP,EP,USP
Physical form/Appearance
It is a white to pale yellow crystalline powder with
sulfate salt.
Description
Atazanavir is an aza-dipeptide analogue with a bis-aryl
substituent on the (hydroxethyl) hydrazine moiety with
activity against both wild type and mutant forms of HIV
protease. Atazanavir does not elevate serum lipids, a
common problem with other protease inhibitors.

Structure
Chemical class
Phenethylamines
BCS Class
Class II (high permeability, low solubility)
Categories/Therapeutic class
Antiretroviral, HIV Protease Inhibitors (PI)
Chemical Formula

State Solid
Melting point 195-209 °C
Experimental
properties
Log P:- 4.36
Mol. Weight
Average: 802.934 g/mol
Monoisotopic: 802.35712729
IUPAC Name
(Methyl N-[(2S)-1-[[(2S,3S)-3-hydroxy-4-[[[(2S)-2-(methoxy
carbonylamino)-3,3-dimethylbutanoyl]amino]-[(4-pyridin-2-
yl phenyl)methyl]amino]-1-phenylbutan-2-l]amino]-3,3-
dimethyl-1-oxobutan-2-yl]carbamate

Solubility
It is slightly soluble in water (4-5 mg/ml, free base equivalent
with the pH of a saturated solution in water being about 1.9 at 24
± 3°C, DMSO: 104mg/ml; H2O: <1mg/ml; EtOH: 20mg/ml
Approval
The U.S. Food and Drug Administration (FDA), June 20, 2003,
CDSCO,2006
ATC Classification J05AE08 (WHO)
CAS NO. 229975-97-7

PHARMACOLOGY
Indications
Used in combination with other antiretroviral agents for the treatment of
HIV-1 infection, as well as post exposure prophylaxis of HIV infection in
individuals who have had occupational or non occupational exposure to
potentially infectious body fluids of a person known to be infected with HIV
when that exposure represents a substantial risk for HIV transmission.
Classes HIV protease inhibitor; Azapeptide inhibitor of HIV-1 protease
Pharmacody
namic
Atazanavir (ATV) is an azapeptide HIV-1 protease inhibitor (PI) with activity
against Human Immunodeficiency Virus Type 1 (HIV-1). HIV-1 protease is
an enzyme required for the proteolytic cleavage of the viral polyprotein
precursors into the individual functional proteins found in infectious HIV-1.
Atazanavir binds to the protease active site and inhibits the activity of the
enzyme. This inhibition prevents cleavage of the viral polyproteins resulting
in the formation of immature non-infectious viral particles.

Mechanism of action HIV protease is a viral enzyme responsible for the cleavage of polyproteins
into structural proteins and certain enzymes that are required for the final
assembly of the new infectious virions. Protease inhibitors act by binding to
the viral protease, in this way preventing the correct cleavage of viral
proteins. Thus, they prevent HIV from being successfully assembled and
released from the infected cells
Figure 12 :- Mechanism of Protease Inhibitors (PI)

Side effects
fever; nausea, vomiting, stomach pain, diarrhoea; headache, muscle pain;
depressed mood, sleep problems (insomnia); numbness, tingling, or
burning pain in your hands or feet; or. changes in the shape or location of
body fat (especially in your arms, legs, face, neck, breasts, and waist).
Adverse drug effects
Mild rash (redness and itching), Chronic kidney disease, Kidney stones,
Gall bladder problems, Diabetes and high blood sugar (hyperglycemia),
Lactic acidosis, severe hepatomegaly, hepatic steatosis.
Contraindication
chronic hepatitis B, chronic hepatitis C, diabetes., increased blood acidity
due to high levels of lactic acid.
Hemophilia, atrioventricular block, a type of slow heart rhythm disorder,
gallstones.
Dose
300mg once daily, boosted with lower dose of 100 mg ritonavir once
daily

Pharmacokinetic Profile
Absorption highly dependent on gastric pH
Protein binding 86% bound to human serum proteins (alpha-1-acid glycoprotein and
albumin). Protein binding is independent of concentration
Distribution
86% bound to human serum proteins and protein binding is independent of
concentration
Metabolism
Liver (CYP3A4-mediated), Atazanavir is extensively metabolized in humans,
primarily by the liver. The major biotransformation pathways of atazanavir
in humans consisted of monooxygenation and dioxygenation. Other minor
biotransformation pathways for atazanavir or its metabolites consisted of
glucuronidation, N-dealkylation, hydrolysis, and oxygenation with
dehydrogenation. In vitro studies using human liver microsomes suggested
that atazanavir is metabolized by CYP3A
Excretion
Unchanged drug accounted for approximately 20% and 7% of the
administered dose in the feces and urine, respectively.

Excretion half life
6.5 hours, Elimination half-life in adults (healthy and HIV
infected) is approximately 7 hours (following a 400 mg daily
dose with a light meal). Elimination half-life in hepatically
impaired is 12.1 hours (following a single 400 mg dose)
Tmax
2.5 Hours
Bioavailability
60-68%

6.2 List of marketed formulation of Atazanavir and its
combination or combined dosage forms & Its impurities60,69-71
Brand name of the
dosage form
Name of the company Dosage form Strength of the dosage
form
Atazor Emcure (ARV) Capsule 100mg, 150mg, 200mg,
300mg
Atavir Cipla Capsule 100mg, 150mg, 200mg,
300mg
Virataz Hetero HC (GenX) Capsule 300mg
6.2.1 List of single component marketed formulations of Atazanavir Sulfate

