spectrophotometric determination of in sodium

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Spectrochimica Acta Part A 60 (2004) 30073012

Spectrophotometric determination of ampicillin sodium in pharmaceuticalproducts 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, 463 = 1.14 104.The absorbance of ampicillin sodium from 2.080g ml1 obeys Beers law. The linear regression equation of the calibration graph isC = 40.24A 2.603, with a linear regression correlation coefficient is 0.9997, the detection limit is 1.5 g ml1, recovery is from 97.23 to104.5%. Effects of pH, surfactant, organic solvents, and foreign ions on the determination of ampicillin sodium have been examined. Thismethod is rapid and simple, and can be used for the determination of ampicillin sodium in the injection solution of ampicillin sodium. Theresults 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

Ampicillin sodium is one of the important penicillinantibiotics used to treat or prevent bacterial infections. Inview of its pharmacological importance, considerable workhas been done for its detection and quantification. Thecontent of ampicillin sodium was determined by HPLCin United States Pharmacopoeia, British Pharmacopoeiaand Chinese Pharmacopoeia [13]. Various analyticaltechniques have been employed for the determination ofampicillin sodium in serum, plasma, urine, pharmaceuticaldosage and so on, such as HPLC method [47], spec-trophotometry [811], spectrofluorimetry [12], colorimetricmethod [13], capillary electrophoresis [14] and micro-biological assay [15,16]. HPLC method can be used todetermine the ampicillin sodium in the serum, but it haspoor reproducibility [47] (R.S.D.: 2.613.9%). H. Mah-goub reported the determination of ampicillin sodium byspectrophotometric method [811], but its inconvenient.Use of 1,2-naphthoquinone-4-sulfonic acid sodium as a

Corresponding author. Tel.: +86-531-6619466;fax: +86-531-2615258.

E-mail address: [email protected] (H. Wang).

colored reagent for determination of ampicillin sodiumby spectrophotometry has not been reported. This pa-per reports a rapid spectrophotometric method for de-termining the content of ampicillin sodium in injection,which is based on a replace reaction [17], i.e. sodium1,2-naphthoquinone-4-sulfonic acid reacts with amino ofampicillin sodium molecule to form a salmon pink com-pound. max of the compound is at 463 nm. The reactionequation reads as follows:

O

O

so3H

NH2

O

NH

N

O

S

O Na+

O

O

O

NH

O

NH

N

O O Na+

S

+

o

Product I

1386-1425/$ see front matter 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.saa.2004.02.018


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3008 L. Xu et al. / Spectrochimica Acta Part A 60 (2004) 30073012

Fig. 1. Absorbance spectra of product I. Concentration of ampicillinsodium: 40g ml1. Line (a): absorption spectrum of product I againstreagent 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 compoundthat 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 compendiousmethods for the determination of ampicillin sodium in phar-maceutical products and biological samples. The maximumabsorption wavelength of product I was at 463 nm, whichshifted 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 bedetermined in the range of visible light, much potential in-terference may be avoided in the determination of ampicillinsodium of biological samples and hemanalysis.

The principal advantage of our method is that the maxi-mum absorption wavelength of ampicillin sodium shifts tothe range of visible light from the range of ultraviolet light,so that ampicillin sodium may be determined in the rangeof visible light. In addition, the method is simple, and canbe used for determining ampicillin sodium in the injectionsolution of ampicillin sodium. The results obtained by themethod agreed with those of the official method [3].

2. Experimental

2.1. Apparatus

All of the spectrophotometric measurements were madewith a Shimadzu UV-265 ultraviolet visible recording spec-trophotometer with matched 1 cm quartz cells. In order tocompare all spectrophotometric measurements and ensurereproducible experimental conditions, the UV-265 spec-trophotometer was checked daily. All pH measurementswere made with a pHs-3C digital pH-meter (Shanghai

Lei Ci Device Works, Shanghai, China) with a combinedglass-calomel electrode.

2.2. Reagents

Ampicillin sodium (99.89%) was purchased from Lu

Kang Drugs and Reagents Company (Shandong, China) andused as standards. Ampicillin sodium was assayed, accord-ing to English Pharmacopoeia [1] by HPLC. All reagentswere of analytical-reagent grade, unless stated otherwise.Double-distilled water was used in all experiments.

