• FEBRINA AMELIA SAPUTRI Faculty of Pharmacy, Universitas Indonesia, Jalan Margonda Raya, Depok, Indonesia https://orcid.org/0000-0001-8753-6868
  • RIMADANI PRATIWI Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia




4-Chloro-7-nitrobenzofurazan, chromatography, pre column derivatization, post column derivatization


Derivatization is a modification process to produce a new compound that can be detected using suitable detector in chromatography analysis. Derivatization reagent is needed to chemically modify a compound. One of a derivatization reagent that is widely used as a fluorogenic and chromogenic reagent in amine groups analysis using chromatography is 4-Chloro-7-nitrobenzofurazan (NBD-Cl). NBD-Cl is considerable attraction because it plays a role in pre and post-column derivatizations fused in chromatography for increasing the selectivity and sensitivity. This review provides an overview of many papers that have been presented regarding the application of 4-Chloro-7-nitrobenzofurazan (NBD-Cl) in pre and post-column derivatization in amine groups analysis and its chromatographic condition from the literature between 2000 to 2022.


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K. Kuśmierek, G. Chwatko, R. Głowacki, P. Kubalczyk, and E. Bald, “Ultraviolet derivatization of low-molecular-mass thiols for high performance liquid chromatography and capillary electrophoresis analysis,” Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences. 2011. doi: 10.1016/j.jchromb.2010.10.035.

M. Moulin, C. Deleu, F. R. Larher, and A. Bouchereau, “High-performance liquid chromatography determination of pipecolic acid after precolumn ninhydrin derivatization using domestic microwave,” Anal Biochem, 2002, doi: 10.1016/S0003-2697(02)00202-6.

C. Agius, S. von Tucher, B. Poppenberger, and W. Rozhon, “Quantification of glutamate and aspartate by ultra-high performance liquid chromatography,” Molecules, 2018, doi: 10.3390/molecules23061389.

M. P. Frank and R. W. Powers, “Simple and rapid quantitative high-performance liquid chromatographic analysis of plasma amino acids,” J Chromatogr B Analyt Technol Biomed Life Sci, 2007, doi: 10.1016/j.jchromb.2007.01.002.

A. A. Ahmed Elbashir, “A High-Performance Liquid Chromatographic (HPLC) Method for the Determination of Amlodipine Drug in Dosage form Using 1,2-Naphthoquine-4-Sulfonate (NQS),” J Anal Pharm Res, 2018, doi: 10.15406/japlr.2017.06.00163.

R. Dubey and R. Bhushan, “A Rapid, Robust and Ultra-Sensitive HPLC Enantioseparation of β-Amino Alcohols,” J Chromatogr Sci, 2015, doi: 10.1093/chromsci/bmv026.

A. A. El-Zaher and M. A. Mahrouse, “Utility of experimental design in pre-column derivatization for the analysis of tobramycin by hplc fluorescence detection: Application to ophthalmic solution and human plasma,” Anal Chem Insights, 2013, doi: 10.4137/ACI.S11612.

P. Sherovski, M. Stefova, and N. Ristovska, “Simultaneous RP-HPLC-dad determination of dansyl amino acids in chemically treated human hair,” Macedonian Journal of Chemistry and Chemical Engineering, 2018, doi: 10.20450/mjcce.2018.1594.

A. H. Rageh, S. R. El-Shaboury, G. A. Saleh, and F. A. Mohamed, “Spectophotometric method for determination of certain cephalosporins using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl),” Nat Sci (Irvine), 2010, doi: 10.4236/ns.2010.28104.

A. A. Elbashir, F. E. O. Suliman, and H. Y. Aboul-Enein, “The application of 7-chloro-4-nitrobenzoxadiazole (NBD-Cl) for the analysis of pharmaceutical-bearing amine group using spectrophotometry and spectrofluorimetry techniques,” Appl Spectrosc Rev, 2011, doi: 10.1080/05704928.2011.557121.


P. B. Ghosh and M. W. Whitehouse, “Potential Antileukemic and Immunosuppressive Drugs. II. Further Studies with Benzo-2,1,3-oxadiazoles(Benzofurazans) and Their N-Oxides(Benzofuroxans),” J Med Chem, 1969, doi: 10.1021/jm00303a606.

P. B. Ghosh and M. W. Whitehouse, “7-chloro-4-nitrobenzo-2-oxa-1,3-diazole: a new fluorigenic reagent for amino acids and other amines,” Biochemical Journal, 2015, doi: 10.1042/bj1080155.