Brand name of
the dosage
form
Name of the
company
Dosage form Strength of the
dosage form
Synthivan Cipla Tablet Atazanavir
300mg, Ritonavir
100mg
Virataz – R Hetero HC (GenX) Tablet Atazanavir
300mg, Ritonavir
100mg
6.2.2 List of combination formulations of Atazanavir Sulfate

6.3 List of known impurities profile 70,71
There are several impurities profile in atazanavir sulphate like Atazanavir Impurity C,
Atazanavir Impurity B, Atazanavir S,S,R,S-Diastereomer, Atazanavir R,S,S,R-Diastereomer
Atazanavir R,S,S,S-diastereomer, Atazanavir R,R,R,R Isomer, Atazanavir S,S,S,R-
diastereomer , Atazanavir Di-tert-butyl Analogue, Atazanavir related compound A, Dealkyl
Atazanavir, Atazanavir Hydrazine Analog Trihydrochloride, Atazanavir Impurity 1, Atazanavir
Impurity 7, Atazanavir Impurity 8 ,Atazanavir Impurity 9, rac-Atazanavir Impurity, Atazanavir
Impurity 5, Atazanavir Impurity 14, Atazanavir Impurity 16, Atazanavir Impurity 17 (RSSSS),
Atazanavir Impurity 18 (SRS), Atazanavir Impurity 19, Atazanavir diol Impurity, Atazanavir-
D5, Atazanavir Benzylidenehydrazine Carbamate (RSS),, Atazanavir Benzylidenehydrazine
Analogue (RS), Atazanavir Hydrazine Analog Trihydrochloride (RS), Rs8 + Rs9 (Mixture),
Atazanavir RS9 Impurity, Atazanavir RS3 Impurity, Atazanavir RS8, Impurity Atazanavir
RS12 Impurity, 5-(Hydroxymethyl)Furan-2-Carbaldehyde, Atazanavir Impurity 9 (4R,5R-
Diasteroisomer of DIBOC), Atazanavir Impurity 10 (4S,5R-Diasteroisomer of DIBOC),
Atazanavir Impurity 6 (4R,5S-Diasteroisomer of DIBOC)

6.3.1 Impurities used in this project
Table 3 :- Atazanavir related compound A (Impurity 1)
Atazanavir related compound A
1. CAS no. 162537-11-3
2. Molecular structure C8H15NO4
3. Molecular formula
4. Molecular weight 189.21
5. CAT no. SZ-A009003
6. Catalogue number PA USP1044356
7. Category USP standards

Table 4 :- Atazanavir Impurity 5 (Impurity 2)
Atazanavir Impurity 5
1. CAS no. N/A
2. Molecular structure
3. Molecular formula C24H29NO11
4. Molecular weight 507.5
5. CAT no. SZ-A009D01
6. Category USP standards

7. Materials and Instrumentals Specification:
7.1 Apparatus and Instruments:
Table 5: Apparatus and Instruments List
Sr. No. Name Description
1. UV Spectrophotometer Systronics 119
2. HPLC Shimadzu [with power stream]
Column:- C18 Hypersil BDS
(250mm × 4.6mm × 5μm)
Pump:- LC-20 AT
Syringe:- Rheodyne injector valve with 20.0 μl
loop
Detector:- PDA 600 UV Detector
Software:- Spinchrom

3. pH Meter Systronics PH361
4. Digital Balance Mettler Toledo ML 204
5. Glass wares Borosile
6. Ultrasonicator Toshcon
7. Melting Point
Apparatus
Veego VMP-01

Sr. No.
Name Description
1. Water HPLC Grade
2. Methanol HPLC Grade
3. Acetonitrile HPLC Grade
4. Potassium dihydrogen
orthophosphate
HPLC Grade
5. Sodium hydroxide AR Grade
6. Hydrochloric acid AR Grade
7.2 Reagents and Materials:
Table 6: List of Reagents and Materials

Sr. No. Name Source
1. Atazanavir sulphate Emcure pharmaceuticals limited,
Ahmedabad
2. Impurity A Medvin pharmaceutical limited,
Ahmedabad
3. Impurity 5 Medvin pharma, Ahmedabad
7.3 Detail of Drug Procurement:
Table 7: Drug Procurement Detail

8.Identification of Drugs:
8.1 Identification by Melting Point:
Taken the atazanavir sulphate in capillary and place into the melting point apparatus .
Melting point observed and compared with the reference.
Sr. No. Drug Reported Melting
Point
Observed Melting
Point
1. Atazanavir sulphate 195-209°C 60 194 – 207 °C
Table 8: Melting Point comparison

8.2 Identification by Solubility:
The sample of atazanavir was taken in test tubes and observed for solubility in various
solvents like water, methanol, 0.1 N HCl and 0.1 N NaOH.
Solvent Solubility
Water Slightly soluble
0.1 N NaOH Slightly soluble
0.1 N HCl Insoluble
Methanol Soluble

8.3 Identification by IR Spectra:
• Potassium Bromide IR disc was prepared placing 1mg of Drug on Hydraulic Pellet
Press at a pressure of 7-10 tones. This disc was scanned in the region of 1800–600 cm-1
in FTIR and obtained IR spectrum was compared with the reference spectrum of
atazanavir sulfate. Following peaks were observed.
Figure 13 :- Interpretation from IR spectra of sample ATZ

Figure 14 :- Interpretation from IR spectra of standard ATZ 72

Type of vibration Observed frequency value (cm-1) Standard Value (cm-1) [72]
C=O stretching 1731 1870-1540
C=C stretching 1540 1670-1600
N-H bending 1600 1650-1580
C-O Stretching 1069 1400-1000
Table 10:- Interpretation from IR spectra of sample atazanavir

9.0 Selection of wavelength:-
• Taking a Standard stock solution of ATZ 50 mg in 100 ml of methanol (500 μg/ml)
and again to take this 1 ml from ATZ standard stock solution (50μg/ml) in
methanol.
• It was scanned between 200-400 nm using UV-visible spectrophotometer.
• Wavelength was selected from the overlay spectra of above solutions.
• ATZ and its impurities A and 5 both shows reasonably good response at 225 nm in
Methanol. 225 nm is found as λmax. Hence, 225 nm wavelength has been chosen for
quantification of impurities due to satisfactory sensitivity and optimum response.

