Antibiotics

Spectrophotometric method for determination of

certain cephalosporins using 4-chloro-7-nitrobenzo-
2-oxa-1,3-diazole (NBD-Cl)
Rageh et al.-Natural Science 2 (2010) 828-840
Na-Benzylpenisilin Na-ampisilin
Penisilin
Na-Kloksasilin
Cefalexin Cefradin Cefadroxil
The official method of cephalosporin: HPLC using
spectrophotometric detector (EP, USP).
Sefalosporin
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cephalosporin penicillin
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CEFACLOR
CEFADROXIL CEFALEXIN
CEFOTAXIME CEFRADINE
CEFIXIME
The official procedures in pharmaceutical preparations
utilize high-performance liquid chromatography (HPLC)
[3]

which is expensive. Other reported procedures include
spectrophotometric
[4-9]
, spectrofluorimetric
[10-13]
,
chemiluminescence
[14-16]
, chromatographic
[17-20]
and
electrochemical methods
[21-24]
and most of them are
lengthy (lama) and/or tedious (jenuh).

---
3- American Pharmaceutical Association. (2008) United States Pharmacopoeia 31 and NF 26. Washington, DC.
4- Abdel-Hamid, M.E. (1998). Il Farmaco, 53(2), 132-138.
10-Aly, F.A., Hefnawy, M.M. and Belal, F. (1996). Analytical Letters, 29(1), 117-130.
14-Li, Y. and Lu, J. (2006). Luminescence, 21(4), 251-255.
17-Shinde, V.M. and Shabadi, C.V. (1997). Indian Drugs, 34, 399-402.
21-zkan, S.A., Erk, N., Uslu, B., Yilmaz, N. and Biryol, I. (2000). J. Pharmaceutical and Biomedical Analysis, 23(2-3),
263-273.
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NBD-Cl
The hydrolytic degradation of cephalosporins a
preliminary step in the analytical procedure used for their
determinations
[25-32]
. The literature reveals that many
spectrophotometric methods based on hydrolysis of
these drugs using alkaline degradation and subsequent
reaction of the formed sulfide ions with chromogenic
reagents
[26,27]
. NBD-Cl (4-chloro-7-nitrobenzo-2-oxa-1,3-
diazole) has been reported as fluorogenic reagent for
determination of amines
[33]
and for spectrophotometric
determination of many compounds
[34-41]
.

---
25-El-Obeid, H.A., Gad-Kariem, E.A., Al-Rashood, K.A., Al-Khames, H.A., El-Shafie, F.S. and Bawaseer, G.A.M. (1999)
Analytical Letters, 32(14), 2809-2823.
26-Mohamed, F.A. (1998). In Proceedings of Assiut University 1st Pharmaceutical Science Conference, Faculty of
Pharmacy, Assiut, 1998, 1-18.
33-Omai, K., Toyo'oka, T. and Miyano, H. (1984). The Analyst (London), 109(11), 1365-1372..
34-Olgun, N., Erturk, S. and Atmaca, S. (2002). J. Pharmaceutical and Biomedical Analysis, 29(1-2), 1-5.
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Thiocompounds have been reported to form intensely
colored products in an alkaline medium with NBD-Cl
which could be used for their colorimetric determination.
On the basis of the aforementioned reasons, it was
decided to develop a quantitative method for the
determination of the studied cephalosporins based on
their alkaline hydrolysis and subsequent reaction of the
resulting hydrolysates with NBD-Cl, which may be used
for their analysis either in pure forms or in pharm.
formulations.
This method is selective for cephalosporins, since other -
lactam antibiotics such as penicillins do not give sulfide
ions under the degradation conditions employed.
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Stock solutions: 100 mg mL
-1
of
each cephalosporin in double
distilled water (methanol was
used for cefalexin, cefadroxil,
cefaclor, cefradine, cepodoxime
proxetil & cefixime).
Working standard solutions: 0.5-
2.5 mg mL
-1
>cefadroxil and
cefalexin, 1-6 mg mL
-1
>cefradine,
2-8 mg mL
-1
>cefaclor, cefazolin,
cefotaxime, ceftriaxone and
cefpodoxime proxetil, 2-10 mg
mL
-1
>cefixime & 2-16 mg mL
-1

