Upload
others
View
3
Download
0
Embed Size (px)
Citation preview
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1685
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT
AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF
CEFTAZIDIME AND AVIBACTAM IN BULK AND
PHARMACEUTICAL DOSAGE FORM USING UPLC
Juaveria Salam1* and Sh. Rizwan
2
1Student, Osmania University, Telangana.
2Student of Deccan School of Pharmacy Hyderabad, Telangana.
ABSTRACT
This research work describes simple, sensitive, accurate, precise UPLC
method for the simultaneous determination of Ceftazidime and
Avibactam in pharmaceutical dosage form. The sample was analyzed
by using C18 column (Waters Acquity C18 130A (100x2.2mm ID)
1.8µm with mobile phase using methanol, acetonitrile & water in the
ratio of 60:20:20 in the flow rate of 0.5 ml/min. Detection wavelength
was achieved at 248 nm. The retention time for Ceftazidime and
Avibactam was found to be 2.365 and 4.460 minute respectively. The
linearity for Ceftazidime and Avibactam was obtained in the
concentration range of 100-300 µg/ml and 25-75 µg/ml respectively. Ceftazidime and
Avibactam API and market formulation were subjected to acid and base hydrolysis, peroxide,
thermal and photolytic forced degradation. In the forced degradation study Ceftazidime and
Avibactam showed degradation in peroxide and acid degradation. The developed method was
simple, specific, sensitive, rapid, and economic and can be used for estimation of Ceftazidime
and Avibactam in bulk and pharmaceutical dosage form for routine analysis and stability
studies.
KEYWORDS: Cetazidime, Avibactam, UPLC method, Methodvalidation, Forced
degradation.
1. INTRODUCTION
Ceftazidime is used to treat lower respiratory tract, skin,blood-stream, joint, and abdominal
infections, and meningitis.[6]
The drug is given intravenously (IV) or intramuscularly
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 7.632
Volume 10, Issue 11, 1685-1699 Research Article ISSN 2278 – 4357
*Corresponding Author
Juaveria Salam
Student, Osmania
University, Telangana.
Article Received on
21 Sept. 2021,
Revised on 11 October 2021,
Accepted on 31 October 2021
DOI: 10.20959/wjpps202111-20399
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1686
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
(IM) every 8–12 hours (two or three times a day), with dose and frequency varying by the
type of infection, severity, and/or renal function of the person. Ceftazidime is also commonly
prescribed off-label for nebulization in people with cystic fibrosis for the suppression
of Pseudomonas aeruginosa in the lungs
IUPAC Name
(6R, 7R, Z)-7-(2-(2-aminothiazol-4-yl)-2-(2-carboxypropan-2-yloxyimino) acetamido)-8-oxo-
3-(pyridinium-1-ylmethyl)-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate
Chemical formula
C22H22N6O7S2
Molecular weight
546.57 g·mol−1
Avibactam
Is a non-β-lactam β-lactamase inhibitor[2]
developed by Actavis (now Teva) jointly
with AstraZeneca. A new drug application for avibactam in combination with
ceftazidime (branded as Avycaz) was approved by the FDA on February 25, 2015, for
treating complicated urinary tract (cUTI) and complicated intra-abdominal infections (cIAI)
caused by antibiotic resistant-pathogens, including those caused by multi-drug
resistant Gram-negative bacterial pathogens.
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1687
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
IUPACName: [(2S, 5R)-2-Carbamoyl-7-oxo-1, 6-diazabicyclo[3.2.1]octan-6-yl] hydrogen
sulfate
Chemical formula: C7H11N3O6S
Molecular weight: 265.24 g·mol−1
2. MATERIALS AND METHODS
I. Instrument
UV-Visible Spectrophotometer Thermo Electron
UPLC software OpenLab EZ Chrome
UPLC Agilent technology Infinity 1290
Ultra sonicator Citizen, Digital Ultrasonic Cleaner
pH meter Thermo scientific corporation.
Chromatographic conditions
Chromatographic separations were achieved on an ACQUITY UPLC C18 column (100 mm
× 2.2 mm, 1.8 μm) with a mobile phase consisting of 2.72 gm of Potassium Di hydrogen
orthophosphate Methanol: ACN: Water (60 : 20: 20v/v/v) isocratically pumped at a flow rate
of 0.8mL/min−1, and detection was monitored at 248 nm at room temperature. Filtration of
the mobile phase was performed using a. +e injection volumes were 10 ul. With run time 10
min. The Ceftazidime & Avibactam peaks was observed at 2.249 mins with good efficiency
(>2000) and peak shape and good resolution and tailing factor ( < 2).