Atazanavir Sulfate Standard at 5 ppm
Retention time
[min]
Area
[mV.s]
Height
[mV]
Area
[%]
5.280 205.425 22.231 100

Selection of Impurities 30-57,74
• Potential impurities of Atazanvir sulfate (ATZ) was not separated from main
analytes in the reported methods and It is the most common in their dosage forms.
It has produces genotoxicity.74
• Hence, no liquid chromatographic methods (HPLC and UPLC) were reported for
the determination of ATZ impurities in their fixed dosage forms.
• RP-HPLC system enables improved sensitivity, selectivity, rapid analysis,
environment friendly due to lower solvent consumption, RP-HPLC equipment was
chosen for the determination of ATZ and its impurities in the fixed dose products.
• Finally, I have selected two impurities in this project Impurity A and Impurity 5
respectively.

Impurities Standard A and 5 at 5 ppm
Impurities Retention time
[min]
Area
[mV.s]
Height
[mV]
Area
[%]
A 6.223 300.757 27.610 53.736
5 14.503 258.940 10.242 46.264

Compounds Retention time
[min]
Area
[mV.s]
Height
[mV]
Area
[%]
ATZ 5.297 19230.092 2061.316 24.722
Impurity-A 6.240 313.022 27.995 0.402
Impurity-5 14.547 261.166 10.285 0.336
Atazanavir Sulfate Standard (500 ppm) and its impurities A and 5 (5 ppm)

Atazanavir Sulfate Test (500 ppm) and its impurities A and 5 (5 ppm)
Compounds Retention time
[min]
Area
[mV.s]
Height
[mV]
Area
[%]
Unknown 3.243 16.513 2.210 0.085
ATZ 5.300 19231.523 2060.645 99.204
Impurity-A 6.270 86.373 7.433 0.446
Impurity-5 14.507 51.360 2.045 0.265

10. Method developments (MD)
10.1 Development and optimization of the RP-HPLC method:-
Method development and optimization has been carried out in a systematic approach by
considering various aspects which will play major role in the separation. Different
factors such as buffer pH, column chemistry, organic solvent and other
chromatographic parameters were chosen as summarized below.
10.2 Selection of buffer strength and pH
Optimum buffer strength shall be maintained for attaining reproducible separation
between the impurities. Since phosphate buffer is having wide range of pKa values,
0.05M concentration potassium dihydrogen orthophosphate (KH2PO4) buffer was
selected for initial study. Considering the presence of “amine” functional groups in both
the drug components, initial trials were taken by adjusting the buffer pH to 4.0 ± 0.05.
However, better separation of all the desired peaks with good resolution was achieved
at pH 4.0.

Hence, the buffer pH was fixed at 4.0 to ensure improved column performance at this acidic
pH. In order to attain baseline resolution of impurities and for eluting late eluting non-polar
impurities, methanol was used as organic solvent and found satisfactory separation with
reduced retention times. Also, sharp peaks with good responses were observed. Hence,
Methanol and 0.05M Potassium dihydrogen phosphate buffer (pH-4.0) were used as organic
solvent.
Preparation of Mobile Phase:- After considering the varying combinations of various
mobile phases, Buffer: Methanol used by gradient RP-HPLC method. [Buffer (0.05 M
KH2PO4, pH 4.0) Take 6.8 gm KH2PO4 in to a 1000 ml beaker, add 800 ml water and
dissolve, adjust pH 4.0 with 0.1N NaOH, Make up Volume 1000 ml with water] was
finalized as it was showing good peak shapes and a significant amount of resolution.

10.3 Evaluation of stationary phase
Selection of stationary phase (column) plays critical role in the separation of impurities
along with both the drug components. Since impurities of ATZ and the main drug is
having different polarities, column used in the method shall separate all these compounds
with satisfactory resolution. Available RP-HPLC column chemistries such as High
Strength Silica (HSS) and Hypersil Base Deactivated Silica (BDS) column were tried for
this purpose. Among these, hypersil BDS C18 (250 x 4.6 mm, 5μ) column column shows
optimum separation between all the desired peaks. Hypersil BDS C18 column contains
trifunctional ligand bonded C18 ligand chemistry which produce superior low pH
stability and ultra low column bleed. This low pH stability is combined with the high pH
stability of the 5 μ BDS particle to deliver the widest usable pH range. This new
chemistry also utilize new, proprietary end capping process which produce outstanding
peak shape for bases. Hence, this column was considered for the entire study.

10.4 Optimization of gradient program
Since impurities present in ATZ having wide range of polarities, it is necessary to adopt
gradient elution mode instead of isocratic elution mode. Trials were taken by changing
the composition of buffer and methanol at fixed flow rate of 1 ml/min. Among different
trials performed, gradient program was finalized in which, all the required components
were well separated.
Time(min) 0-7 7-20 20-25
Flow rate (ml/min) 1 1 1
% Buffer 60 15 60
% Methanol 40 85 40
10.5 Gradient program

10.6 Evaluation of flow rate and column oven temperature
For optimum retention times of all impurities, flow rate of 1 ml/min was adopted. In order to
have symmetric peak shapes and optimum resolution between the impurities, column oven
temperature set at 45°C.
10.7 Selection of detector wavelength
Impurities of ATZ show spectral absorption maxima at ~225 nm. Hence, wavelength of 225
nm has been chosen for quantification of impurities due to satisfactory sensitivity and
optimum responses.
10.8 Selection of sample concentration and injection volume
Considering the solubility of ATZ in methanol, mixture of methanol and buffer (pH 4.0) in
the ratio set by gradient technique at different interval time. It was set as diluents and found
satisfactory solubility for impurities of ATZ. Sufficient responses were observed for
impurities at 20 μL injection volume hence the same was finalized to attain reproducible area
counts. In the finalized conditions, standard solutions were injected to check the system
suitability.

