Int. J. Pharm. Sci. Rev. Res., 34(1), September – October 2015; Article No.

03, Pages: 12-16 ISSN 0976 – 044X

Research Article

Investigation of Spectrophotometric for Determination of Cloxacillin Sodium in

Different Brands of Pharmaceutical Preparations
a b,c,* c,d a* a
Sajjad Ali Ilyas , Muhammad Imran , Naresh Kumar , Jasmin Shah , M. Rasul Jan
a
Institute of Chemical Sciences, University of Peshawar, Peshawar, Pakistan.
b
Department of Chemistry, University of Azad Jammu & Kashmir, Muzaffarabad, Pakistan.
c
UCG Thar Project, Islamkot, Sindh, Pakistan.
d
Institute of Chemistry, University of São Paulo, São Paulo, Brazil.
*Corresponding author’s E-mail: imjaral@[Link]

Accepted on: 10-05-2015; Finalized on: 31-08-2015.

ABSTRACT
This article comprises a very effective, sensitive economical and precise spectrophotometric method for the fortitude of cloxacillin
sodium in different brands of pharmaceutical preparations. Studied method is based on complexation of cloxacillin sodium with Cu
ions by heating in water bath. The method has investigated the parameters of wavelength 450nm concentration of Cu ions (1000
ppm) and volume of the Cu ions 20 mL with incubation time of 10 minutes, for 40 ppm of cloxacillin sodium. The limit of detection
was found 0.3099 with quantification limit of 1.033. The standard deviation was found 0.1033, and relative standard deviation of
1.033. The method was applied and found very promising for the determination of cloxacillin sodium in various pharma brands with
labeled claims of the assay. The investigated method is recommended for the quality control analysis of the cloxacillin sodium in
pharma industry.
Keywords: Spectrophotometric method, Cloxacillin sodium, Pharmaceutical Preparations.

INTRODUCTION Chemical formula: C19H17ClN3NaO5S, H2O.4,5

C
loxacillin is semi-synthetic antibiotic related to Molecular weight: 475.94,5
penicillin,1 and is also isoxazolyl penicillin.2
Characters
Cloxacillin is used in treatment of infections due to
staphylococci resistance to benzyl penicillin. It is A white, hygroscopic, crystalline powder, soluble in
administered by orally or by injection as sodium salt.2 methanol, alcohol and freely soluble in water. In the
present study an attempt was made to develop an
Cloxacillin is a bactericidal with mode of action similar to
innovative, less expensive, a simple and more authentic
benzyl penicillin. It’s active against pancillinase- producing
method for the determination of Cloxacilin Sodium in
and non-pencillinase-producing staphylococci. It’s activity
pure state and its pharmaceutical preparations.5
against streptococci such as Streptococcus pneumonia
and str. Pyogenes is less than that of benzylpenicilline, Spectrophotometric determination method
but sufficient to be useful when these organisms are development strategy for Cloxacilin sodium
present with penicillin resistant staphylococci. Cloxacillin
The presence of carboxylic acid functional group in the
is virtually ineffective against Enterococcus faecalis
2 formula of drug suggests the possibility of complexation
Resistance.
with transition metal offering a possibility for its
Cloxacillin is incompletely absorbed from the spectrophotometric determination. Therefore in the
gastrointestinal tract, after an oral dose of 500mg, peak proposed method Cloxacilin sodium was tried for its
plasma concentration of 7-15 µg/mL in fasting subjects in complex formation with various transition metals such as
1-2 hours. Absorption is more complete when given Cr, Ni, V, Cu to give a colored product. The colored
intramuscular injection and peak plasma concentration of product thus formed was used for the determination of
about 15 µg/mL is observed in 30 minutes after a dose of Cloxacilin sodium.
500mg. drug has been reported to have plasma half life of
MATERIALS AND METHODS
0.5-1 Hour.2
Preliminary Study of the chelation potential for
The chemical structure of Cloxacilin Sodium is shown in
3-5 spectrophotometric determination of Cloxacilin sodium
Figure 1.
Preliminary determinations were conducted to explore
the possibility of the formation of expected colored
complex between metal ion and Cloxacilin sodium.
Initially higher concentration of Cloxacilin sodium
(1000ppm) was treated with different metal ion solution
Figure 1: The structural formula of cloxacillin sodium along with heating to check the formation of expected