2.2.1. Stock standard solution of ampicillin sodium

(1000g ml1)

An accurately weighed 0.10 g standard sample of ampi-cillin sodium was dissolved in water, transferred into a100 ml standard flask and diluted to the mark with water andmixed well. The solution was stable for at least 2 months at4 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 mlstandard flask and diluted to the mark with water and mixedwell. The solution was stable for at least 2 weeks at 4 C.

2.2.3. S rensen buffer solution (pH 9.00

Na2HPO4KH2PO4)

A total of 1/15 moll1 Na2HPO4 and 1/15moll1

KH2PO4 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 inwater, transferred into 250 ml standard flask, diluted to themark with water and mixed well. Triton X-120 and sodiumlaurylsulfonate were prepared, according to the same proce-dure.

2.3. Determination of ampicillin sodium by HPLC

According to The English Pharmacopoeia [1], ampicillinsodium was determined. The result showed that the contentof ampicillin sodium was 99.89%; this sample was used asstandard sample. In addition, 20.0 ml of injection solutionof 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-sulfonicwas transferred into a 10 ml standard flask, 0.1 ml ethanol,1.0 ml of 0.1% CTPB, 0.40 ml of 1000g ml1 ampicillinsodium and 2.0 ml pH 9.00 Na2HPO4KH2PO4 buffer so-lution were added sequentially, diluted to the mark with


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L. Xu et al. / Spectrochimica Acta Part A 60 (2004) 30073012 3009

water and mixed well. The absorbance of the solutionwas measured at 463 nm against a reagent blank preparedwith the same reagent concentration, but no ampicillinsodium.

3. Result and discussion

3.1. Absorption spectrum of product I

According to the procedure, the absorption spectrum ofproduct I was recorded. As can be seen (Fig. 1), the max-imum absorption wavelength of product I was at 463 nmagainst a reagent blank (line a). Obviously, the absorptionspectrum of reagent blank overlapped partially at 463 nmagainst water blank although this superposition was little.Fortunately, this interference can be eliminated, when themeasurement was carried out at 463 nm against the reagentblank. An excellent linear relationship existed between theabsorbance 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 250500 nm (line c), andits maximum absorption wavelength was at 200 nm. There-fore, ampicillin sodium can be determined conveniently at463 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 Ichanges with pH. The variation of the pH from 1.00 to 13.0was investigated. The result showed in Fig. 2.

0 2 4 6 8 10 120.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

A

pH

Fig. 2. Effect of pH on absorbance of product I. Concentration of ampicillin sodium: 40 g ml1.

It can be seen (Fig. 2) that the absorbance of product Iincreased in the range of pH 1.008.60, and then reducedsharply, when pH above 9.40. The tests showed that theabsorbance of product I was maximal at pH 9.00 and anexcellent linear relationship existed between the absorbanceof product I and the concentration of ampicillin sodium at pH

9.00. Therefore, pH 9.00 Na2HPO4KH2PO4 buffer solutionwas selected to control the pH of system. Obviously, buffersolution had no effect on the determination of ampicillinsodium.

3.3. Effect of surfactant

The effects of the surfactants, CTPB, sodium laurylsul-fonate and Triton X-100, were tested on the determinationof ampicillin sodium. As we all know that the surfactant waswidely used as sensitizing reagent in the spectrophotome-try. Because of the formation of micelle, the absorbance wasenlarged in the presence of the surfactant. The tests showed

that CTPB was a sensitizing reagent in the determination ofampicillin sodium. The result was showed in Fig. 3. As canbe seen (Fig. 3), the absorbance increased with the amountof CTPB below 1.0 ml. However, the absorbance decreased,when the amount of CTPB above 1.0 ml. It indicated thatthe 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 mlCTPB was selected as the optimum.

3.4. Effect of organic solvents

Organic solvents, including methanol, ethanol, dimethyl-

sulfoxide (DMSO), dimethylformaide (DMF), were testedfor determining ampicillin sodium. The result was shown inFig. 4.


