Sensitive Chemiluminescence Determination of Thirteen Cephalosporin Antibiotics with LuminolCopper(II) Reaction

JIANXIU DU* and HONG LI

Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Materials Science, Shaanxi Normal University, Xian 710062, China (J.D.); and Weinan Vocational and Technical College, Weinan 714000, China (H.L.)

A new chemiluminescence reaction, the luminolCu2 reaction, was investigated for the determination of thirteen (13) cephalosporin antibiotics, namely cefalexin, cefadroxil, cefradine, cefazolin sodium, cefaclor, cefuroxime axetil, cefotaxime sodium, cefoperazone sodium, ceftriaxone sodium, ceftazidime, cefetamet pivoxil hydrochloride, cexime, and cefpodoxime. It was found that, without adding any special oxidant, strong chemiluminescent (CL) signal could be produced from the reaction of the alkaline luminol with the above-mentioned antibiotics in the presence of Cu2. The experimental conditions for the reaction were carefully optimized with ow-injection mode. The detection limits are 0.3 ng/mL cefalexin, 3 ng/mL cefadroxil, 0.3 ng/mL cefradine, 0.02 lg/mL cefazolin sodium, 0.8 ng/mL cefaclor, 0.02 lg/mL cefuroxime axetil, 5 ng/ mL cefotaxime sodium, 0.02 lg/mL cefoperazone sodium, 0.8 ng/mL ceftriaxone sodium, 1 ng/mL ceftazidime, 0.08 ng/mL cefetamet pivoxil hydrochloride, 0.8 ng/mL cexime, and 2 ng/mL cefpodoxime. The proposed method was validated by direct application to commercial formulations and spiked milk samples containing cefradine. A possible reaction mechanism is also discussed. Index Headings: Cephalosporin antibiotics; Chemiluminescence; Flow injection.

INTRODUCTION
Cephalosporin antibiotics, referred to as the b-lactam antibiotics, are the most frequently prescribed class of antibiotics against infections. They have signicant activity against both gram-positive and gram-negative bacteria.1 Cephalosporin antibiotics are grouped into generations based on their spectrum of antimicrobial activity. The therapeutic importance of these antibiotics requires the development of sensitive, rapid analytical methods for industrial quality control and clinical monitoring. A wide variety of analytical methods have been reported for the determination of cephalosporin antibiotics in pharmaceutical preparations and in biological uids, including spectrophotometry,24 uorimetry,5,6 electrochemical methods,7,8 liquid chromatography,911 and capillary electrophoresis.12,13 Recently, a comprehensive review of the analysis of cephalosporin antibiotics was presented by El-Shaboury.14 The chemiluminescence (CL) method with the advantages of simplicity, rapidity, and sensitivity has also been explored for the analysis of cephalosporin antibiotics.1528 The involved CL systems and the analytical merits of the reported CL methods for the determination of cephalosporin antibiotics are summarized in Table I. Luminol (5-amino-2,3-dihydrophalazine-1,4-dione), one of the best-known and widely used liquid-phase CL agents, is oxidized to produce CL in the presence of an oxidant in an alkaline solution. Many familiar oxidants, such as H2O2,
Received 2 March 2010; accepted 2 July 2010. * Author to whom correspondence should be sent. E-mail: jxdu@snnu. edu.cn.

K3Fe(CN)6, KIO4, and KMnO4, have been employed for this purpose. These CL reactions are the basis for the determination of a variety of both inorganic and organic species, which act as a catalyst, an enhancer, or an inhibitor during the reaction. Unfortunately, these determinations suffer from high background signal because strong CL emission usually accompanies the reaction between luminol and the oxidant. This, to some extent, became a hindrance in improving the sensitivity of detection. We here found that CL was directly produced by the reaction of cephalosporin antibiotics, including cefalexin, cefadroxil, cefradine, cefazolin sodium, cefaclor, cefuroxime axetil, cefotaxime sodium, cefoperazone sodium, ceftriaxone sodium, ceftazidime, cefetamet pivoxil hydrochloride, cexime, and cefpodoxime, with luminol in an alkaline condition without adding any special oxidants. In the presence of Cu2 the CL signal was signicantly enhanced. This reaction system has been established as a ow-injection analysis for the determination of these antibiotics. The experimental conditions for the reaction are optimized, and the possible reaction mechanism is discussed. A direct application to commercial drug formulations and spiked milk samples is presented.

