• 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, Jatinangor, Indonesia




4-Chloro-7-nitrobenzofurazan, Amine groups, Chromatography, Pre column derivatization, Post column derivatization


Derivatization is a modification process to produce a new compound that can be detected using the 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 group’s 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 group’s analysis and its chromatographic condition from the literature between 2000 to 2022.


Download data is not yet available.


Kusmierek K, Chwatko G, Głowacki R, Kubalczyk P, Bald E. Ultraviolet derivatization of low-molecular-mass thiols for high-performance liquid chromatography and capillary electrophoresis analysis. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879(17-18):1290-307. doi: 10.1016/j.jchromb.2010.10.035, PMID 21112259.

Moulin M, Deleu C, Larher FR, Bouchereau A. High-performance liquid chromatography determination of pipecolic acid after precolumn Ninhydrin derivatization using domestic microwave. Anal Biochem. 2002;308(2):320-7. doi: 10.1016/S0003-2697(02)00202-6, PMID 12419346.

Agius C, von Tucher S, Poppenberger B, Rozhon W. Quantification of glutamate and aspartate by ultra-high performance liquid chromatography. Molecules. 2018;23(6). doi: 10.3390/molecules23061389, PMID 29890641.

Frank MP, Powers RW. Simple and rapid quantitative high-performance liquid chromatographic analysis of plasma amino acids. J Chromatogr B Analyt Technol Biomed Life Sci. 2007;852(1-2):646-9. doi: 10.1016/j.jchromb.2007.01.002. PMID 17254851.

Ahmed Elbashir AA. A High-performance liquid chromatographic (HPLC) method for the determination of amlodipine drug in dosage form using 1,2-naphthoquine-4-sulfonate (NQS). JAPLR 2017;6(1). doi: 10.15406/japlr.2017.06.00163.

Dubey R, Bhushan R. A rapid, robust and ultra-sensitive HPLC enantioseparation of β-amino alcohols. J Chromatogr Sci. 2015;53(8):1366-72. doi: 10.1093/chromsci/bmv026, PMID 25824571.

El-Zaher AA, Mahrouse MA. 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;8:9-20. doi: 10.4137/ACI.S11612. PMID 23700362.

Sherovski P, Stefova M, Ristovska N. Simultaneous RP-HPLC-dad determination of dansyl amino acids in chemically treated human hair. Maced J Chem Chem Eng. 2018;37(2). doi: 10.20450/mjcce.2018.1594.

Rageh AH, El-Shaboury SR, Saleh GA, Mohamed FA. Spectophotometric method for determination of certain cephalosporins using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). NS. 2010;2(8):828-40. doi: 10.4236/ns.2010.28104.

Elbashir AA, Suliman FEO, Aboul-Enein HY. 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;46(3):222-41. doi: 10.1080/05704928.2011.557121.

Onal A. Spectrophotometric and spectrofluorimetric determination of some drugs containing secondary amino group in bulk drug and dosage forms via derivatization with 7-chloro-4-nitrobenzofurazon. Quim Nova. 2011.

Ghosh PB, Whitehouse MW. Potential antileukemic and immunosuppressive drugs. II. Further studies with benzo-2,1,3-oxadiazoles (benzofurazans) and their N-oxides (benzofuroxans). J Med Chem. 1969;12(3):505-7. doi: 10.1021/jm00303a606, PMID 5788169.

Ghosh PB, Whitehouse MW. 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole: a new fluorigenic reagent for amino acids and other amines. Biochem J. 1968;108(1):155-6. doi: 10.1042/bj1080155, PMID 5657448.

Elbashir AA, Suliman FEO, Aboul-Enein HY. 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 Univ J Sci. 2011.

Duran Meras I, Galeano Diaz T, Alexandre Franco M. 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 Talanta. 2005;65(1):7-14. doi: 10.1016/j.talanta.2004.03.008. PMID 18969757.

Al-Majed AA, Belal F, Abounassif MA, Khalil NY. 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;141(1-2):1-6. doi: 10.1007/s00604-002-0920-x.

Walsh M, Belal F, El-Enany N, Elmansi H. Spectrofluorimetric determination of paroxetine HCl in pharmaceuticals via derivatization with 4-chloro-7- nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). J Fluoresc. 2011;21(1):105-12. doi: 10.1007/s10895-010-0693-2, PMID 20593227.

Ahmed Elbashir A. Application of spectrophotometric methods for the determination of thiamine (VB1) in pharmaceutical formulations using 7-chloro-4-nitrobenzoxadiazole (NBD-Cl). JAPLR 2016;2(3). doi: 10.15406/japlr.2016.02.00018.

Walash MI, El-Enany N, Askar H. 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;30(7):1119-24. doi: 10.1002/bio.2868, PMID 25783013.

El-Enany NM, Belal F, Rizk MS. 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 Anal. 2004.

Niu LY, Zheng HR, Chen YZ, Wu LZ, Tung CH, Yang QZ. Fluorescent sensors for selective detection of thiols: expanding the intramolecular displacement based mechanism to new chromophores. Analyst. 2014;139(6):1389-95. doi: 10.1039/c3an01849k, PMID 24466567.

