Spectrochimica Acta Part A 60 (2004) 3007–3012

Spectrophotometric determination of ampicillin sodium in pharmaceutical

products using sodium 1,2-naphthoquinone-4-sulfonic as the
chromogentic reagent
Lixiao Xu, Huaiyou Wang∗ , Yan Xiao
Chemical Engineering and Materials Science, College of Chemistry, Shandong Normal University, Jinan 250014, PR China

Received 25 September 2003; accepted 24 February 2004

Abstract

Spectrophotometric determination of ampicillin sodium is described. The ampicillin sodium reacts with sodium 1,2-naphthoquinone-4-sul-
fonic in pH 9.00 buffer solution to form a salmon pink compound, and its maximum absorption wavelength is at 463 nm, ε463 = 1.14 × 104 .
The absorbance of ampicillin sodium from 2.0–80 ␮g ml−1 obeys Beer’s law. The linear regression equation of the calibration graph is
C = 40.24A − 2.603, with a linear regression correlation coefficient is 0.9997, the detection limit is 1.5 ␮g ml−1 , recovery is from 97.23 to
104.5%. Effects of pH, surfactant, organic solvents, and foreign ions on the determination of ampicillin sodium have been examined. This
method is rapid and simple, and can be used for the determination of ampicillin sodium in the injection solution of ampicillin sodium. The
results obtained by this method agreed with those by the official method (HPLC).
© 2004 Elsevier B.V. All rights reserved.

Keywords: Ampicillin sodium; Sodium 1,2-naphthoquinone-4-sulfonic; Spectrophotometry

1. Introduction colored reagent for determination of ampicillin sodium

by spectrophotometry has not been reported. This pa-
Ampicillin sodium is one of the important penicillin per reports a rapid spectrophotometric method for de-
antibiotics used to treat or prevent bacterial infections. In termining the content of ampicillin sodium in injection,
view of its pharmacological importance, considerable work which is based on a replace reaction [17], i.e. sodium
has been done for its detection and quantification. The 1,2-naphthoquinone-4-sulfonic acid reacts with amino of
content of ampicillin sodium was determined by HPLC ampicillin sodium molecule to form a salmon pink com-
in United States Pharmacopoeia, British Pharmacopoeia pound. λmax of the compound is at 463 nm. The reaction
and Chinese Pharmacopoeia [1–3]. Various analytical equation reads as follows:
techniques have been employed for the determination of
ampicillin sodium in serum, plasma, urine, pharmaceutical O +
O O Na
dosage and so on, such as HPLC method [4–7], spec- O O
O
trophotometry [8–11], spectrofluorimetry [12], colorimetric + N
method [13], capillary electrophoresis [14] and micro-
biological assay [15,16]. HPLC method can be used to so3 H NH S
NH2
determine the ampicillin sodium in the serum, but it has
+
poor reproducibility [4–7] (R.S.D.: 2.6–13.9%). H. Mah- O O O Na
goub reported the determination of ampicillin sodium by O o
spectrophotometric method [8–11], but it’s inconvenient. N
NH
Use of 1,2-naphthoquinone-4-sulfonic acid sodium as a
NH S
O
∗ Corresponding author. Tel.: +86-531-6619466;

fax: +86-531-2615258.
E-mail address: wanghuaiyou@sdnu.edu.cn (H. Wang). Product I