EXPERIMENTAL
Apparatus. Chemiluminescent measurements were carried out on an IFFM-D type ow-injection CL analyzer (Xian Remex Analysis Instrument Co., Ltd, China). PTFE tubing (0.8-mm inner diameter) was used as the connection material in the ow system. CL spectra were scanned on a 970CRT uorescence spectrophotometer (Shanghai Analysis Instrument Plant) with the excitation source shutter closed. Absorption spectra were taken on a TU-1901 ultravioletvisible (UV-Vis) spectrophotometer (Beijing Currency Instrumental Ltd, China). pH measurements were made with a pHS-3C(A) pH meter (Shanghai Dapu Instruments Co., Ltd, China). Chemicals. All chemicals were of analytical grade; doubly distilled deionized water was used to prepare the solutions. The standards of cephalosporin antibiotics were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Luminol was synthesized by the Institute of Analytical Science of Shaanxi Normal University (Xian, China). CuSO45H2O and other reagents were purchased from Xian Chemical Reagent Factory (Xian, China). Stock solutions of cephalosporin antibiotics (50.0 lg/mL) were prepared by dissolving 0.0500 g of each standard in water. These stock solutions were stored at 4 8C in a refrigerator and protected from light. Working solutions of cephalosporin antibiotics were freshly prepared by diluting the corresponding stock solution with water. Luminol solution (5.0 3 104 mol/L) was prepared by diluting the appropriate amount

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TABLE I.

CL methods reported for the determination of cephalosporin antibiotics. CL systems K3Fe(CN)6luminol system H2O2luminol system KIO4luminol system Detection limits 0.04 lg/mL 0.08 lg/mL 0.1 lg/mL 6.0 ng/mL 0.9 ng/mL 0.4 ng/mL 0.01 lg/mL 0.01 lg/mL 0.025 lg/mL 1.6 lg/mL 0.03 lg/mL 0.03 lg/mL 0.08 lg/mL 0.08 lg/mL 0.08 lg/mL 0.06 lg/mL 5 ng/mL 0.096 lg/mL 0.10 lg/mL 0.025 lg/mL 0.01 lg/mL 2 ng/mL 2 ng/mL 0.05 lg/mL 8 ng/mL 0.03 lg/mL 0.035 lg/mL 0.038 lg/mL 0.039 lg/mL 0.04 lg/mL 0.06 lg/mL milk granules capsules, tablets Matrix Ref. 15 16 17

Analytes Cefradine Cefadroxil Cefalexin Cefaclor Cefalexin Cefaclor Cefradine Cefadroxil Ceftriaxone sodium Cefazolin sodium Cefoxitin Cefazolin Cephalexin Cefadroxil Cefaclor Cefoperazone Cefprozil Cephalexin Cephradine Cefadroxil Cefalexin Cefadroxil Cefazolin sodium Cefadroxil Ceftriaxone sodium Cefradine Cefuroxime sodium Cefotaxime sodium Ceftriaxone sodium Cefoperazone sodium Cephalexin

KMnO4polyphosphoric acid system KMnO4polyphosphoric acid system KMnO4tris(2,2 0 -bipyridyl) ruthenium(II) system

dosage forms injections injections, capsules, oral suspension

18 19 20

KMnO4tris(2,2 0 -bipyridyl) ruthenium(II) system KMnO4HCHO system KMnO4HCHO system KMnO4glyoxal system KMnO4quinine system KMnO4acridine orange system Ce(IV)rhodamine 6G system

tablets capsules capsules injections, capsules, tablets capsules, suspension, spiked urine, and plasma samples powder injections synthetic samples

