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ISSN-1996-918X
Pak. J. Anal. Environ. Chem. Vol. 9, No. 1 (2008) 43 – 48
Spectrophotometric Determination of Ceftriaxone Using
4-Dimethylaminobenzaldehyde
F.M.A. Rind*1,M.G.H. Laghari1, A.H. Memon1, U.R. Mughal, F. Almani1,
N. Memon1, M.Y. Khuhawar2and M.L. Maheshwari2
*1Faculty of Pharmacy, University of Sindh, Jamshoro, Pakistan.
2Dr. M.A. Kazi Institute of Chemistry, University of Sindh, Jamshoro, Pakistan.
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Abstract
A new spectrophotometric method has been developed for the determination of the potent
antibiotic ceftriaxone (CF) by derivatization with 4-dimethylaminobenzaldehyde (DAB). The
derivative indicated molar absorptivity of 5.3 x 103L mol-1 cm-1 at max 397 nm and obeyed the
Beer’s law within 20-100 µgmL-1. The color reaction was highly stable and did not show any
change in absorbance up to 24 h. The method was successfully applied for the determination of CF
from various pharmaceutical preparations available in local market. The amounts of CF found in
various pharmaceutical preparations were within 237.4-990 mg ampoule-1 with standard deviation
(SD) ± 0.0004-0.044 (n=3) respectively.
Keywords: Ceftriaxone, 4-dimethylaminobenzaldehyde; spectrophotometry.
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Introduction
Ceftriaxone (CF) (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-
2-(methoxyimino)acetamido)-3-((6-hydroxy-2-methyl-
5-oxo-2,5-dihydro-1,2,4-triazin-3-ylthio)methyl)-8-oxo-
5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid
is a third-generation cephalosporin antibiotic. Like other
third-generation cephalosporins, it has broad spectrum
activity against Gram negative and Gram positive
bacteria [1]. CF is often used (in combination with
macrolide and/or aminoglycoside antibiotics) for the
treatment of community-acquired pneumonia. It is also
a drug of choice for the treatment of bacterial
meningitis. In pediatrics, it is commonly used in febrile
infants. It has also been used in the treatment of
leptospirosis [2], lyme disease and gonorrhea. It is also
used as a routine prophylactic antibiotic for the patients
undergoing orthopedic surgery [3]. Several analytical
methods have been reported for the analysis of CF,
based on spectrophotometric [4-10], derivative
spectrophotometric [11], FIA [12], flourimetric [13,
14], thin layer chromatographic [15-17], ion selective
electrodes [18], ion exchange chromatographic [19],
high performance liquid chromatographic [20, 21], ion-
pair liquid chromatographic [22] and polarographic
[23, 24] techniques. For spectrometric analysis, the
determination is carried out using suitable reagents such
as metol-chromium(VI) reagent, mixture of Fe(III) and
hexacyanoferate(III) ions, leuco crystal violet and 3-
methyl-2-benzothiazoline hydrazone hydrochloride and
ferric chloride [4-10, 25]. The derivatization either
increases the molar absorptivity or produces
bathochromic shift in the absorbance. Therefore, 4-
dimethylaminobenzaldehyde, (DAB) is reported for the
first time as a derivatizing reagent for
spectrophotometric determination of CF in
pharmaceutical preparations.
Experimental
Materials and reagents
All the chemicals and reagents used were of
analytical grades. The double distilled water was used
throughout the study. Pure ceftriaxone (CF), 4-
dimethylaminobenzaldehyde (DAB) and acetic acid
were obtained from E. Merck (Germany), sodium
acetate from Fluka (Switzerland) and ethanol from BDH
(U.K) were used. Buffer solutions between pH 1-10 at
unit internal were prepared from hydrochloric acid
(0.1M), potassium chloride (1M), acetic acid (1M),
ammonium acetate (1M), sodium acetate (1M), sodium
carbonate (saturated solution), sodium bicarbonate
*Corresponding Author: falmani@hotmail.com
Pak. J. Anal. Environ. Chem. Vol. 9, No. 1 (2008)
44
(1M), potassium chloride (1M), ammonium chloride
(1M) and ammonia solution (1M).
