Journal of Al-Nahrain University

Vol.15 (4), December, 2012, pp.22-30

Science

Kinetic Spectrophotometric Methods for the Determination of

Chloramphenicol in Pharmaceutical Preparations
Hind S. Al-Ward
Department of Chemistry, College of Sciences, University of Baghdad, Baghdad-Iraq.
Abstract
Simple and sensitive kinetic methods are described for the determination of chloramphenicol in
pure form and pharmaceutical preparations. The methods are based on oxidative coupling reaction
between reduced chloramphenicol (by zinc powder and concentrated hydrochloric acid) with
promethazine hydrochloride in the presence of sodium periodate yielding a highly colored product
at room temperature, the reaction is followed spectrophotometriclly at max= 590 nm. Initial rate and
fixed time (at 20 minutes) methods are utilized for concentration determination. The calibration
graphs were linear in the concentration ranges (2-20 g.ml-1) and (0.5-30 g.ml-1) respectively. The
results were validated statistically and checked through recovery studies, and have been applied
successfully for the determination of chloramphenicol in commercial dosage forms.
Keywords: chloramphenicol, kinetic spectrophotometry, oxidativecoupling reaction.
idiosyncratic (rare, unpredictable, and
unrelated to dose) and generally fatal. CAP is
a non-irritant and is used by local application
for the treatment of a variety of infections of
the skin, ear and eye including trachoma [3].
Various methods have been reported for
the determination of CAP in pharmaceutical
preparations, including HPLC [4], LC-Mass
spectrometry [5-7], Polarographic
[8],
electrogenerated chemiluminescence [9],
Fluorescent [10], enzymatic method [11],
colorimetric and spectrophotometric methods
[12-19]. The literature is still poor in analytical
procedures based on kinetics, especially for
drugs in pharmaceuticals or biological fluids.
However, some specific advantages in the
application of kinetic methods can be expected
such as, selectivity due to the measurement of
the evolution of the absorbance with the time
of the reaction instead of the measurement of
absorbance value. Potassium permanganate
has been frequently utilized for kinetic
measurements in the field of pharmaceutical
analysis. Many pharmaceutical compounds
have been determined kinetically through this
approach such as tetracycline hydrochloride
[20], cephalosporins [21]. A norfloxacine [22]
was determined by its reaction with
acetaldehyde and 2,3,5,6 tetrachloro 1, 4 benzoquinone to give a colored product.
Ketoprofen [23] was determined kinetically by
oxidative coupling reaction of the drug with

Introduction
Chloramphenicol (CAP) is 2,2 dichloroN-[(1R,2R)-2-hydroxy-1-hydroxymethyl-2-(4nitrophenyl)ethyl]acetamide, C11H12Cl2N2O5 ,
whereas its chemical structure is:

Its molecular weight is 323.1 g mol-1, It is

a white, greyish-white or yellowish-white, fine
crystalline powder or fine crystals, needles or
elongated plates, freely soluble in methanol,
ethanol, butanol, ethyl acetate, acetone, and in
propylene glycol, slightly soluble in water, and
ether, insoluble in benzene, and petroleum
ether, it melts at 150.5151.5C [1].
Chloramphenicol is a bacteriostatic
antimicrobial. It is considered a prototypical
broad-spectrum antibiotic, alongside the
tetracyclines. Chloramphenicol is effective
against a wide variety of Gram-positive and
Gram-negative bacteria, including most
anaerobic organisms. It is widely used because
it is inexpensive and readily available [2]. The
most serious adverse effect associated with
chloramphenicol treatment is bone marrow
toxicity, which may occur in two distinct
forms: bone marrow suppression, which is a
direct toxic effect of the drug and is usually
reversible, and aplastic anemia, which is
22

Hind S. Al-Ward

Sodium periodate solution (3.0910-2M).

Prepared by dissolving 0.6609 gm with
distilled water then completed the volume to
100 ml with the same solvent.

MBTH reagent in the presence of Ce (IV) in

acidic medium. Ramipril has also determined
kinetically based on the reaction of the
carboxylic group of the drug with a mixture of
potassium iodate and potassium iodide and the
reaction was followed spectrophotometrically
[24]. The aim of the present work was to study
the reaction between reduced chloramphenicol
and promethazine hydrochloride in the
presence of sodium periodate; kinetically in an
attempt to evaluate the drug in pharmaceutical
preparations. Initial-rate and fixed-time
methods were adopted after a full
investigation.