1386-1425/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2004.02.018
3008 L. Xu et al. / Spectrochimica Acta Part A 60 (2004) 3007–3012
Fig. 1. Absorbance spectra of product I. Concentration of ampicillin
sodium: 40 ␮g ml−1 . Line (a): absorption spectrum of product I against
reagent blank. Line (b): absorption spectrum of sodium 1,2-naphtho-
quinone-4-sulfonic against water. Line (c): absorption spectrum of ampi-
cillin sodium against water blank.
As we all know, druggery is mostly organic compound
that possesses strong absorption in the range of ultraviolet.
Obviously, when druggery is determined in the range of ul-
traviolet, interference will occur if absorption of other com-
pounds lies near the absorption wavelength of the druggery.
Therefore, many analytical chemists try to find compendious
methods for the determination of ampicillin sodium in phar-
maceutical products and biological samples. The maximum
absorption wavelength of product I was at 463 nm, which
shifted 263 nm to long wave compared to the maximum ab-
sorption wavelength of ampicillin sodium (200 nm) (see line
c, Fig. 1). What is more, because ampicillin sodium can be
determined in the range of visible light, much potential in-
terference may be avoided in the determination of ampicillin
sodium of biological samples and hemanalysis.
The principal advantage of our method is that the maxi-
mum absorption wavelength of ampicillin sodium shifts to
the range of visible light from the range of ultraviolet light,
so that ampicillin sodium may be determined in the range
of visible light. In addition, the method is simple, and can
be used for determining ampicillin sodium in the injection
solution of ampicillin sodium. The results obtained by the
method agreed with those of the official method [3].
2. Experimental
2.1. Apparatus
All of the spectrophotometric measurements were made
with a Shimadzu UV-265 ultraviolet visible recording spec-
trophotometer with matched 1 cm quartz cells. In order to
compare all spectrophotometric measurements and ensure
reproducible experimental conditions, the UV-265 spec-
trophotometer was checked daily. All pH measurements
were made with a pHs-3C digital pH-meter (Shanghai
Lei Ci Device Works, Shanghai, China) with a combined
glass-calomel electrode.
2.2. Reagents
Ampicillin sodium (99.89%) was purchased from Lu
Kang Drugs and Reagents Company (Shandong, China) and
used as standards. Ampicillin sodium was assayed, accord-
ing to English Pharmacopoeia [1] by HPLC. All reagents
were of analytical-reagent grade, unless stated otherwise.
Double-distilled water was used in all experiments.
2.2.1. Stock standard solution of ampicillin sodium
(1000 µg ml−1 )
An accurately weighed 0.10 g standard sample of ampi-
cillin sodium was dissolved in water, transferred into a
100 ml standard flask and diluted to the mark with water and
mixed well. The solution was stable for at least 2 months at
4 ◦ C.
2.2.2. Sodium 1,2-naphthoquinone-4-sulfonic (from Sigma)
solution, 0.2% (w/v)
A weighed 0.20 g of sodium 1,2-naphthoquinone-
4-sulfonic was dissolved in water, transferred into a 100 ml
standard flask and diluted to the mark with water and mixed
well. The solution was stable for at least 2 weeks at 4 ◦ C.
2.2.3. S φ rensen buffer solution (pH 9.00
Na2 HPO4 –KH2 PO4 )
A total of 1/15 mol l−1 Na2 HPO4 and 1/15 mol l−1
KH2 PO4 were mixed well, according to volume ratio of
63:1.
2.2.4. Cetylpyridine bromide (CTPB, 0.1%, (w/v))
An accurately weighed 0.25 g of CTPB was dissolved in
water, transferred into 250 ml standard flask, diluted to the
mark with water and mixed well. Triton X-120 and sodium
laurylsulfonate were prepared, according to the same proce-
dure.
2.3. Determination of ampicillin sodium by HPLC
According to The English Pharmacopoeia [1], ampicillin
sodium was determined. The result showed that the content
of ampicillin sodium was 99.89%; this sample was used as
standard sample. In addition, 20.0 ml of injection solution
of ampicillin sodium (same batch number) was measured,
according to the same procedure.
2.4. Procedure
A 1.0 ml of 0.2% sodium 1,2-nathoquinone-4-sulfonic
was transferred into a 10 ml standard flask, 0.1 ml ethanol,
1.0 ml of 0.1% CTPB, 0.40 ml of 1000 ␮g ml−1 ampicillin
sodium and 2.0 ml pH 9.00 Na2 HPO4 –KH2 PO4 buffer so-
lution were added sequentially, diluted to the mark with
 L. Xu et al. / Spectrochimica Acta Part A 60 (2004) 3007–3012 3009