21 22 23 24 25 26 27

Ce(IV)rhodamine 6G system

capsules, tablets

28

of 1.0 3 102 mol/L luminol stock solution (prepared in 1.0 3 102 mol/L NaOH solution) with 0.1 mol/L carbonate buffer solution to provide a nal pH of 12.5. A 5.0 3 105 mol/L Cu2 solution was prepared by dissolving the appropriate amount of CuSO45H2O in water. Procedure. Figure 1 depicts the schematic diagram of the CL ow system employed. Flow lines were connected with luminol solution, Cu2 solution, and cephalosporin antibiotic solution, respectively. A peristaltic pump was started to wash the ow system to offer a stable baseline. Then 50 lL of luminol solution was injected into the merged stream of Cu2 solution with cephalosporin antibiotic solution with the aid of a six-way valve for producing CL. The CL emission produced was collected by a CR105 photomultiplier tube (Beijing Hamamatsu Photo Techniques Inc.). The concentration of cephalosporin antibiotics was quantied by the increment in the CL intensity, calculated as DI I Ib, where I is the CL

signal in the presence of cephalosporin antibiotics and Ib is the blank signal.

RESULTS AND DISCUSSION

Cefradine was selected as a typical analyte for the investigation of the CL reaction mechanism and the optimization of the experimental conditions. Chemiluminescence of Alkaline Luminol and Cefradine in the Presence of Cu2. The CL of alkaline luminol and cefradine with and without Cu2 was thoroughly investigated using a batch method. As shown in Fig. 2, no signicant difference has been observed by injecting 1.0 mL alkaline solution of 5.0 3 104 mol/L luminol into 1.0 mL of H2O (peak a) and into 1.0 mL of 5.0 3 105 mol/L Cu2 solution (peak c), which suggested no CL reaction took place between alkaline luminol and Cu2. The replacement of H2O by 1.0 lg/mL cefradine solution gave a weak CL emission (peak b). This phenomenon indicated that a direct CL reaction could be

FIG. 1. Schematic diagram of the CL ow system.

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FIG. 2. CL peaks of the injection of 1.0 mL alkaline solution of 5.0 3 104 mol/L luminol into 1.0 mL of (a) H2O, (b) 1.0 lg/mL cefradine solution, (c) 5.0 3 105 mol/L Cu2 solution, and (d) the mixture of 1.0 lg/mL cefradine with 5.0 3 105 mol/L Cu2.

FIG. 4. UV-Vis absorption spectra of (a) 5.0 3 105 mol/L Cu2 solution, (b) 10.0 lg/mL cefradine solution, and (c) the mixture of 10.0 lg/mL cefradine and 5.0 3 105mol/L Cu2.

occurring between alkaline luminol and cefradine. When alkaline luminol solution was injected into the mixture of 1.0 lg/mL cefradine with 5.0 3 105 mol/L Cu(II), a very strong CL signal was recorded (peak d), which indicated Cu2 has a catalytic effect on the weak CL reaction between alkaline luminol and cefradine. It was found that the rate of the luminolCu2cefradine CL reaction was very fast; from reagent mixing to the peak maximum, only 3 s was needed and it took 4 s for the signal to decline to baseline. Discussion of the Chemiluminescence Reaction Mechanism. The CL mechanism of the luminol system has been extensively investigated and many research works have conrmed that 3-aminophthalate anion was the emitter in the luminol system, regardless of the medium and the oxidant being used. To illuminate the CL emitter, CL spectra were obtained by continuously pumping solutions of luminol, Cu2, and cefradine solution into a mixing Y-piece positioned prior to the ow cell of the 970CRT spectrouorimeter with the excitation source shutter closed and in the scanning range from

FIG. 3. CL spectra of the reaction of 5.0 3 104 mol/L luminol with 5.0 3 105 mol/L Cu2 in the presence of 0.5 lg/mL cefradine.

