1. The document discusses the development and validation of an analytical method to detect and quantify levels of three genotoxic nitrosamine impurities - N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), and N-nitrosodiisopropylamine (NDIPA) - in the drug rifapentine and its dosage forms. 2. Rifapentine is an antibiotic used to treat tuberculosis, but certain samples were found to contain unacceptable levels of nitrosamine impurities. The FDA set interim limits of 0.3 ppm for nitrosamines in rifapentine. 3. The project aims to develop an accurate analytical method and

1. ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF THE CONTENT
ESTIMATION OF GENOTOXIC IMPURITIES OF N-NITROSODIMETHYLAMINE,
N-NITROSODIETHYLAMINE AND N-NITROSODIISOPROPYLAMINE IN
RIFAPENTINE IN BULK AND ITS DOSAGE FORMS
PROJECT REPORT
Submitted for the partial fulfillment
for the award of degree of
BACHELOR OF PHARMACY
FRANKLIN SYLVESTER C (188021601011)
JAWITH SAHUL A (188021601018)
MOHAMMED MEERAN MASTHAN K (188021601035)
RIZWANA PARVEEN N (188021601043)
SWETHA S (188021601054)
Under the supervision of
Dr. Y. Ismail M. Pharm., Ph.D.
Associate Professor
Crescent School of Pharmacy

2. INTRODUCTION

3. GENOTOXIC
IMPURITIES
henomenon
ability of an
s to destruct the
of a cell .
ame of the
destruction
c material of the
e loss of
g to cancer,
defects
3
As per the International Council for
Harmonization (ICH) S2 (R1)
Guideline, genotoxic impurities can be
broadly defined as impurities that
have been demonstrated to cause
deleterious changes in the genetic
material regardless of the
mechanism.
Nitrosamines form a large group of
genotoxic chemical carcinogens and
can be formed endogenously in the
human body. N-Nitroso compounds
can induce cancer
NITROSAMINE
&
TYPES
bable human carcinogens
7(R1), a lifetime intake of
e less than one additional
line as Class 1 impurities,
t carcinogenicity and
FORMATION
OF
NITROSAMINE
TO
CANCER

4. PROBLEM STATEMENT

5. PROBLEM STATEMENT
The pharmaceutical industry is under intense pressure to increase productivity and release new drugs into the
market. Analytical chemists are challenged to find faster ways of delivering quality data across a range of
project needs. Rifapentine is a drug indicated for the treatment of tuberculosis. Rifapentine can be used by
adults and children of 12 years age for the treatment of active pulmonary tuberculosis (TB) caused by
Mycobacterium tuberculosis. On September 2020, Food and Drug Administration announced nitrosamine
impurities in certain samples of Rifapentine due to contamination of unacceptable level of the N-nitrosamine
analogue impurities like N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) and
N-nitrosodiisopropylamine (NDIPA), These N-nitrosamine Impurities are under organic compounds with
genotoxic, carcinogenic and mutagenic activity. In general, they can be found in the natural environment, food
and water, as well as in industrial manufacturing during drug processing. In Industry, it is deducted that
N-nitrosamine is formed because of the reaction between sodium nitrite or another nitrosating agent and the
presence of amine groups in their substances.
Due to the likely danger to the human body, limit set for nitrosamine in Rifapentine were regulated and the
FDA established at interim limit where by acceptable intake of N-nitrosamine in Rifapentine is 0.3 ppm. There
is a need for the development of analytical method and validation for the detection and quantification of
N-nitrosamine impurities in Rifapentine is of great interest. Hence, the research work embodied in this project
work is to develop an analytical method and validation for the content estimation of N-nitrosamine impurities
in Rifapentine in bulk and its dosage forms.
5

6. DRUG PROFILE

7. DRUG PROFILE
RIFAPENTINE:
Synonyms : Cyclopentyl rifampicin, Rifapentinum
Molecular formula : C47H64N4O12
Chemical name : 3-(((4-Cyclopentyl-1-piperazinyl)imino)methyl)rifamycin
Molecular Weight : 877.031 g/mol
Figure 1: Structure of Rifapentine