Solutions of pharmaceutical preparations.

1-Capsules samples (Aphenicol / 250 mg
ChloramphenicolAjanta Pharma
limited, India):
The contents of ten capsules were weighed
and the powder was mixed. An accurately
weighed portion of the powder equivalent to
50 mg of CAP was dissolved in to 30 ml of
ethanol. The solution was filtered into a 50 ml
volumetric flask, the residue was washed with
ethanol and diluted to volume with the same
solvent to obtain 1000 g ml-1 of CAP. This
solution was transferred into 125 ml beaker
and was reduced as described above.

Experimental
Apparatus
All spectral and absorbance measurements
were carried out on a Shimadzu UVVisble-260 digital double-beam recording
spectrophotometer (Tokyo-Japan), using 1-cm
quartz cells.

2-Eye drops samples -10 ml (0.5%

chloramphenicol/ 0.005% cetrimide-SDI,
Sammara, Iraq):
The contents of three bottles of eye drops
were mixed. An aliquot corresponding to
50 mg of CAP (10 ml) was diluted to 50 ml
with ethanol in a volumetric flask to obtain
1000 g.ml-1 of CAP. This solution was
transferred into 125 ml beaker and was
reduced as described above.

Reagents:
All chemicals used were of analytical
reagent grade. Chloramphenicol standard
material was provided from the state company
for drug industries and medical appliances
(SDI) Sammara-Iraq.
Chloramphenicol (CAP) solution
(500 g ml-1)= 1.547 10-3M [25].
Prepared by dissolving 0.0500 g of CAP in
ethanol transferred into 50 ml volumetric
flask, and diluted to the mark with the
same solvent. The solution was transferred
into a beaker of 125 ml. A 20 ml of distilled
water, 20 ml of concentrated hydrochloric acid
(11.64 N) and 3 g of zinc powder were added.
The beaker was allowed to stand for 15 min at
room temperature, then the solution was
filtered into 100 ml volumetric flask, washed
the residue with distilled water, and diluted to
the mark volume with distilled water to obtain
500 g.ml-1 of CAP reduced solution. More
dilute solutions were prepared daily by
appropriate dilution using distilled water.

3-Ointment samples - 5 gm (Betaphenicol

sterile ophthalmic / 0.5% chloramphenicol
0.2 % betamethasone - Delta for
medicaments, Syria):
The contents of five tubes of ointment
were mixed. An accurately weighed amount of
ointment equivalent to 50 mg of CAP was
extracted three times with 10 ml of ethanol.
The solution was filtered into a 50 ml
volumetric flask, the residue was washed with
ethanol and diluted to volume with the same
solvent to obtain 1000 g ml-1 of CAP. This
solution was transferred into 125 ml beaker
and was reduced as described above.
Results and Discussion
Preliminary investigations
Throughout the preliminary investigations
of oxidative coupling reaction between
reduced CAP with promethazine HCl in the
presence of sodium periodate to give a soluble
purple colour dye that have a maximum
absorbance at 590 nm. The absorbance of the
colored product was measured versus reagent

Promethazine Hydrochloride solution

(3.0910-2M).
Prepared freshly by dissolving 0.9915 gm
of pure promethazine hydrochloride in small
amount of distilled water then completed to
100 ml with the same solvent.
23

Journal of Al-Nahrain University

Vol.15 (4), December, 2012, pp.22-30

blank increases with time and then remains

stable for at least 120 min. This was used as
a basis for a useful kinetic method for
the determination of CAP in pharmaceutical
preparations. Initial studies were directed
towards the optimization of the experimental
conditions in order to establish the optimum
conditions necessary for quantitative formation
of the product with maximum sensitivity.

Science

precipitate the dye formed with decreasing in

absorbance after 10 mins.
Absorption spectra
After obtaining the optimum conditions for
the formation of the product, the absorption
spectra of the product solution versus reagent
blank and reagent blank versus distilled water
were recorded within 300 to 700 nm (Fig.(1)).
The maximum absorption of the product was
found at 590 nm, which was the same as found
in the preliminary investigations, and it was
used in all subsequent experiments.

