JOURNAL CLUB PRESENTATION (20L81S0701-PA) Presented by: P.NARESH (Department of pharmaceutical analysis).RIPER, anantapur

1. Journal club presentation
As a part of Curricular requirement for
M.Pharmacy II year III Semester
Presented by
P.Naresh
Reg.No.20L81S0701
Department of Pharmaceutical Analysis
Under the guidance of
Dr. K. Vinod Kumar Ph.D.
Associate Professor & HOD
Department of Pharmaceutical Analysis
1

2. Title, Authors and Affiliations:
2

3. Contents:
❖ Introduction
❖ Aim and Objectives
❖ Materials and Methods
❖ Results and Discussion
❖ Author conclusion
❖ My conclusion
❖ References
3

4. Introduction:
• Various undesired side products or compounds may be generated during the
production process of active pharmaceutical ingredients (APIs) in the
pharmaceutical industry.
• Studies show that among these undesirable impurities (generated due to
degradation or synthesis), a number of them may have a potentially
genotoxic effect in vivo and in vitro genotoxicity tests having the ability to
interact with DNA and lead to DNA damage.
• These compounds are also called potentially genotoxic impurities (PGIs).
4

5. • The ability to induce chromosomal breaks, genetic mutations and
rearrangements of chromosomes and are therefore, potentially cancerogenic
for humans .
• Therefore, any trace amount of exposure to these types of undesirable
compounds (impurities) existing in API can be a considerable toxicological
issue .
• Recently, sensitive analysis of these genotoxic impurities (GTIs) in APIs
has received great interest from researchers from different disciplines, such
as pharmacology, chemistry, biology and various guidelines.
5

6. • The Viracept accident is a good example of the importance of GTIs in
pharmaceutical compounds.
• The Swiss pharmaceutical company Roche, which is one of the largest
companies conducting research in this area, produced a new product named
Viracept for the treatment of AIDS in 2007.
• However, researchers realized that an excess amount of a GTI ethyl
mesylate formed in the product due to an undesirable reaction between
methyl sulfonic acid.
6

7. • Which is a starting material, and the residual solvent ethanol in the
production tank.
• After the detection of GTI ethyl mesylate in the final product, all related
products were recalled globally .
• It is therefore important for process researchers to explore alternative ways
to avoid the generation and exposure of these GTIs during the synthesis of
APIs.
7

8. • On the other hand, aminopyridines are extensively used as starting
compounds for the production of APIs, and they are considered as potential
GTIs, which may exist in the final product.
• Various chromatographic studies for the analysis of a number of potentially
genotoxic impurities (PGIs), such as methyl p-toluene sulfonate, ethyl p-
toluene sulfonate, acrolein, 2 aminopyridine and 4-aminopyridine in APIs .
• They were successfully analyzed using high-performance liquid
chromatography with a UV (HPLC-UV), LC-MS and GC-MS.
8

9. • Aminopyridines are commonly used as starting compounds in the synthesis
of APIs and are PGIs that can be present in trace levels in pharmaceutical
compounds in this study.
• Tenoxicam (TNX) was examined because its molecular structure is similar
to piroxicam (PRX).
• The analysis of PGI 5-amino-2-chloropyridine (5A2Cl) in a model API
such as TNX has not been investigated so far.
9

10. • In this research, a new HPLC-UV method is developed for the
efficient analysis of PGI 5A2Cl in a model API TNX that may be
generated during the production process of APIs, which is extremely
important in the pharmaceutical industry.
• The target GTI 5-Amino-2-chloropyridine and model API TNX.
10

11. Aim and Objective:
• The Research work is aimed on the determination of Potential Genotoxic
Impurity ,5- Amino-2-Chloropyridine ,in Active Pharmaceutical Ingredient
using the HPLC-UV System.
• The main objective of the research is identification of PGI obtained from 5-
Amino-2-Chloropyridine .
• The present study deals with experimental Retention time of API & GTI &
predicted results were compared and calculating the % Recovery, %RSD,
as well as LOD,LOQ.
11