8. Description:
Rifapentine is a rifamycin antibiotic that is similar in structure and activity to rifampin and rifabutin and that is used in
combination with other agents as therapy of tuberculosis, particularly in once or twice weekly regimens.
Rifapentine is a long-acting, cyclopentyl-substituted derivative of rifamycin used to treat mycobacterium
infections.Rifapentine is a N-alkylpiperazine, a N-iminopiperazine and a member of rifamycins.
It has a role as an antitubercular agent and a leprostatic drug.
Solubility:
Freely soluble in chloroform and dimethyl sulfoxide; soluble in ethyl acetate, methanol, tetrahydrofuran; slightly soluble in
acetone, water, carbon tetrachloride.
Category:
Anti-Bacterial Agents , Anti-Infective Agents, Antibiotics, Anti-tubercular, Anti-infectives for Systemic Use , Anti-
mycobacterials, Cytochrome P-450 CYP2B6 Inducers, Cytochrome P-450 CYP2B6 Inducers (strength unknown),
Cytochrome P-450 CYP2C19 Inducers, Cytochrome P-450 CYP2C19 Inducers (strong).
8

9. Possible Impurities:
1-cyclopentyl-4-nitrosopiperazine (CPNP) (CAS 61379-66-6).
Indication:
Rifapentine is indicated in adults and children 12 years and older for the treatment of active pulmonary
tuberculosis (TB) caused by Mycobacterium tuberculosis.
Rifapentine must always be used in combination with one or more anti-tuberculosis (anti-TB) drugs to which
the isolate is susceptible.
Brand: Priftin
Available dosage forms: Tablets, Injectable powder.
Recommended dosage: Tablet – 150mg, 300mg
Injectable powder – 600mg
Uses:
For the treatment of pulmonary tuberculosis caused by Mycobacterium tuberculosis in combination with 1 or more
anti-tuberculosis drugs.
9

10. LITERATURE REVIEW

11. LITERATURE REVIEW
1. Khaja Moinuddin Shaik et al., Research on Regulatory Updates and Analytical Methodologies for Nitrosamine Impurities Detection in
Sartans, Ranitidine, Nizatidine, and Metformin along with Sample Preparation Techniques
2. Maria Kristina Parr et al., Research on NDMA Impurity in Valsartan and other Pharmaceutical Products: Analytical Methods for the
Determination of N-Nitrosamines
3. Sebastian Schmidtsdorf et al., Analytical life cycle management for comprehensive and universal nitrosamine analysis in various
pharmaceutical formulations by supercritical fluid chromatography
4. Claudia Giménez-Campillo et al., Development of a new methodology for the determination of N-5 nitrosamines impurities in ranitidine
pharmaceuticals using 6 microextraction and gas chromatography-mass spectrometry
5. Jie Liu et al., Development of a sensitive and stable GC-MS/MS method for simultaneous determination of four
N-nitrosamine genotoxic impurities insartan substances.
6. Mikhail Khorolskiy et al., Development and Validation of four Nitrosamine Impurities Determination Method in Medicines of Valsartan,
Losartan, and Irbesartan with HPLC-MS/MS (APCI)
7. Sayaka Masada et al., Rapid and efficient high-performance liquid chromatography analysis of N-nitrosodimethylamine impurity in
valsartan drug substance and its products.
8. Pornchai Rojsitthisak et al., Development of a Sensitive Headspace Gas Chromatography−Mass Spectrometry Method for the
Simultaneous Determination of Nitrosamines in Losartan Active Pharmaceutical Ingredients
9. Fahad S. Aldawsari et al., HS-SPME-GC-MS as an alternative method for NDMA analysis in ranitidine products
10. Hassan Y. Aboul-Enein et al., Determination of Potential Genotoxic Impurity, 5-Amino-2-Chloropyridine, in Active Pharmaceutical
Ingredient Using the HPLC-UV System
11. Jinjian Zheng et al., A Full Evaporation Static Headspace Gas Chromatography Method with Nitrogen Phosphorous Detection for
Ultrasensitive Analysis of Semi-volatile Nitrosamines in Pharmaceutical Products.

