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UHPLC Method for Simultaneous Estimation of Cefpodoxime and Azithromycin
1. ANALYTICAL MEHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF
CEFPODOXIME AND AZITHROMYCIN BY UHPLC IN SOLID DOSAGE FORMS
A Dissertation submitted to
JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY ANANTAPUR
ANDHRA PRADESH, INDIA
SESHACHALA COLLEGE OF PHARMACY
NAGARI ROAD, PUTTUR -517 583, A.P
DECEMBER- 2021
I
n partial fulfillment of the requirements
For the award of degree in
MASTER OF PHARMACY
(PHARMACEUTICAL ANALYSIS)
Submitted By
GOWDUCHERUVU KOWSALYA(18CP1S0703)
Under the guidance of
Mrs. .M.PRIYANKA,M. Pharm.
Assistant Professor
Department of Pharmaceutical analysis
2. INTRODUCTION
• Pharmaceutical analysis:
• Pharmaceutical analysis is a branch of practical chemistry that
involves a series of process for identification, determination,
quantification and purification of a substance, separation of the
components of a solution or mixture, or determination of structure
of chemical compounds.
• Based on literature survey no method is reported for estimation of
CEFPODOXIME AND AZITHROMYCIN by UHPLC.
• The sample to be analysed is called as analyse and on the basis of
size of sample.
• They can be classified as macro(0.1 g or more), semi micro (0.01 g
to 0.1 g), micro(0.001 g to 0.01 g), sub micro (0.0001 g to 0.001 g),
ultramicro (below 10-4 g), trace analysis(100 to 10000 ppm).
Among all, the semi micro analysis is widely used.
3. Types of Analysis:
• There are main two types of chemical analysis.
1. Qualitative (identification)
2. Quantitative (estimation)
• 1. Qualitative analysis is performed to establish composition
of natural/synthetic substances.
• 2. Quantitative analytical techniques are mainly used to
quantify any compound or substance in the sample.
4. Various types of Qualitative analysis:
Chemical methods
a) volumetric or titrimetric methods
b) gravimetric methods
c) gasometric analysis
Electrical methods
Instrumental methods
Biological and microbiological
5. DRUG PROFILE:
• Name: AZITHROMYCIN
• Structure:
Chemical Formula:C38H72N2O12
• In March 2020, a small study was funded by the French government to
investigate the treatment of COVID-19 with a combination of azithromycin
and the anti-malaria drug hydroxychloroquine. The results were positive,
all patients taking the combination were virologically cured within 6 days
of treatment, however, larger studies are required.
6. REVIEW OF LITERATURE:
Shahed Mirza, Mohammed Imran, Khateeb Muntasiruddin:
• SIMULTANEOUS ESTIMATION OF AZITHROMYCIN AND CEFPODOXIME
PROXETIL FROM ITS TABLET DOSAGE FORM BY UV VISIBLE
SPECTROSCOPIC METHODS.
• Method – I is simultaneous equation method in which wavelength
selected for azithromycin is 251 nm (λmax) and cefpodoxime shows
maximum absorption at 234 nm.
• Method – II is derivative spectroscopic method in which zero order (D 0)
and first order (D 1) derivative spectroscopic methods were explicated.
Shaikh Javed , Shaikh Afzal, Mohd Arif:
• RP-HPLC ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR
AZITHROMYCIN AND CEFPODOXIME IN TABLET DOSAGE FORM:
• A simple, rapid and accurate reversed phase high-performance liquid
chromatographic (RP-HPLC) method has been developed and
subsequently validated for the simultaneous determination of
Azithromycin (AZT) and Cefpodoxime(CEF) in pharmaceutical dosage form.
7. AIM:
Present work is aimed to develop a new, simple, fast, rapid, accurate,
efficient and reproducible UHPLC method for the simultaneous analysis
of Cefpodoxime and Azithromycin. The developed method will be
validated according to ICH guidelines.
OBJECTIVE:
The analytical method for the simultaneous estimation of
Cefpodoxime and Azithromycin will be developed by UHPLC method
by optimizing the chromatographic conditions.
The developed method is validated according to ICH guidelines for
various parameters specified in ICH guidelines.
9. Diluent:
Based up on the solubility of the drugs, diluent was selected,
mobile phase used as diluent.