Optimization of the experimental conditions

The effect of various variables on the color
development was tested to establish the
optimum conditions for determination of CAP.
In subsequent experiments, 500 g ml-1
of CAP was taken to a 25 ml final volume
and the absorbance was measured at room
temperature (25C) for series of solutions by
varying one and fixing the other parameters at
590 nm versus reagent blank after 20 min from
the beginning of the reaction.

1- Effect of volume of Promethazine HCl

(3.09 10-2 M).
The effect of volume of the reagent
solution was investigated by carrying out
the reaction using different volumes of
promethazine HCl ranging from (0.5-2.5 ml).
The maximum absorbance was obtained upon
using 1 ml of (3.09 10-2 M) promethazine
HCl solution.

B
300

Wave length

Fig.(1) The absorbance spectra of (A) the

colored dye against blank and (B) the blank
against distilled water.
Analytical procedure for calibration
In to a series of 25 ml volumetric flask,
transfer increasing volumes of standard stock

2-Effect of volume of Sodium periodate

solution (3.09 10-2 M).
The effect of volume of the oxidant
solution was studied by carrying out the
reaction using different volumes of sodium
periodate solution ranging from (0.5-2.5 ml).
An increase in absorbance was obtained
upon using 1.5 ml of oxidizing solution
(3.0910-2 M).

solution (500 g.ml-1=1.54710-3M) containing

(0.1-1 ml) of reduced CAP to cover the range

of the calibration graph (2-20 g.ml-1) for
the initial-rate method and (0.25-1.5 ml) of
reduced CAP to cover the range of the
calibration graph (0.5-30 g.ml-1) for the
fixed-time method, to this solutions added
1.5 ml of Sodium periodate (3.0910-2 M)
shake thoroughly, then 1 ml of (3.0910-2 M)
of promethazine HCl was added and the
contents were dilute to the mark with distilled
water and shake well and transferred to a
spectrophotometer cell. The absorbance of the
colored product was measured as a function of
time (after 5 minutes and after 20 minutes for
the two methods respectively), at 590 nm
against a reagent blank prepared in the same
way but containing no CAP at room
temperature (25 Co). The initial rate of the
reaction at different concentration was

3-Effect of order of addition

To optimum results, the order of addition
of reagents should be followed as given under
the analytical procedure, otherwise a loss in
color intensity and stability was observed.
4- Effect of temperature
The effect of temperature on the oxidative
coupling reaction study show that the
absorbance of the dye remains constant
at room temperature (25 Co) for more than
120 min, and decrease at (0-5Co). Heating of
the mixture of reaction over 45 Co will
24

Hind S. Al-Ward

obtained from the slop of the tangent to the

absorbance time curve as shown in (Fig.(3)),
and analytical values of statistical treatments
for the calibration graph for the fixed time
method was shown in (Fig.(6)).

HO

-2

-1

y = 0.4978x + 2.0798

-2

-0.1

y = 0.4662x + 1.1805
-0.2

N
H

CH3
N

.HCl
R

N
S

colored-product

R=-CHOHCH(CH2OH)NHCOCHCl2

Scheme (1) Reaction scheme for the reaction

between chiorawphneeal and promethazine
HCl.
Evaluation of the kinetic methods
The quantitation of CAP under the
optimized experimental conditions outlined
above would result in a pseudo-first order with
respect to its concentrations where promethazine
HCl, were at least 20 time of the concentration

of CAP. However, the rate was directly

proportional to CAP concentration in a
pseudo-first order equation as follows:
Rate = k' [CAP] ............................................ (1)
where k' is the pseudo-first order rate constant.
Several experiments were then carried out
to obtain CAP concentration from the rate data
according to equation (1). Initial rate, fixed
time methods [27,28] were tried and the most
suitable analytical method was selected taking
into account the applicability, the sensitivity,
the intercept and the correlation coefficient (r).

