15
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 Salam 1 * and Sh. Rizwan 2 1 Student, Osmania University, Telangana. 2 Student 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

STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

  • Upload
    others

  • View
    3

  • Download
    0

Embed Size (px)

Citation preview

Page 1: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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

Page 2: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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.

Page 3: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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.

Page 4: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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.

Page 5: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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

Page 6: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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

Page 7: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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.

Page 8: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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

Page 9: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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).

Page 10: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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

Page 11: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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

Page 12: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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

Page 13: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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.

Page 14: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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.

Page 15: STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT …

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.