Parameter Method
Types of chromatography Reverse phase (RP)
Mode of operation Gradient
Stationary phase (Column) Hypersil BDS C18 column (250mm X 4.6mm i.d.,5µ)
Mobile phase Methanol: 0.05M potassium phosphate buffer (pH 4.0)
Flow rate (mL/min) 1.0
Run time (Minutes) 15
Column temperature ( OC) : 40
Volume of injection loop (µL) 20
Detection wavelength (nm) 210
Diluent Methanol
Detector PDA
10.9 Optimized chromatographic conditions for proposed method:-

Experimental Works
11. Experimental works
11.1 Preparation of stock solution
• Weigh and powdered 20 capsule. Disperse the content of capsules
containing about 20mg ATZ with 60 ml of the methanol in the 100 ml
volumetric flask. Sonicate for 15 minutes and make up volume with
methanol. Filter this solution with whatman filter paper no-1. (ATZ-
200mcg/ml)
11.2 Preparation of standard working solution
• From the stock solution pipette out 1ml into 10 ml volumetric flask and
makeup the final volume with mobile phase (ATZ-20μg/ml).

11.3 Preparation of mobile phase
• Mobile Phase A: 0.05M Potassium dihydrogen phosphate buffer (pH-4.0)
• Take 6.8gm potassium dihydrogen orthophosphate (KH2PO4) into a 1000 ml
beaker. Add 800ml water and dissolve with water. Adjust pH 4.0 with 1%
orthophosphoric acid. Make up volume with water.
• Mobile phase B: Methanol
Preparation of working sample solution
• Take 1ml from sample stock solution into a 10ml and make up with mobile phase.
(ATZ-20mcg/ml)

10.10. Selection of Mobile phase
Sr.No
Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir sulphate
Remarks
Trials for Atazanavir sulphate by using isocratic Method
1. Water: Methanol 1 30:70 7.88 Single peak observed at 7.88
along with tailing
Figure 12: Trial 1 - Chromatogram of Atazanavir sulphate (AZT) in Water: Methanol (30:70v/v)

2. Water: Methanol 1 20:80 7.88 Single peak
observed at 7.88
along with tailing
Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks

Sr.No
Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
3. Water: Methanol 1 10:90 6.30 Retention time
reduced along with
tailing

Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
Figure 12: Trial 4 - Chromatogram of Atazanavir sulphate (AZT) in Buffer (pH 7.0) : Methanol (20:80v/v)
4. Buffer(pH 7.0)
:Methanol
1 20:80 7.88 Single peak
observed at 7.88
along with tailing

Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
5. Buffer(pH 6.0)
:Methanol
observed at 9.23
along with tailing

Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
6. Buffer(pH 5.0)
:Methanol
observed at 7.47 and
retention time
reduced

Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
7. Buffer(pH 5.0)
:Methanol
1 10:90 4.393 Retention time
reduced.
Figure 12: Trial 7 - Chromatogram of Atazanavir sulphate (AZT) in Buffer (pH 5.0) : Methanol (10:90 v/v)

Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
Figure 12: Trial 8 - Chromatogram of Atazanavir sulphate (AZT) +Impurity 1 in Buffer (pH 5.0) : Methanol (60:40v/v)
Trials for Atazanavir sulphate and its impurities by using isocratic methods
8. Buffer(pH 5.0)
:Methanol
1 60:40 4.393 and 4.567 Two merge peaks
observed (ATZ +
Impurity 1)

Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
9. Buffer(pH 5.0)
:Methanol
1 60:40 4.393 Only one peak
observed
(ATZ).Impurity 2
peak is not observed
Figure 12: Trial 9 - Chromatogram of Atazanavir sulphate (AZT)+ Impurity 2 in Buffer (pH 5.0) : Methanol (60:40v/v)

Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
Figure 12: Trial 10 - Chromatogram of Atazanavir sulphate (AZT)+ Impurity 1 + Impurity 2 in Buffer (pH 5.0) : Methanol
(60:40v/v)
10. Buffer(pH 5.0) :Methanol 1 60:40 4.567 Only ATZ peaks
detected.

Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
11 Buffer(pH 5.0)
:Methanol
1 40:60 2.457 and 15.703 AZT Two peaks are
separated and
observed (ATZ +
Impurity 2)
Figure 12: Trial 11 - Chromatogram of Atazanavir sulphate (AZT)+ Impurity 2 in Buffer (pH 5.0) : Methanol (40:60v/v)

Here above the trials, we can conclude that three peaks are not separated by using
isocratic method. Therefore another option for going to take trial for separation of
AZT and its impurities peaks by gradient method.

Trials for Atazanavir sulphate and its impurity-A
& 5 separated by using Gradient method

Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
12. Buffer (pH 5.0)
Methanol
1 1) 0-10 min :-
40:60
2) 10-25 min:-
25:75
ATZ:- 4.370
Impurity A:-4.540
Impurity 5 :-
18.443
ATZ and Impurity 1
peaks merged and
impurity 2 separated
at delayed.