Chattopadhyay A. Chemistry and biology of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: fluorescent probes of biological and model membranes. Chem Phys Lipids. 1990;53(1):1-15. doi: 10.1016/0009-3084(90)90128-E.

Adegoke OA. An overview of applications of pre-column derivatization reactions for the liquid chromatographic analysis of pharmaceuticals and other compounds. Afr J Pure Appl Chem. 2012. doi: 10.5897/AJPAC12.038.

Colin R, Le Fur E, Charreteur C, Dufau C, Peron JJ. 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;28(9):819-24. doi: 10.1051/analusis:2000149.

Tatar S, Atmaca S. Determination of amlodipine in human plasma by high-performance liquid chromatography with fluorescence detection. J Chromatogr B Biomed Sci Appl. 2001;758(2):305-10. doi: 10.1016/s0378-4347(01)00197-9, PMID 11486841.

Bahrami G, Mirzaeei Sh. 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;36(1):163-8. doi: 10.1016/j.jpba.2004.05.016, PMID 15351061.

Saputri FA, Hasanah AN, Mutakin, Rusdiana T, Surono IS, Abdulah R. A sensitive bioanalytical method for the simultaneous determination of amlodipine and glibenclamide. Pharm Sci Asia. 2022;49(4):381-9. doi: 10.29090/psa.2022.04.22.095.

El-Emam AA, Hansen SH, Moustafa MA, El-Ashry SM, El-Sherbiny DT. 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;34(1):35-44. doi: 10.1016/j.japna.2003.08.021, PMID 14738917.

Bahrami G, Mohammadi B. 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;837(1-2):24-8. doi: 10.1016/j.jchromb.2006.03.056, PMID 16644294.

Erturk S, Cetin SM, Atmaca S, Ersoy L, Baktr G. A sensitive HPLC method for the determination of fluoxetine and norfluoxetine in human plasma with fluorescence detection. Ther Drug Monit. 2005;27(1):38-43. doi: 10.1097/00007691-200502000-00009, PMID 15665745.

Yang C, Jiang X, Guo L, Zhang H, Liu M. 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;30(18):3154-63. doi: 10.1002/jssc.200700160, PMID 18008275.

Farshchi A, Ghiasi G, Bahrami G. High-performance liquid chromatography determination of sertraline in human; 2009.

Wang Y, Yang J, Cai Y, Lin X, Li H. Determination of baclofen using micellar electrokinetic chromatography with laser induced fluorescence detection. Se Pu. 2011;29(12):1230-5. doi: 10.3724/SP. PMID 22500452.

Ulu ST, Tuncel M. Determination of bupropion using liquid chromatography with fluorescence detection in pharmaceutical preparations, human plasma and human urine. J Chromatogr Sci. 2012;50(5):433-9. doi: 10.1093/chromsci/bms020, PMID 22454390.

Milosheska D, Vovk T, Grabnar I, Roskar R. Simple and sensitive high-performance liquid chromatography method with fluorescence detection for therapeutic drug monitoring of topiramate. Acta Chim Slov. 2015;62(2):411-9. doi: 10.17344/acsi.2014.382, PMID 26085425.

Yilmaz EM, Aydogmus Z, Aboul-Enein HY. Determination of nilotinib in spiked plasma, urine, and capsules by high-performance liquid chromatography with fluorimetric detection. Acta Chromatogr. 2016;28(3):313-31. doi: 10.1556/1326.2016.28.3.3.

El-Bagary RI, Abo-Talib NF, Mohamed MA. Novel liquid chromatographic methods for the determination of varenicline tartrate. Talanta. 2016;146:83-92. doi: 10.1016/j.talanta.2015.07.077. PMID 26695238.

Omer M, Omar M, Thiel A, Elbashir A. “High performance liquid chromatographic methods for analysis of taurine in energy drinks after pre-column derivatization. Eura Asian Journal of Analytical Chemistry. 2018;13(5). doi: 10.29333/ejac/93422.

Gao PF, Zheng JS, Zhang SM, Wen XJ. 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;223. doi: 10.1088/1755-1315/223/1/012056.

Elbashir AA, Suliman FOE, Aboul-enein HY. 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.

Maroulis M, Monemvasios I, Vardaka E, Rigas P. 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. 2008 Dec;876(2):245-51. doi: 10.1016/j.jchromb.2008.10.053, PMID 19026599.

Zacharis CK, Tzanavaras PD, Mouroutis PN, Rigas PG. NBD-Cl as a post-column reagent for primary and secondary amines after separation by ion-exchange chromatography. Anal Lett. 2011 Jan;44(10):1821-34. doi: 10.1080/00032719.2010.526263.

Rigas PG. Post-column labeling techniques in amino acid analysis by liquid chromatography amino acid analysis. Analytical and Bioanalytical Chemistry. 2013 Oct;405(25):7957-92. doi: 10.1007/s00216-013-7127-3, PMID 24013667.



How to Cite

SAPUTRI, F. A., & PRATIWI, R. (2023). 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), 20–23. https://doi.org/10.22159/ijap.2023v15i1.46461



Review Article(s)

Most read articles by the same author(s)