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A New Spectrophotometric Method for the
Simultaneous Multicomponent Analysis
Resolution of Cefixime and Dicloxacillin
Sodium in Their Tablet Dosage Form
Heena J. Patel*1, Bharat G. Chaudhari1
Department of Quality Assurance, S. K. Patel College of Pharmaceutical Education and Research, Ganpat
1
University, Ganpat Vidyanagar, Mehsana, Gujarat, India
*1 heena2206@yahoo.com; 1bharat.chaudhari@ganpatuniversity.ac.in
Abstract
The present manuscript describes simple, sensitive, rapid, accurate, precise and economical ratio spectra derivative method for
the simultaneous determination of Cefixime and Dicloxacillin sodium in tablet dosage form. The method is based on the use of
the first derivative of the ratio‐spectra obtained by dividing the absorption spectrum of binary mixtures by a standard spectrum
of one of the compounds. The cefixime was estimated directly at the 289 nm (λ‐max of cefixime) without any interference of
dicloxacillin sodium. The first derivative amplitudes at 227.2 nm was selected for estimation of dicloxacillin sodium. The
wavelength interval (Dl) was selected as 8 nm. Methanol was used as the solvent. Both the drugs showed linearity in the range
of 3‐16 μg/ml. The method was successfully applied for the determination of these two drugs in tablet dosage form. No
interference was observed from excipients present in the tablet. The suitability of this method for the quantitative determination
of Cefixime and Dicloxacillin sodium was proved by validation. The proposed method was found to be simple and sensitive for
the routine analysis of these two drugs in tablet dosage form. The results of analysis have been validated statistically by
recovery studies.
Keywords
Cefixime; Dicloxacillin Sodium; Spectrophotometric Method; Ratio Spectra Derivative Method; Validation
Introduction
Cefixime (CEF) is chemically (6R,7R)‐7‐[(2Z)‐2‐(2‐amino‐1,3‐thiazol‐4‐yl)‐2[(carboxymethoxy) imino]acetamido]‐3‐
ethenyl‐8‐oxo‐5‐thia‐1‐azabicyclo‐oct‐2‐ene‐2 carboxylic acid [1] (Figure‐1). Cefixime (CEF), an antibiotic, is a third
generation cephalosporin. The antibacterial effect of cefixime results from inhibition of mucopeptide synthesis in
the bacterial cell wall [2]. Cefixime (CEF) is official in Indian Pharmacopeia (IP) [3], British Pharmacopeia (BP) [4], and
United States Pharmacopeia (USP) [5]. These three pharmacopeias describe liquid chromatography method for its
estimation. Literature survey reveals UV spectrophotometry [6‐9], HPLC [10‐13] methods for determination of CEF
alone. Literature survey also reveals UV [14‐25], HPLC [26‐40] methods for the determination of CEF with other drugs
combination. Dicloxacillin sodium (DIC) is chemically (2S,5R,6R)‐6‐[3‐(2,6‐dichlorophenyl)‐5‐methyl‐1,2‐oxazole‐4‐
amido]‐3,3‐dimethyl‐7‐oxo‐4‐thia‐1‐azabicycloheptane‐2‐carboxylic acid [41] (Figure 2). Dicloxacillin sodium (DIC)
is an anti‐bacterial agent. It is official in Indian Pharmacopoeia (IP) [42], British Pharmacopoeia (BP) [43], and United
States Pharmacopeia (USP) [44]. IP, BP, USP describe HPLC method for its estimation. Literature survey reveals
HPLC [45‐46] methods for estimation of DIC alone. Literature survey also reveals UV [47‐49], HPLC [50‐56] methods for
determination of DIC with other drugs in combination. The combination of these two drugs is not official in any
pharmacopoeia; hence no official method is available for the simultaneous estimation of CEF and DIC in their
combined synthetic mixture or dosage forms. Literature survey reveals only HPLC [57‐60] and HPTLC [61] methods
for CEF and DIC in combined dosage forms. The present manuscript describes simple, sensitive, rapid, accurate,
precise and cost effective spectrophotometric method based on zero order and ratio spectra first derivative for
simultaneous estimation of both drugs in tablet dosage form.