Figure 12: Trial 12 - Chromatogram of Atazanavir sulphate (AZT)+ Impurity 1+ Impurity 2 in Gradient 1 technique

Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
13. Buffer (pH
5.0)
Methanol
1 1) 0-10 min :-
50:50
2) 10-25 min:-
15:85
ATZ:- 5.863
Impurity A:-
6.250
Impurity 5:-
17.337
ATZ and Impurity
1 peaks merged
but resolution
increased and
impurity 2
separated at
delayed

Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
14.
Buffer (pH
5.0)
Methanol
1
1) 0-7 min :-
60:40
2) 7-20min:-
15:85
3) 20-25 min:-
60:40
ATZ:-6.320
Impurity A:-
7.410
Impurity 5 :-
17.807
ATZ and
Impurity 1 and
impurity 2 peaks
are separated.
Retention time is
so long for
impurity 2

Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
15.
Buffer (pH 4.0)
Methanol
1
1) 0-7 min :- 70:30
2) 7-20min:- 15:85
3) 20-25 min:-60:40
ATZ:-4.807
Impurity A:-
5.253
Impurity 5:-
13.123
Retention time is
less than the #14
trial but ATZ and
impurity 1 peak
merged.

Sr.No Mobile
Phase A and B
Flow
Rate
(ml/min)
Ratio
Retention
Time (min)
Atazanavir
sulphate
Remarks
16.
Buffer (pH
4.0)
Methanol
1
1) 0-7 min :- 60:40
2) 7-20min:- 15:85
3) 20-25 min:-
60:40
ATZ:-5.313
Impurity A:-
6.230
Impurity 5 :-
14.537
Due to change of
buffer pH. Three
peaks are valid
as per SST
parameter.
Final Method

Optimized mobile phase
Parameters Atazanvir
sulphate
Impurity A Impurity 5 Limit
Retention
Time
5.313 6.230 14.537 -
Efficiency 6951 7157 7317 > 2000
Tailing/Asym
metry
1.343 1.341 1.376 < 2.0
Resolution -- 3.336 17.051 > 2.0

10.11 System Suitability Test (SST) :- It is an integral part of chromatographic method.
These tests are used to verify that the resolution and reproducibility of the system are
adequate for the analysis to be performed. System suitability tests are based on the
concept that the equipment, electronics, analytical operations and samples constitute an
integral system that can be evaluated as a whole. System suitability testing provides
assurance that the method will provide accurate and precise data for its intended use.
System suitability (SST) is an essential part of the analytical procedure.
The below mentioned system suitability criteria was adopted from standard
solution:
A. The column efficiency as determined from ATA and its impurities peaks is not <2000
plate count.
B. The Tailing factor for ATA and its impurities are not more than 2.0.
C. RSD for peak areas of six injections of the standard solution is not more than 2.0%

Final Analytical Method
• Analytical method was developed using HPLC Shimadzu [with power stream] gradient
chromatographic technique. Data were passed through the spinchrom software.
Separation was achieved on hypersil BDS C18 (250 x 4.6 mm, 5 μm) column using
mobile phase composition of 0.05M potassium phosphate buffer: methanol (60:40 v/v),
(15:85 v/v),(60:40 v/v), pH adjusted to 4 with 1% orthophosphoric acid (OPA). Make up
volume with water. Flow rate was maintained at 1 ml/min with 225 nm UV detection.
The retention time (RT) obtained for atazanavir sulphate (ATZ) , impurity A and
impurity 5 was at 5.3 min, 6.23 min and 14.53 min respectively with injection volume
20 μL and the detection was made at 225 nm.

Forced Degradation study

Forced Degradation study
• Degradation studies are performed on drug product under acidic, alkali, oxidative,
thermal and photolytic stress conditions. Each stress condition samples are analyzed
in the proposed method and peak purity data is recorded to check the homogeneous
nature of the drug.

1) Hydrolytic degradation:
• Hydrolytic study under acidic and basic condition involves canalization of ionisable functional
groups present in the molecule. 0.1 N HCl and 0.1 N NaOH are employed for generating acidic
and basic stress samples, respectively.
2) Oxidative degradation:
• Many drug substances undergo auto oxidation i.e. oxidation under normal storage condition and
involving ground state elemental oxygen.
• Therefore it is an important degradation pathway of many drugs. Auto- oxidation is a free
radical reaction that requires free radical initiator to begin the chain reaction. Hydrogen
peroxide, metal ions, or trace level of impurities in a drug substance act as initiators for auto-
oxidation
• The mechanism of oxidative degradation of drug substance involves an electron transfer
mechanism to form reactive anions and cations.
• Amines, sulphides and phenols are susceptible to electron transfer oxidation to give N-oxides,
hydroxylamine, sulphones and sulphoxide.
• 3% Hydrogen peroxide is very common oxidant to produce oxidative degradation products
which may arise as minor impurities during long term stability studies.

3) Thermal degradation:
• In general, rate of a reaction increase with increase in temperature. Hence, the drugs are
susceptible to degradation at higher temperature (105ºC). Many APIs are sensitive to heat or
tropical temperatures. For example, vitamins, peptides, etc. Thermal degradation involves
different reactions like pyrolysis, hydrolysis, decarboxylation, isomerisation, rearrangement
and polymerization
4) Photolytic degradation:
• The rate of photodegradtion depends upon the intensity of incident light and quantity of light
absorbed by the drug molecule. The photolytic degradation can occur through non-oxidative or
oxidative photolytic reaction.
• Photolytic degradation is carried out by exposing the drug substance or drug product to a
combination of visible and UV light.
The non-oxidative photolytic reactions include isomerisation, dimerization, cyclization,
rearrangements & decarboxylation etc. and while oxidative photolytic reactions occur through
either singlet oxygen (1O2) or triplet oxygen (3O2) mechanism.