300 nm to 700 nm. No obvious CL peak was observed in the absence of cefradine due to very weak blank signal. In the presence of cefradine, the CL spectrum of the reaction showed one peak band within 375;600 nm, with a maximum emission wavelength of ;425 nm (Fig. 3). This emission peak was the typical CL peak reported for the luminol CL reaction, in which the excited state of 3-aminophthalate anion was suggested as the emitter.29 Therefore, it was concluded that 3-aminophthalate anion was also the emitter of this reaction. Figure 4 depicts the UV-Vis absorption spectra of (Fig. 4a) 5.0 3 105 mol/L Cu2 solution, (Fig. 4b) 10.0 lg/mL cefradine solution, and (Fig. 4c) the mixture of 10.0 lg/mL cefradine with 5.0 3 105 mol/L Cu2. The UV-Vis spectrum taken from the cefradine solution showed one absorbance peak at 262 nm, which was attributed to the structure of OCNC C in 7-aminocephalosporanic acid.30 In the presence of Cu2, the absorbance peak of cefradine at 262 nm decreased obviously, which revealed that a reaction took place between cefradine and Cu2 and the structure of OCNCC in 7aminocephalosporanic acid was destroyed. After dissolved oxygen in all solutions was removed by bubbling with N2 gas for 40 min, the CL signal signicantly decreased: about 60% of the CL signal was inhibited. These results indicated that dissolved oxygen took an important role in the CL reaction. It is well known that the b-lactam skeleton with the fourmembered ring structure in cephalosporin antibiotics was easily hydrolyzed31 and electro spin resonance has proved that hydroxyl and superoxide radicals were generated from the hydrolysis of b-lactam antibiotics in the alkaline solution.32 These radicals have been reported as important intermediates in the luminol CL reaction.33,34 To examine whether these reactive oxygen species participated in the CL reaction, the effect of scavengers was tested. The CL signal was completely inhibited by the common radical scavenger ascorbic acid (1 mmol/L) and the hydroxyl radical scavenger thiourea (0.035 mol/L). About 40% of the CL signal was inhibited by the addition of other hydroxy radical scavengers, including sodium benzoate (0.4 mol/L), methanol (10%), and mannitol (0.4 mol/ L). Dimethylfuran (0.05 mol/L), an effective scavenger for singlet oxygen, had no inuence on the CL signal. Therefore, it

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SCHEME 1. Suggested CL reaction mechanism.

was concluded that hydroxyl and superoxide radicals were involved in the CL reaction and singlet oxygen was excluded. Based on the above discussion, a possible CL reaction mechanism is suggested in Scheme 1. Optimization of Experimental Conditions. It is known that metal ions often work as catalysts in luminol CL reactions

and in the hydrolysis of cephalosporin antibiotics;35,36 they are expected to cause an enhancement on the present reaction. Eight metal ions, including Cu2, Cr3, Mn2, Co2, Cd2, Mg2, Ni2, and Zn2, at same concentration of 1.0 3 104 mol/L were tested. The results indicated that the presence of Cu2, Mn2, and Co2 signicantly enhanced the CL signal.

FIG. 5. Effect of Cu2 concentration on the CL reaction. Conditions: 0.05 lg/mL cefradine, 5.0 3 104 mol/L luminol at pH 12.5.

FIG. 6. Effect of luminol concentration on the CL reaction. Conditions: 0.05 lg/mL cefradine, 5.0 3 105 mol/L Cu2, pH 12.5.

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TABLE II. Parameters of calibration curves and detection limits. Linear ranges (lg/mL) 0.007;5.0 0.007;1.0 0.0007;0.7 0.1;10.0 0.001;0.4 0.07;1.0 0.01;1.0 0.07;10.0 0.004;1.0 0.04;1.0 0.0004;0.7 0.001;0.1 0.004;1.0 Regression equation (102 lg/mL) DI DI DI DI DI DI DI DI DI DI DI DI DI 6.00c 89.0 7.27c 5.24 13.01c 18.3 3.53c 21.9 22.9c 25.9 0.96c 5.57 50.2c 7.98 0.13c 13.1 7.21c 22.0 3.62c 9.57 16.4c 18.3 13.6c 5.45 6.64c 15.5 Correlation coefficient (r2) 0.9997 0.9997 0.9977 0.9985 0.9985 0.9998 0.9993 0.9988 0.9993 0.9991 0.9994 0.9986 0.9984 Detection limits (ng/mL) 0.3 3 0.3 20 0.8 20 5 20 0.8 1 0.08 0.8 2