The solution of DAB (2% w/v) was prepared in
ethanol (100mL). The spectrophotometric studies were
carried out with a double beam spectrophotometer UV-
1601 (Shimadzu Corporation, Japan) with fused silica
1cm cells.
General procedure
The aqueous solution (0.2-1.0mL) containing
CF (200-1000 µg) was transferred to 10mL calibrated
flask and were added 3mL DAB (2% in ethanol w/v)
and acetate buffer pH 5 (1mL). The contents were
heated on water bath at 90 0C for 15 min. The solutions
were cooled at room temperature and the volume was
made up to the mark with ethanol. The absorbance was
measured at 397 nm against reagent blank which was
prepared in a similar way only omitting the addition of
CF.
UV method (Un-derivatized)
The solution (0.2-1.0mL) containing CF (20-
100µg) was transferred to 10mL calibrated flask and the
volume was made up to the mark with ethanol. The
absorbance was measured at 272 nm against ethanol.
The molar absorptivity was calculated as 3.4x104
L mol-1 cm-1.
Analysis of CF from pharmaceutical preparations
Thirty samples of different pharmaceutical
companies were collected and analyzed for the contents
of CF. The sample (0.1g) from each of the CF
preparations, Acmex (Acme Laboratories (PVT) Ltd.
Lahore, Pakistan), Aczon (Global pharmaceuticals
Islamabad, Pakistan), Bestrix (Asian Pharmaceuticals
Karachi, Pakistan), Broadced (Kalbe Pharma
International (PVT) Ltd. Lahore, Pakistan), Cef-3
(Shazal'z Pharmaceuticals Rawalpindi, Pakistan), Cefcin
(Cirin Pharmaceuticals (PVT) Ltd. Rawalpindi,
Pakistan), Cefin (Macter International (PVT) Ltd.
Karachi, Pakistan), Cefotrim (Pharmedic Laboratories
(PVT) Ltd. Lahore, Pakistan), Ceftison (Qureshi
Pharma (PVT) Ltd. Karachi, Pakistan), Ceftrex
(Polyfine Chempharma (PVT) Ltd. Peshawar, Pakistan),
Ceftridex (Rex Pharmaceuticals Karachi, Pakistan),
Cefxone (Bosch Pharmaceuticals (PVT) Ltd. Karachi,
Pakistan), Cepox (MBL Pharma Karachi, Pakistan),
Cerixon (Genesis Pharmaceutical (PVT) Ltd. Lahore,
Pakistan), Chef (Al-Habib Pharmaceuticals / Al-Habib
Corporation Karachi, Pakistan), Chroncef (English
Pharmaceuticals Industries Lahore, Pakistan), C-trox
(Mediceena Pharma (PVT) Ltd. Lahore, Pakistan),
Cyforon (Ali Gohar Pharmaceuticals (PVT) Ltd.
Karachi, Pakistan), Dayzone (High-Q International
Karachi, Pakistan), Eftriax (Pharmatec (PVT) Ltd.
Karchi, Pakistan), Efxone (Candid Pharmaceuticals
Lahore, Pakistan), Elxone (Ethel Pharma International
Karachi, Pakistan), Farcef (Pulse Pharmaceuticals
Lahore, Pakistan), Fotamin (Vision Pharmaceuticals
Islamabad, Pakistan), Inocef (Barrett Hodgson (PVT)
Ltd. Karachi, Pakistan), Lozon (Zesion Pharmaceuticals
(PVT) Ltd. Islamabad, Pakistan), Macxone (Macquins
International Karachi, Pakistan), Maxcef (Indus Pharma
(PVT) Ltd. Karachi, Pakistan), Oxidil (Sami
Pharmaceuticals (PVT) Ltd. Karachi, Pakistan) and
Rociphin (Roche (PVT) Ltd. Karachi, Pakistan) was
dissolved in 100mL of distilled water. The solution
(0.6mL) was transferred to 10mL calibrated flask and
the content of CF was determined following the general
procedure as described in section B.