Procedure
Preparation of Phosphate buffer Ph 6.8
2.72 gm of Potassium Di hydrogen orthophosphate was weighed and dissolved in 1000mL of
water. Adjust the pH to 6.8 ± 0.02 using diluted orthophosphoric acid. Buffer was filtered
through 0.45μm filters to remove all fine particles and gases.
Preparation of standard solution
Standard stock solution prepared by dissolving 10 mg of Ceftazidime & Avibactam dissolved
in sufficient mobile phase. Further dilution is prepared by adding 0.1 ml of stock solution to
10 ml of mobile phase.
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1688
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
Method validation parameters
1. System Suitability & System precision
To verify that the analytical system is working properly and can give accurate and precise
results were evaluated by 200µg/ml of Ceftazidime & Avibactam was injected six times and
the chromatograms were recorded for the same. The plate count and tailing factor results
were found to be within the limits and the % RSD was found to be 2.0% so system is suitable
and giving precise result.
Injection RT Peak
area
Theoreti
cal
plates
(TP)
Tailing
factor
(TF)
1 2.355 3158.91 14996 1.20
2 2.356 3167.73 14569 1.22
3 2.357 3167.58 14255 1.24
4 2.356 3170.77 14586 1.23
5 2.354 3171.91 14563 1.20
6 2.356 3173.17 14563 1.22
Mean 2.36 3168.35 - -
SD 0.00 5.14 - -
%RSD 0.04 0.2 -
-
-
Injection Retention
time
Peak
area
Theoretical
plates
Tailing
factor
Resolution
1 4.46 1282.04 13569 1.30 6.90
2 4.457 1282.00 13585 1.33 6.98
3 4.457 1281.71 13541 1.36 6.96
4 4.454 1282.47 13596 1.34 6.90
5 4.458 1283.28 13569 1.31 6.99
6 4.458 1283.81 13569 1.31 6.90
Mean 4.46 1283 - -
SD 0.00 0.82 - - -
%RSD 0.0 0.1 - - -
System suitability for Ceftazidime & Avibactam
2. Method precision
Method precision was determined by injecting sample solutions of concentration Ceftazidime
(200μg/mL) & Avibactam(50 μg/mL) for six times are prepared separately. The % RSD of
Area and Retention times for 6 Sample determinations of Ceftazidime &Avibactam found to
be within the acceptance criteria (less than 2.0%). Hence method is precise.
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1689
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
Injection Ceftazidime Avibactam
%Assay %Assay
1 100.3 100.3
2 99.6 100.5
3 99.9 99.9
4 100.3 99.8
5 99.9 100.3
6 99.4 100.5
Average 100.6 100.6
SD 0.4 0.3
%RSD 0.4 0.3
Chromatogram method precision 1
Chromatogram of method precision 2
Chromatogram of method precision 3
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1690
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
Chromatogram of method precision 4
Chromatogram of method precision 5
Chromatogram of method precision 6
3. Linearity and Range
Preparation of standard stock solution: weigh accurately 400mg of CEFTAZIDIME and 100
mg of AVIBACTUM in 200 ml of volumetric flask and dissolve in 70ml of mobile phase and
make up the volume with mobile phase.
Preparations Volume from
standard stock
transferred in
mL
Volume made
up in mL (with
mobile phase)
Conc. obtained (µg/mL)
Ceftazidime Avibactam
1 2.5 100 100.0 25.0
2 4 100 160.0 40.0
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1691
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
3 5 100 200.0 50.0
4 6 100 240.0 60.0
5 7.5 100 300.0 75.0
Linearity graph of ceftazidime
Linearity graph of avibactam
Results: The correlation coefficient for linear curve obtained between concentration vs. Area
for standard preparation for ceftazidime & avibactam was found to be 0.9998 & 0.9991.
4. Specificity
Blank solution was injected, and the chromatogram was recorded for the same as given in
figure below .Placebo solution was prepared, and it was injected, and the chromatogram was
recorded for the same as given in fig.
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1692
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
Chromatogram of placebo
Chromatogram of blank
Result: It was observed that diluent or excipient peaks do not interfere with the ceftazidime
& avibactam peaks.
5. Accuracy
Accuracy of the method was determined by Recovery studies. To the formulation
(preanalysed sample), the reference standards of the drugs (50µg/ml, 150µg/ml and
250µg/ml) were added at the level of 50%, 150%, 250%. The recovery studies were carried
out three times and the percentage recovery and percentage mean recovery were calculated
for drug.