Species Cefalexin Cefadroxil Cefradine Cefazolin sodium Cefaclor Cefuroxime axetil Cefotaxime sodium Cefoperazone sodium Ceftriaxone sodium Ceftazidime Cefetamet pivoxil hydrochloride Cefixime Cefpodoxime

Cu2 was nally employed since it offered the maximum enhancement. The effect of Cu2 concentration on the CL reaction was further examined in the range of 1.0 3 105 to 1.0 3 104 mol/L. As shown in Fig. 5, the increment in CL intensity continuously increased by increasing Cu2 concentration and reached its maximum at 5.0 3 105 mol/L Cu2. Therefore, 5.0 3 105 mol/L Cu2 was employed. The effect of luminol concentration on the CL reaction over the range from 5.0 3 105 mol/L to 1.0 3 103 mol/L was examined and the results are shown in Fig. 6. The increment in CL intensity increased with increase in luminol concentration in the range of 5.0 3 105 to 5.0 3 104 mol/L, whereas higher concentrations of luminol reduced the increment. Thus, 5.0 3 104 mol/L luminol was selected. Preliminary experiments showed that carbonate buffer solution (0.1 mol/L) was a suitable medium for the reaction. The effect of pH of carbonate buffer solution on the CL reaction was examined in the range of 10.5 to 13.0. Both the CL intensity and blank signal increased with increase in the pH of the carbonate buffer solution. The maximum signal-to-blank was obtained at pH 12.5. Thus, 0.1 mol/L carbonate buffer solution at pH 12.5 was used as the reaction medium. The distance between the valve and the ow cell inuences the time that it takes for the reagents to move from the nal mixing point to the ow cell. Too short or too long a distance would result in the maximum CL emission occurring outside the range of detection. The effect of the distance between the valve and the ow cell was examined over a range of up to 30 cm by xing the ow rate at 2 mL/min. The maximum CL
TABLE III. Determination of cefradine in pharmaceutical preparations. Labeled (g) 0.5 0.125 0.25 0.25 Determined (ng/mL) 38.9 39.0 41.8 41.2

signal was observed at 5 cm. Therefore, the distance between the valve and the ow cell was set at 5 cm. Analytical Performance. Under the experimental conditions described above, the calibration curves for 13 cephalosporin antibiotics were constructed. The parameters of calibration curves and the calculated detection limits (3sb) are summarized in Table II. The relative standard deviations for the eleven replicate measurements of 0.1 lg/mL cefalexin, 0.1 lg/ mL cefadroxil, 0.05 lg/mL cefradine, 1.0 lg/mL cefazolin sodium, 0.05 lg/mL cefaclor, 0.05 lg/mL cefuroxime axetil, 0.1 lg/mL cefotaxime sodium, 0.4 lg/mL cefoperazone sodium, 0.1 lg/mL ceftriaxone sodium, 0.7 lg/mL ceftazidime, 0.04 lg/mL cefetamet pivoxil hydrochloride, 0.04 lg/ mL cexime, and 0.07 lg/mL cefpodoxime are 1.0%, 1.4%, 1.0%,1.2%, 1.4 %, 1.1%, 1.3%, 1.6%, 1.1%, 1.0%, 1.2%, 1.0%, and 1.6%, respectively. Interference. The effect of some inorganic ions and excipients commonly used in pharmaceutical preparations was investigated on the determination of 0.05 lg/mL cefradine solution. The tolerance level was established as the maximum amount of foreign species that produced a relative error in the measurement not exceeding 65%. No interference has been found when including 1000-fold albumin, glucose, sucrose, lactose, dextrin, starch, saccharine, Na, K, Ca2, Ba2, Zn2, SO42-, NO3, Cl, Br, or PO43; 500-fold NH4, Sr2, Pb2, or CO32-; 200-fold fructose; 100-fold Mg2 or Cd2; 50-fold sorbitol or citric acid; or 10-fold Ni2 or Cr3. Equal amounts of Mn2 and Co2 produced a positive interference on the

Samples Injection (No. B080531813)a Granules (No. 090313)b Capsules (No. A08117001)a Capsules (No. 055080301)d
a