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FIG. 1 CHEMICAL STRUCTURE OF CEFIXIME FIG. 2 CHEMICAL STRUCTURE OF DICLOXACILLIN
1. Apparatus
A shimadzu model 1700 (Japan) double beam UV/Visible spectrophotometer with spectral width of 2 nm,
wavelength accuracy of 0.5 nm and a pair of 10 mm matched quartz cell was used to measure absorbance of all the
solutions. Spectra were automatically obtained by UV‐Probe 2.31 system software. A Sartorius CP224S analytical
balance (Gottingen, Germany), an ultrasonic bath (Frontline FS 4, Mumbai, India) was used in the study.
2. Reagents and Materials
Cefixime (CEF) and Dicloxacillin sodium (DIC) were kindly supplied as a gift samples from Brussels Laboratories
Pvt. Ltd, Changodar, Ahmedabad, Gujarat, AR grade Methanol (S.D. Fine Chemical Ltd., Mumbai, India.) as a
solvent and Whatman filter paper no. 41 (Millipore, USA) were used in the study.
3. Preparation of Standard Stock Solution
An accurately weighed CEF and DIC powder (10 mg) were transferred to 100 ml separate volumetric flasks and
dissolved in methanol. The flasks were shaken and volumes were made up to mark with methanol to give a
solution having concentration 100 μg/ml for both of drugs.
III. Methodology
The proposed method, works on two mechanisms viz. (1) Zero order and (2) Ratio Spectra Derivatization. In this
method, CEF is estimated directly at the 289 nm (λ‐max of CEF) from zero order spectra of mixture without any
interference of DIC (Fig. 3). For the estimation of DIC, the mixture spectra are divided with the standard spectra of
CEF (divisor) and first derivative spectra of these ratio spectra are generated. The main advantage of the ratio
spectra derivative spectrophotometry is the chance of doing easy measurements in correspondence of peaks so it
permits the use of the wavelength of highest value of analytical signals (a maximum or a minimum). Moreover, the
presence of a lot of maxima and minima is another advantage by the fact that these wavelengths give an
opportunity for the determination of active compounds in the presence of other compounds and excipients which
possibly interferes the assay. The spectra of DIC at increasing concentrations in methanol were divided by
previously stored absorption spectrum standard solution of CEF (3 μg/ml) to obtain the corresponding ratio
spectra. Then the first derivatives of the obtained ratio spectra were traced with interval of 8 nm. In the binary
mixtures, content of DIC was determined by measuring the first derivative amplitude at 227.2 nm, where therewas
no contribution or interference from CEF. First‐derivative technique (D1) traced with Δλ=8 nm was used to resolve
the spectral overlapping. The calibration curves were checked for linearity and linear behavior was observed in the
concentration range of 3‐16 μg/ml, for both drugs.
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FIG. 3 OVERLAIN ABSORPTION SPECTRA OF CEF AND DIC SHOWING Λ‐MAX OF CEFIXIME AT 289 NM IN METHANOL
FIG.4 OVERLAIN OF RATIO SPECTRA OF SOLUTION OF DIC (3, 4, 6, 8, 10, 12, 14, 16 ΜG/ML) WHERE 3 ΜG/ML SOLUTION OF CEF WAS
USED AS DIVISOR AT ΔΛ=8 NM
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FIG. 5 OVERLAIN FIRST DERIVATIVE OF RATIO SPECTRA FORSOLUTION OF DIC (3, 4, 6, 8, 10, 12, 14, 16 ΜG/ML ) AT 227.2 NM
1. Linearity (Calibration Curve)
The calibration curves were plotted over a concentration range of 3 to 16 μg/ml for both CEF and DIC. Accurately
measured standard solutions of CEF (0.3, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 ml) and DIC (0.3, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6
ml) were transferred to a series of 10 ml of volumetric flasks and diluted to the mark with methanol. The
absorbances of the standard solutions were measured at 289 nm against methanol as blank. All the spectra of DIC
divided by spectra of CEF (3 μg/ml) to obtain corresponding ratio spectra. Then the first derivatives of the obtained
ratio spectra were traced with interval of 8 nm and DIC determined by measuring the first derivative amplitude at
227.2 nm. The calibration curves were constructed by plotting absorbance versus concentrations and the regression
equations were calculated.