Preparation of Standard solution:Weighed accurately
and transferred about 62.5 mg of Azithromycin standard
and about 50 mg Cefpodoxime standard in 50 ml
volumetric standard flask. Dissolved and madeup to the
volume with mobile phase. Diluted 5 ml of this solution to
25 ml with mobile phase.
Preparation of Sample solution: Weighed accurately and
transferred powdered sample equivalent to about 20mg of
Cefpodoxime in 100 ml volumetric standard flask.
Dissolved and madeup to the volume with mobile phase.
10. METHOD DEVELOPMENT :
• Numerous trails were conducted for optimized method with alteration in
mobile phase concentration and flow rate.
Validation:
The validation of the optimized method was carried out using the validation
parameters
1. Specificity
2. Linearity
3. Precision
4. Accuracy
5. Robustness
6. Assay
11. Method development: Method development was done by changing various,
mobile phase ratios, buffers ,Wavelength etc.
Trial 1:
• Chromatographic conditions:
• Mobile phase : Buffer: Acetonitrile: Methanol (20:40:40)
• Flow rate : 0.7ml/min
• Column : Waters symmetry C 18; 50mm x 4.6mm; 3.5 µm
• Detector wave length : 220 nm
• Injection volume: 2L
• Run time : 5 min
• Diluent : Mobile phase
• Results :Improper peak elution and more run time so, further
trial is carried out.
Trial chromatogram 1
12. • Chromatographic conditions:
• Mobile phase : Buffer: Acetonitrile: Methanol (30:50:20)
• Flow rate : 0.7ml/min
• Column : Waters symmetry C 18; 50mm x 4.6mm; 3.5 µm
• Detector wave length : 220 nm
• Injection volume : 2L
• Run time : 5 min
• Diluent : Mobile phase
• Results :Peak was eluted but peak shape was not good .so, further trial is
carried out.
Trial chromatogram 2
Trial 2:
13. TRAIL 3:
• Mobile phase : Buffer: Acetonitrile: Methanol (40:30:30)
• Flow rate : 0.7ml/min
• Column :Waters symmetry C 18; 50mm x 4.6mm; 3.5 µm
• Detector wave length : 220 nm
• Injection volume : 2L
• Run time : 5 min
• Diluent : Mobile phase
• Results : Retention time is more and peak separation not good
so, further trial is carried out.
Trial chromatogram
14. Optimized conditions : TRAIL 4:
Chromatographic conditions:
Mobile phase : Buffer: Acetonitrile: Methanol (30:30:60)
Flow rate : 0.7ml/min
Column : Waters symmetry C 18; 50mm x 4.6mm; 3.5 µm
Detector wave length : 220 nm
Injection volume : 2L
Run time : 5 min
Diluent : Mobile phase
Result: : Retention time and peak shape is good. Cefodoxime was eluted at 3.16 min
and Azithromycin was eluted at 3.76 min with good resolution. Plate count and tailing factor
was very satisfactory, so this method was optimized and to be validated.
16. 1.Specificity
Table for Cefpodoxime and Azithromycin
Sample ID
Cefpodoxime Azithromycin
RT AREA RT AREA
BLANK 3.157 No peak observed 3.752 No peak observed
STANDARD 3.157 1.5478 3.752 651.2345
PLACEBO 3.157 No peak observed 3.752 No peak observed
18. 2.Linearity:
Linearity Data of Cefpodoxime
72.90 %
1
linearity stock dilution:
70.00 mg 50 ml
Soln. ID
50.0% : 2.5 ml 25 ml 102.06 mcg/ml
75.0% : 3.75 ml 25 ml 153.09 mcg/ml
100.0% : 5.0 ml 25 ml 204.12 mcg/ml
125.0% : 6.25 ml 25 ml 255.15 mcg/ml
150.0% : 7.5 ml 25 ml 306.18 mcg/ml
102.06 153.09 204.12 255.15 306.18
310.2533 465.3698 620.6965 775.6988 945.3565
(Stock Soln.)