0
-1

.HCl

H3C

0
log abs

-3

NH2

H3C

-0.5

-4

CH3

NaIO4

-0.4

Log molar of [R]

Promethazine HCl

-0.1

-0.3

Reducing form

Reducing form

-0.2

NH2

N
R

log abs

-3

N
H3C

0
-4

Zn powder

H
H3C

log molar of [D]

-5

NO2 +2 HCl Reduction

HO
Cl 2HCOCHN

Stoichiometry of the reaction

The stoichiometry of the reaction,
combining ratio between promethazine HCl
and CAP, was established by limiting the
logarithmic method [26], using two sets of
experiments. In the first set, the CAP
concentration was varied while keeping a
constant promethazine HCl concentration
(3.0910-2 M); in the second set, the
promethazine HCl concentration was varied
while keeping a constant concentration of CAP
(1.547x10-3M).
A plot of log absorbance versus log [CAP]
and log [promethazine HCl] gave straight
lines; the values of the slopes were 0.4978
and 0.466, respectively (Fig.(2)). Hence, it
is concluded that, the molar reactivity of
the reaction is 0.4978 / 0.4462, i.e. the
reaction proceeds in the ratio of 1:1 (CAP:
promethazine HCl).

-6

1- Initial rate method

The initial rates of the reaction were
determined by measuring the slopes of the
initial tangents the absorbance time curves
for the first 5 min (Fig.(3)). Furthermore,
logarithmic analysis of the reaction rate (R)
was plotted against log concentration of the
drug (Fig.(4)).

-0.3
-0.4
-0.5

Fig.(2) Limiting logarithmic plots for

the molar ratio.
Based on the obtained molar reactivity, A
reaction subsequent based on the above results
is shown in Scheme (1).
25

Journal of Al-Nahrain University

Vol.15 (4), December, 2012, pp.22-30

Log (rate) = 2.915 + 0.9305 log C

Where (r = 0.9969).
Hence k = 822 min-1 = 14 sec-1 and the
reaction is first order (n = 0.9305) with respect
to CAP concentration.
The analytical values of statistical treatments
for the calibration graphs are summarized in
(Table (1)).

Abs.

2.5
2

(5)

1.5

(4)

(3)
(2)

0.5

(1)
B

0
0

10

15

20

25

30

35

40

45

Table (1)
analytical values of statistical treatments for
the calibration graph of the initial-rate
method (at 5 mins.).
Parameters
value
Correlation
9.973X10-1
coefficient, r
Linearity percentage,
99.73
r2%
Test for a significant
23.528
correlation ,t*
Regression equation
y=0.0835x+0.0545
-1
Slop, b (ml.g )
8.35 X 10-2
Intercept, a
5.45 X 10-2
Standard deviation of
5.00 X 10-2
the residuals, Sy/x
Standard deviation of
3.424 X 10-2
the slop, Sb
Standard deviation of
7.963 X 10-3
the intercept, Sa
Linearity range (ppm)
2-20
Molar absorptivity
2.697 X 103
- 1
-1
(l.mol .cm )
Sandell's sensitivity S
1.197 X 10-1
(g.cm-2)
Limit of detection,
1.796
LOD (g.ml-1)
Limit of
quantification, LOQ
5.988
-1
(g.ml )

Time (min)

Fig.(3) Absorbance verses time graph

showing the dependence of the reaction on
CAP concentration. B: blank, (1) 6.19 10-6
M, (2) 15.6 10-6 M, (3) 31.2 10-6 M,
(4) 4.68 10-5 M and (5) 6.24 10-5 M.
Log [CAP]
0

Log (rate)

y = 0.9305x + 2.9125 -3
R2 = 0.9969
-0.5

-3.5

-4

-4.5

-5

Science

-5.5

-1
-1.5
-2
-2.5

Fig.(4) Log (rate) versus log[CAP] graph.