12. 12. R.Gonzalez et al., Development of an analytical method for the determination and quantification of N-
nitrosodimethylamine in olmesartan by HPLC-MS/MS
13. Farzad Malihi et al., An improved analytical method for quantitation of nitrosamine impurities in ophthalmic solutions using
liquid chromatography with tandem mass spectrometry.
14. Sebastian Schmidtsdorff et al., Simultaneous detection of nitrosamines and other sartan-related impurities in active
pharmaceutical ingredients by supercritical fluid chromatography
15. Krista L.Dobo et al., Practical and Science-Based Strategy for Establishing Acceptable Intakes for Drug Product N-
Nitrosamine Impurities.
16. Minju Lee et al., Analysis of Nnitrosamines and other nitro(so) compounds in water by high-performance liquid
chromatography with post-column UV photolysis/Griess reaction
17. Kartheek Srinivas Chidella et al., Ultra-Sensitive LC-MS/MS Method for the Trace Level Quantification of Six Potential
Genotoxic Nitrosamine Impurities in Telmisartan.
18. Yuyuan Chen et al., Development and Validation of LC-MS/MS for Analyzing Potential Genotoxic Impurities in Pantoprazole
Starting Materials.
19. Qing Lv et al., High resolution GC–Orbitrap MS for nitrosamines analysis: Method performance, exploration of solid phase
extraction regularity, and screening of children’s products.
20. Ho-Sang Shin et al., High resolution GC–Orbitrap MS for nitrosamines analysis: Method performance, exploration of solid
phase extraction regularity, and screening of children’s products
21. Scott W. Roberts et al., N-Nitrosamine Impurity Control Strategies in the Pharmaceutical and Biotechnology Industries.
12

13. 22. Lee C.Winchester et al., Determination of the rifamycin antibiotics rifabutin, rifampin, rifapentine and their major metabolites in
human plasma via simultaneous extraction coupled with LC/MS/MS.
23. Amitkumar J. Vyas et al., A Review on Carcinogenic Impurities Found in Marketed Drugs and Strategies for its Determination by
Analytical Methods.
24. K. C. Graham et al., High Performance Liquid Chromatographic Analysis of Rifampin and Related Impurities in Pharmaceutical
Formulations.
25. Kàroly Vékey et al., Comparison of mass spectrometric methods for studying thermally labile compounds: Rifapentine.
26. Santosh Patil et al., Quantification and Validation of a HPLC-UV Method for Simultaneous Analysis of Nitrosoamine Impurities
(NDMA, NDEA and NDIPA) in Losartan.
27. Shuhong et al., Simultaneous and trace level quantification of two potential genotoxic impurities in valsartan drug substance using
UPLC-MS/MS
28. Pallavi M. Nawale et al., Determination of Impurity Profile in Rifapentine.
29. Hye S.Lee et al., High-performance liquid chromatographic determination of rifapentine in serum using column switching.
30. Bodin Tuesuwan et al., Nitrosamine Contamination in Pharmaceuticals: Threat, Impact, and Control.
31. Mohana Krishna Mudiam et al., Development, Validation, and Estimation of Measurement Uncertainty for the Quantitative
Determination of Nitrosamines in Sartan Drugs Using LC-APCI-MS/MS.
32. Mychelle Alves Monterio et al., Development and Validation of Methods Based on High-Performance Liquid Chromatography-
Tandem Mass Spectrometry for Determining N-Nitrosamines Impurities in Sartan Pharmaceutical Products for Monitoring Program.
33. Madhushudhan Reddy Bethi et al., Analytical Method Development and Validation of Impurity Profile in Rifapentine
34. Priyanshu Jain et al., Review on Various Analytical Methods Developed for Rifapentine: An Antitubercular Drug.
35. Mohammad Al-Kaseem et al., Validated RP-HPLC Method for the Determination of Seven Volatile N-Nitrosamines in Meat.
13

14. AIM AND OBJECTIVE

15. AIM AND OBJECTIVE
AIM:
Develop an analytical method and validation for the content estimation of N-nitrosodimethylamine (NDMA), N-
nitrosodiethylamine (NDEA) and N-nitrosodiisopropylamine (NDIPA) impurities in Rifapentine in bulk and its
dosage forms by RP-HPLC technique.
OBJECTIVE:
The Literature survey reveals that there are no analytical methods available for content estimation of
N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) and N-nitrosodiisopropylamine (NDIPA)
impurities in Rifapentine in bulk and its dosage forms by RP-HPLC technique.
Hence our present plan is to develop a new, simple, precise & accurate RP-HPLC analytical method development
and validation for content estimation of N-nitrosodimethylamine(NDMA), N-nitrosodiethylamine(NDEA) and
N-nitrosodiisopropylamine(NDIPA) impurities in Rifapentine in bulk and its dosage forms.
15