standard diluted to
Std purity :
Stock soln diluted to
Concentrations
Observed Area-->
VALIDATION PARAMETER : LINEARITY
LABEL CLAIM:
Stock soln diluted to
Stock soln diluted to
Stock soln diluted to
Stock soln diluted to
Linearity solution dilutions:
Linearity Concentrations-->
CEFPODOXIME
Factor :
200 mg
CEFPODOXIME
310.2533; 102.06
465.3698; 153.09
620.6965; 204.12
775.6988; 255.15
945.3565; 306.18
y = 0.3228x + 2.8887
R² = 0.9997
0.00
50.00
100.00
150.00
200.00
250.00
300.00
350.00
0.0000 200.0000 400.0000 600.0000 800.0000 1000.0000
CEFPODOXIME
19. Linearity Data of Azithromycin
250 mg 94.60 %
65.63 mg 50 ml
Soln. ID
50.0% : 2.5 ml 25 ml 124.17 mcg/ml
75.0% : 3.75 ml 25 ml 186.26 mcg/ml
100.0% : 5.0 ml 25 ml 248.34 mcg/ml
125.0% : 6.25 ml 25 ml 310.43 mcg/ml
150.0% : 7.5 ml 25 ml 372.52 mcg/ml
124.17 186.26 248.34 310.43 372.52
325.566 487.588 650.255 812.700 985.636
AZITHROMYCIN
Observed Area-->
standard diluted to (Stock Soln.)
Linearity solution dilutions: Concentrations
Stock soln. diluted to
Stock soln. diluted to
Stock soln. diluted to
Stock soln. diluted to
Stock soln. diluted to
Linearity Concentrations-->
linearity stock dilution:
VALIDATION PARAMETER : LINEARITY
LABEL CLAIM:
AZITHROMYCIN Std purity :
124.17; 325.566
186.26; 487.588
248.34; 650.255
310.43; 812.700
372.52; 985.636
y = 2.6500x - 5.7525
R² = 0.9998
0.000
200.000
400.000
600.000
800.000
1000.000
1200.000
0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00
AZITHROMYCIN
20. Discussion:
Five linear concentrations of Cefpodoxime and Azithromycin were
injected in a duplicate manner. Average areas were mentioned above
and linearity equations obtained for Cefpodoxime was y = 0.3228x +
2.8887 and Azithromycin was y = 2.6500x - 5.7525. Correlation
coefficient obtained was R² = 0.9997 for Cefpodoxime and R² = 0.9998
for Azithromycin respectively.
21. 3. Precision:
3.1.Method precision table of Cefpodoxime
mg
:
: %
70.00 50 5 25 ml.
622.3630
SAMPLE DILUTIONS :
100 1 1 ml.
Spl. Area 5 100 1
621.883 25 Spl. Wt. 1
Conc. level
72.90
Average :
CEFPODOXIME
Factor 1.0000
622.3565
STANDARD DILUTIONS : Purity of std.
mg diluted to ml, further ml diluted to
STANDARD VALUES :
621.2547
0.484
% RSD : 0.08
1
ml diluted to
Sample diluted to ml
700.00
VALIDATION PARAMETER - METHOD PRECISION
LABEL CLAIM: Avg. weight of a tablet : 700
200 mg
621.5855 621.8575
621.8834
X
50
Content in mcg
X
70.00
X X X X
72.900
100.000
X
Standard Deviation :
Sample ID Sample wt. (mg) Sample Area
Calculated Assay
(in mg)
Calculated Assay
(in percentage )
Sample -01 70.63 612.2121 199.153 99.58
Sample -02 70.42 613.6599 200.219 100.11
70.60 612.7855 199.424 99.71
Sample -04 70.63 611.1555 198.810
612.4154 200.611 100.31
SD. : 1.222 0.609
Sample -06 70.14
MIDDLE
LEVEL
(100%)
Sample -03 70.64 621.4587 202.144 101.07
99.41
Sample -05
Average : 200.060 100.03
22. Method precision table of Azithromycin
mg
:
65.63 50 5 25 ml.
651.2525
SAMPLE DILUTIONS :
100 1 1 ml.