A. A. Elbashir, F. E. O. Suliman, and H. Y. Aboul-Enein, “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,” Gazi University Journal of Science. 2011.

I. Durán Merás, T. Galeano Díaz, and M. Alexandre Franco, “Simultaneous fluorimetric determination of glyphosate and its metabolite, aminomethylphosphonic acid, in water, previous derivatization with NBD-Cl and by partial least squares calibration (PLS),” Talanta, 2005, doi: 10.1016/j.talanta.2004.03.008.

A. A. Al-Majed, F. Belal, M. A. Abounassif, and N. Y. Khalil, “Fluorimetric determination of gentamicin in dosage forms and biological fluids through derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl),” Mikrochim Acta, 2003, doi: 10.1007/s00604-002-0920-x.

M. Walsh, F. Belal, N. El-Enany, and H. Elmansi, “Spectrofluorimetric determination of paroxetine HCl in pharmaceuticals via derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl),” J Fluoresc, 2011, doi: 10.1007/s10895-010-0693-2.

A. Ahmed Elbashir, “Application of Spectrophotometric Methods for the Determination of Thiamine (VB1) in Pharmaceutical Formulations Using 7-Chloro-4-Nitrobenzoxadiazole (NBD-Cl),” J Anal Pharm Res, 2016, doi: 10.15406/japlr.2016.02.00018.

M. I. Walash, N. El-Enany, and H. Askar, “Validated spectrofluorimetric method for the determination of carbamazepine in pharmaceutical dosage forms after reaction with 4-chloro-7 - Nitrobenzo-2-oxa-1,3-diazole (NBD-Cl),” Luminescence, 2015, doi: 10.1002/bio.2868.

N. M. El-Enany, F. Belal, and M. S. Rizk, “Spectrophotometric determination of salbutamol in bulk and dosage forms after its derivatisation with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl),” Chem Analityczna, 2004.

L. Y. Niu, H. R. Zheng, Y. Z. Chen, L. Z. Wu, C. H. Tung, and Q. Z. Yang, “Fluorescent sensors for selective detection of thiols: expanding the intramolecular displacement based mechanism to new chromophores,” Analyst, 2014, doi: 10.1039/c3an01849k.

A. Chattopadhyay, “Chemistry and biology of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: fluorescent probes of biological and model membranes,” Chemistry and Physics of Lipids. 1990. doi: 10.1016/0009-3084(90)90128-E.

O. A. Adegoke, “An overview of applications of pre-column derivatization reactions for the liquid chromatographic analysis of pharmaceuticals and other compounds,” African Journal of Pure and Applied Chemistry, 2012, doi: 10.5897/AJPAC12.038.

R. Colin, E. Le Fur, C. Charrêteur, C. Dufau, and J.-J. Péron, “Determination of glyphosate herbicide and (aminomethyl)phosphonic acid (AMPA) in water by liquid chromatography and fluorescence detection. Part II: Direct determination using pre-column derivatization with NBD-Cl,” Analusis, 2000, doi: 10.1051/analusis:2000149.

S. Tatar and S. Atmaca, “Determination of amlodipine in human plasma by high-performance liquid chromatography with fluorescence detection,” Journal of Chromatography B, vol. 758, no. 2001, pp. 305–310, 2001.

G. Bahrami and S. Mirzaeei, “Simple and rapid HPLC method for determination of amlodipine in human serum with fluorescence detection and its use in pharmacokinetic studies,” J Pharm Biomed Anal, 2004, doi: 10.1016/j.jpba.2004.05.016.

F. A. Saputri, A. N. Hasanah, Mutakin, T. Rusdiana, I. S. Surono, and R. Abdulah, “A sensitive bioanalytical method for the simultaneous determination of amlodipine and glibenclamide,” Pharmaceutical Sciences Asia, vol. 49, no. 4, pp. 390–398, 2022, doi: 10.29090/psa.2022.04.22.095.

A. A. El-Emam, S. H. Hansen, M. A. Moustafa, S. M. El-Ashry, and D. T. El-Sherbiny, “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, 2004, doi: 10.1016/j.japna.2003.08.021.

G. Bahrami and B. Mohammadi, “Sensitive microanalysis of gabapentin by high-performance liquid chromatography in human serum using pre-column derivatization with 4-chloro-7-nitrobenzofurazan: Application to a bioequivalence study,” J Chromatogr B Analyt Technol Biomed Life Sci, 2006, doi: 10.1016/j.jchromb.2006.03.056.