12. Material and Method:
❖ Chemicals and materials:
• TNX, 5-amino-2-chloropyridine (5A2Cl), ACN, MeOH, HPLC grade
water and orthophosphoric acid were supplied by Sigma Aldrich .
• Analytical reagent grade chemicals (all were HPLC grade or ≥99% purity)
were utilized in all of the experiments.
• Ultrasonic bath Jeio Tech, Lab companion (Republic of Korea) was used
for degassing the mobile phase and all solutions.
• AVortex(Germany) was used for themixing process.
12

13. ❖ Instrumentation and chromatographic conditions:
• A Shimadzu Nexera-i HPLC system combined with an ultraviolet detector was
used in this study.
• Separation was performed using a Restek Raptor C18 (150 mm î 4.6 mm î
2.7 μm particles)column.
• All dilution processes were conducted using a mobile phase consisting of
water; MeOH, (50:50, v/v) (with 0.01% orthophosphoric acid). All analyses
were detected at a 254 nm wavelength.
• The mobile phase flow rate was 0.7 ml/ min−1 and the HPLC-UV column
temperature was maintained at 40◦C.
13

14. • A total of 10 μL volume (all standards and samples) was injected into the HPLC-
UV system. 5A2Cl and TNX retention times were 2.30 and 2.80 min.
❖ Sample and standards preparations:
• Standard stock solutions of 10 mg 5A2Cl and TNX were dissolved at a
concentration of 1 mg mL−1 and 10 mg was dissolved in 10 mL 60% ACN,
containing 0.01% orthophosphoric acid at a concentration of 1 mg mL−1.
• Both stock solutions were stored in a refrigerator at +4◦C.
14

15. • The obtained standard concentration were 1, 5, 10, 20 and 40 μg mL−1,
respectively. The sample mixture consisted of 250 μg mL−1 TNX spiked with
20 μg mL−1 5A2Cl.
• The obtained chromatogram of containing 20 μg mL−1 of 5A2CI and 250
μg mL−1 of TNX solution.
15

16. Results:
❖ Optimization and Conditions of the HPLC-UV method:
• Several analyzes were performed under different chromatographic conditions
to find the optimum method conditions for mobile phase content and flow rate,
column temperature, injection volume and detector wavelength ,Retention times
and parameters of system suitability were investigated and used to decide on
optimum conditions.
• First of all, mixtures of water, methanol and acetonitrile were tested as the
mobile phase containing different percentages of solutions.
16

17. • Water (with 0.01% with orthophosphoric acid: MeOH, (50:50, v/v) provided
good peak symmetry with short elution times for the target compounds.
• In addition, a different mobile phase pH (such as 5 and 8) was used for shorter
elution times and better peak symmetry.
• Finally, the best chromatogram was obtained in the mentioned mobile phase
conditions.
• The flow rate of the mobile phase was performed at 0.7 mL min−1 for low
system pressure and best elution times.
17

18. • The effect of column temperature was investigated at 25, 30 and 40◦C.
• The best peak symmetry and elution time were observed at 40◦C, and this
temperature was selected as the optimum condition.
• An HPLC-UV analysis was conducted at a wavelength of 254 nm for the
determination of 5A2Cl and TNX, System Suitability Testing parameter.
18

19. ❖ Validation of the HPLC-UV method:
• The proposed method was validated in terms of selectivity, linearity, limit
of detection (LOD), limit of quantification (LOQ), precision, accuracy and
robustness, in accordance with the International Conference on
Harmonization guidelines (ICH Q2(R1) .
➢ Selectivity:
• The selectivity of the HPLC-UV method was observed by comparing
chromatograms from blank samples; blank samples spiked with 5A2Cl.
• It was determined that there is no interference of peaks.
19

20. ➢ Linearity:
• Linearity was determined for 5A2Cl in the concentration range of 1-40 μg
mL−1.
• Linearity was achieved using the mean values of the results obtained from
analyses of the standard solutions of 5A2Cl repeated three times prepared in five
different concentrations.
• Calibration sets were prepared and these were also analyzed three times (n = 3)
on different days.
20

21. • Calibration curves were found to be linear y = 40766x − 11256
• Concentration levels with determination coefficients R2 = 0.999
for 5A2Cl.
• The closeness of this value to 1 shows that the correlation was extremely
good for this developed HPLC-UV analytical method.
21

