A novel method for the spectrophotometric determination of

cefradine by using sodium nitroprusside as chromogenic reagent

Hua Zhang a,b, Ling Li Wu a,c, Quan Min Li a,*, Xin Zhen Du b

a College of Chemistry and Environmental Science, Henan Normal University, Xinxiang 453007, Chinab College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070, China

c Henan Radio and Television University, Zhengzhou 453008, China

Received 2 June 2008

Abstract

A novel method is developed for the determination of cefradine by using sodium nitroprusside as chromogenic reagent. The

experiment indicates that a russety product is formed by the reaction of cefradine with sodium nitroprusside in basic solution, and

the maximum absorption wavelength (lmax) of russety product is 505 nm. And the sensitization of tetradecyl benzyl dimethyl

ammonium chloride for the reaction of cefradine with sodium nitroprusside is remarkable. The apparent molar absorption

coefficient (e505) is 2.81 � 103 L/mol cm. The linear equation is A = 0.0657 + 0.00804C (mg/mL) in the range of 1.50–55.0 mg/mL

of cefradine with a correlation coefficient r = 0.9992, and the detection limit is 1.38 mg/mL. This method has been applied to

determine cefradine in capsule and tablet samples.

# 2008 Quan Min Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Keywords: Cefradine; Sodium nitroprusside; Chromogenic reagent; Spectrophotometry

High-performance liquid chromatography [1], flow injection analysis [2], fluorimetric method [3], molecularly

imprinted solid phase extraction method [4], etc. have been used for the determination of cefradine. In this paper, a

novel rapid, simple and economic method is developed for the determination of cefradine by using sodium

nitroprusside as chromogenic reagent. The experiment indicates that cefradine could react with sodium nitroprusside

by nucleophilic substitution reaction in basic solution, and N-nitrosamines compound is formed. And tetradecyl

benzyl dimethyl ammonium chloride (Zeph) can sensitize the reaction of cefradine with sodium nitroprusside. By

determining the absorbance of solution, the amount of cefradine can be obtained. This method has been applied to

determine cefradine in capsule and tablet samples.

1. Experimental

1.1. Reagents and apparatus

All reagents used were of analytical grade. Cefradine: 1.000 g/L, 10% (m/v) sodium nitroprusside (A.R., Beijing

Chemical Plant, Beijing, China), 10% (m/v) potassium ferricyanide (A.R., Beijing Chemical Plant, Beijing, China),

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Chinese Chemical Letters 19 (2008) 1470–1474

* Corresponding author.

E-mail address: mercury6068@126.com (Q.M. Li).

1001-8417/$ – see front matter # 2008 Quan Min Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

doi:10.1016/j.cclet.2008.09.014

10% (m/v) sodium hydroxide (A.R., TianJin Chemical Plant, Tianjin, China), 0.1% (m/v) tetradecyl benzyl dimethyl

ammonium chloride (Zeph) (A.R., Tokyo Kasei Kogyo Co., Tokyo, Japan), a model T6 UV–vis spectrophotometer

(Beijing Purkinje General Instrument Co., Beijing, China).

1.2. Preparation of chromogenic reagent (SNP)

Respectively, take 100 mL of 10% sodium nitroprusside, 10% potassium ferricyanide and 10% NaOH to a plastic

container, and then dilute them to 600 mL. The concentration of sodium nitroprusside and that of NaOH are

5.6 � 10�2 mol/L and 0.40 mol/L in chromogenic reagent (SNP), respectively.

1.3. Determination of cefradine

Add 1.00 mL of 500 mg/mL cefradine, 4.00 mL of SNP, and 1.60 mL of 0.1% Zeph to a 10 mL color comparison

tube. Then dilute to scale with distilled water and mix well. Stand for 40 min at 30 8C. The absorbance of the solution

is measured at 505 nm against reagent blank.

H. Zhang et al. / Chinese Chemical Letters 19 (2008) 1470–1474 1471

Scheme 1.

Fig. 1. Absorption spectrum: (a) cefradine against water; (b) SNP-Zeph (reagent blank) against water; (c) SNP-cefradine against reagent blank; (d)

SNP-cefradine-Zeph against reagent blank, the conditions as mentioned in the determination of cefradine section.

2. Results and discussion

2.1. Absorption spectrum

The absorption spectrum is shown in Fig. 1. The absorption peak of product I (see Scheme 1) is the same as that of

product II (see Scheme 1) at 505 nm, and the absorbance of product II is much bigger than that of product I, which

indicates that the sensitization of Zeph for the reaction of SNP with cefradine is remarkable. Corresponding to the

blank reagent solution, there is no maximum absorption peak between 400 and 650 nm.

2.2. Influence of NaOH

The results show that the absorbance is decreased with the rise of the amount of NaOH (Fig. 2). This is due to fact

that OH� can react with the electron-deficient center group (–N O) of sodium nitroprusside anion ([Fe(CN)5NO]2�)

to form [Fe(CN)5NO2H]3� [5], which results in that the reaction degree of sodium nitroprusside with cefradine is

declined, and the absorbance of solution is reduced.

2.3. Influence of surfactants

When the concentration of cefradine is 50 mg/mL (1.43 � 10�4 mol/L), 1.43 � 10�4 mol/L of product I is

formed (see Scheme 1). Fig. 3 shows that emulsor OP-10 and sodium laurylsulfonate (SLS) almost have no effect

on the determination of cefradine. Remarkably, the absorbance is increased with the increase of the amount of

H. Zhang et al. / Chinese Chemical Letters 19 (2008) 1470–14741472

Fig. 2. Influence of NaOH. NaOH: 1.5 mol/L, other conditions as mentioned in the determination of cefradine section.