S. Ertürk, S. M. Çetin, S. Atmaca, L. Ersoy, and G. Baktir, “A sensitive HPLC method for the determination of fluoxetine and norfluoxetine in human plasma with fluorescence detection,” Ther Drug Monit, 2005, doi: 10.1097/00007691-200502000-00009.

C. Yang, X. Jiang, L. Guo, H. Zhang, and M. Liu, “Analysis of free amino acids in islets of Langerhans by high-performance liquid chromatography using pre-column derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole,” J Sep Sci, 2007, doi: 10.1002/jssc.200700160.

A. Farshchi, G. Ghiasi, and G. Bahrami, “High Performance Liquid Chromatography Determination of Sertraline in Human,” 2009.

Y. Wang, J. Yang, Y. Cai, X. Lin, and H. Li, “Determination of baclofen using micellar electrokinetic chromatography with laser induced fluorescence detection,” Chinese Journal of Chromatography (Se Pu), 2011, doi: 10.3724/SP.J.1123.2011.01230.

S. T. Ulu and M. Tuncel, “Determination of bupropion using liquid chromatography with fluorescence detection in pharmaceutical preparations, human plasma and human urine,” J Chromatogr Sci, 2012, doi: 10.1093/chromsci/bms020.

D. Milosheska, T. Vovk, I. Grabnar, and R. Roškar, “Simple and sensitive high performance liquid chromatography method with fluorescence detection for therapeutic drug monitoring of topiramate,” Acta Chim Slov, 2015, doi: 10.17344/acsi.2014.382.

E. M. Yilmaz, Z. Aydoğmuş, and H. Y. Aboul-Enein, “Determination of nilotinib in spiked plasma, urine, and capsules by high-performance liquid chromatography with fluorimetric detection,” Acta Chromatogr, 2015, doi: 10.1556/1326.2016.28.3.3.

R. I. El-Bagary, N. F. Abo-Talib, and M. A. El-Wahab Mohamed, “Novel liquid chromatographic methods for the determination of varenicline tartrate,” Talanta, 2016, doi: 10.1016/j.talanta.2015.07.077.

M. Omer, M. Omar, A. Thiel, and A. Elbashir, “High Performance Liquid Chromatographic Methods for Analysis of Taurine in Energy Drinks after Pre-column Derivatization,” Eurasian Journal of Analytical Chemistry, 2018, doi: 10.29333/ejac/93422.

P. F. Gao, J. S. Zheng, S. M. Zhang, and X. J. Wen, “Fluorescence detection of Trace Dimethylamine and Diethylamine in Natural Water by HPLC with O-phthalaldehyde and 7-chlore-4nitrobenzo-2-oax-1, 3-diazole,” IOP Conf Ser Earth Environ Sci, 2019, doi: 10.1088/1755-1315/223/1/012056.

A. A. Elbashir, F. O. Eldin SULIMAN, and H. Y. Aboul-enein, “REVIEW 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,” 2011.

M. Maroulis, I. Monemvasios, E. Vardaka, and P. Rigas, “Determination of domoic acid in mussels by HPLC with post-column derivatization using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and fluorescence detection,” J Chromatogr B Analyt Technol Biomed Life Sci, vol. 876, no. 2, pp. 245–251, Dec. 2008, doi: 10.1016/j.jchromb.2008.10.053.

C. K. Zacharis, P. D. Tzanavaras, P. N. Mouroutis, and P. G. Rigas, “NBD-Cl as a post-column reagent for primary and secondary amines after separation by ion-exchange chromatography,” Anal Lett, vol. 44, no. 10, pp. 1821–1834, Jan. 2011, doi: 10.1080/00032719.2010.526263.

P. G. Rigas, “Post-column labeling techniques in amino acid analysis by liquid chromatography Amino Acid Analysis,” Analytical and Bioanalytical Chemistry, vol. 405, no. 25. pp. 7957–7992, Oct. 2013. doi: 10.1007/s00216-013-7127-3.



How to Cite

SAPUTRI, F. A., & PRATIWI, R. (2022). 4-CHLORO-7-NITROBENZOFURAZAN (NBD-CL) AS PRE AND POST COLUMN DERIVATIZATION REAGENT FOR AMINE GROUPS ANALYSIS USING CHROMATOGRAPHY: A MINI REVIEW. International Journal of Applied Pharmaceutics, 15(1). https://doi.org/10.22159/ijap.2023v15i1.46461



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