water and mixed well. The absorbance of the solution
was measured at 463 nm against a reagent blank prepared
with the same reagent concentration, but no ampicillin
sodium.
3. Result and discussion
3.1. Absorption spectrum of product I
According to the procedure, the absorption spectrum of
product I was recorded. As can be seen (Fig. 1), the max-
imum absorption wavelength of product I was at 463 nm
against a reagent blank (line a). Obviously, the absorption
spectrum of reagent blank overlapped partially at 463 nm
against water blank although this superposition was little.
Fortunately, this interference can be eliminated, when the
measurement was carried out at 463 nm against the reagent
blank. An excellent linear relationship existed between the
absorbance and the concentration of the ampicillin sodium
(R = 0.9997).
In addition, the ampicillin sodium solution was colorless,
it has no absorption in the range of 250–500 nm (line c), and
its maximum absorption wavelength was at 200 nm. There-
fore, ampicillin sodium can be determined conveniently at
463 nm against a reagent blank.
3.2. Influence of pH
According to the procedure, effect of pH on the ab-
sorbance of product I was tested, comparative tests at
various pH values showed that the absorbance of product I
changes with pH. The variation of the pH from 1.00 to 13.0
was investigated. The result showed in Fig. 2.
It can be seen (Fig. 2) that the absorbance of product I
increased in the range of pH 1.00–8.60, and then reduced
sharply, when pH above 9.40. The tests showed that the
absorbance of product I was maximal at pH 9.00 and an
excellent linear relationship existed between the absorbance
of product I and the concentration of ampicillin sodium at pH
9.00. Therefore, pH 9.00 Na2 HPO4 –KH2 PO4 buffer solution
was selected to control the pH of system. Obviously, buffer
solution had no effect on the determination of ampicillin
sodium.
3.3. Effect of surfactant
The effects of the surfactants, CTPB, sodium laurylsul-
fonate and Triton X-100, were tested on the determination
of ampicillin sodium. As we all know that the surfactant was
widely used as sensitizing reagent in the spectrophotome-
try. Because of the formation of micelle, the absorbance was
enlarged in the presence of the surfactant. The tests showed
that CTPB was a sensitizing reagent in the determination of
ampicillin sodium. The result was showed in Fig. 3. As can
be seen (Fig. 3), the absorbance increased with the amount
of CTPB below 1.0 ml. However, the absorbance decreased,
when the amount of CTPB above 1.0 ml. It indicated that
the 1.0 ml of 0.1% CTPB reacted with product I to form mi-
celle. Triton X-100 and sodium laurylsulfonate were aban-
doned because of no formation of micelle. Therefore, 1.0 ml
CTPB was selected as the optimum.
3.4. Effect of organic solvents
Organic solvents, including methanol, ethanol, dimethyl-
sulfoxide (DMSO), dimethylformaide (DMF), were tested
for determining ampicillin sodium. The result was shown in
Fig. 4.

0.40

0.35

0.30

0.25

A
0.20

0.15

0.10

0.05
0 2 4 6 8 10 12
pH
Fig. 2. Effect of pH on absorbance of product I. Concentration of ampicillin sodium: 40 ␮g ml−1 .
3010 L. Xu et al. / Spectrochimica Acta Part A 60 (2004) 3007–3012

0.4 0.6

B
F 0.5
F
0.3 B C
D 0.4 D

A
0.2 0.3
A

0.2
0.1

0.1
0.0 0.2 0.4 0.6 0.8 1.0

0.0
0.0 0.5 1.0 1.5 2.0
v(ml)
Fig. 3. Effect of surfactant on absorbance of product I. Concentration
of ampicillin sodium: 40 ␮g ml−1 . Line (F): sodium laurylsulfonate; Line
(B): Triton X-100; Line (D): CTPB.
It was found (Fig. 4) that the absorbance of product I de-
creased with the presence of DMF and DMSO. But the ab-
sorbance was maximum in presence of 0.1 ml ethanol and
then decreased with a rise in ethanol. In addition, methanol
was abandoned because of its toxicity. Obviously, 0.1 ml
ethanol is more effective to increase the absorbance of the
product I, therefore, 0.1 ml ethanol was selected as the op-
timum.
v(ml)
Fig. 4. Effect of organic solvents on the absorbance of product I. Con-
centration of ampicillin sodium: 40 ␮g ml−1 . Line (B): DMSO; Line (F):
DMF; Line (C): ethanol; Line (D): methanol.
der the different volume of sodium 1,2-naphthoquinone-4-
sulfonic, the result was showed in Fig. 5. As can be seen
(Fig. 5), the absorbance of the mixture increased with the
rise of sodium 1,2-naphthoquinone-4-sulfonic in the range
of 0.2–0.6 ml; the absorbance of the mixture solution was
stable from 0.6 to 0.8 ml of sodium 1,2-naphthoquinone-4-
sulfonic, but it decreased above 1.0 ml. So 1.0 ml sodium
1,2-naphthoquinone-4-sulfonic was enough in this paper.
3.6. Effect of heating time

3.5. Effect of concentration of sodium
1,2-naphthoquinone-4-sulfonic
According to procedure, the absorbance of mixture so-
lution of 40 ␮g ml−1 ampicillin sodium was measured un-
According to the procedure, the absorption of the mixture
solution of 40 ␮g ml−1 ampicillin sodium was measured af-
ter heated in boiling water bath for different time; tests found
that the absorbance decreased when heated. Therefore, room
temperature was recommended.