% Recovery of CF from pharmaceutical samples by
standard addition technique
CF powder (0.1g) was dissolved in 100mL
water. Two portions, each consisting 0.6mL were taken
in two different 10mL calibrated flask. One was added
with 0.2mL containing 200g CF solution and the
derivatization procedure was followed for both solutions
as described in B.
The % recoveries were calculated from the
increase in the absorbance with added standard.
Results and Discussion
Ceftriaxone (CF) reacts with 4-dimethylami-
nobenzaldehyde (DAB) to form an azomethine
derivative 7 - ( 2 - { 2 -[(4-Dimethylamino-benzylidene)
–amino]–thiazol–4–yl } – 2 -methoxyiminocetylamino)-
3-(6-hydroxy – 2 – methyl l - 5-oxo-2,5-dihydro -[1,2,4]
triazin -3-ylsulfanylmethyl)-8-oxo-5-thia-1-aza-bicyclo
[4.2.0]oct-2-ene-2-carboxylic acid (CF-DAB) (Fig. 1)
which has maximum absorbance (max) at 397 nm with
molar absorptivity of 5.3x103 L mol -1 cm-1. DAB was
then tested as a derivatizing reagent for the spectroph-
otometric determination of CF. The effects of pH, effect
of reagent (DAB), heating time and temperature on the
formation of (CF-DAB) derivative were studied.
Optimization of analytical parameters
Absorption spectra for wavelength selection
For the quantitative analysis, the wavelength of
maximum absorbance plays an important role. It is
necessary to select the wavelength where the
derivatizing reagent indicates minimum absorbance and
the analyte derivative shows maximum absorbance
value.
Pak. J. Anal. Environ. Chem. Vol. 9, No. 1 (2008)
45
N
S
O
OH O
S
NN
N
O
HO
O
NH
NO
S
N
NH2
H
(6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-
2-(methoxyimino)acetamido)-3-((6-hydroxy-2-methyl-5-oxo-
2,5-dihydro-1,2,4-triazin-3-ylthio)methyl)-8-oxo-5-thia-
1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid
+
O
N
4-dimethylaminobenzaldehyde.
N
S
O
OH O
S
NN
N
O
HO
O
NH
NO
S
N
N
H
N
7-(2-{2-[(4-Dimethylamino-benzylidene)-amino]-thiazol-4-yl}-2-methoxyimino-acetylamino)-3-(6-hydroxy-2-methyl-5-oxo-2,5-dihydro-
[1,2,4]triazin-3-ylsulfanylm ethyl)-8-oxo-5-thia-1-aza-bic yclo[4.2.0]oct-2-ene-2-carbox ylic acid
Figure 1. Formation of CF derivative using 4-dimethyl aminobenzaldehyde as derivatizing reagent
The absorbance value of 20 µg mL-1 of CF as
DAB derivative was recorded at different wavelengths
between 250-500 nm after heating for 15 min at 900C
using buffer pH 5. It is evident that the maximum
absorbance occurred in visible region at 397 nm against
reagent blank and was selected as optimum.