Table 1: The recovery results of ceftazidime.
Recovery
level
Accuracy CEFTAZIDIME Average
%
Recovery
Amount
taken
(mcg/ml)
Area Average
area
Amount
recovered(mcg/ml) %Recovery
50%
100.0 1594.90
1590.88
100.68
100.4 100.4 100.0 1588.33 100.26
100.0 1589.41 100.33
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1693
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
100%
200.0 3169.99
3178.66
200.10
100.3 200.0 3180.89 200.79
200.0 3183.17 200.94
150%
300.0 4767.38
4773.11
300.94
100.4 300.0 4773.64 301.33
300.0 4778.31 301.63
6. Limit of detection & Limit of quantification (LOD & LOQ)
The LOD is the lowest amount of an analyte in a sample which can be detected by an
analytical method. The concentration at LOD should give a signal-to-noise ratio (S/N) of3
The LOD for this method was found to be 3.53 µg/ml for Ceftazidime & 2.03 µg/ml for
Avibactam The LOQ for this method was found to be 11.67µg/ml for Ceftazidime & 6.72
µg/ml for Avibactam
7. Robustness
The Robustness of the method was determined. The results obtained by deliberate variation in
method parameters are summarized below in Table
Chromatogram of Ceftazidime and Avibactam Robustness (0.4mL/min)
Chromatogram of Ceftazidime and Avibactam Robustness (0.6mL/min).
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1694
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
Chromatogram of Ceftazidime and Avibactam for Robustness (284nm)
Chromatogram of Ceftazidime and Avibactum Robustness (294nm)
Chromatographic
changes
Theoretical Plates Tailing factor
Ceftazidime Avibactum Ceftazidime Avibactum Resolution %RSD for
05
Replicate
injections
Wavelength
(nm)
284 13290 12682 1.20 1.31 7.25 0.60
294 13690 12525 1.23 1.36 7.50 0.31
Flow rate
mL/min
0.4 13692 12862 1.25 1.33 7.64 0.25
0.6 13652 12362 1.20 1.32 7.25 0.75
Result: The tailing factor and theoretical plates was found to be within the limits on small
variation of flow rate and temperature.
8. Ruggedness
The ruggedness of the method was studied by the determining the analyst to analyst
variationc by performing the Assay by two different analysts
From the above results % Assay and %RSD obtained acceptance criteria 2% so method is
rugged.
Ceftazidime %Assay Avibactam %Assay
Analyst 01 99.4 Analyst 01 98.9
Analyst 02 99.7 Analyst 02 100.1
%RSD 0.14 %RSD 0.87
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1695
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
Results: From the above results % Assay and %RSD obtained acceptance criteria 2% so
method is rugged.
Forced degradation studies
Forced degradation is a technique where different stress conditions are applied over drug
Product and which in turn different degradation products are produced. These studies are also
called as stress testing or stress degradation studies. These methods are mainly used for the
determination of stability of molecule under accelerated conditions.
It is known that regulatory documentation process, selection of proper storage and package
conditions, and selection of formulation are dependent on the stability of molecules.] In
forced degradation process, general conditions such as light, heat, humidity, and oxidation are
accelerated individually or in combination with automated stress to accelerate the degradation
of the molecule by physical or chemical means.
Studies on forced degradation of drug molecules are very important in the following aspects.
1. To develop methods to determine stability
2. To determine the degradation pathways.
3. For determination of intrinsic stability of drug in dosage forms.
4. To study the chemical properties of molecules
5. For production of stable formulations.
6. To determine the structure of decomposition products.
7. To solve problems related to stability.
8. To generate a degradation profile under ICH conditions.
Injection Ceftazidime Avibactum
Condition %Assay Area %Assay
1 Thermal 99.9 100.1
2 Photolytic 100.2 99.6
3 Acid Hydrolysis 100.1 94.1
4 Base Hydrolysis 94.8 100.4
5 Peroxide Hydrolysis 92.2 100.1
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1696
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
Peroxide degradation
Photolytic degradation
.
Acidic degradation
Base degradation
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1697
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
Thermal degradation
Result: In above all conditions 8.8% of Ceftazidime degradation was achieved in Peroxide
Degradation and 5.9% of Avibactam degradation achieved in Acid degradation, Peak purity
of main analyte was passed so this method proved as a stability indicating method.