16. PLAN OF WORK

17. PLAN OF WORK
 Study of physicochemical properties of drug and N-
nitrosamine impurities
 (pH, pKa, solubility and molecular weight.
 Selection of chromatographic condition (mobile phase,
column, flow rate, Detection Wavelength, etc.).
 Optimization of method.
 Study of system suitability parameters.
 Validation of proposed method.
 Applying developed method to marketed formulation.
17

18. EXPERIMENTAL PART

19. SL. No Instrument Model
1 HPLC
WATERS, software: Empower, 2695
separation module, UV 2489 detector.
2 UV/VIS spectrophotometer LABINDIA UV 3000+
3 pH meter Adwa – AD 1020
4 Weighing machine Afcoset ER-200A
5 Pipettes and Burettes Borosil
6 Beakers Borosil
19
SL. No Chemical Company Name
1 NDMA,NDEA Hetero drugs
2 NDIPA Hetero drugs
3 Rifapentine Hetero drugs
4 KH2PO4 FINER chemical LTD
5 Water and Methanol for HPLC LICHROSOLV (MERCK)
6 Acetonitrile for HPLC MOLYCHEM
7 Ortho phosphoric Acid MERCK
List of Instruments Used
List of Chemicals Used

20. METHOD DEVELOPMENT TRAILS
TRIAL 1:
Chromatographic conditions:
Column :symmetry, C18
4.6150mm × 5µm
Mobile phase ratio :MeOH: H2O(50:50%v/v)
Detection wavelength :280 nm
Flow rate :1.0 ml/min
Injection volume :20µl
Run time :5.0min
Observation:
The trial did not show pure peak in the
chromatogram, so more trials were obtaining for
obtaining peaks.
20
Chromatogram Showing Trial-1

21. TRIAL 2:
Chromatographic conditions:
Column : ZodiacSil C18
4.6x150mm 5µm
Mobile phase ratio : ACN: H2O(50:50%v/v)
Detection wavelength : 280 nm
Flow rate : 1.0ml/min
Injection volume : 20µl
Run time : 5.0 min
Observation:
In this trail one peak only eluted, still more trials
were required to elute other components.
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Chromatogram showing Trial-2

22. TRIAL-3:
Chromatographic conditions:
Column : Hypersil RP C8
4.5×150mm 5.0 µm
Mobile phase ratio : ACN: pH 6.8 phosphate
buffer (50:50)
Detection wavelength : 280 nm
Flow rate : 1.0ml/min
Injection volume : 20µl
Run time : 10.0 min
Observation:
In this trial both peaks were eluted but there is no
proper resolution in peaks and elute other
components.
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Chromatogram showing Trial-3

23. TRIAL-4:
Chromatographic conditions
Column : Inertsil RPC18
4.6×250 mm 5µm
Mobile phase ratio : 30% 0.1% OPA
buffer: 70% Methanol
Detection wavelength : 280 nm
Injection volume : 20µl
Run time : 8 min
Observation:
The separation was good, Second peak shape
was not good; still more trials were required to
elute other components.
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Chromatogram showing Trial-4

24. TRAIL - 5
Chromatographic conditions
Column : Endoversil C18
(2.1 x 150mm,4.6m)
Flow rate : 1.0 ml/min .
Injection volume : 20 µl.
Wavelength : 280 nm.
Run time : 5 min.
Mobile phase preparation: 0.1% OPA :
Acetonitrile (30:70)
Diluents : mobile phase
Observation:
The separation was good but still more trails
required to elute the Rifapentine.
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Chromatogram showing Trial-5

25. TRIAL-6 (Optimized chromatogram)
Chromatographic conditions:
Column : Zorbax SB C18, 150 x 4.6 mm, 3.5 µm or
equivalent
Flow rate : 1.0 mL/min
Injection volume : 20L
Column temperature : 250 Auto sampler temperature ±5°C
Buffer : 0.10% Formic acid in water. Filter and
degas through 0.22 µm membrane
filter paper
Solution A : Buffer
Solution B : Methanol
Mobile Phase : Solution A: Solution B (5:95) v/v
Run time : 15 min
Diluent : Mobile phase
25
System Suitability Parameter Results of NDMA, NDEA, NDIPA,
and Rifapentine
Chromatogram showing peak of NDMA, NDEA, NDIPA, and
Rifapentine