Proposed methodd (g) 0.491 6 0.013 0.122 6 0.002 0.259 6 0.006 0.253 6 0.005

Added (ng/mL) 20.0 40.0 20.0 40.0 20.0 40.0 20.0 40.0

Found (ng/mL) 59.8 78.7 59.4 80.1 61.1 80.3 60.1 81.7

Recovery (%) 104.5 99.5 102.0 103.0 101.5 98.8 94.5 101.3

Spectrophotometry (g) 0.496 0.129 0.256 0.260

General Pharmaceutical Factory of Harbin Pharmaceutical Group. Beijing Yongzheng Pharmacy. Ltd. c Beijing Ouyi Pharmaceutical Co., Ltd. d Three replicate measurements.
b

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TABLE IV. Determination of cefradine in spiked milk samples. Added (ng/mL) 0.0 30.0 50.0 70.0 Found (ng/mL) 0.0 28.9 51.1 64.5 R.S.D (n 5) 3.0% 2.9% 3.6% Recovery (%) 96.3 102.2 92.1

ACKNOWLEDGMENTS This work was supported by the Fundamental Research Funds for the Central Universities (Program No. GK200902012) and Excellent Preresearch Projects of Science and Technology of Shaanxi Normal University (Program No. 200902016). 1. Q. D. You, Medicinal Chemistry (Chemical Industry Press, Beijing, 2004). 2. G. A. Saleh, S. R. El-Shaboury, F. A. Mohamed, and A. H. Rageh, Spectrochim. Acta, Part A 73, 946 (2009). 3. G. A. Saleh, H. F. Askal, M. F. Radwan, and M. A. Omar, Talanta 54, 1205 (2001). 4. I. F. Al-Momani, J. Pharm. Biomed. Anal. 25, 751 (2001). 5. M. A. Omar, O. H. Abdelmageed, and T. Z. Attia, Talanta 77, 1394 (2009). 6. L. I. Bebawy, K. E. Kelani, and L. A. Fattah, J. Pharm. Biomed. Anal. 32, 1219 (2003). 7. E. S. Jamasbi, A. Rouhollahi, S. Shahrokhian, and S. Haghgoo, Talanta 71, 1669 (2007). 8. T. M. Reddy, M. Sreedhar, and S. J. Reddy, J. Pharm. Biomed. Anal. 31, 811 (2003). 9. Chinese Pharmacopoeia Commission, Chinese Pharmacopoeia (Part II) (Chemical Industry Press, Beijing, 2005). 10. Y. Cai, Y. Q. Cai, S. F. Mou, and Y. Q. Lu, Chin. J. Anal. Chem. 34, 745 (2006). 11. V. F. Samanidou, E. A. Hapeshi, and I. N. Papdoyannis, J. Chromatogr., B 788, 147 (2003). 12. M. Andrasi, A. Gaspar, and A. Klekner, J. Chromatogr., B 846, 355 (2007). 13. P. Puig, F. Borrull, C. Aguilar, and M. Calull, Chromatographia 65, 501 (2007). 14. S. R. El-Shaboury, G. A. Saleh, F. A. Mohamed, and A. H. Rageh, J. Pharm. Biomed. Anal. 45, 1 (2007). 15. W. Liu, Z. J. Zhang, and Z. Q. Liu, Anal. Chim. Acta 592, 187 (2007). 16. S. F. Li, X. W. Wei, X. J. Lu, L. Zhang, and W. Y. Liu, Fenxi shiyanshi 25, 9 (2006). 17. H. Yao, Y. Tang, Y. H. Li, and Y. Y. Sun, Anal. Lett. 36, 2975 (2003). 18. D. Y. Zhang, Y. J. Ma, M. Zhou, L. Li, and H. Chen, Anal. Sci. 22, 183 (2006). 19. D. Y. Zhang, Y. J. Ma, M. Zhou, Y. Q. Yang, X. Y. Zhou, and H. Chen, Fenxi shiyanshi 25, 44 (2006). 20. C. Thongpoon, B. Liawruangrath, S. Liawruangrath, R. A. Wheatley, and A. Townshend, Anal. Chim. Acta 553, 123 (2005). 21. N. A. Alarfaj and S. A. Abd El-Razeq, J. Pharm. Biomed. Anal. 41, 1423 (2006). 22. J. W. Wang and H. Y. Xiong, Fenxi shiyanshi 23, 9 (2004). 23. C. Thongpoon, B. Liawruangrath, S. Liawruangrath, R. A. Wheatley, and A. Townshend, J. Pharm. Biomed. Anal. 42, 277 (2006). 24. Y. Y. Sun, Y. H. Tang, H. Yao, and X. H. Zheng, Talanta 64, 156 (2004). 25. F. A. Aly, N. A. Alarfaffj, and A. A. Alwarthan, Talanta 47, 471 (1998). 26. B. Liu, J. Z. Wang, and Y. Man, Fenxi Ceshi Xuebao 22, 45 (2003). 27. J. D. Yang, Y. M. Huang, and S. P. Liu, Yaowu Fenxi Zazhi 22, 352 (2002). 28. S. L. Fan, C. Lu, J. Wang, and Q. X. Zhou, Fenxi shiyanshi 20, 61 (2001). 29. E. H. White and M. M. Bursey, J. Am. Chem. Soc. 86, 941 (1964). 30. W. Y. Liu, Pharmaceutical Analysis (Peoples Medical Publishing House, Beijing, 1999), 4th ed. 31. R. Broderson, Acta Pharmacol. Toxicol. 3, 345 (1947). 32. H. Kubo, M. Saitoh, S. Murase, T. Inomata, Y. Yoshimura, and H. Nakazawa, Anal. Chim. Acta 389, 89 (1999). 33. G. Merenyi, J. Lind, and T. E. Ericksen, J. Biolumin. Chemilumin. 5, 53 (1990). 34. K. Faulkner and I. Fridovich, J. Free Radic. Biol. Med. 15, 447 (1993). 35. N. P. Gensmantel, P. Proctor, and M. I. Page, J. Chem. Soc., Perkin Trans. 2 11, 1725 (1980). 36. J. V. Uri, P. Actor, L. Philips, and J. A. Weisbach, Experientia 31, 54 (1975). 37. M. Wu and X. Lou, Chin. J. Antibiot. 26, 226 (2001).

