World Journal of Analytical Chemistry
ISSN (Print): 2333-1178 ISSN (Online): 2333-1283 Website: Editor-in-chief: Raluca-Ioana Stefan-van Staden
Open Access
Journal Browser
World Journal of Analytical Chemistry. 2013, 1(2), 18-22
DOI: 10.12691/wjac-1-2-1
Open AccessArticle

Development and Validation of Spectrophotometric Method for Determination of Penicillamine (PA) in Pharmaceutical Formulation Using 4-Choro-Nitrobenzo-2-Oxa-1, 3-Diazol (NBD-CL)

Abdalla Ahmed Elbashir1, and Amira Anwar Babikir Alfadil2

1University of Khartoum, Faculty of Science, Chemistry Department, Khartoum, Sudan

2University of Sudan for Science & Technology, Faculty of Science, Department of chemical Laboratory, Khartoum, Sudan

Pub. Date: April 19, 2013

Cite this paper:
Abdalla Ahmed Elbashir and Amira Anwar Babikir Alfadil. Development and Validation of Spectrophotometric Method for Determination of Penicillamine (PA) in Pharmaceutical Formulation Using 4-Choro-Nitrobenzo-2-Oxa-1, 3-Diazol (NBD-CL). World Journal of Analytical Chemistry. 2013; 1(2):18-22. doi: 10.12691/wjac-1-2-1


A sensitive and simple spectrophotometric method has been proposed for the determination of D-Penicillamine (PA) in pharmaceutical formulations. The proposed method is based on the reaction between the PA and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-CL) at alkaline medium (pH 10.5) to form deep brown-purple adduct, exhibiting maximum absorption (λmax) at 468nm. Under optimized reaction condition, the method was linear in the concentration range 1-15µg mL-1. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.11µg mL-1, 0.38µg mL-1, respectively. The method was applied successfully to the determination of PA in pharmaceutical dosage form. A proposal of the reaction pathway has been postulated. The results were in a good agreement with those obtained with the official USP method. The method is useful for routine analysis of PA in quality control laboratories.

D-Penicillamine spectrophotometric pharmaceutical formulation method validation NBD-CL