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0.0 0.5 1.0 1.5 2.00.0

0.1

0.2

0.3

0.4

F

B

D

A

v(ml)

Fig. 3. Effect of surfactant on absorbance of product I. Concentration

of ampicillin sodium: 40g ml1. 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 andthen decreased with a rise in ethanol. In addition, methanolwas abandoned because of its toxicity. Obviously, 0.1 mlethanol is more effective to increase the absorbance of theproduct I, therefore, 0.1 ml ethanol was selected as the op-timum.

3.5. Effect of concentration of sodium1,2-naphthoquinone-4-sulfonic

According to procedure, the absorbance of mixture so-lution of 40g ml1 ampicillin sodium was measured un-

0 1 2 3 40.1

0.2

0.3

0.4

0.5

A

v(ml)

Fig. 5. Effect of concentration of sodium 1,2-naphthoquinone-4-sulfonic. Concentration of ampicillin sodium: 40 g ml1.

0.0 0.2 0.4 0.6 0.8 1.0

0.1

0.2

0.3

0.4

0.5

0.6

B

F

C

D

A

v(ml)

Fig. 4. Effect of organic solvents on the absorbance of product I. Con-centration of ampicillin sodium: 40 g ml1. 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 therise of sodium 1,2-naphthoquinone-4-sulfonic in the rangeof 0.20.6 ml; the absorbance of the mixture solution wasstable 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 sodium1,2-naphthoquinone-4-sulfonic was enough in this paper.

3.6. Effect of heating time

According to the procedure, the absorption of the mixturesolution of 40g ml1 ampicillin sodium was measured af-ter heated in boiling water bath for different time; tests foundthat the absorbance decreased when heated. Therefore, roomtemperature was recommended.


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L. Xu et al. / Spectrochimica Acta Part A 60 (2004) 30073012 3011

0 20 40 60 80 1000.00

0.05

0.10

0.15

0.20

0.25

0.30

A

T(min)

Fig. 6. Effect of standing time on determining ampicillin sodium. The concentration of ampicillin sodium: 40 g ml1.

3.7. Formation and stability of product I

According to the procedure, the absorbance of the mixturesolution of 40g ml1 ampicillin sodium was measured af-ter standing for different time. The results were showed inFig. 6.

From Fig. 6, it can be seen that ampicillin sodium reactsimmediately with sodium 1,2-naphthoquinone-4-sulfonic atroom temperature. The absorbance was no longer changedafter standing for 50 min. Therefore, 50 min was selected asthe optimum. In addition, the absorbance of the procedure Iwas stable at least 2 h at room temperature.

3.8. Order of reagents

According to the procedure, the order of the addition of thereagents was tested. It was found that the excellent order wassodium 1,2-naphthoquinone-4-sulfonic acid, ethanol, CTPBand 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 40g ml1 ampi-cillin sodium. A 500mgml1 level of each potentially in-terfering ion was tested first. If interference occurred, theratio was reduced progressively, until interference ceased.The tolerance level was defined as an error not exceeding5% 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 of2.080g ml1, the linear regression equation of the cal-ibration graph is C = 40.24A 2.603, with a correlation

Table 1

Effect of foreign ions on the determination of ampicillin sodium

Foreign ions or species Tolerance level(g ml1)

K+, Na+, NO3, PO4, Cl, H2PO4, SO42 500CO32 100Ba2+ 50Mg2+ 25Cu2+, Ni2+, Zn2+ 5

coefficient of linear regression of 0.9997. The unit of C isg ml1 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 at463nm, 463 = 1.14 104 lmol1 cm1 was estimated.

3.11. Reproducibility and detection limit

A portion of the sample solution of ampicillin sodiumwas transferred into a 10 ml volumetric flask. The reagentswere added and measured, according to the procedure. Thissample solution was measured 10 times (n = 10); the meanvalue was 40.12g ml1 with a R.S.D. of 4.2%. Accordingto the procedure, a reagent blank was measured 10 times(n = 10), the detection limit was 1.5 g ml1, based on theblank 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 (differentbatch number) were diluted to different concentrations withwater. 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 agreedwith those of the HPLC [1]. The experimental data of the


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Table 2Determination of ampicillin sodium sample

Sampleno.