Spl. Area 5 100 1
650.980 25 Spl. Wt. 1
Conc. level
VALIDATION PARAMETER - METHOD PRECISION
LABEL CLAIM: Avg. weight of a tablet : 700
AZITHROMYCIN 250 mg
STANDARD DILUTIONS : Purity of std. 94.60 %
mg diluted to ml, further ml diluted to
STANDARD VALUES :
651.4787 650.0255 651.3666 650.7744
Average : 650.9795
Standard Deviation : 0.597
% RSD : 0.09
Sample diluted to ml ml diluted to
Content in mcg
X
65.63
X X X X
94.600
X 700.00
50 100.000
MIDDLE
LEVEL
(100%)
Sample -01 70.63 650.4755 245.94
Sample ID Sample wt. (mg) Sample Area
Calculated Assay
(in mg)
Calculated Assay
(in percentage )
98.38
Sample -02 70.42 658.5855 249.75 99.90
Sample -04 70.63 659.5252 249.36 99.74
Sample -03 70.64 651.4858 246.30 98.52
Sample -06 70.14 650.4775 247.66 99.06
Sample -05 70.60 665.3636 251.67 100.67
% RSD : 0.89 0.89
Average : 248.45 99.38
SD. : 2.211 0.88
23. Discussion: From a single volumetric flask of working standard
solution six injections were given and the obtained areas were
mentioned above. Average area, standard deviation and % RSD
was calculated for Cefpodoxime and Azithromycin % RSD
obtained as 0.61 % for Cefpodoxime and 0.89 % for Azithromycin
respectively. As the limit of Precision was less than “2” the
method precision was passed in this method.
24. 3.2.Intermediate precision: (Ruggedness: ) Day 1
Intermediate precision table for Cefpodoxime
Conc. level Sample ID Sample Area Calculated Assay (in
percentage )
MIDDLE
LEVEL
(100%)
Sample -01 620.3589 100.87
Sample -02 615.3027 100.48
Sample -03 609.6478 98.68
Sample -04 612.3087 99.89
Sample -05 618.6475 100.27
Sample -06 613.2842 98.43
Average :99.77
SD. :0.99
% RSD :0.99
26. Discussion:
Multiple sampling from a sample stock solution was done and six working
sample solutions of same concentrations were prepared, each injection from each
working sample solution was given and obtained areas were mentioned in the
above table. Average area, standard deviation and % RSD was calculated for
Cefpodoxime and obtained as 1.00 % and % RSD was calculated for
Azithromycin and obtained as 0.85 %. As the limit of Precision was less than “2”
the system precision was passed in this method.
27. 3.3.Intermediate precision: Day 2
Intermediate precision table for Cefpodoxime
Conc. level Sample ID Sample Area Calculated Assay (in
percentage )
MIDDLE
LEVEL (100%)
Sample -01 615.3147 100.52
Sample -02 609.6517 99.47
Sample -03 612.3018 99.56
Sample -04 620.6532 101.27
Sample -05 618.3541 100.63
Sample -06 613.5521 99.81
Average :100.21
SD. :0.71
% RSD :0.71
28. Intermediate precision table for Azithromycin
Conc. level Sample ID Sample Area Calculated Assay
(in percentage )
MIDDLE
LEVEL
(100%)
Sample -01 662.3148 99.37
Sample -02 669.3218 100.30
Sample -03 664.3982 99.22
Sample -04 665.2019 99.68
Sample -05 670.3159 100.19
Sample -06 670.9648 100.24
Average :99.83
SD. :0.47
% RSD :0.47
Discussion: Multiple sampling from a sample stock solution was done and six working
sample solutions of same concentrations were prepared, each injection from each working
sample solution was given and obtained areas were mentioned in the above table. Average
area, standard deviation and % RSD was calculated for Cefpodoxime and obtained as 0.71
% and % RSD was calculated for Azithromycin and obtained as 0.47 %. As the limit of
Precision was less than “2” the system precision was passed in this method.
29. 4.ACCURACY:
• The accuracy study was performed for 80%, 100%, 120% for , Cefpodoxime
Azithromycin Each level was injected in triplicate into chromatographic system.
The area of each level was used for calculation of % recovery.
Accuracy table of Cefpodoxime
30. Accuracy table of Azithromycin
Discussion: Three levels of Accuracy samples were prepared by standard addition
method. Triplicate injections were given for each level of accuracy and mean % Recovery
was obtained as 99.85% and 100.68% for Cefpodoxime and 98.65 % and 100.18% for
Azithromycin.
31. 5.Robustness:
S.No. Condition
% RSD of
Cefodoxime
assay
% RSD of
Azithromycin
assay
1 Flow rate minus 0.46 0.42
2 Flow rate plus 0.6 0.55
3 Wavelength
plus
0.86 0.91
4 Wavelength
minus
0.61 0.67
DISCUSSION: Robustness conditions like Flow minus (0.6ml/min), Flow plus
(0.8ml/min), Wavelength minus (218 nm) and Wavelength plus (222nm) was
maintained and samples were injected in duplicate manner. System suitability
parameters were not much affected and all the parameters were passed. %RSD was
within the limit.