Table :- Force degradation studies of ATZ standard and sample with both impurities.
Sr.
no
Stress
condition
and time
% Standard
Degradation
% drug
recovered
Std.
Mean
Peak area
% Sample
Degradation
% drug
recovered
Sample.
Mean
Peak
Area
Area of standard - 19133.117
1. Acid
Hydrolysis
(4 hours)
17.99 82.01 14872.066 19.61 76.35 14608.917
2. Alkaline
Hydrolysis
( 3 Hours)
14.04 85.96 15605.437 13.84 86.16 15795.364
3. Thermal
degradation
(2 hours)
21.17 78.83 14243.767 21.80 78.20 14123.621
4. Oxidative
degradation
(48 Hours)
11.76 88.24 16190.821 12.85 87.15 15968.178
5 Photolytic
degradation
(48 hours)
8.45 91.55 16796.639 7.37 92.63 16970.265

ATZ Standard Acid degradation
Sr.no Retention time
(min)
% Area Asymmetry Theoretical
plates
1. 5.293 94.777 1.382 7216
2. 6.240 2.831 1.341 7180
3. 14.540 2.393 1.366 7444

ATZ Sample Acid degradation
(min)
plates
1. 5.267 94.975 1.382 7144
2. 6.277 2.720 1.341 7264
3. 14.623 2.305 1.376 7404

ATZ Standard Base degradation
(min)
plates
1. 5.283 94.888 1.382 7189
2. 6.260 2.770 1.400 7226
3. 14.593 2.342 1.366 7498

ATZ Sample Base degradation
(min)
plates
1. 5.283 95.821 1.382 7189
2. 6.240 2.269 1.341 7180
3. 14.540 1.909 1.380 7444

ATZ Standard Oxidative degradation
(min)
plates
1. 5.287 95.901 1.382 7198
2. 6.243 2.227 1.400 7188
3. 14.553 1.872 1.366 7457

ATZ Sample Oxidative degradation
(min)
plates
1. 5.283 95.767 1.382 7171
2. 6.250 2.297 1.341 7203
3. 14.563 1.935 1.366 7468

ATZ Standard Photodegradation
(min)
plates
1. 5.300 95.888 1.314 7234
2. 6.283 2.231 1.366 7280
3. 14.643 1.881 1.376 7425

ATZ Sample Photodegradation
(min)
plates
1. 5.293 95.748 1.382 7216
2. 6.267 2.308 1.366 7241
3. 14.607 1.944 1.366 7512

ATZ Standard Thermal degradation
(min)
plates
1. 5.273 94.441 1.353 7162
2. 6.253 3.010 1.341 7211
3. 14.570 2.549 1.366 7474

ATZ Sample Thermal degradation
(min)
plates
1. 5.300 94.391 1.314 7234
2. 6.250 3.038 1.341 7203
3. 14.583 2.571 1.366 7468

Linearity of ATZ, Impurity A and Impurity 5:
 Preparation of Stock solution of ATZ: - Weighed 5 mg of ATZ and diluted in 100 ml
volumetric flasks to obtain 50 ppm solution.
 Preparation of Standard solution of ATZ: - Taken 1 ml of the stock solution of ATZ and
diluted up to the 10 ml volumetric flask to obtain 5 ppm solution

The linearity and Range: - The linearity of the method was determined at six
concentration levels. The linearity data obtained for the calibration curve of ATZ
(concentration 0.5-7.5 μg/ml) & its Impurity A (concentration 0.5-7.5 μg/ml) and
Impurity 5 (concentration 0.2-7.5 μg/ml) were linear over the concentration range of
LOQ to 150 % respectively shown in below Table. 20 µl of each solution was injected in
to the HPLC system and the peak area of the chromatogram obtained was noted. Then, a
linear regression equation was derived by plotting the graph between the sample dissolved
and recovered by the method. A calibration curve was drawn by taking the concentration
on the x-axis and the corresponding peak area on the y-axis shown in Figure.

Linearity of Atazanavir sulphate, Impurity A and Impurity 5
Atazanvir Sulphate Impurity A Impurity 5
Sr.No Level Conc.
( µg/ml)
Response
(Area)
Conc.
(µg/ml)
Response
(Area)
Conc.
( µg/ml)
Response
(Area)
1. LOQ
0.5 31.087 0.5 55.993 0.2 34.86
2. Linearity-1
2.5 101.133 2.5 153.158 2.5 130.737
3. Linearity-2
3.75 156.255 3.75 222.875 3.75 190.671
4. Linearity-3
5 207.854 5 302.874 5 260.029
5. Linearity-4
6.25 255.678 6.25 378.95 6.25 325.376
6. Linearity-5
7.5 311.047 7.5 452.428 7.5 388.485
Slope
41.540 60.369
150.434
Correlation
Coefficient
0.9997 0.9974
0.9998

Figure:- Linearity of Atazanavir sulphate
y = 40.199x + 6.3305
R² = 0.9987
0
50
100
150
200
250
300
350
0 1 2 3 4 5 6 7 8
Concentration ( µg/ml)
Response
(Area)

Figure:- Linearity of Impurity A
y = 57.416x + 17.03
R² = 0.9974
0
50
100
150
200
250
300
350
400
450
500
0 1 2 3 4 5 6 7 8
Response
(Area)

y = 50.877x + 5.4661
R² = 0.9998
0
50
100
150
200
250
300
350
400
450
0 1 2 3 4 5 6 7 8
Figure:- Linearity of Impurity 5
Response
(Area)

Accuracy:- Accuracy Sample of ATZ was spiked with known impurities at five
different levels: LOQ, 80%, 100% and 120 % of the specification limit in
triplicate (total 12 determinations) and then proceed with sample preparation as
described under Methodology.
The Acceptance Criteria of Mean Recovery should be in the range of 90.0% to
110.0% for LOQ, 80%, 100% and 120% levels. Recovery studies were carried
out in triplicate and the percentage recovery and standard deviation of the
percentage recovery was calculated. The Mean Recovery for known Impurities
is within limits. Therefore, the HPLC Method for the determination of ATZ,
impurity A and Impurity 5 are accurate.