2. Method Precision (Repeatability)
The precision of the instrument was checked by repeated scanning and measuring the absorbance of solutions (n =
6) of CEF and DIC (10 μg/ml for both drugs) without changing the parameters of the proposed Method. The results
were reported in terms of relative standard deviation (% RSD).
3. Intermediate Precision (Reproducibility)
The intra‐day and inter‐day precision of the proposed method was evaluated by analyzing the corresponding
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responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of
sample solutions of CEF and DIC (8, 10, and 12μg /ml). The results were reported in terms of relative standard
deviation (% RSD).
4. Limit of Detection (LOD) & Limit of Quantification (LOQ)
The limit of detection (LOD) and limit of quantification (LOQ) of the method were calculated by using the
following equations.
LOD = 3.3 X σ/S
LOQ = 10 X σ/S
Where, σ = the standard deviation of the response
S = slope of the calibration curve
5. Accuracy (% Recovery Study)
The accuracy of the method was determined by calculating recoveries of CEF and DIC by the standard addition
method. Known amounts of standard solution of CEF and DIC were added at three levels (50 %, 75 % and 100 %)
to pre‐quantified sample solutions of CEF and DIC.
6. Analysis of Tablet Sample
Twenty tablets were weighed individually and powdered. Labelled claim: 200 mg CEF and 500 mg DIC. Quantity
of the powder equivalent to 10 mg CEF was transferred to 100 ml volumetric flask containing 50 ml methanol, cork
it and sonicated for 20 min. The solution was filtered through Whatman filter paper No. 41 and the volume was
adjusted up to the mark with distilled water. From this solution, 0.6 ml was taken in to a 10 ml volumetric flask
and the volume was adjusted up to mark with methanol to get a concentration of CEF (6.0 μg/ml) and DIC (15.0
μg/ml). The absorbance of sample solution was measured against methanol as blank at 289 nm for quantification of
CEF and for DIC, spectra was divided by CEF (3 μg/ml) and obtained ratio spectra were derivatized, absorbance at
227.2 nm was taken. The amount of CEF and DIC present in the sample solutions was determined by regression
equation.
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The proposed validated method was successfully applied for the simultaneous determination of CEF and DIC in
their tablet dosage form without interference of any excipients. The % assay of CEF and DIC in tablet samples was
calculated compared with label claim and recorded in Table‐3. The proposed method can be applicable for the
routine simultaneous analysis of CEF and DIC in tablet dosage form.
TABLE 1 REGRESSION ANALYSIS DATA AND SUMMARY OF VALIDATION PARAMETERS FOR CEF AND DIC
TABLE 2 RECOVERY DATA OF CEF AND DIC
S.D.Standard deviation and n is number of replicate
TABLE 3 ANALYSIS OF CEF AND DIC IN TABLET DOSAGE FORM
S. D. is standard deviation and n is number of replicate
VI.Conclusion
Based on the results which have been obtained from the analysis using proposed method, it can be concluded that
the method has linear response in the range 3 to 16 μg /ml for both the drugs, CEF and DIC. Moreover, the
proposed method was found to be simple, accurate, precise and sensitive. The result of the analysis of different
marketed tablet dosage form by the proposed method is highly reproducible, reliable, as well as in agreement with
label claim of the drugs. The additive present in the formulation dose not interfere in the analysis. The method can
be used for the routine analysis of both drugs in their combination drug products.
ACKNOWLEDGMENT
The authors are thankful to Brussel Laboratories Pvt. Ltd, Changodar, Ahmedabad, India for providing gift sample
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of CEF and DIC for carry out the research work. The authors are highly thankful to Shree S. K. Patel College of
Pharmaceutical Education and Research, Ganpat University, Ganpat Vidyanagar – 384012, Mehsana, Gujarat, India
for providing all the facilities to carry out the research work.
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