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Int. J. Pharm. Sci. Rev. Res., 34(1), September – October 2015; Article No. 03, Pages: 12-16 ISSN 0976 – 044X

colored product. The colored product indicates the Investigation of the effect of metal ion solution and
chances of reaction and subsequent formation of incubation time on the formation and absorbance
Cloxacilin sodium complex by this method. Further behavior of complex.
studies were focused on the optimization of various
Instrument: The same as mention before
parameters for maximum complexation and are discussed
below. Reagent: The same as mention before
Investigation of appropriate wavelength for the Solution: The same as mention before
estimation of Cloxacilin metal complex
Procedure
Instruments
Standard Cloxacilin sodium solutions 2 mL from (1000
Digital analytical balance, thermostatic water bath and ppm) stock solutions were transferred to six separate 50
UV/VIS spectrophotometer were used during this mL volumetric flasks and to each of these flasks, varied
investigation. volumes of metal ion solution from (1000 ppm) stock
solution was added along with little dilution with distilled
Reagents
water and was incubated in water bath for 20 minute.
Analytical reagent grade copper nitrate trihydrate and The resulting colored complex was allowed to cool and
Cloxacilin sodium were used during this work. diluted to 50 mL with distilled water. Blank was prepared
in the same manner without the addition of Cloxacilin
Solution Preparation
sodium. The absorbance of the resulting complex was
Metal Ion Solution measured at 450 nm using Genesys 5 spectrophotometer.
The results are given in Table 2 and are shown in Figure 3.
Metal Ion solution (1000ppm) was prepared by dissolving
For studying the effect of incubation time Standard
0.380g of Cu(NO3)2.3H2O in distilled water and diluted to
Cloxacilin sodium solution 2 mL from 1000 ppm stock
100 mL with distilled water.
solution was transferred to six separate 50 mL volumetric
Standard Cloxacilin Sodium solution flasks. To each of these flask 20 mL of metal ion solution
form stock solution and little volume of water was added
Cloxacilin sodium (1000ppm) stock solution was prepared
followed by incubation in boiling water bath for varied
by dissolving 0.1 g of authentic standard Cloxacilin
times, in a range of 0-25 minutes. The resulting yellow
sodium in distilled water and diluted upto 100 mL with
colored complex was cooled and diluted to 50 mL with
distilled water.
distilled water. Blank was prepared in the same manner
Procedure without the addition of Cloxacilin sodium. The
absorbance of the resulting complex was measured at
Cloxacilin sodium 2 mL from (1000 pm) stock solution was
450 nm using Genesys 5 spectrophotometer. The results
slowly transferred to 50 mL volumetric flask. To this 10
are given in Table 3 and are shown in Figure-4.
mL of metal ion solution from (1000 ppm) stock solution
of metal ion was added with little dilution with water
followed by incubation in boiling water bath for 20
minute. The contents were allowed to cool to room
temperature in tape water tub, and diluted to 50 mL with
distilled water. Blank solution was prepared by the same
procedure without addition of Cloxacilin sodium. The
absorbance of yellow colored complex was measured
from 400-570 nm, using Genesys 5 spectrophotometer.
Each time after changing wavelength the instrument
calibrated with blank solution.
Figure 3: Optimization of the incubation time for the
The results are given in Table 1 and are shown in Figure 2. formation of complex

Figure 2: Wavelength optimization for Figure 4: Optimization of volume of metal ion solution for
spectrophotometric determination of cloxacillin sodium formation of complex

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Int. J. Pharm. Sci. Rev. Res., 34(1), September – October 2015; Article No. 03, Pages: 12-16 ISSN 0976 – 044X