Effect of reagent concentration
The effects of adding various amounts of DAB
solution on absorbance of 20µg mL-1 CF was examined
(Fig. 2). The concentration of reagent DAB was varied
between 1-6mL of 2% in ethanol with an interval of
1mL. A similar absorbance was observed with addition
of 2 and 3mL and the addition of 3mL (2% w/v) DAB
solution was selected. Figure 2. Effect of volume of reagent on absorbance of CF
derivative
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
0 2 4 6 8
Volume of reagent (DAB) mL
Absorbance
Pak. J. Anal. Environ. Chem. Vol. 9, No. 1 (2008)
46
Effect of order of mixing the reagents
The order of adding reagents during
derivatization process has important role in accuracy of
results and enhancement of absorbance. In the present
study, it was observed that the addition of buffer pH 5
(1mL) to 200 µg CF solution (0.2mL) followed by
3mL DAB reagent (2% w/v) resulted in a decrease in
absorbance value. Taking the reagent first and then
adding the buffer, followed by CF solution also had
lower absorbance value. The maximum absorbance
value was observed when 3mL of reagent DAB was
added to the standard solution of CF followed by buffer
(1mL) pH 5. The contents were then heated on water
bath and the volume was adjusted to 10mL with
ethanol.
Optimization of heating time and temperature for the
formation of derivative
To achieve the maximum absorbance value for
an analyte by the formation of stable derivative, the
selection of optimum time and temperature is essential.
The effect of time on the formation of derivative was
checked at 397 nm from 0-30min with an interval of 5
min. A similar absorbance was observed after heating
for 15 min at 900C and was considered as optimal.
Effect of solvents
The effect of various solvents such as
methanol, 1-propanol, 1-butanol, amyl alcohol, isoamyl
alcohol, acetonitrile, ethyl acetate, toluene, nitrobenzene
and carbon tetrachloride on the absorbance was
examined. From each of the solvents 1and 2 mL was
added after the addition of 2% ethanolic solution of
DAB, and 1 mL acetate buffer pH 5 followed by heating
for 15 min at 90°C. The ethanol proved to be the best
choice.
Effect of pH
The effect of adding 1mL of 1M buffers of pH
range 1-10 on the absorbance at optimized conditions
was studied.
It is evident from Fig. 3 that the absorbance
increased gradually from buffer pH 1 and reaches to
maximum value at pH 5. Addition of buffer of pH 8 and
above produced precipitation. Therefore, the acetate
buffer of pH 5 was selected as optimal.
Interference study
The effect of possible presence of associated
materials such as mannitol, sorbitol, sucrose, glucose,
galactose and fructose was investigated at 10 times the
concentration of CF and it was observed that none of
these substances interfered with no change in
absorbance of more than ±5% (Table. 1).
Figure 3. Effect of pH on derivatization of CF
Table 1. Effects of different possible additives on the absorbance
of 20 µg mL-1 CF derivative.
S.
No. Chemical
added Absorbance Relative error (%)
1. ------------- 0.190 00
2. Mannitol. 0.191 0.5
3. Sorbitol. 0.188 -1.0
4. Glucose 0.190 00
5. Galactose. 0.192 1.0
6. Fructose. 0.186 -2.1
7. Sucrose. 0.180 -5.0
Stability of the derivative
The stability of CF-DAB derivative was
examined in terms of absorbance at the concentration of
20 µg mL-1 CF, but no change in absorbance of more
than 5% was observed within 48 h.
Calibration graph (Beer’s Law)
The effect of variation in the concentration of
CF on its absorbance was studied. A linear calibration
curve was obtained which obeyed the Beer’s law within
the concentration range 20-100µg mL-1 of CF with
coefficient of determination r2 0.9996 (Fig. 4).
