validated spectrophotometric assay of cefepime...

ISSN: 0973-4945; CODEN ECJHAO

E-Journal of Chemistry

http://www.ejchem.net 2012, 9(4), 2261-2267

Validated Spectrophotometric Assay of

Cefepime Hydrochloride and Cefuroxime

Sodium Using a Tetrazolium Salt

MARWA S. ELAZAZY*

AND ABDALLA SHALABY

Analytical Chemistry Department, Faculty of Pharmacy,

Zagazig University, Zagazig, Egypt

[email protected]

Received 29 August 2011; Accepted 28 October 2011

Abstract: A simple, rapid and sensitive spectrophotometric method for the

determination of micro amounts of cefepime hydrochloride and cefuroxime sodium

is described. The method is based on reduction of 2,3,5-triphenyltetrazolium

chloride (TTC) by the cited drugs in slightly alkaline medium leading to formation

of a highly colored formazan derivative. Different variables affecting the color

development were investigated and optimized. Absorbance measurements were

made at 483 nm. Under the proposed conditions, this method is applicable over

concentration range of 4 – 50 µg ml-1 with molar absorpitivities ranging from 5.208

x 103 – 1.217 x 104 L.mol-1.cm-1 and Sandell’s sensitivities ranging from 1.007 x 10-3

– 2.727 x10-3 µg cm-2. The proposed method was successfully applied for analysis of

the cited drugs in formulations and the recovery percentages ranged from 99.47 to

99.8%. The results obtained demonstrated that the proposed method is equally

accurate, precise and reproducible as the reported methods thus it is recommended

for quality control and routine analysis where time, cost effectiveness and high

specificity of analytical techniques are of great importance.

Keywords: Spectrophotometry, Triphenyltetrazolium Chloride, Pharmaceutical Analysis, Validation.

Introduction

Cephalosporins are penicillinase – resistant antibiotics derived from moulds of

“Cephalosporium” species. They differ in their antimicrobial spectrum, resistance to

β – lactamase and method of administration1. In this work two cephalosporins have been

determined; cefepime HCl and cefuroxime Na. Several procedures were reported in

literature for their determination e.g. spectrophotometry2-5

, fluorometry6,7

, polarography8, IR

spectroscopy9, HPLC

10, potentiometry

11 and FIA

12.

The present work develops a colorimetric method for the determination of the studied

drugs using TTC as an oxidizing agent. The drugs under study were previously determined

using ammonium molybdate13

and 1,10 – phenanthroline – ferric reagent14

as oxidizing

agents depending on their reducing properties – the author’s previous work.

The purpose of this study was to determine the two drugs in their bulk and dosage forms

without prior extraction by simple and selective assays for quality control and routine

analysis purposes using TTC as an oxidizing agent. This paper presents a validated

MARWA S. ELAZAZY et al. 2222

colorimetric method. The validation was performed in respect of the ICH Q2R1

regulations15

for analytical procedures. The following parameters were taken into account:

specificity, linearity, limits of detection and quantification, accuracy, precision.

Experimental

A Shimadzu 260 - UV recording spectrophotometer with 10 mm quartz cell was used for all

absorbance measurements.

Materials and reagents

All chemicals were of analytical grade and doubly distilled water was used throughout all

measurements.Cefepime hydrochloride (Bristol Myers Squibb, Egypt), Cefuroxime

sodium (Glaxo Wellcome Egypt), Triphenyltetrazolium chloride (TTC) (Aldrich,

Germany), 0.5% w/v aqueous solutions, prepared and stored in dark closed bottles,

Sodium hydroxide (El-Nasr Chemical Co., Egypt); 0.2 N ethanolic solution. Standard

solutions of antibiotics were freshly prepared by dissolving the appropriate amounts of

each in methanol to form 1 mg mL-1

solutions. Working solutions of 20 µg mL-1

were also

prepared in methanol.

Procedure

For pure pharmaceuticals

Accurately measured aliquots of the standard drug solutions ranging from 40 - 500 µg mL-1

were transferred into separate 10 mL volumetric flasks, followed by 0.3 mL aliquots of 0.5%

w/v TTC reagent and 0.3-1 mL of 0.2 N NaOH. The reaction mixture was heated at 75 °C in

water bath for 10 minutes. Volume was made up to the mark with methanol. The absorbance

was measured at the specified wavelength (Table 1) and the concentration was then

determined from calibration graphs or the corresponding regression equations.

For vials

The contents of two vials were mixed well. An amount of powder equivalent to 50 mg of the

active ingredient was transferred into 50 mL volumetric flask and the volume was made up to the

mark with methanol. Twenty ml of this solution were further diluted to 100 mL with methanol.

Drug content in the obtained solution was determined following the general procedures.