Fig. 3. Effect of surfactant. 0.1% (m/v) Zeph, OP, SLS, other conditions as mentioned in the determination of cefradine section.

Zeph. The absorbance is maximal when the amount of Zeph is 1.60 mL (4.38 � 10�4 mol/L), and then it is

unchanged with any further increase of amount of Zeph. It is due to reason that 4.38 � 10�4 mol/L of Zeph can

react with 1.43 � 10�4 mol/L of product I completely to form product II (see Scheme 1), thereby 1.60 mL of Zeph

is selected. And the reaction stoichiometric ratio of product I with Zeph is near to 1:3 (1.43 � 10�4 mol/

L:4.38 � 10�4 mol/L).

2.4. Influence of the amount of SNP, the temperature and standing time

The absorbance is maximal when the amount of SNP, the temperature and standing time are 4.00 mL, 30 8C and

40 min, respectively. And the maximal absorbance can be stable at least 60 min at this condition.

2.5. Calibration curve

Good calibration curve of the concentration of cefradine versus the absorbance is observed (see Fig. 4). The linear

equation is found to be A = 0.0657 + 0.00804C (mg/mL) in the range of 1.50–55.0 mg/mL of cefradine with a

correlation coefficient r = 0.9992. The detection limit is 1.38 mg/mL, the apparent molar absorption coefficient e505 is

2.81 � 103 L/mol cm.

2.6. Reaction mechanism

Due to electrophilic of nitroso-group, the nitroso-group of sodium nitroprusside can react with nucleophile to form

the colored N-nitrosamines compounds by nucleophilic substitution reaction in basic solution [6]. Similarly, amine of

cefradine can react with sodium nitroprusside by nucleophilic substitution reaction to form N-nitrosamines

compound (product I). The reaction stoichiometric ratio of cefradine with sodium nitroprusside is 1:2 proved by the

continuous variation method of equivalent mole and the slope ratio method. When Zeph is added, Zeph is likely to

coordinate with product I to form a bigger conjugated system (product II). And the reaction stoichiometric ratio of

product I with Zeph is 1:3 as mentioned above (see Section 2.3). It seems reasonable that the reaction mechanism is as

given in Scheme 1.

2.7. The study of the kinetics property

The concentration of sodium nitroprusside is much more than that of cefradine in solution, and the concentration of

sodium nitroprusside variation is relatively lesser, hence this reaction can be regarded as a pseudo-first order reaction,

and the reaction rate equation can be express as d[reaction product]/dt = k0[cefradine]. The apparent rate constant

(k030 �C) can be obtained by initial rate method, and k030 �C is 5.08 � 10�4 s�1. The apparent activation energy (Ea) of the

determination cefradine can be calculated, and it is 20.9 kJ/mol.

H. Zhang et al. / Chinese Chemical Letters 19 (2008) 1470–1474 1473

Fig. 4. Calibration curve. The conditions as mentioned in the determination of cefradine section.

2.8. Influence of potential interference

The effects of some familiar excipients, diluents substances and common ions on the determination of cefradine are

investigated. The tolerance levels are defined as an error less than �5%. A conclusion is drawn as follows: 2%

(amylum), 10 mg/mL (citric acid) and large numbers of Zn2+, K+, Ca2+, Na+, Mg2+, SO42�, NO3

�, Cl� and Br� do not

affect the determination. But dextrose (5%) and arginine (10 mg/mL) affect the determination.

2.9. Sample analysis

In order to demonstrate the feasibility of the described method, the determination of cefradine is carried out in its

capsule and tablet samples. The results are presented in Table 1, and agree well with that obtained by the previous

method [1].

References

[1] M.C. Hsu, Y.S. Lin, H.C. Chung, J. Chromatogr. A 692 (1995) 67.

[2] U.J. Meyer, Z.L. Zhi, E. Loomans, Analyst 124 (1999) 1605.

[3] B. Tang, X.W. He, H.X. Shen, Chin. J. Anal. Chem. 22 (1994) 1089.

[4] S.G. Wu, E.P.C. Lai, P.M. Mayer, J. Pharm. Biomed. Anal. 36 (2004) 483.

[5] N.E. Katz, M.A. Blesa, J.A. Olabe, J. Inorg. Nucl. Chem. 42 (1980) 581.

[6] O.R. Leeuwenkamp, W.P. Van Benneekom, A. Bult, Analytical Profile of Drug Substances, Academic Press, New York, 1986.

H. Zhang et al. / Chinese Chemical Letters 19 (2008) 1470–14741474

Table 1

Determination results of samples and recovery (n = 5, t0.05, 4 = 2.78)

Sample Samples contents

(mg/mL)

Proposed method

(mg/mL)

Pharmacopeia

method (mg/mL) [1]

Added

(mg/mL)

Found

(mg/mL)

Recovery (%) R.S.D

(%) n = 5

Quality

percent (%)

1 250.0 250.5 249.9 50.00 301.1 101 1.1 99.6

2 250.0 249.7 249.9 100.0 347.4 97.7 0.96 99.7

3 250.0 250.3 250.2 150.0 401.3 101 0.83 101

4 250.0 249.9 250.1 200.0 449.2 99.7 1.4 99.9

5 250.0 250.1 249.8 250.0 500.6 100.2 0.67 100.3