22. ➢ LOD and LOQ:
• LOD and LOQ values were found to be 3.3 and 10 times the ratio of
standard deviation (SD) at low concentration of 5A2Cl solution to the
slope of the regression equation.
• LOD and the LOQ values were found to be 0.015 and 0.048 μg mL−1.
➢ Precision and accuracy:
• The accuracy of the applied HPLC-UV method was performed by recovery
studies.
22

23. • The recovery of the method was determined by adding a known amount of
standard 5A2Cl to the samples, and all of the solutions were analyzed using
the same method.
• The recovery values were determined by comparison with their
concentrations without a target genotoxic compound.
Recovery (%) = (observed amount − amount)/spiked amount × 100
• The recovery values were calculated and found to be between 98.80 and
100.03%,with the SD of recovery values being reported as between 0.13 and
0.58.
23

24. • The precision of the developed analytical method is demon- strated by the
results of intraday and interday reproducibility.
• The results were obtained after replicate (n = 3) injections of standard
solutions (25 μg mL−1).
• Repeatability and Intermediate Precision Values for the (25 μg mL−1 ) 5A2Cl
Standard Solutions.
24

25. ➢ Robustness:
• Robustness studies were performed with small changes to the flow rate of the
mobile phase, column temperature, pH and percentage concentrations of
organic solvent.
• The observed RSD% values are 0.82, 0.24, 0.88 and 0.67.
• It was observed that the obtained values are statistically acceptable;
(recommended reference RSD% value < 2).
25

26. Discussion:
• The present study describes a sensitive, accurate and reproducible HPLC-UV
method for the determination of the potential GTI 5A2Cl in the model API TNX.
• Aminopyridines are commonly used as starting compounds in the synthesis of
APIs and they are potential GTIs, which can be present in trace levels in
pharmaceuticalcompounds.
• To obtain optimum conditions, various pH values and chemical buffers at
different concentrations were tested with the best chromatographic separation
being obtained with water(with0.01% with orthophosphoric acid: MeOH),
(50:50, v/v) for the mobile phase.
26

27. • There is no need to use an internal standard as a >90% system recovery was
achieved.
• The results of the validated method were found to be within the range of
statistically appropriate values.
• It was reported that the system suitability testing results were also within
the limits of the quite satisfactory values.
• The obtained retention times for 5A2Cl and TNX were 2.3 and 2.8 min.
• The LOD and the LOQ values for the 5A2Cl were found to be 0.015 and
0.048 μg mL−1,
27

28. Author conclusion:
• In conclusion, the developed novel HPLC-UV-based analytical method
was successfully applied for the analysis of 5-amino-2- chloropyridine and
TNX.
• The achieved LOD and the LOQ values for the target compound 5A2Cl
were 0.015 and 0.048 μg mL−1.
• The obtained results from the HPLC-UV analyses confirm that the
developed and validated.
28

29. • The analytical method is sensitive, facile, fast, robust and accurate, and that
it could easily be applied with great success for the sensitive analysis of
potential GTIs in pharmaceutical products.
29

30. My conclusion:
• I concur with present research , It is necessary to decrease these GTIs to
acceptable levels or limits , if they still exist in the final APIs and Finished
products.
• GTI decrease can be carried out by applying one or more purification
stages after the synthesis of API and Finished products.
• At the moment, there is no comprehensive list of GTI compounds.
• Overall, It’s the right platform to learn & gain about method development
and validation parameters .
30

31. References:
1. Bolt, H.M., Foth, H., Hengstler J.G., Degen, G.H.; Carcinogenicity
categorization of chemicals-new aspects to be considered in a European
perspective; Toxicology Letters, (2004); 151(1): 29-41.
2. Dobo, K.L., Greene, N., Cyr, M.O., Caron, S., Ku, W.W.; The application
of structure-based assessment to support safety and chemistry diligence to
manage genotoxic impurities in active pharmaceutical ingredients during
drug development; Regulatory Toxicology and Pharmacology, (2006);
44(3): 282-293.
3. Muller, L., Singer, T.; EMS in Viracept-the course of events in 2007 and
2008 from the non-clinical safety point of view; Toxicology Letters,
(2009); 190(3): 243-247.
4. Jacobson-Kram, D., McGovern, T.; Toxicological overview of impurities
in pharmaceutical products; Adv Drug Deliv Rev, (2007); 59(1): 38-42.
31

32. 32