>cefoperazone and ceftazidime S
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must be freshly prepared
Twenty tablets or the contents of 20 capsules were
weighed, finely powdered and mixed thoroughly.
An accurately weighed amount of the powder obtained
from tablets or capsules equivalent to 250 mg of each
drug was transferred into a 25-mL volumetric flask,
dissolved in about 10 mL solvent, sonicated for 15 min,
diluted to the mark with solvent, mixed well and filtered;
the first portion of the filtrate was rejected.
Further dilutions with the same solvent were made to
obtain sample solution containing the specified
concentration for each drug as mentioned under the
preparation of standard solutions and then the general
procedure was followed.
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An accurately weighed amount of powder equivalent to
250 mg of each drug was transferred into a 25-mL
volumetric flask, then the procedure was followed as
under tablets and capsules beginning from (dissolved in
about 10 mL double distilled water etc.)
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Accurately measured one milliliter aliquot volume of
the standard or sample solutions was transferred into
10-mL volumetric flask.
weighing
Five mL of 0.5 M NaOH were added and the flask was
heated in a boiling water-bath for 30 min, cooled to
room temperature and completed to volume with
double distilled water.
hydrolysis
One milliliter of the resulting drug hydrolysate was
pipetted into 10-mL volumetric flask, 1.0 mL of 3
10
-3
M NBD-Cl was added followed by 1 mL of
concentrated HCl.
working
tube
The resulting solution was mixed well and the flask
was completed to volume with ethanol. The
absorbance was measured at 390 nm against reagent
blank treated similarly.
measure
Optimasi Metode
The influence of NaOH concentration on producing the
maximum absorption intensity was investigated using
0.1-1.0 M NaOH keeping other factors constant. Maximum
absorption readings were obtained upon using 0.5 M
NaOH; above this concentration and up to 1 M NaOH, the
absorbance remains constant.

The effect of hydrolysis time on the absorption intensity
was studied using different heating times in a boiling
water bath (at 100C) starting from 10 min until 2 hours
and the reaction was carried out as usual. The maximum
absorption intensity was attained after 20 min and
remained stable for at least 100 min. Thirty minutes
hydrolysis time was used in all subsequent experiments.
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The concentration of NBD-Cl, for the maximum color
development was varied in the range of 0.75 10
-3
4
10
-3
M. It was found that 1 mL of 3 10
-3
M NBD-Cl was
the most suitable concentration for determination of the
studied drugs. Owing to the presence of labile chloride, a
daily fresh solution is recommended.
Different acids such as sulfuric, hydrochloric, perchloric,
nitric and acetic acids were tested to determine the most
suitable acid for the reaction. One mL of concentrated
hydrochloric acid was selected in this study as it gave the
highest absorbance readings taking cefalexin anhydrous
(15 g mL
-1
). As a result of the most suitable
concentration of hydrochloric acid investigations.
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The reaction between the investigated drugs hydrolysates
and NBD-Cl was very rapid and the interaction colored
product can survive before dilution unchanged for at
least 1 hour. However, measurements were achieved
instantaneously.

Stability time was obtained by following the absorbance
readings of the developed reaction product for 24 hours at
room temperature (25 5C). It was found that the
produced color was stable for 24 hours for all studied
drugs.
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Different solvents were tested in order to select the most
appropriate solvent for optimum color development. The
results given in Table 3 show small shifts in the position
of the maximum absorption peak. The absorption
intensities were slightly influenced. Ethanol was used
throughout this work because it gave the highest
absorbance readings and the most reproducible results.
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LOD and
LOQ
the limits of detection and
quantitation
Accuracy
by investigating the recovery of
each of the studied drugs at
three concentration levels
Precision
the small values of SD and %
RSD point to high precision
Selectivity
the effect of the presence of
common excipients was studied
Robustness
by evaluating the influence of
small variation in the
experimental parameters
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The visible spectrophotometric methods for
cephalosporins are based on their interaction with
different reagents, such as imidazole in the presence of
ferrihydroxamate, mercury (II) (2), ninhydrine (3),
N,N-dimethyl-D-phenylendiamhe and ferric ions (4),
ammonium vanadate (5), phenol with sodium
nitroprusside and hypochlorite (6), 4--
dimethylaminocinnam aldehyde (7), 3-bromo-4,4-
dimethyl-2-oxazolidinon(8), O-hydroxyhydroquinone
phtalein (9), paramolybdate anion (10), sodium nitrite
(11), chloranilic acid (12), molybdophosporic acid
(13), copper (II) acetate (14), oxidized heamatoxylin
(15), 2-nitro-phenyl-hydrazine hydrochloride (16),
mercurochrome (17)
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2. Sengun F.I., Ulas K. Talanta. 1986; 33:363
3. MarriniD., DiCoccoM.E. Rass.Chim. 1980;32:231
4. Abdall M.A., Fogg A.G., Burges C. Analyst(London) 1982;107:213
5. Abdel-Khalek M.M., Maihrais M.S. Talanta 1983;30:792
6. Foog A.G., Abdulla A.M. J.Pharm.Biomed.Anal.1985;3:315
7. Rao G.R., Raghweer S. J.Inst.Chem.(India) 1983;55:61
8. Kaminski J.J., Bdoor N. Int.J.Pharm. 1975; 3:151
9. Moris I., Fujita Y., Saraguchi K. Chem.Pharm.Bull. 1982;30:2599
10. Issopoulos P.B. J.Pharm.Biomed.Anal. 1988;6:97
11. Uri J.V., Jain T.C. J.Antibiot. 1986;39:669
12. Sastry C.S.P., Divauor T.E. Chem.Anal.(Warsaw)l987;32:301
13. Issopoulos P.B. Analyst(London) 1988;113:1083
14. Issopoulos P.B. J. Pharm.Biomed.Anal. 1988;6:321
15. Sastry C.S.P., Salyarayana P., Rao A.R., SinghN.R.P. Microchim.Acta. 1989;
1:17
16. Koraky M.A., Abdel-Hay M.H. Bedair M.M. Talanta 1989;36:1253
17. Galal S.M. Acta Pharm.Jugosl.1991;41:25
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A Spectrophotometric Method for The
Determination of Penicillin
Herriott-J. Biol. Chem. 1946, 164:725-736
Cefalexin Cefradin Cefadroxilxib
The official method of aceclofenac, diclofenac, ketorolac was
potentiometric titration method with sodium hydroxide, perchloric
acid, tetrabutylammonium hydroxide, respectively
(Pharmacopoeia, 2004)
Sefalosporin
Penisilin
Na-Benzylpenisilin Na-ampisilin Na-Kloksasilin
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cephalosporin penicillin
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N