26. 1. SYSTEM PRECISION
Procedure:
Take each 20mg of NDMA, and NDIPA std solutions into a 20mL
vol flask containing 5mL diluent and make up with diluent. Transfer
0.75mL of above sol. Into 25mL vol flask containing diluent and
make up with diluent.
Take 20mg of NDEA std solutions into a 20mL vol flask containing
5mL diluent and make up with diluent. Transfer 0.75mL of above
sol. Into 25mL vol flask containing diluent and make up with diluent.
Transfer 1.0mL of std stock solution-I and std stock solution-ii in to a
100mL vol flask containing 25mL
Take1.0 mL of standard solution into a headspace vial immediately
the vial.
Inject blank solution into the system and record the chromatogram.
Inject standard solutions into the system separately for six times
Observation:
%RSD for the peak areas of NDMA, NDEA, and NDIPA,
were 0.48, 6.80 and 8.47 respectively.
Acceptance Criteria:
Not More than 15.0
System Precision data for NDMA, NDEA, and NDIPA
26
Injection
No.
Areas of
NDMA
Areas of NDEA Areas of NDIPA
1 7950 2522 4884
2 7923 2394 4543
3 7918 2139 4747
4 7863 2594 4456
5 7850 2329 4034
6 7892 2500 3952
Average 7899.333 2413 4436
SD 38.1663 164.1365 375.5354
% RSD 0.48 6.80 8.47

27. 27
Chromatogram showing system precision of 6 injection levels

28. 2. SPECIFICITY
Procedure:
Weigh accurately about 10 mg of the test sample into a 10 ml VF and add 7.0 mL of diluent and make up the
solution upto 10 ml(1mg/ml). Further Pipette out 0.1 ml from the above solution and make up with Same solution
(0.1mg/ml).
Inject blank solution and conclude no interference due to blank at the retention time of NDMA, NDEA and NDIPA.
Inject test solution and blend solution and record the chromatograms.
Observation:
There were no interference observed.
Acceptance criteria:
If interference is observed the area (or) response of interference should not be more than 5.0%.
Chromatogram Showing Blank Solution Impurity Mix Chromatogram
28

29. Chromatogram Showing Rifapentine Chromatogram Showing NDMA,
NDEA, NDIPA, and Rifapentine
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30. 3. DETECTION LIMIT:
Procedure:
Prepare DL solution containing NDMA, NDEA, and NDIPA with respect to test concentration by diluting NDMA,
NDEA, NEIA and NDIPA stock solutions so as to get S/N ratio about 3:1 or by visual method.
Inject blank solution into the system and record the chromatograms. Inject DL solution into the system and record the
chromatograms. Calculate the signal to noise ratio of NDMA, NDEA, and NDIPA peaks from DL solution through
software.
Observation:
Detection limit NDMA, NDEA, and NDIPA impurity peak were visualized.
Acceptance criteria:
The S/N ratio should be about 3:1 (or) report the DL level values by visual method.
30
Name of the
Compound
Conc. w.r.t. Test (ppm)
NDMA and NDIPA 0.03
NDEA 0.02
Chromatogram showing Detection Limit of NDMA, NDEA,
NDIPA and Rifapentine

31. 4. QUANTITATION LIMIT:
Procedure:
Based on the signal to noise ratio obtained from DL solution, derive QL concentration of NDMA, NDEA and NDIPA
so as to get S/N ratio about 10:1 or by visual method.
Inject blank solution into the system and record the chromatograms.
Inject QL solution into the system for six times (for precision at QL) and record the chromatograms. Calculate the
signal to noise ratio of NDMA, NDEA and NDIPA peak from QL solution (first injection) through software.
Calculate %RSD for the peak areas of NDMA, NDEA and NDIPA from Six replicate injections.
Observation:
Quantitation limit for NDMA, NDEA and NDIPA impurity peak were visualized.
Acceptance criteria:
The S/N ratio should be about 10:1 (or) report the QL level values by visual method.
31
Name of the
Compound
Conc. w.r.t. Test
(ppm)
NDMA and NDIPA 0.09
NDEA 0.06
Chromatogram showing Quantitation Limit of NDMA, NDEA, NDIPA and Rifapentine

32. 4.1. Precision at QL:
32

33. QL Level
Peak Areas of
NDMA
Peak Areas of
NDEA
Peak Areas of
NDIPA
Injection-1 2423 495 1386
Injection-2 2214 444 1352
Injection-3 2103 463 1349
Injection-4 2590 433 1391
Injection-5 2191 476 1369
Injection-6 2208 485 1311
Average 2288.167 466 1359.667
SD 181.6253 24.01666 29.33712
%RSD 7.94 5.15 2.16
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Observation: QL level were within the limit.
Acceptance criteria:
The % RSD for the six replicate injections of QL level should not to be more than 20.0.