32. Assay:
%
70.00 50 5 25 ml.
70.00 100 1 1 ml.
620.3563
618.5445
619.512 70.00 5 100 1 72.90
620.025 50 25 70.00 1 100.00
.=
98 % to 102 %
.=
0.526
SD :
Equivalent Factor : 1
ml, further ml diluted to
ml diluted to
STANDARD VALUES :
% RSD : 0.22
SAMPLE VALUES :
619.5788
mg diluted to
Average wt.of a tablet :
101.98 %
Limit :
X X
X
Content in %
203.951 mg
% RSD : 0.08
CEFPODOXIME
620.7885
ml
Content in mg
VALIDATION PARAMETER : ASSAY
STANDARD DILUTIONS :
SAMPLE DILUTIONS :
700.00
Name of standard :
Purity of standard :
CEFPODOXIME 200
LABEL CLAIM:
mg
mg
72.90 ( as such)
mg diluted to
619.7477 619.6556
Average :
SD :
620.0254
620.4785
X
1.37
X 700.00
X X
Average : 619.512
33. %
65.63 50 5 25 ml.
70.00 100 1 1 ml.
650.5485
648.6555
649.207 65.63 5 100 1 94.60
650.897 50 25 70.00 1 100.00
.=
98 % to 102 %
.=
Average wt.of tablet : 700.0 mg
VALIDATION PARAMETER : ASSAY
LABEL CLAIM:
AZITHROMYCIN 250 mg
STANDARD DILUTIONS :
Name of standard : AZITHROMYCIN
Purity of standard : 94.60 ( as such)
mg diluted to ml, further ml diluted to
SAMPLE DILUTIONS :
mg diluted to ml ml diluted to
STANDARD VALUES :
651.8568 651.2323 650.4788 650.3669
Average : 650.8967
SD : 0.635
% RSD : 0.10
SAMPLE VALUES :
649.7587
Average : 649.207
SD : 0.78
% RSD : 0.12
Content in mg
X X X X X 700.00
247.700 mg
Content in % Limit :
99.08 %
X
34. Discussion: Assay was performed with the above
formulation. Average % Assay for Cefpodoxime 101.98 %
and Azithromycin 99.08 % respectively
.
36. Conclusion:
A simple, Accurate, precise method was developed for the simultaneous estimation of the
Cefpodoxime and Azithromycin in solid dosage form. Retention time of Cefpodoxime and
Azithromycin were found 3.16 min and 3.76 min respectively. %RSD of the Cefpodoxime
and Azithromycin were found 0.07 and 0.42 respectively. % accuracy were obtained as
100.29 % for Cefpodoxime and 99.35% for Azithromycin. Regression equation of
Cefpodoxime is y = 0.3228x + 2.8887.Regression equation of Azithromycin is y = 2.6500x -
5.7525. Retention times were decreased and that run time was decreased, so the method
developed was simple and economical.
37.
38. • https://www.chromatographytoday.com/news/hplc-uhplc/31/breaking-
news/what-is-the-difference-between-uhplc-and-uplc/30754
• https://www.britannica.com/science/chromatography
• https://www.sciencedirect.com/topics/chemistry/pharmaceutical-analysis
• https://go.drugbank.com/salts/DBSALT001354
• https://go.drugbank.com/drugs/DB01416
• https://www.1mg.com/generics/cefpodoxime-proxetil-azithromycin-
400866
• Dr.S. Ravi Shankar, Text book of Pharmaceutical analysis, Fourth edition,
• ICH, Validation of analytical procedures: Text and Methodology.
International Conference on Harmonization, IFPMA , Geneva , (1996)
• https://www.chromatographytoday.com/news/hplc-uhplc/31/breaking-
news/what-is-the-difference-between-uhplc-and-uplc/30754
• Lindholm.J, Development and Validation of HPLC Method for Analytical
and Preparative purpose. Acta Universitatis Upsaliensis, pg. . 13-14,
(2004).
• https://www.britannica.com/science/chromatography
• https://dyadlabs.com/hplc-vs-uplc