P
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N

P
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P
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P
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P
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P
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Spectrophotometric Method for Assay
of Penicillins
Hiremath & Mayanna-Mikrochim. Acta [Wien]
1986 I, 265--270
PRINCIPLE
Ninhydrin in 0.2 M citrate buffer (pH 5) containing
SnCl
2
is proposed as a new reagent for
spectrophotometric determination of penicillins and
analysis of their pharmaceutical formulations. The
method is based on acid hydrolysis of penicillins with 5
M HCl and subsequent treatment with ninhydrin. The
resulting violet colour exhibits maximum absorption at
570 nm.
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On acid hydrolysis (pH <3) penicillin gives penaldic acid
and penicillamine [21]. It is known that compounds
containing a free amine group react with ninhydrin (I) to
give the violet diketohydrindylidene diketohydrindamine
(II) [15] only in presence of a reducing agent capable of
producing hydrindantin [22].
Others: titrimetric methods have been reported in the
literature for the assay of penicillins, based on use of
iodine [1], N-bromosuccinimide [2] and chloramine-T
[3] as titrants. Conductometric [4], polarographic [5]
and HPLC [6] procedures are also employed for the assay
of penicillins.

The spectrophotometric methods used are based on
determination of the hydroxamic acid formed by reaction
with hydroxylamine [8], or the penicillenic acid
mercury(II) mercaptides formed by reaction with
imidazole in the presence of mercury(II) chloride [9], or
involve ammonium vanadate [10], chromium(VI) with
metol [11] and Azure B [12].
P
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Ninhydrin, on reduction with SnCl
2
[13] at pH 5 or with
ascorbic acid [14] gives a colour reaction with amino-
acids. The colour is stabilized by using pyridine-phenol
[15], cyanide [16] or cyanide/acetate buffer [17].
Ninhydrin has also been used for qualitative detection of
penicillin [18] and for spectrophotometric assay of
chloramphenicol [19] and D-cycloserine [20].

In several pharmaceutical formulations, penicillin is associated with
flavouring agents, diluents, excipients such as sucrose and glucose,
colours such as tartrazine and Sunset Yellow FCF, and is buffered with
calcium carbonate and sodium phosphate, etc.
In preliminary experiments, these compounds were tested with the
ninhydrin reagent and found not to interfere in the assay of
penicillins.
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Method of quantitative estimation of aceclofenac from
tablet formulation using FolinCiocalteu reagent.
Aceclofenac forms a blue coloured chromogen with the
reagent, which shows absorbance maxima at 642.6 nm
and linearity in the concentration range of 80160 gmL
-1

of drug (Singhvi and Goyal, 2007).

The extracted complex with orange G shows absorbance
maxima at 481 nm and linearity in the concentration
range of 1080 g mL
-1
. The extracted complex with
naphthol green shows absorbance maxima at 633.6 nm
and linearity in the concentration range of 0.21.0 g mL
-1
(Goyal and Singhvi, 2006).
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