For tablets

Ten tablets were accurately weighed and ground into a fine powder. A weight of powder

equivalent to 100 mg of the drug was transferred into 100 mL volumetric flask and extracted

with 3x25 mL portions of methanol. These portions were filtered into 100 mL calibrated

flasks then the volume was made up to the mark with the suitable solvent. The drug content

in the obtained extract was determined following the general procedures.

Results and Discussion

Tetrazolium salts have been used for colorimetric determination of many compounds of

pharmaceutical interest. Examples of these compounds included steroids (based on presence

of a side chain with a reducible α-ketol group)16

, carbidopa, methyldopa and ethamsylate

(based on presence of two phenyl hydroxy groups)17-19

, the reduction of tetrazolium blue is

represented by the following (Scheme 1)20,21

:

Validated Spectrophotometric Assay of Cefepime Hydrochloride 2222

Wavelength, nm

Ab

sorb

ance

Scheme 1. Reduction of tetrazolium blue to formazan red.

The absorption spectra of the reaction product were scanned in the range of 300 – 650 nm

against a reagent blank, characteristic absorption maxima at 483 nm were observed (Figure 1).

The rate of reaction is indicated by the rate of color development, which depends on the rate

of reduction of TTC by the studied drugs. To optimize conditions, we have investigated a

number of parameters such as alkalinity, temperature, time, reagent volume and solvent.

Figure 1. Absorption spectra of the reaction product resulting from reaction of (1) 50 µg/mL

cefepime HCl measured against reagent blank (3) and (2) 20 µg/mL cefuroxime Na

measured against reagent blank (4) with TTC.

To investigate the effect of alkalinity on the absorbance of reaction product (formazan

red), sodium carbonate, sodium bicarbonate, potassium hydroxide and sodium hydroxide

were tested. Sodium hydroxide was the best for maximum color development and stability.

The optimum basic media was achieved using 0.3 – 1 mL of 0.2 N NaOH. It was found that

the oxidation – reduction reaction does not occur in acidic and neutral media.

As an assay solvent, methanol afforded maximum sensitivity. For the other solvents

tested (ethanol, acetone, propanol, water and water – methanol mixture), the color

development does not achieve the same wavelength, color intensity and stability. The color

obtained was found to be stable for at least 30 minutes at room temperature (Table 1).


Table 1. Analytical data for determination of investigated drugs.

Drug max,

nm

Reaction

Time, min

Reaction

temperature, °C

Volume of

TTC, mL

Volume of

NaOH, mL

Stability,

min

Cefepime HCl

Cefuroxime Na

483

483

10

10

75

75

0.3

0.3

1

0.3

30

60

Identification of validation characteristics

Linearity

A linear correlation was found between absorbance and concentration in the range given in

Table 2. The correlation coefficients, intercepts and slopes for the calibration data for the

tested drugs were calculated using least – squares method.

Table 2. Optical characteristics for the reaction of cited drugs with triphenyltetrazolium

chloride (TTC).

*Regression equation :- A = a+ bc a = intercept b = slope c = concentration (µg mL-1) A = absorbance

unit.

Sensitivity

The limit of detection (LOD) and limit of quantization (LOQ) for the proposed methods

were calculated using the following equations15

:

LOD = 3.3 σA / B LOQ = 10 σA / B,

where σA is the standard deviation of replicate determination values under the same

conditions as for the sample analysis in the absence of the analyte and B is the slope of the

calibration line. In accordance with the formula, the LODs were found to be 0.298 and

0.263 μg mL−1

for cefepime HCl and cefuroxime Na, respectively. The LOQs were 0.903

and 0.796 μg mL−1

for cefepime HCl and cefuroxime Na, respectively. These values indicate

high sensitivity of the proposed methods, Table 2.

Quantification, accuracy and precision

The mean Sandell sensitivity (S) and molar absorpitivity (ε) for each drug were calculated,

Table 2.

The accuracy was studied using known amounts of the drugs in the range of 4 - 50 μg/mL,

via the recovery coefficient, which was in the range of 99.43 - 100.42%. The validity of the

proposed methods was assessed by applying both the calibration method and the standard

addition technique, Tables 3,4.

Parameter Cefepime HCl Cefuroxime Na

Beer’s law limits, µg mL-1

10-50 4.0 -30

Molar absorptivity, L moL-1

cm-1

0.521x104 1.217x10

4

Sandell’s sensitivity, µg cm-2

1.007x10-3

2.727x10-3

Regression equation (A)

Slope, b* 0.0106 0.0272

Intercept, a* - 0.0138 2.798x10

-4

Correlation coefficient (r)* 0.99995 0.99995

Limit of detection, µg mL-1

1.097 0.280

Limit of quantification, µg mL-1

3.656 0.934


Table 3. Comparative analytical results of the proposed and the reported methods for the

tested drugs in pure forms:

Statistical parameters**

Cefepime Hydrochloride Cefuroxime Sodium

Proposed

method

Reference

method14

Proposed

method

Reference

method13

*Mean% Recovery *n

*Variance

*S.D.