Recovery
Level
area of
recovery
spiked
with Test
Area of
imp in
Test
Net
area of
std
area of std
amount
added(mcg/
ml)
amount
recovered(mcg/ml)
%recovery
Mean ±
SD
%RSD
LOQ
114.428 38.965 75.463 150.809 2.500 2.502 100.078
100.098 ±
1.788
1.786
LOQ
115.799 38.965 76.834 150.809 2.500 2.547 101.896
LOQ
113.103 38.965 74.138 150.809 2.500 2.458 98.320
80%
158.779 38.965 119.814 150.809 4.000 3.972 99.309
99.585 ±
1.196
1.201
80%
160.691 38.965 121.726 150.809 4.000 4.036 100.894
80%
157.863 38.965 118.898 150.809 4.000 3.942 98.550
100%
189.281 38.965 150.316 150.809 5.000 4.984 99.673
99.371 ±
1.175
1.183
100%
190.326 38.965 151.361 150.809 5.000 5.018 100.366
100%
186.870 38.965 147.905 150.809 5.000 4.904 98.074
120%
218.674 38.965 179.709 150.809 6.000 5.958 99.303
100.114 ±
0.713
0.712
120%
220.654 38.965 181.689 150.809 6.000 6.024 100.397
120%
221.098 38.965 182.133 150.809 6.000 6.039 100.642
Table:- Recovery result of Impurity A

Recovery
Level
area of
recovery
spiked
with Test
Area of
imp in
Test
Net area
of std
Area of std
amount
added(mcg
/ml)
amount
recovered(mcg/ml)
%recovery Mean SD % RSD
LOQ 89.396 25.836 63.56 128.524 2.500 2.473 98.908
99.791 0.898 0.900
LOQ 89.945 25.836 64.109 128.524 2.500 2.494 99.762
LOQ 90.55 25.836 64.714 128.524 2.500 2.518 100.703
80% 130.337 25.836 104.501 128.524 4.000 4.065 101.636
100.613 1.065 1.058
80% 128.152 25.836 102.316 128.524 4.000 3.980 99.511
80% 129.366 25.836 103.530 128.524 4.000 4.028 100.691
100% 156.591 25.836 130.755 128.524 5.000 5.087 101.736
101.022 0.732 0.725
100% 155.720 25.836 129.884 128.524 5.000 5.053 101.058
100% 154.710 25.836 128.874 128.524 5.000 5.014 100.272
120% 182.207 25.836 156.371 128.524 6.000 6.083 101.389
100.564 1.118 1.112
120% 178.971 25.836 153.135 128.524 6.000 5.957 99.291
120%
181.624 25.836 155.788 128.524 6.000 6.061 101.011
Table:- Recovery result of Impurity 5

Precision
• The intra-day precision of the assay method was evaluated by carrying out 9
independent assays of a test sample of Atazanavir sulphate and its impurities at
three levels (LOQ, 100% and 150%) against the qualified reference standard.
The %RSD of three obtained assay values at three different concentration
levels was calculated. The interday precision study was performed on three
different days i.e. day 1, day 2 and day 3 at three different concentration levels
(LOQ, 100% and 150%, n=3). The % RSD of three obtained assay values on
three different days was calculated.
• The low% RSD values of intra-day and inter-day of ATZ, impurity A and
impurity 5 (2.561,1.217 and 1.288%) for Ataznavir sulphate and its both
impurities reveal that the proposed method is precise.

Intraday Precision (Ruggedness)
Atazanavir Suphate Impurity A Impurity 5
Sr.no Level Conc.
(µg/ml)
Mean ±
SD
%RSD Conc.
(µg/ml)
Mean ±
SD
%RSD Conc.
(µg/ml)
Mean ±
SD
%RSD
1. LOQ 0.5 31.855
± 1.364
4.282 0.5 63.004±
2.469
3.919 0.2
48.351±1
.363
2.818
2. 100 5 215.779
± 5.527
2.561 5 311.464
±11.659
3.743 5
262.182±
5.889
2.246
3. 150 7.5 318.499
±
10.657
3.346 7.5 456.746
±12.732
2.787 7.5
390.022±
5.550
1.423
 Mean of three replicate

Interday Precision
Atazanavir Suphate Impurity A Impurity 5
Sr.no Conc.
(µg/ml)
Mean ± SD %RSD Conc.
(µg/ml)
Mean ±
SD
%RSD Conc.
(µg/ml)
Mean ± SD %RSD
1. 0.5
36.263 ±
1.338
3.689
0.5
67.287 ±
2.741
4.073
0.2
51.527 ±
1.712
3.323
2. 5
219.824 ±
7.470
3.398
5
310.250
±
13.095
4.221
5
263.964 ±
7.814
2.960
3. 7.5
320.181 ±
9.034
2.822
7.5
448.087
±
5.452
1.217
7.5
386.250 ±
4.975
1.288
 Mean of three replicates

Robustness:- The robustness of an analytical procedure refers to its ability to remain
unaffected by small and deliberate variations in method parameters and provides an
indication of its reliability for routine analysis. The robustness of the method was
evaluated by performing the assay of Atazanavir sulphate and its impurities A and 5 both
under different analytical conditions deliberately changing from the original condition.
Slight changes in mobile phase composition, flow rate, and pH affects the
chromatographic response such as retention time and peak area as given in Table. The %
RSD obtained for peak area was 0.64 – 2.68 indicating that the proposed method is
robust.