The rate of reaction was also found to be
dependent on CAP concentrations; the rates
were followed at room temperature (25C)
with various concentration of CAP in the
range of 220 g ml-1 keeping the reagent
and the oxidant concentrations constant. The
reaction rate was found to obey the following
equation:
Rate = k' [CAP]n ...........................................(2)
Where k' is the pseudo-order rate constant and
n is the order of the reaction. The rate of the
reaction may be estimated by the variable-time
method [29] (differential initial rate method)
[30] as A / t, where A is the absorbance and
t is the time in minutes. Taking logarithms of
rates and concentration, equation (3) is
transformed into:
Log(rate)=logA/t=logk'+nlog[CAP]. ........(3)

*t-tabulate =4.303 at confidence

(n 2) = 3 degrees of freedom

level

95%

and

2- Rate Constant Method

The best way to obtain an average K` value
for the reaction, is to plot the log (A)
versus time for CAP in the concentration range
2.0 - 20.0 g.ml-1 (6.2110-6 6.2410-5 M)
(Fig.(3)), obtained pseudo first rate constant K`
corresponding to different CAP concentrations.
These K` values were calculated from the slops
of curves multiplied by -2.303, (Table (2)).

Regression of log (rate) versus log [CAP]

gave the regression equation:

26

Hind S. Al-Ward

It is clear that the slope increases with time

and the most acceptable values of the
correlation coefficient (r) and the intercept
were obtained for a fixed time of 20 min,
which was therefore, chosen as the most
suitable time interval for measurement. After
optimizing the reaction conditions, the fixed
time method was applied to the determination
of CAP in pure form over the range 0.5
30 g.ml-1, (Fig.(6)), and analytical values of
statistical treatments for the calibration graphs
are summarized in (Table (4)).

Table (2)
Values of K` calculated from slops of Log A
versus t graphs at 590 nm.
[Drug]

Equation

K`/min-1

6.20 X 10-6

LogA= 0.0040t-0.6695

-9.212x10-3

15.6 X 10-6

LogA= 0.0025t-0.3015

-5.757x10-3

31.2 X 10-6

LogA= 0.0021t-0.0386

-4.836x10-3

4.68 X 10-5

LogA= 0.0011t-0.1473

-2.533x10-3

6.24 X 10-5

LogA= 0.0008t-0.2353

-1.842x10-3

Regression of [CAP] versus K` gave the

following equation:
K`=121.82[Drug]0.0088
Where r = 0.9483 as shown in (Fig.(5)).

2
1.8

y = 0.0566x + 0.0948
R2 = 0.9992

1.6
1.4
Abs.

1.2
[CAP]

-0.001
-0.002
-0.003

0.00001

0.00002

0.00003

0.00004

0.00005

0.00006

0.00007

0.6
0.4

y = 121.82x - 0.0088
R2 = 0.8994

0.2
0

-0.004
K

1
0.8

10

-0.005

15

20

25

30

35

Conc of CAP (ppm)

-0.006

Fig.(6) Calibration graphs of CAP at fixed

time 20 min.

-0.007
-0.008
-0.009
-0.01

Table (4)
Analytical values of statistical treatments for
the calibration graph of the fixed time
method.

Fig.(5) A plot of rate constant K'

versus [CAP].
3-Fixed-time method
At a pre-selected fixed time, Calibration
graphs of absorbance versus initial concentration
of CAP were established at fixed times of 5,
10, 15, 20, 25, 30, 35, and 40 min with
regression equations assembled in (Table (3)).
Table (3)
Regression equations for CAP at different
fixed time over range 1.54710-6to9.28510-5M
at room temperature.
Time (min)
5
10
15
20
25
30
40

Regression equation
A=0.0545+0.0835X
A=0.0553+0.089X
A=0.0570+0.0927X
A=0.0650+0.0961X
A=0.0670+0.095X
A=0.0852+0.0943X
A=0.0936+0.0945X

R
0.9947
0.9936
0.9968
0.9991
0.9974
0.9942
0.9936

Parameters

value

Correlation coefficient, r
Linearity percentage , r2%
Test for a significant correlation, t*
Regression equation
Slop, b (ml.g-1)
Intercept, a
Standard deviation of the
residuals, Sy/x
Standard deviation of the slop,Sb
Standard deviation of the
intercept, Sa
Linearity range (ppm)
Molar absorptivity (l.mol- 1.cm-1)
Sandell's sensitivity S (g.cm-2)
Limit of detection, LOD (g.ml-1)
Limit of quantification, LOQ
(g.ml-1)

9.997 X 10-1
99.97
132.245
y=0.0566x+0.0947
5.658 X 10-2
9.466 X 10-2
8.001 X 10-3
0.02139
0.009525
0.5-30
1.822
1.767 X 10-2
0.4285
1.428

*t-tabulate = 2.447 at confidence level 95% and

(n 2) = 7 degrees of freedom.