*S.E.

*R.S.D.

*”t” * f

100.01

7

0.167

0.409

0.155

0.409

1.06(2.228)

2.83 (4.53)

99.81

5

0.059

0.243

0.109

0.243

100.12

8

0.555

0.745

0.263

0.744

0.447(2.201)

3.85 (4.12)

99.98

5

0.144

0.380

0.170

0.380

**n = Number of experiments S.D. = Standard Deviation S.E. = Standard Error R.S.D.= Relative

Standard Deviation t = “t” test of unpaired data F = Variance test.

Table 4. Comparative analytical results of the proposed and the reported methods for tested

drug in some pharmaceutical formulations:

Formulation Statistical

parameters**

Proposed method Reference

method Calibration

method

Standard

addition method

Maxipime® vials

(500 mg cefepime

HCl/ vial, Bristol

Myers Squibb,

Egypt)

Mean % recovery

n

Variance

S.D.

S.E.

R.S.D.

“t”

F

100.33

6

0.264

0.514

0.210

0.512

1.94 (2.228)

1.74 (4.53)

100.31

7

0.602

0.776

0.293

0.774

1.497 (2.228)

3.96 (4.53)

99.8 14

5

0.152

0.390

0.174

0.391

Zinnat®

tablets

(250 mg

cefuroxime Na/

tablet, Glaxo

Wellcome Egypt)

Mean %recovery

n

Variance

S.D.

S.E.

R.S.D.

“t”

F

99.43

6

0.543

0.737

0.301

0.741

0.097 (2.262)

1.368 (5.19)

99.73

6

1.309

1.144

0.467

1.147

0.477 (2.262)

0.912 (5.19)

99.47 13

5

0.397

0.630

0.282

0.633

Zinnat® vials (250

mg cefuroxime

Na/ vial, Glaxo

Wellcome Egypt)

Mean % recovery

n

Variance

S.D.

S.E.

R.S.D.

“t”

F

99.95

6

0.186

0.431

0.176

0.431

0.517 (2.262)

1.430 (5.19)

100.42

6

0.236

0.486

0.198

0.484

2.038 (2.262)

1.127 (5.19)

99.8 13

5

0.266

0.516

0.231

0.517

**n = Number of experiments S.D. = Standard Deviation S.E. = Standard Error R.S.D.= Relative

Standard Deviation t = “t” test of unpaired data F = Variance test.


Reproducibility

The reproducibility of the proposed methods was determined by replicate analysis of five

separate solutions of the working standards. The methods gave satisfactory results; the

relative standard deviations were 4.33 and 3.83% for cefepime HCl and cefuroxime Na,

respectively, Table 5, indicating good reproducibility of the proposed methods. This

precision level is adequate for the precision and routine analysis of the cited drugs in quality

control laboratories.

Table 5. Replicate analysis of the examined drug solutions by the proposed method.

Sample Number

Absorbance

Cefepime HCl,

50 μg mL−1

Cefuroxime Na,

20 μg mL−1

1 0.210 0.583

2 0.211 0.543

3 0.198 0.576

4 0.200 0.550

5 0.220 0.594

Mean 0.208 0.569

SD 0.009 0.0218

RSD 4.33 3.83 **S.D. = Standard Deviation R.S.D.= Relative Standard Deviation.

Robustness

Robustness was assessed as the method was developed. Reliable results can be obtained with

small variations in the methodology by evaluating the influence of small change in the

experimental parameters on the analytical performance of the proposed methods22

. As a

general rule, one parameter was changed whereas the others were kept unchanged, and the

recovery percentage was calculated each time. Variations in the quantities of reagents added

over the range 6% had no effect on optical density, which indicates the robustness of the

proposed methods.

Conclusion

The present method besides its sensitivity, selectivity and simplicity, offers many

advantages such as:

a. No prior extraction or separation of the studied drugs is necessary,

b. The precision, accuracy and reproducibility of the results are comparable to those of the

official and reference methods

These advantages render the proposed method eminently suited to routine determination

of the studied drugs in various pharmaceutical formulations with low cost depending on

availability of chemicals and equipments.

References

1. Martindale, The Complete Drug Reference, 32nd

Ed., 1999.

2. Morelli B, J Pharm Biomed Anal., 1988, 6, 199 - 209.

3. Morelli B, Anal., 1988, 113, 1077 - 1082.

4. Rodenas V, Parra A, Garcia – Villanova J and Gomez M D, J Pharm Biomed Anal.,

1995, 13 (9), 1095 –1099.