Atazanavir suphate Impurity A Impurity 5
Sr.No Parameter Condition Mean ±SD %RSD Mean
±SD
%RSD Mean ±SD %RSD
1. Flow rate
+0.2 ml
1.2
201.3923 ±
4.656829
2.312317 291.4833
±
3.422955
1.174323 251.55467 ±
4.142761
1.6468633
2. Flow rate
-0.2 ml
0.8
219.704 ±
4.659113
2.120632 319.11866
±
2.755327
0.863418 269.3927 ±
3.963373
1.471225
3. Mobile
phase +2%
62:38
17:83
62:38
201.4623 ±
2.17482
1.079517 296.288 ±
7.948018
2.682531
2
251.767 ±
3.575102
1.4200044

4. Mobile
phase -2%
58:42
13:87
58:42
221.637 ±
1.698928
0.766536 319.674
3 ±
3.45390
1
1.080444 271.61
±
1.73909
4
0.640291
5. pH +0.2 unit 4.2
209.229 ±
3.920163
1.873623 299.018
7 ±
4.53986
1
1.518253 259.437
7 ±
1.67752
1
0.646599
6. pH -0.2 unit 3.8
206.2097
±
5.16774
2.506061 302.065
±
2.80977
4
0.930188 259.249
3 ±
4.15985
6
1.604577

Repeatability
ATZ ATZ IMP 1 ATZ IMP 2
at100% 5 µg/ml at100% 5 µg/ml at 100% 5 µg/ml
Std area Std area Std area
1 205.206 1 296.875 1 256.403
2 208.988 2 304.97 2 263.618
3 207.37 3 311.882 3 270.28
4 214.41 4 303.515 4 261.543
5 210.381 5 321.496 5 277.167
6 203.358 6 326.784 6 263.966
avg 208.286 avg 310.920 avg 265.496
sd 3.924 sd 11.419 sd 7.256
%RSD 1.884 %RSD 3.673 %RSD 2.733
Limit:%RSD for area NMT 5.0% Limit:%RSD for area NMT 5.0% Limit:%RSD for area NMT 5.0%

Analysis of commercial formulations (Capsules):
The proposed method was applied for the determination of ATZ impurities
estimation in marketed capsules results of its impurities RSD < 5.0 %. The
results indicate that the method is selective for the assay of ATZ without
interference from the excipients used in these capsules.
1. 2.

Stock and Sample preparation
• Preparation of Stock solution of ATZ: - Weighed 50 mg of ATZ and
diluted in 100 ml volumetric flasks to obtain 500 ppm solution.
• Preparation of Mix Standard solution of ATZ: - Taken 1 ml of the above
stock solution of ATZ and diluted up to the 10 ml volumetric flask to obtain
50 ppm solution.
• Preparation of sample solution:-Weighed 50 mg of ATZ and diluted in
100 ml volumetric flasks to obtain 500 ppm solution. Again Take 1 ml of
this prepared solution diluted up to the 10 ml volumetric flask to obtain 50
ppm solution.

Formulation Impurities A Impurities 5 Total impurities
Brand name (Mean ±SD) %RSD (Mean ±SD) %RSD (Mean ±SD) %RSD
1. Atavir 300
(Cipla)
0.254±0.003 1.190
0.195 ±
0.005
2.453 0.449 ±
0.007
1.561
2. ATAZOR-
300
(Emcure)
0.256 ± 0.006 2.473
0.199
±0.002
1.189 0.455 ±
0.008
1.704
* Mean ± SD (n=3)

Impurities A and Impurities 5 standard
Sr.no Retention
time (min)
plates
1. 6.223 53.736 1.375 7142
2. 14.503 46.264 1.380 7406

(min)
plates
1. 3.240 0.085 1.194 4273
2. 5.293 99.253 1.353 7216
3. 6.260 0.396 1.385 7512
4. 14.480 0.265 1.400 7508
Atavir 300 mg - Formulation 1 (Run 1)

(min)
plates
1. 3.217 0.085 1.194 4463
2. 5.257 99.254 1.353 7451
3. 6.217 0.396 1.385 7126
4. 14.380 0.265 1.378 7532

(min)
plates
1. 3.240 0.085 1.194 4246
2. 5.277 99.262 1.382 7171
3. 6.243 0.396 1.350 7472
4. 14.437 0.256 1.333 7591

Impurities A and Impurities 5 standard
Sr.no Retention
time (min)
plates
1. 6.223 53.736 1.375 7142
2. 14.503 46.264 1.380 7406

Atazor 300 mg - Formulation 2 (Run 1)
(min)
plates
1. 3.213 0.085 1.161 4454
2. 5.247 99.260 1.382 7089
3. 6.207 0.390 1.275 7385
4. 14.353 0.265 1.378 7504

(min)
plates
1. 3.227 0.085 1.161 4238
2. 5.267 99.244 1.424 7144
3. 6.233 0.406 1.262 7448
4. 14.410 0.265 1.389 7436

(min)
plates
1. 3.237 0.084 1.161 4264
2. 5.283 99.256 1.382 7189
3. 6.250 0.394 1.385 7488
4. 14.453 0.265 1.400 7480

Estimation of ATZ
Label claim Mean±SD
1. Atavir 300
(Cipla)
99.73 ± 1.66
2. ATAZOR-300
(Emcure)
100.57 ± 1.39

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