34. 5. LINEARITY:
Preparation of Linearity Level-1 Solution (QL):
Consider first two injections of precision at QL.
Preparation of Linearity level-2 (50 %) standard solution:
Dilute 0.5 mL of standard stock solution in to a 25 mL volumetric flask dilute to Volume with diluents and mix well.
Preparation of Linearity level-3 (75 %) standard solution:
Dilute 0.75 mL of standard stock solution in to a 25 mL volumetric flask dilute to Volume with diluents and mix well.
Preparation of Linearity level-4 (100 %) standard solution:
Dilute 1.0 mL of standard stock solution in to a 25 mL volumetric flask dilute to volume with diluents and mix well.
Preparation of Linearity level-5 (125 %) standard solution:
Dilute 1.25mL of standard stock solution in to a 25 mL volumetric flask dilute to volume with diluent and mix well.
Preparation of Linearity level-6 (150 %) standard solution:
Dilute 1.5 mL of standard stock solution in to a 25 mL volumetric flask dilute to volume with diluent and mix well.
Inject linearity level (Level 2-6) in duplicate runs and record the chromatograms. Plot linearity graph between average
peak areas from duplicate injections against Concentration and determine the correlation coefficient values for NDMA,
NDEA and NDIPA by using Microsoft excel.
34

35. Chromatogram showing Linearity
35

36. Level Concentration of NDMA (ppm) Average Areas of NDMA
Level-1 0.093 2391
Level-2 0.155 3985
Level-3 0.232 5964
Level-4 0.309 7944
Level-5 0.387 9949
Level-6 0.464 11929
Correlation Coefficient 0.999
36
2391
3985
5964
7944
9949
11929
y = 25709x + 1E-11
R² = 0.9999
0
2000
4000
6000
8000
10000
12000
14000
0 0.1 0.2 0.3 0.4 0.5
LINEARITY GRAPH OF NDMA
Linearity data for NDMA
Level Concentration of NDEA (ppm) Average Areas of NDEA
Level-1 0.062 497
Level-2 0.154 1234
Level-3 0.231 1850
Level-4 0.308 2467
Level-5 0.384 3076
Level-6 0.461 3692
Correlation Coefficient 0.999
497
1234
1850
2467
3076
3692
y = 25709x + 1E-11
R² = 0.9999
0
500
1000
1500
2000
2500
3000
3500
4000
0 0.1 0.2 0.3 0.4 0.5
LINEARITY GRAPH OF NDEA
Linearity data for NDEA

37. Level Concentration of NDIPA (ppm) Average Areas of NDIPA
Level-1 0.088 1345
Level-2 0.146 2231
Level-3 0.220 3362
Level-4 0.293 4478
Level-5 0.366 5594
Level-6 0.439 6709
Correlation Coefficient 0.999
1345
2231
3362
4478
5594
6709
y = 25709x + 1E-11
R² = 0.9999
0
1000
2000
3000
4000
5000
6000
7000
8000
0 0.1 0.2 0.3 0.4 0.5
LINEARITY GRAPH OF NDIPA
Linearity Data for NDIPA
Observation:
Correlation coefficient value of NDMA, NDEA and NDIPA derived from respective linearity graph were
within the limit.
Acceptance criteria:
Correlation coefficient value derived from the linearity graph should be not less than 0.999.

38. 6. ACCURACY:
Procedure:
Inject blank solution into the system and record the chromatograms.
Inject test solution into the system and record the chromatograms.
Inject each accuracy level-2 and level-6 in triplicate preparations and accuracy
Level-4 in six preparations (method precision) and record the chromatograms.
Consider first three preparations for accuracy study.
Calculate the % recovery of NDMA, NDEA and NDIPA separately from each preparation by using the formula given
below and report the results in the range.
Calculate the NDMA, NDEA and NDIPA content in test sample using the following formula:
AT CS
NDMA content (ppm) = --------x -------- x P x10000
AS CT
Where,
AT = Peak area of NDMA in test solution.
AS = Average peak area of NDMA in standard solutions.
CS = Concentration of NDMA in standard preparation (mg/mL).
CT = Concentration of test solution (mg/mL).
P = Purity or Assay of NDMA standard (%).
Similarly calculate for the NDEA and NDIPA by using the above formula.
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39. Chromatogram Showing Accuracy 50%:
39