The effect of concentration on the absorbance behavior Sample preparation

of Cloxacillin Cu complex
Procedure
Instruments: The same as mentioned before
Weigh 20 capsules and take contents of powder
Reagents: The same as mentioned before equivalent to 100 mg cloxacillin in 100 mL volumetric
flask. Dissolved and made the volume with distilled
Solutions: The same as mentioned before
water, shaked well sonicated for 5 minutes. Filtered the
Procedure solution using filter paper # 1, Pipette out 3 mL of the
filtrate into 50 mL volumetric flask, to this 20 mL of (1000
Varied amount of standard Cloxacilin sodium solution
ppm) Cu++ ion solution was added along with little volume
with final concentration after dilution ranging from 2-120
of water followed by incubation in water bath for 10
ppm taken in fifteen separate 50 mL volumetric flasks. To
minutes, allow it to cool and diluted upto mark by
each of these flask 20mL of metal ion solution from (1000
distilled water and measure the absorbance at 450 nm. In
ppm) stock solution and little volume of distilled water
cause of injection proceed same as for standard
was added followed by the incubation in boiling water
preparation.
bath for 10 minutes. The resulting colored complex was
cooled and diluted to 50 mL with distilled water. Blank Calculations
solution was prepared in same manner without the Mg/caps/injection of cloxacillin
addition of Cloxacilin sodium. The absorbance of the Au x wt of std x 3 x100 x 50 x 5 x potency of standard x average weight
=
resulting colored complex was measured at 450 nm using As x 100 x 50 x Wt of sample x 3 x 100
Genesys 5 spectrophotometer to find out absorbance Where Au= Absorbance of Sample
behavior. The results are given in Table 4 and are shown
in Figure 5. As= Absorbance of Standard
Percentage Label Claim = (mg per caps/inj x 100) / (L.C mg
per caps/inj)
Determination of cloxacillin sodium by official method
(HPLC method)
Buffer
Prepared a 0.2 M solution of monobasic potassium
phosphate in water, and adjusted with 2N NaOH to pH of
6.8.
Mobile phase
Figure 5: The effect of concentration on the absorbance
Prepared a mixture of buffer and acetonitrile (80:20).
behavior for cloxacillin sodium at lower concentration
using spectrophotometric method Standard preparation
Analysis of Cloxacilin sodium in various pharmaceutical Prepared a solution of USP cloxacillin sodium in buffer
preparations using investigated method and its having a concentration of 0.55 µg/mL.
comparison with official method
Sample Preparation
Instruments: The same as mentioned before
Transferred 110 mg of cloxacillin sodium in 200 mL
Reagents: The same as mentioned before volumetric flask diluted with buffer to volume, and mixed
using magnetic stirrer for five minutes to dissolve.
Solutions: The same as mentioned before
Chromatograph parameters
Procedure
Wavelength : 225 nm
Accurately weigh 100 mg of reference standard cloxacillin
sodium in 100 mL volumetric flask. Dissolved it in distilled Flow rate : 1 mL/min
water by continues shaking and made up to mark. Pipette
Column : 4.6 mm x 25 cm (Contains
out 3 mL of the solution into 50 mL volumetric flask, to
packing L1)
this 20 mL of (1000 ppm) Cu++ ion solution was added
after little dilution and incubation was carried out in Procedure
water bath for 10 minutes. The solution was allowed
Separately injected equal volume (20 microlitres) of
cooling in tape water tub and diluted upto mark with
standard and sample into the 200(CE / W) (ru / rs)
distilled water. Blank solution was prepared in the same
manner without the addition of Cloxacilin sodium. The C = concentration in mg/ mL
absorbance of the resulting colored complex was
E = cloxacillin equivalent, in microgram per mL
measured at 450 nm using Genesys 5 spectrophotometer.

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W = weight in mg Limits of quantifications (for concentration) = 10 x S

ru and rs = cloxacillin peak response obtained from assay Standard deviation. S /( − 1)
and standard preparation respectively.
Relative standard deviation. R.S.D= S/X- x 100
RESULTS AND DISCUSSION
Where as
The proposed method involves the complexation of the
cloxacillin sodium with Cu++ ions leading to a yellow X = Concentration in (ppm) found.
colored complex. X- = Average founded concentration (ppm) of six samples.
Various parameters like wavelength, volume of Cu++ ion n= X- X
-

solution, incubation time were optimized for the

formation of utmost colored complex. Applications of investigated method for the
determination of cloxacillin sodium in different
After the preliminary experiment the complex formed, pharmaceutical preparations and comparison with
was investigated for the optimal wavelength. The results official method
are given in Table 1 and are shown in Figure 2 as can be
seen from Table 1 that resulting complex has a maximum The investigated method was applied for determination
absorbance at 450 nm, and was used as optimum of cloxacillin sodium in different formulations of capsules
wavelength for further investigation of Cloxacillin sodium and injections. The method used for determination and
determination. calculations has been discussed above and was compared
++
with official method, while the results of comparison
Cu solution (1000 ppm) was used for the complexation. have been shown in Table 7.
Various volumes of (1000 ppm) Cu++ ion solution in the
range of 5 to 30 mL were tried with 40 ppm of cloxacillin Table 1: Wavelength optimization for spectrophotometric
sodium. The results are given in Table 2 and are shown in determination of cloxacillin sodium
Figure 3. As can be seen from the Table 2 that 20 mL of Wavelength Absorbance Wavelength Absorbance
Cu++ ion (1000 ppm) solution was found to be optimum (nm) (nm)
400 0.111 490 0.061
volume for the formation of maximum complex with 40
ppm of cloxacillin sodium. 410 0.122 500 0.042
420 0.133 510 0.030
It was observed that incomplete complexation could be
achieved at room temperature and it was necessary to 430 0.140 520 0.020
heat the solutions in hot water bath. Therefore 440 0.146 530 0.016
incubation time in range of 0-25 minute was investigated 450 0.147 540 0.014
for maximum complexation in boiling water bath and the
460 0.136 550 0.013
results are given in Table 3 and are shown in Figure 4. It
470 0.118 560 0.012
was found that 10 minute incubation time in boiling
water bath was the optimum incubation time for 480 0.085 570 0.010
maximum complexation.
Table 2: Optimization of the incubation time for the
Effect of concentration at lower level on the absorbance formation of complex
behavior of cloxacillin Cu++ was investigated to calculate
Time in 0.00 5.0 10.00 15.00 20.00 25.00
the limit of detection (LOD) and limit of quantification
minutes
(LOQ) at optimum conditions. Cloxacillin sodium 2 ppm Absorbance 0.004 0.101 0.179 0.172 0.148 0.139
was selected for investigation of detection limit as this
Table 3: Optimization of volume of metal ion solution for
was the minimum concentration for which the
formation of complex
absorbance could be noted. Six replicate readings were
taken for this concentration. The results are given in Volume
Table 6. The following formulas were used for calculation used in mL 05 10 15 20 25 30
of LOD, LOQ, S.D and R.S.D. (1000 ppm)
Absorbance 0.178 0.172 0.197 0.218 0.211 0.200
Limits of detection (for concentration) = 3 x S
Table 4: The effect of concentration on the absorbance behavior for cloxacillin sodium at lower concentration using
spectrophotometric method
Cloxacillin sodium Conc. (ppm) 2 4 8 12 16 20 24 28