RESULTS AND DISCUSSIONS
A simple and selective UPLC method is described for the determination of Ceftazidime &
Avibactam in bulk and tablet dosage forms. Chromatographic separation was achieved on
mobile phase consisting of a mixture of Methanol: Acetonitrile: Water in ratio 60: 20:
20v/v/v with detection of 248nm. Linearity was observed in the range 100-300µg/ml for
Ceftazidime (r2 =0.9998) & range 25-75µg/ml for Avibactam drug estimated by the proposed
methods was in good agreement with the label claim.
The proposed methods were validated. The accuracy of the methods was assessed by
recovery studies at three different levels. Recovery experiments indicated the absence of
interference from commonly encountered pharmaceutical additives. The method was found to
be precise as indicated by the repeatability analysis, showing %RSD less than 2. All
statistical data proves validity of the methods and can be used for routine analysis of
pharmaceutical dosage form.
CONCLUSION
From the above experimental results and parameters it was concluded that, this newly
developed method for the simultaneous estimation of Ceftazidime & Avibactam was found to
be simple, precise, accurate and high resolution and shorter retention time makes this method
more acceptable and cost effective and it can be effectively applied for routine analysis in
research institutions, quality control department in meant in industries, approved testing
laboratories studies in near future.
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1698
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
BIBLIOGRAPHY
1. Skoog DA, West DM, Holler FJ. Introduction of analytical chemistry. Sounder college of
publishing, Harcourt Brace college publishers, 1996; 1-5.
2. Sharma B K. Instrumental method of chemical analysis Meerut, 1999; 175-203.
3. Breaux J and Jones K: Understanding and implementing efficient analytical method
development and validation. Journal of Pharmaceutical Technology, 2003; 5: 110-114.
4. Willard H, et al. Instrumental methods of analysis” CBS publisher and, 7.
5. Patel A, Pluim T, Helms A, Bauer A, Tuttle RM, Francis GL: Enzyme expression profiles
suggest the novel tumor-activated fluoropyrimidine carbamate capecitabine (Xeloda)
might be effective against papillary thyroid cancers of children and young adults. Cancer
Chemother Pharmacol, 2004; 53(5): 409-14. [PubMed:15132128 ]
6. Eliason JF, Megyeri A: Potential for predicting toxicity and response of
fluoropyrimidines in patients. Curr Drug Targets, 2004; 5(4): 383-8. [Pub Med:
15134221 ]
7. Carlini LE, Meropol NJ, Bever J, Andria ML, Hill T, Gold P, Rogatko A, Wang H,
Blanchard RL: UGT1A7 and UGT1A9 polymorphisms predict response and toxicity in
colorectal cancer patients treated with capecitabine/irinotecan. Clin Cancer Res, 2005; 1,
11(3): 1226-36. [PubMed:15709193 ]
8. British national formulary: BNF 69 (69 ed.). British Medical Association, 2015; 585:
588. ISBN 9780857111562.
9. "Capecitabine". The American Society of Health-System Pharmacists. Retrieved
December, 2016; 8.
10. Caudle, KE; Thorn, CF; Klein, TE; Swen, JJ; McLeod, HL; Diasio, RB; Schwab, M
(December). "Clinical Pharmacogenetics Implementation Consortium guidelines for
dihydropyrimidine dehydrogenase genotype and fluoropyrimidine dosing.". Clinical
pharmacology and therapeutics, 2013; 94 (6): 640–5. PMC 3831181 . PMID 23988873.
doi:10.1038/clpt.2013.172.
11. Fischer, Janos; Ganellin, C. Robin Analogue-based Drug Discovery. John Wiley & Sons,
2006; 511. ISBN 9783527607495.
12. "Gemcitabine International Brands". Drugs.com. Retrieved, 2017; 6.
13. "Gemcitabine Hydrochloride". The American Society of Health-System Pharmacists.
Retrieved 8 December, 2016.
14. National Cancer Institute. "FDA Approval for Gemcitabine Hydrochloride". National
Cancer Institute. Retrieved, 2017; 22.
www.wjpps.com │ Vol 10, Issue 11, 2021. │ ISO 9001:2015 Certified Journal │
1699
Salam et al. World Journal of Pharmacy and Pharmaceutical Sciences
15. Fischer, Janos; Ganellin, C. Robin Analogue-based Drug Discovery. John Wiley & Sons,
2006; 511. ISBN 9783527607495.
16. Myers, Calisha (March). "Gemcitabine from Actavis launched on patent expiry in EU
markets". Fierce Biotech, 2009; 13.
17. "Press release: Hospira launches two-gram vial of gemcitabine hydrochloride for
injection". Hospira via News-Medical. Net, 2010; 16.