40. Chromatogram Showing Accuracy 100%:
40

41. Chromatogram Showing Accuracy 150%:
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42. Accuracy
level
Preparations
%
Recovery
of NDMA
%
Recovery
of NDEA
%
Recovery
of NDIPA
50%
1 107.1 102.6 114.4
2 109.0 116.9 117.1
3 103.9 117.5 121.2
100 %
1 108.7 108.1 117.1
2 107.1 106.8 115.7
3 106.1 102.9 109.9
150%
1 108.0 106.9 113.7
2 107.5 104.1 113.7
3 108.2 102.2 112.1
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Accuracy (recover) data for NDMA, NDEA, and NDIPA
Observation:
The % recovery of NDMA, NDEA and NDIPA were within the limit.
Acceptance criteria:
The % recovery should be between 70 to 130 for 50%, 100% & 150% level.

43. 7. METHOD PRECISION:
• Perform the analysis by spiking the sample with NDMA, NDEA and NDIPA at 100 % of the
specified limit with respect to the sample concentration.
• Prepare in six replicates as per method and calculate the content of NDMA, NDEA and NDIPA and
determine the %RSD.
Chromatogram showing Method Precision:

44. Chromatogram showing Method Precision
44
44

45. Method
Precision NDMA (ppm) NDEA (ppm) NDIPA (ppm)
Preparation-1 0.336 0.333 0.343
Preparation-2 0.331 0.329 0.339
Preparation-3 0.328 0.317 0.322
Preparation-4 0.335 0.320 0.324
Preparation-5 0.335 0.308 0.323
Preparation-6 0.340 0.337 0.333
Average 0.334 0.324 0.331
% RSD 1.3 3.4 2.7
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Observation:
The % RSD for the results obtained from method precision study were within the limit.
Acceptance criteria:
The % RSD for the results obtained from method precision study should not be more than 15.0.
Summary results of Method Precision data for NDMA, NDEA, and NDIPA

46. 8. INTERMEDIATE PRECISION:
 Establish the system suitability as per method. Inject blank solution and record the chromatogram.
Inject standard solution in to the system in six replicates and record the chromatogram. Inject test
solution and record the chromatogram.
 Inject spiked test sample solution in six preparations (which is analyzed like as method
 Precision study) and record the chromatograms.
 Calculate the %RSD for each NDMA, NDEA and NDIPA content from each preparation and report the
results.
 Calculate the cumulative %RSD for the results obtained from method precision study
 And intermediate precision study.
Variation Study of Method for Intermediate Precision
46
Variation of Study Method Precision
Intermediate
Precision
Analyst to Analyst Franklin Swetha
Day 20/05/2022 21/05/2022

47. Chromatogram showing Intermediate Precision
47

48. 48
Intermediate
Precision NDMA(ppm) NDEA(ppm) NDIPA(ppm)
Preparation-1 0.319 0.323 0.324
Preparation-2 0.317 0.321 0.323
Preparation-3 0.316 0.336 0.299
Preparation-4 0.319 0.314 0.321
Preparation-5 0.321 0.333 0.317
Preparation-6 0.313 0.302 0.310
Average 0.318 0.322 0.316
% RSD 0.9 3.9 3.0
Summary Results of Intermediate Precision for NDMA,
NDEA and NDIPA

49. Preparations
Content(ppm)ND
MA
Content(ppm)NDEA Content(ppm)NDIPA
Method Precision
Preparation-1
0.336 0.333 0.343
Method Precision
Preparation-2
0.331 0.329 0.339
Method Precision
Preparation-3
0.328 0.317 0.322
Method Precision
Preparation-4
0.335 0.320 0.324
Method Precision
Preparation-5
0.335 0.308 0.323
Method Precision
Preparatton-6
0.340 0.337 0.333
Intermediate Precision
Preparation-1
0.319 0.323 0.324
Intermediate Precision
Preparation-2
0.317 0.321 0.323
Intermediate Precision
Preparation-3
0.316 0.336 0.299
Intermediate Precision
Preparation-4
0.319 0.314 0.321
Intermediate Precision
Preparation-5
0.321 0.333 0.317
Intermediate Precision
Preparation-6
0.313 0.302 0.310
Average 0.326 0.323 0.323
Cumulative % RSD 2.9 3.5 3.7
49
Summary results for Cumulative % RSD of Method Precision and
Intermediate Precision
Observation:
The cumulative %RSD for the results obtained from
method precision study and intermediate precision study
together were within the limit.
Acceptance criteria:
System suitability should comply as per the method.
The % RSD for the results obtained from Intermediate
precision study should not be more than 15.0
The cumulative % RSD for the results obtained from
method precision study and Intermediate precision study
should not be more than 20.0