Absorbance 0.012 0.017 0.034 0.045 0.070 0.089 0.098 0.115

Cloxacillin sodium Conc. (ppm) 32 36 40 60 80 100 120

Absorbance 0.140 0.164 0.206 0.270 0.365 0.438 0.532

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Table 5: Replicate readings for 2 ppm concentration of cloxacillin sodium

Absorbance Concentration (ppm) found (X)
0.012 2.2
0.011 2.0
0.012 2.2
0.012 2.2
0.011 2.0
0.012 2.2

Table 6: Results of investigated method

Linear range 1-120µ
λ max 450nm
standard deviation 0.1033
R.S.D 4.849
Correlation coefficient 0.996
-5
Molar absorptivity 5.04ˣ10
3
∑ 1.08ˣ10
L.O.D 0.3099
L.O.Q 1.033

Table 7: Application of investigated method for the analysis of cloxacillin sodium in various pharmaceutical preparations
and comparison with official method

Name of Drug Label Claim Develop method Official method

Auropen capsules 250 mg/capsule 251.31±1.13 mg/ capsule 251.83±1.41 mg/ capsule
Auropen inj 250 mg/vial 2.49.8±0.59 mg/vial 250.4±0.87 mg/vial
Cloxazan Capsules 250 mg/capsule 253.2±1.64 mg/ capsule 252.9±0.92 mg/ capsule

CONCLUSION good conformity with labeled claim in pharmaceutical

preparations.
Spectrophotometric method for the determination of
cloxacillin sodium involves complexation of cloxacillin Acknowledgement: The authors gratefully acknowledge
sodium with Cu++ ions followed by heating in water bath. the financial support from the Institute of Chemical
Various analytical parameters like wavelength, Sciences, University of Peshawar for the research project.
concentration and volume of metal ion solution and
REFERENCES
incubation time in water bath were optimized for
spectrophotometric determination of cloxacillin sodium 1. “The Merck Index. An encyclopedia of chemicals, Drugs and
th
and were found to be 459nm, 20 ml (1000 ppm), and 10 biological” 15 ed. White house station: Merck and Co, Inc,
minutes respectively for 40 ppm of cloxacillin sodium. The NJ, U.S.A. 432, 2013.
limit of quantification and limit of detection for the 2. S. C. Sweetman. “Martindail the complete drug reference”
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investigated method were calculated using authentic .34 ed. London, Chicago (PhP). 198, 213, 2005.
reference standard and were found to be 1.033 and 3. USP 37 NF32. “The united state pharmacopeia The national
0.3099 respectively. The relative standard deviation and formulary” volume 3. The united state pharmacopeial
standard deviation were established to be 1.033 and convention 12609 twin brook parkway, Rockville, MD
0.1033 respectively. 20852. 2438, 2014.
The method was found linear in range of 1-120 ppm. The 4. “British pharmacopoeia” Volume I. The stationery office
developed method was effectively useful for London. I-594- I-595, 2014.
determination of cloxacillin sodium in various 5.
th
European pharmacopoeia” 7 ed. Volume 2. Council of
pharmaceutical formulations and method was found in Europe 67075 Strasbourg Cedex, France. 1735-1736, 2010.

Source of Support: Nil, Conflict of Interest: None.

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