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit


Figure of 6


[1]  Suliman, F.O., AL-Hinai, M.M., AL-Kindy, S.M.Z., and Salama, S.B, “Enhancement of the chemiluminescence of penicillamine and ephedrine after derivatization with aldehydes using tris(bipyridyl)ruthenium(II) peroxydisulfate system and its analytical application,” Talanta, 74(5) 1256-1264. Feb.2008.
[2]  Gotti, R., Pomponio, R., Anderson, V., and Cavrini, V, “ Analytical study of penicillamine in pharmaceuticals by capillary zone electrophoresis,” J. Chromatogr. A, 844(1-2).361-369. Jun.1999.
[3]  AL-Majed, A.A, “Spectrophotometric estimation of D-penicillamine in bulk and dosage forms using 2,6-dichloroquinone-4-chlorimide, (DCQ),” J Pharm Biomed Anal. 21(4).827-833.Dec.1999.
[4]  Yang, X., Yuan, H., Ch Wang, Su, X., Hu, L., and Xiao, D, “Determination of penicillamine in pharmaceuticals and human plasma by capillary electrophoresis with in-column fiber optics light-emitting diode induced fluorescence detection,” J. Pharm. Biomed. Anal,45(2).362-366. Oct.2007.
[5]  Rojanarata, T., Opanasopit, P., Ngawhirunpat, T., Saehuan, C, “Ninhydrin reaction on thiol-reactive solid and its potential for the quantitation of d-penicillamine,”Talanta, 82(2).444-449. Jul.2010.
[6]  Elbashir,A.A., Awad, S. F. “A New Spectrophotometric Method for Determination of Penicillamine in Pharmaceutical Formulation Using 1, 2-naphthoquine-4-sulfonate (NQS),” J. Pharmacovigilance1, 1-5, March.2013
[7]  AL-Majed, A.A, “Specific spectrofluorometric quantification of D-penicillamine in bulk and dosage forms after derivatization with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole,” Anal Chim. Acta, 408(1-2), 169-175. Mar.2000.
[8]  Russell J., Mckeown, J.A., Hensman, C., Smith, WE., and Reglinski, J, “HPLC determination of biologically active thiols using pre-column derivatisation with 5,5'-dithio-(bis-2-nitrobenzoic acid),” J. Pharm. Biomed. Anal,15(11).1757-1763. Jul.1997.
[9]  Taha, E.A., Salama, N.N., and Fattah, L, “Spectrofluorimetric and spectrophotometric stability-indicating methods for determination of some oxicams using 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-CL),” Chem Pharm Bull.,54(5).653-658. May.2005.
[10]  El-Enany, N., El-Sherbiny, D., and Belal, F, “Spectrophotometric, spectrofluorometric and HPLC determination of desloratadine in dosage forms and human plasma,” Chem Pharm Bull., 55(12)1662-1670. Dec.2007.
[11]  Saleh, H.M., EL-Henawee, M.M., Ragab, G.H., and El-Hay, S.S.A., “Utility of NBD-CL for the spectrophotometric determination of some skeletal muscle relaxant and antihistaminic drugs,” Spectrochim Acta A ,67(5).1284-1289. Oct.2007.
[12]  Shehata, M.A., El-Sayed, G.M., and Abdel-Fattah, L.E, “Utilization of 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-CL) for kinetic spectrophotometric assay of befunolol hydrochloride in its pharmaceutical formulation,” J AOAC Int., 89(3).646-650. May-Jun.2006.
[13]  Olojo, RO., Xia, R.H., and Abramson, J.J, “Spectrophotometric and fluorometric assay of superoxide ion using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole,” Anal Biochem. 339(2). 338-344. Apr. 2005.
[14]  Olgun, N., Erturk, S., and Atmaca, S, “Spectrofluorimetric and spectrophotometric methods for the determination of vigabatrin in tablets,” J. Pharm. Biomed. Anal., 29(1-2).1-5. Jun.2002.
[15]  Onal, A., Kepekci, S.E., and Oztunc, A, “Spectrophotometric methods for the determination of antidepressant drug paroxetine hydrochloride in tablets,” J AOAC Int., 88(2) 490-495. Mar-Apr.2005.
[16]  El-Emam, A.A., Hansen, S.H., Moustafa, M.A., El- Ashry, S.M., and El-Sherbiny, D.T, “Determination of lisinopril in dosage forms and spiked human plasma through derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-CL) followed by spectrophotometry or HPLC with fluorimetric detection,” J. Pharm.Biomed.Anal., 34(1).35-44. Jan.2004.
[17]  Amin, A.S., Ragab, G.H., and Saleh, H, “Colorimetric determination of β-blockers in pharmaceutical formulations,” J. Pharm. and Biomed.Anal., 30(4).1347-1353. Nov.2002.
[18]  Abdellatef, H.E, “Kinetic spectrophotometric determination of tramadol hydrochloride in pharmaceutical formulation,” J. Pharm. Biomed. Anal., 29(5)835-842. Jul.2002.
[19]  Darwish, I.A., Amer, S.M., Abdine, H.H., and AL-Rayes, L.A.I, “ New Spectrophotometric and Fluorimetric Methods for Determination of Fluoxetine in Pharmaceutical Formulations,” Int J Anal Chem. 257(3)1-9. May.2009.
[20]  Önal, A., and Sagirli, O, “A Spectrophotometric and spectrofluorimetric methods for the determination of pregabalin in bulk and pharmaceutical preparation,” Spectrochim Acta A 72(1).68-71. Feb.2009.
[21]  El-Shabrawy, Y., Belal, F., Sharaf, El-Din, M., Shalan, Sh, “Spectrophotometric determination of fenoterol hydrobromide in pure form and dosage forms,” IL Farmaco,58(10)1033-1038.Oct. 2003.
[22]  Elbashir, A.A., Suliman, F.O., Aboul-Enein, H.Y, “The application of 7-chloro-4-nitrobenzoxadiazole (NBD-CL) for the analysis of pharmaceutical-bearing amine group using spectrophotometry and spectrofluorimetry techniques,” Appl Spectros Rev., 46(3). 222-241. Mar.2011.
[23]  Elbashir, A.A., Suliman, F.O., Aboul-Enein, H.Y, “The Application of 7-Chloro-4-Nitrobenzoxadiazole and 4-Fluoro-7-Nitro-2,1,3 Benzoxadiazole for The Analysis of Amines and Amino Acids Using High-Performance Liquid Chromatography,” GU J Sci 24(4).679-697.Oct.2011.
[24]  Job, P, “Advanced Physiochemical Experiments,2nd ed.,Oliner and Boyd, Edinburgh, 1963,p.54 Anal. Chem, 1928; 9, 113.