0.5

0.4

0.3
A

0.2

0.1
0 1 2 3 4

v(ml)
Fig. 5. Effect of concentration of sodium 1,2-naphthoquinone-4-sulfonic. Concentration of ampicillin sodium: 40 ␮g ml−1 .
 L. Xu et al. / Spectrochimica Acta Part A 60 (2004) 3007–3012
0.30
0.25
0.20
0.15
A
0.10
0.05
0.00
0 20
Fig. 6. Effect of standing time on determining ampicillin sodium. The concentration of ampicillin sodium: 40 ␮g ml−1 .
3.7. Formation and stability of product I
According to the procedure, the absorbance of the mixture
solution of 40 ␮g ml−1 ampicillin sodium was measured af-
ter standing for different time. The results were showed in
Fig. 6.
From Fig. 6, it can be seen that ampicillin sodium reacts
immediately with sodium 1,2-naphthoquinone-4-sulfonic at
room temperature. The absorbance was no longer changed
after standing for 50 min. Therefore, 50 min was selected as
the optimum. In addition, the absorbance of the procedure I
was stable at least 2 h at room temperature.
3.8. Order of reagents
According to the procedure, the order of the addition of the
reagents was tested. It was found that the excellent order was
sodium 1,2-naphthoquinone-4-sulfonic acid, ethanol, CTPB
and ampicillin sodium. The buffer solution was added finally.
3.9. Effect of foreign ions
A systematic study was carried out on the effects of com-
monly found ions on the determination of 40 ␮g ml−1 ampi-
cillin sodium. A 500 mg ml−1 level of each potentially in-
terfering ion was tested first. If interference occurred, the
ratio was reduced progressively, until interference ceased.
The tolerance level was defined as an error not exceeding
±5% in the determination of the analyst. Results were sum-
marized in the Table 1.
3.10. Calibration graph of ampicillin sodium
Under the selected conditions, a linear relationship be-
tween the absorbance of compound and the concentra-
tion of ampicillin sodium was obtained in the range of
2.0–80 ␮g ml−1 , the linear regression equation of the cal-
ibration graph is C = 40.24A − 2.603, with a correlation
3011
40 60 80 100
T(min)
Table 1
Effect of foreign ions on the determination of ampicillin sodium
Foreign ions or species Tolerance level
(␮g ml−1 )
K+ , Na+ , NO3 − , PO4 − , Cl− , H2 PO4 − , SO4 2− 500
CO3 2− 100
Ba2+ 50
Mg2+ 25
Cu2+ , Ni2+ , Zn2+ 5
coefficient of linear regression of 0.9997. The unit of C is
␮g ml−1 and A is the absorbance in the regression equation.
In addition, based on the absorbance and concentration of
the ampicillin sodium, molar absorptivity of product I at
463 nm, ε463 = 1.14 × 104 l mol−1 cm−1 was estimated.
3.11. Reproducibility and detection limit
A portion of the sample solution of ampicillin sodium
was transferred into a 10 ml volumetric flask. The reagents
were added and measured, according to the procedure. This
sample solution was measured 10 times (n = 10); the mean
value was 40.12 ␮g ml−1 with a R.S.D. of 4.2%. According
to the procedure, a reagent blank was measured 10 times
(n = 10), the detection limit was 1.5 ␮g ml−1 , based on the
blank plus three times the standard deviation of the blank
[18].
3.12. Comparison of spectrophotometric method and
HPLC method for analysis of sample
The injection solutions of ampicillin sodium (different
batch number) were diluted to different concentrations with
water. These solutions were used as sample solutions. Ac-
cording to the procedure, the sample solutions were mea-
sured. The results are shown in Table 2. As can be seen
(Table 2), the results by spectrophotometic method agreed
with those of the HPLC [1]. The experimental data of the
3012 L. Xu et al. / Spectrochimica Acta Part A 60 (2004) 3007–3012

Table 2 tometry. The results obtained agreed with those of the HPLC
Determination of ampicillin sodium sample method. The principal advantage of the proposed method
Sample Spectrophotometric HPLC method (mg ml−1 ) was that ampicillin sodium could be determined in the visi-
no. method (mg ml−1 ) ble light, so that the potential interference may be avoided.
1 9.75 ± 0.03 9.72 ± 0.21 In addition, the method was simple and wider linear range.
2 14.60 ± 0.01 14.72 ± 0.13
3 19.35 ± 0.12 19.17 ± 0.23
4 24.20 ± 0.07 24.35 ± 0.32
References

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