Sample No. No. No. No. 1 2 3 4

signal possibly because they had an action similar to that of Cu2. Determination of Cefradine in Pharmaceutical Preparations. The proposed method was applied to the determination of cefradine in several pharmaceutical preparations purchased from local drugstores. Five capsules, granules, or injections were accurately weighed to obtained the mean weight. They were nely powdered and homogenized, and a portion of the powder, corresponding to 50 mg of cefradine, was accurately weighed and dissolved in 100 mL of water. The mixture was ltered and the ltrate was appropriately diluted with water for sample analysis. The results are shown in Table III. Statistical analysis of the results by using a t-test showed that there were no signicant differences between the proposed method and spectrophotometry37 at a condence level of 95%. The recovery studies were also carried out on samples to which known amounts of cefradine were added. The results are also listed in Table III. Recoveries with the range of 94.5% to 104.5% were achieved. Determination of Cefradine in Spiked Milk Samples. Known amounts of cefradine standard solution were added into 1.0 mL milk samples, followed by addition of 1.50 mL of 10% aqueous trichloroacetic acid to promote protein precipitation.15 The mixture was vortexed for several minutes and then centrifuged at 4000 rpm for 10 min. The supernatant was collected, ltered through a 0.2 lm membrane lter, and evaporated to near dryness to remove excess trichloroacetic acid. The residual was dissolved into 50 mL of water and determined by the proposed method. Table IV shows the results of the recovery studies of cefradine from spiked milk samples, and recoveries within the range of 92.1% to 102.2% were achieved.

CONCLUSION
A new chemiluminescence method has been suggested for the determination of thirteen cephalosporin antibiotics, based upon their direct CL reaction with alkaline luminol with Cu2 as a catalyst in the absence of extra added oxidant. The absence of an intentionally added oxidant reduces the background signal and leads to better sensitivity. The application of the method to the analysis of some pharmaceutical preparations and spiked milk samples containing cefradine was evaluated. The proposed method is simple, sensitive, and offers an alternative detection method to liquid chromatography and capillary electrophoresis.

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