27

Journal of Al-Nahrain University

Vol.15 (4), December, 2012, pp.22-30

From values of test for a significant

correlation (t-calculate > t-tabulate) and
linearity percentage (> 95%). These calibration
graphs possess excellent linearity.

Table (7)
Application of the proposed method of CAP
In pharmaceutical preparations by the
initial-rate method.

Accuracy and precision

To determine the accuracy and precision of
CAP which was determined in five replacements
of three different concentrations. The results
shown in (Tables (5, 6)), indicate that a
satisfactory precision and accuracy could be
obtained with the proposed method.

Concentration
Drug

Error
%

Rec.
%

R.S.D
%

Present

Found*

4.00

4.05

0.50

100.50

1.509

12.00

12.28

0.33

100.33

0.415

20.00

19.96

0.20

100.20

0.375

Rec.
%

Found*

2.00

1.98

1.00

101.00

1.238

8.00

7.93

1.00

101.00

0.851

16.00

16.03

Drug
sample

R.S.D
%

Present

-0.18

99.810

Rec

R.S.D
%

Present

Found*

5.09

-1.80

98.20

1.06

15

15.25

-1.66

98.33

0.94

4.95

1.00

101.00

0.77

15

15.08

-0.53

99.46

0.31

5.11

-2.20

97.80

2.31

15

15.19

-1.26

98.73

1.79

Table (8)
Application of the proposed method of
CAP In pharmaceutical preparations by the
fixed-time method.

Table (6)
Accuracy and precision of the fixed-time
method.
Error
%

Error

*for five determinations

1- Aphenicol capsule
2- Cetrimide eye drops
3- Betapheni Ointment

* for five determinations.

Concentration of
CAP g.ml-1

of CAP (ppm)

sample

Table (5)
Accuracy and precision of the initialrate 0
method.
Concentration of
CAP g.ml-1

Science

0.424

* for five determinations.

Pharmaceutical applications
The initial-rate and fixed-time methods
were applied to the determination of CAP in
pharmaceutical preparation by the analysis of
two different concentrations of pharmaceutical
preparations using the analytical procedures.
The results are given in (Table (7)) and
(Table (8)).

Concentration of
CAP (ppm)

Error
%

Rec.

R.S.D

Present

Found*

4.93

1.40

101.40

0.85

15

14.84

1.06

101.63

0.62

5.03

-0.60

99.40

1.19

15

14.89

0.73

100.73

1.01

5.02

-0.40

99.60

1.31

15

15.21

-1.40

98.60

0.93

*for five determinations

1- Aphenicol capsule
2- Cetrimide eye drops
3- Betapheni Ointment

The proposed method was compared

successfully with the BP method [1] for both
pure CAP and the pharmaceutical preparations
for both initial-rate and fixed-time method,
good recoveries were obtained as shown in
(Table (9)).

28

Hind S. Al-Ward

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determination
of
chloramphenicolsulphacetamide in eye drops"; Pharmazie.,
33, 721-722, 1978.

Table (9)
Comparison of the proposed methods with
standard method.
Drug
sample
Pure CAP
Aphenicol
capsule
Cetrimideey
e drops
Betapheni
Ointment

Fixedtime
method
101.00

Recovery%*
InitialBP
rate
method
method
100.70
100.00

101.63

98.33

99.53

100.73

99.46

100.60

98.60

98.73

98.67

*for five determinations

Conclusions
The proposed methods are showing good
sensitivity, and low detection limit. In
addition, the proposed procedures show
relevant selectivity allowing analysis without
separation steps, and providing suitable
alternative to the many chromatographic
procedures proposed [4-7]. The proposed
methods are advantageous when they are
compared with colorimetric methods [12-19]
in having higher sensitivity. The data given
above reveal that the proposed methods are
accurate and sensitive with good precision and
accuracy. With this method, one can do the
analysis with speed at low cost without losing
accuracy. The proposed method can be used as
alternative method to reported ones for the
routine determination of CAP in the pure form
and in pharmaceutical preparations depending
upon the availability of chemicals and
equipment.
References
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Churchill Livingstone, London, 1975.
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29

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.

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