5. Amin A S, Khalil H M and Saleh H M, Sci Pharm., 2001, 69, 143-150.


6. Murillo J A, Lemus J M and Garcia L F, J Pharm Biomed Anal., 1994, 12(7), 875 - 881.

7. Omar M A, Abdelmageed O H and Attiaa T Z, Talanta., 2009, 77, 1394-1404.

8. Palucios J J, Mochon C M, Sanches J C and Carranza H J, Electroanalysis (NY).,

2000, 12, 296 - 300.

9. Bugay D E, Newman A W and Findlay W P, J Pharm Biomed Anal., 1996, 15, 49-61.

10. El - Khalili H, Linger L, Monteil H and Jehl F, J Chromatogr B Biomed Sci Appl.,

1997, 690, 181 - 188.

11. Evagelou V, Tsantili-Kakoulidou A and Koupparis M, J Pharm Biomed Anal., 2003,

31, 1119 - 1128.

12. Can N, Altiokka G and Aboul-Enein H Y, Anal Chim Acta., 2006, 576, 246-252.

13. Elazazy M S, Shalaby A, Elbolkiny M N and Khalil H M, Sci Pharm., 2003, 71,

211- 228.

14. Elazazy M S, Shalaby A, Elbolkiny M N and Khalil H M, Sci Pharm., 2004, 72, 73-86.

15. Validation on Analytical Procedures: Text and Methodology – ICH Q2 (R1) –

International Conference on Harmonisation, Geneva, Switzerland, 2005.

16. Short P M and Rhodes C T, Steroids, 1970, 16, 217 - 224.

17. Issopoulos P B, Pharm Weekbl Sci., 1989, 11, 213 - 217.

18. Issopoulos P B, J Pharm Pharmacol., 1992, 4, 1020 - 1022.

19. Tarkhanova O A and Vasyuk S A, Pharm Chem J., 2010, 44, 52-56.

20. Amin A S, Mikrochim Acta., 1997, 126, 105 - 108.

21. Amin A S, Euro J Pharm Biopharm., 2001, 51, 267-272.

22. Heyden Y V, Nijhuis A, Smeyers-Verbeke J and Vandeginste BGM and Massart D L,

J Pharm Biomed Anal., 2004, 24, 723-753.

http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=27983&_origin=article&_zone=art_page&_linkType=scopusAuthorDocuments&_targetURL=http%3A%2F%2Fwww.scopus.com%2Fscopus%2Finward%2Fauthor.url%3FpartnerID%3D10%26rel%3D3.0.0%26sortField%3Dcited%26sortOrder%3Dasc%26author%3DOmar,%2520Mahmoud%2520A.%26authorID%3D15733097700%26md5%3D5cd83555760dd34e864c92dc42f6060b&_acct=C000009958&_version=1&_userid=8194203&md5=be42c92f741e6d8a535244e918245809

http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=27983&_origin=article&_zone=art_page&_linkType=scopusAuthorDocuments&_targetURL=http%3A%2F%2Fwww.scopus.com%2Fscopus%2Finward%2Fauthor.url%3FpartnerID%3D10%26rel%3D3.0.0%26sortField%3Dcited%26sortOrder%3Dasc%26author%3DAbdelmageed,%2520Osama%2520H.%26authorID%3D6507813077%26md5%3D9b3d4e0483df865c4313b343cfd9192e&_acct=C000009958&_version=1&_userid=8194203&md5=3011d4a0a5dc601a4ad07c83ee394dc5

http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=27983&_origin=article&_zone=art_page&_linkType=scopusAuthorDocuments&_targetURL=http%3A%2F%2Fwww.scopus.com%2Fscopus%2Finward%2Fauthor.url%3FpartnerID%3D10%26rel%3D3.0.0%26sortField%3Dcited%26sortOrder%3Dasc%26author%3DAttia,%2520Tamer%2520Z.%26authorID%3D25635138800%26md5%3Da00c85f867fdf39e3ec3f85777b2e02f&_acct=C000009958&_version=1&_userid=8194203&md5=bd4565251c3decf1bb45426554742235

http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=27983&_origin=article&_zone=art_page&_linkType=scopusAuthorDocuments&_targetURL=http%3A%2F%2Fwww.scopus.com%2Fscopus%2Finward%2Fauthor.url%3FpartnerID%3D10%26rel%3D3.0.0%26sortField%3Dcited%26sortOrder%3Dasc%26author%3DAttia,%2520Tamer%2520Z.%26authorID%3D25635138800%26md5%3Da00c85f867fdf39e3ec3f85777b2e02f&_acct=C000009958&_version=1&_userid=8194203&md5=bd4565251c3decf1bb45426554742235

http://www.sciencedirect.com/science/journal/0039128X

Submit your manuscripts athttp://www.hindawi.com

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation http://www.hindawi.com Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttp://www.hindawi.com

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International


CatalystsJournal of

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation http://www.hindawi.com Volume 2014