50. SUMMARY AND CONCLUSION

51. SUMMARY AND CONCLUSION
A new analytical method was established and validated for content estimation of genotoxic impurities like
NDMA (N-nitrosodimethylamine), NDEA (N-Nitrosodiethylamine) and NDIPA (N-
Nitrosodiisopropylamine) in Rifapentine in bulk and its dosage forms by RP-HPLC method.
The instrument used was WATERS HPLC Auto sampler, separation module 2695, UV Detector 2489,
Empower-software version-3. The method was developed on a reversed-phase Zorbax SB C18 (4.6 × 150
mm, 3.5 µm) column with an isocratic elution. The Mobile phase ratio was Buffer (0.10 % Formic acid in
water: Methanol (05:95 % v/v).Detection was done by UV-Spectroscopy at a detection wavelength of 280
nm. The flow rate was 1.0 ml/min. The mobile phase was used as a diluent. The Injection volume was 20
μl. The analytical procedure was validated as per ICH guidelines.
The retention time for N-nitrosodimethyl amine, N-nitrosodiethyl amine, N-nitrosodiisopropyl amine and
Rifapentine in the standard solution were observed to be around 3.117, 3.712, 4.134 and 6.756 minutes
respectively. System suitability parameters were calculated and found within the acceptance criteria. The
limit of detection of N-nitrosamine impurities in Rifapentine ranged from 0.02- 0.03 ppm and the
corresponding limit of quantification were in the range 0.06- 0.09 ppm which met the sensitivity
requirements for the limits set by FDA of United States. The determined LOD and LOQ values are very
low which show the sensitivity performance of the method.
51

52. The proposed method was found to have a high degree of precision and reproducibility.
The Calibration plots of N-nitrosamine impurities in Rifapentine showed good linearity of regression
coefficient (r2> 0.999). The recoveries of nitrosamine impurities in drugs ranged from 102.2 % - 117.1 %.
The system precision and precision at quantitation limit studies, the Relative Standard Deviation of peak
area values of N-nitrosamine impurities were within the acceptance criteria. The method precision and
interday precision studies, the Relative Standard Deviation of content estimation values of N-nitrosamine
impurities were within the acceptance criteria. The accuracy and precision results demonstrated the
efficiency of this method.
Therefore this proposed method exhibit good sensitivity, precision, high accuracy and faster analysis, which
provide a reliable method for quality control of N-nitrosamine impurities in Rifapentine products. The
developed method can be used as a standard method for content estimation of NDMA
(N-nitrosodimethylamine), NDEA (N-Nitrosodiethylamine) and NDIPA (N-Nitrosodiisopropylamine) in
Rifapentine for pharmaceutical companies and researchers.
However, as it is a drug administered with different pathologies, with daily administration and continuous
use, it would be necessary to check the possible consequence of continuous administration, even at very low
concentrations, of a genotoxic impurities such as NDMA (N-nitrosodimethylamine), NDEA
(N-Nitrosodiethylamine) and NDIPA (N-Nitrosodiisopropylamine) in Rifapentine products.
52

53. SCOPE FOR FURTHER WORK

54. SCOPE FOR FURTHER WORK
It is imperative to have safety and efficacy in drug therapy, pharmacists must take into consideration, the
stability of drugs and its therapeutic values. The stability of the drug formulations can be assessed by
using stability indicating methods. A well-designed stress study is important to help develop and
demonstrate the specificity of stability indicating methods. They are also useful for checking rapid and
accurate drug quality during stability testing. Hence there is scope for future work to develop a stability
indicating analytical method and validation for content estimation of N-nitrosodimethylamine (NDMA),
N-nitrosodiethylamine (NDEA) and N-nitrosodiisopropylamine (NDIPA) impurities in Rifapentine in
bulk and its dosage forms and also by using these chromatographic conditions it will try to help us
develop an analytical method in LC-MS technique for more sensitivity.
54

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