Spectrophotometricmethod (mg ml1)

HPLC method (mg ml1)

1 9.75 0.03 9.72 0.212 14.60 0.01 14.72 0.133 19.35 0.12 19.17 0.23

4 24.20 0.07 24.35 0.32

Table 3Results for the recovery of ampicillin sodium from samples

Sampleno.

Sample content(g ml1)

Ampicillin sodium(g ml1)

Recovery (%)

Added Found

1 9.75 5.00 9.982 0.02 99.64 0.42 9.75 10.00 19.95 0.01 99.51 0.13 9.75 15.00 30.05 0.03 100.4 0.24 4.88 10.00 19.72 0.03 97.23 0.35 14.63 10.00 20.45 0.01 104.5 0.1

two methods were tested by the Cochram method [18]. Ata confidence level (P) of 95%, there was no significant dif-ference between the two methods.

3.13. Recovery of ampicillin sodium

The recovery of ampicillin sodium added to the differentconcentration of sample solutions was showed in Table 3.The recovery was from 97.23 to 104.5%. The confidence in-tervals were estimated to results of determination in Table 3based on P = 95% and n = 3. P and n are the confidence

level and times of replicating determination, respectively.

4. Conclusion

The results presented in this paper demonstrated clearlythat ampicillin sodium could be determined by spectropho-

tometry. The results obtained agreed with those of the HPLCmethod. The principal advantage of the proposed methodwas that ampicillin sodium could be determined in the visi-ble light, so that the potential interference may be avoided.In addition, the method was simple and wider linear range.

References

[1] The British Pharmacopoeia, vol. I, British Pharmacopoeia Commis-sion, London, HMSO, 2000, p. 116.

[2] The United States Pharmacopoeial Convention Inc., vol. I, 24threvision, Rockville, ME, p. 140.

[3] The Chinese Pharmacopoeia, vol. 2, fifth ed., Pharmacopoeia Com-mission, Ministry of Health, PRC Chemical Industry Press, Beijing,2000, p. 718.

[4] M. Ishida, K. Kobayashi, N. Awata, F. Sakamoto, J. Chromatogr. B:Biomed. Sci. Appl. 727 (1999) 245.

[5] L.K. Sorensen, L.K. Snor, T. Elkaer, H. Hansen, J. Chromatogr. B:Biomed. Sci. Appl. 734 (1999) 307.

[6] E. Verdon, R. Fuselier, D. Hurtaud-Pessel, P. Couedor, N. Cadieu,M. Laurentie, J. Chromatogr. A 882 (2000) 135.

[7] S.S. Zarapkar, N.P. Bhandari, U.P. Halker, Indian Drugs 37 (2000)421.

[8] H. Mahgoub, F.A. Aly, J. Pharm. Biomed. Anal. 17 (1998) 1273.[9] M. Gibella, B. Tilquin, Analysis 27 (1999) 657.

[10] S.Z. Qureshi, T. Qayoom, M.I. Helalet, J. Pharm. Biomed. Anal. 21(1999) 473.

[11] F. Belal, M.M. El-Kerdawy, S.M. El-Ashry, D.R. El-Wasseef, IlFarmaco. 55 (2000) 680.

[12] P. Gutirez Navarro, A. el Bekkouri, E. Rodrguez Reinoso, Analyst123 (1998) 2263.

[13] H.A. El-Obeid, E.A. Gad-Kariem, K.A. Al-Rashood, H.A.Al-Khamees, F.S. El-Shafie, G.A.M. Bawazeer, Anal. Lett. 32 (1999)2706.

[14] Y.X. Zhu, C. Hoogmartens, A. Van Schepdael, E. Roets, J. Hoog-martens, J. Liquid Chromatogr. Relat. Technol. 22 (1999) 1403.

[15] E. Usleber, S. Litz, E. Martlbauer, Food Agric. Immunol. 10 (1998)317.

[16] L. Basaez, P. Vanysek, J. Pharm. Biomed. Anal. 19 (1999) 183.[17] L. Huang, D.Q. Yu, Application of Ultraviolet Spectra in the Organic

Chemistry, Science Press, Beijing, 1988, pp. 210, 213.[18] Y.X. Zheng, The Method of Mathematical Statistics in Analytical

Chemistry, Science Press, Beijing, 1986, pp. 122, 231, 308.

spectrophotometric determination of in sodium

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