The Sandells sensitivity (0.004) was observed
at 5g cm-2 CF. The validity of the calibration curve
was obtained by the analysis of test solution of CF and
the percent relative error was found ±1-2%.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
0 1 2 3 4 5 6 7
pH
Absorbance
Pak. J. Anal. Environ. Chem. Vol. 9, No. 1 (2008)
47
Table 2. Analysis of CF from pharmaceutical preparations
S. No. Name of drug Amount labeled
per ampoule (mg)
Amount found
(mg) per ampoule
(standard deviations)
±(%)
Relative deviations
from labeled values
Recovery
(%)
01. Acmex. 250 238.7 (0.021) 4.5 95.0
02. Aczon. 250 247.5 (0.002) 1.0 97.8
03. Bestrix. 250 242.5 (0.022) 3.0 100.02
04. Broadced. 1000 990.0 (0.002) 1.0 99.3
05. Cef-3. 250 247.5 (0.031) 1.0 98.3
06. Cefcin. 250 247.6 (0.002) 1.0 100.2
07. Cefin. 500 454.0 (0.040) 9.2 95.0
08. Cefotrim. 250 237.4 (0.030) 5.0 96.0
09. Ceftison. 250 247 .5 (0.028) 1.0 95.0
10. Ceftrex. 250 243.5 (0.015) 2.6 99.9
11. Ceftridex. 500 485.6 (0.020) 2.8 95.3
12. Cefxone. 250 237.8 (0.004) 4.8 98.6
13. Cepox. 500 475.7 (0.043) 4.8 99.8
14. Cerixon. 500 479.3 (0.044) 4.1 99.4
15. Chef. 500 486.4 (0.022) 2.7 99.5
16. Chroncef. 250 245.2 (0.003) 1.9 98.2
17. C-trox. 500 490.0 (0.002) 2.0 98.0
18. Cyforon. 500 494.1 (0.003) 1.1 97.0
19. Dayzone. 250 247.5 (0.001) 1.0 95.8
20. Eftriax. 500 479.1 (0.001) 4.1 99.0
21. Efxone. 250 247.5 (0.003) 1.0 98.2
22. Elxone. 500 482.3 (0.002) 3.5 99.0
23. Farcef. 1000 974.0 (0.003) 2.6 96.0
24. Fotamin. 500 493.2 (0.001) 1.3 98.0
25. Inocef. 250 245.7 (0.005) 1.7 97.0
26. Lozon. 500 487.5 (0.001) 2.5 99.9
27. Macxone. 250 246.6 (0.002) 1.3 98.6
28. Maxcef. 1000 990.0 (0.002) 1.1 100.02
29. Oxidil. 250 245.2 (0.005) 1.9 100
30. Rociphin. 250 247.0 (0.001) 1.2 99.7
Conc. CF g mL-1
Figure 4. Calbration curve of Ceftriaxone 0.1% using 4-dimethyl
aminobenzaldehyde as derivatizing re agent
The pharmaceutical preparations containing CF
available in the local market were analyzed to determine
the amount of CF quantitatively (Table. 2).
The mean observed values were within 238.7-
990 mg ampoule-1 with standard deviation (SD) within
0.0004-0.044 of thirty pharmaceutical brands (Table. 3).
y = 0.0092x
R
2
= 0.9996
0
0.2
0.4
0.6
0.8
1
020 40 60 80 100
Absorbance
Pak. J. Anal. Environ. Chem. Vol. 9, No. 1 (2008)
48
Table 3. Results of optimization, precision and accuracy
S.
No. Parameter (s) Selected Values
01. Wave length max (nm) 397
02. Beer’s law limits (µg mL-1)20-100
03. Molar absorptivity (L mol-1 cm-1)5.3x103
04. Sandells sensitivity (conc. at 0.004
absorbance unit) (g cm-2)5
05. Regression equation (y)a.
Slope (b). Intercept (a). 0.9189
0
06. Coefficient of determination (r2)0.9996
07. Standard deviation. ± 0.0004-0.044
Day to day reproducibility / repeatability
For the determination of intra and interday
reproducibility of the method, the aqueous standard
solution 200g CF was taken in three different
calibrated flasks (10mL) and the procedure was
followed as described in section B. The above
procedure was repeated for three days (n=3). The mean
absorbances of intraday and interday reproducibilities
were observed as 0.191 and 0.19 with (RSD) values
0.0036% and 0.095%, respectively.
Conclusions
The developed method is simple, accurate,
precise, inexpensive and less time consuming in visible
region after derivatization with DAB. The developed
method may avoid the interferences from associated
materials which may absorb in the UV region. The
method was applied for the determination of CF
contents from pharmaceutical preparations
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