Accessible Requires Authentication Published by De Gruyter January 23, 2020

Determination of Melphalan by Micelle Enhanced Spectrofluorimetric Method: Application to Content Uniformity Testing and Human Plasma

Bestimmung von Melphalan mithilfe einer durch Mizellen verstärkten spektrofluorimetrischen Methode: Methodenanwendung zum Test der Homogenität des Wirkstoffes (Content Uniformity Testing) und im menschlichem Blut
Naga Gayatri Sambhani and Valsala Madhavan Nair Biju


This manuscript describes a responsive, rapid, validated and economical spectrofluorimetric method for determination of Melphalan in its pharmaceutical dosage forms and spiked human plasma. The proposed method is relied on the investigation of fluorescence action of Melphalan in Tween-80 micellar system. The Melphalan shows strong native fluorescence at λem 365 nm upon excitation at λex 265 nm in Tween-80 system. There was good enhancement (1.68 fold) in fluorescence intensity in the presence of Tween-80. The proposed approach has a good linearity in the concentration range of 0.2 μg/mL–2 μg/mL. The lower limit of detection and that of quantitation was 0.03 μg/mL and 0.08 μg/mL respectively. The developed method has been validated and applied for content uniformity testing of Melphalan medicines available on the market and Melphalan spiked human plasma samples. The results are statistically compared and were found to be in good agreement to those obtained by comparison methods.


Dieses Manuskript beschreibt eine reaktionsschnelle, validierte und kostengünstige spektrofluorimetrische Methode zur Bestimmung von Melphalan in seinen pharmazeutischen Darreichungsformen und in mit Melphalan aufgestocktem Humanplasma. Die vorgeschlagene Methode beruht auf der Bestimmung der Fluoreszenzwirkung von Melphalan im Tween-80-Mizellensystem. Das Melphalan zeigt im Tween-80-System eine starke Eigenfluoreszenz bei λem 365 nm und nach Anregung eine Fluoreszenz bei λex 265 nm. In Gegenwart von Tween-80 war die Fluoreszenzintensität um das 1,68-fache erhöht. Der vorgeschlagene Ansatz zeigt eine gute Linearität im Konzentrationsbereich von 0,2 μg/ml–2 μg/mL. Die untere Nachweis- und Bestimmungsgrenze betrug 0,03 μg/ml bzw. 0,08 ug/ml. Die entwickelte Methode ist validiert und wird für die Prüfung der Homogenität von Melphalan enthaltenen Arzneimitteln und in mit Melphalan aufgestockten Humanplasmaproben angewendet. Die Ergebnisse wurden statistisch verglichen und stimmten gut mit denen überein, die durch Vergleichsmethoden erhalten wurden.

Correspondence address, Dr. Valsala Madhavan Nair Biju, Associate Professor, Department of Chemistry, National Institute of Technology, Tiruchirappalli, Tamil Nadu 620015, Tel.: +91 0431-2503638, E-Mail: ,

Dr. Valsala Madhavan Nair Biju, PhD: Associate Professor of Analytical Chemistry in the National Institute of Technology, Tiruchirappalli, Tamil Nadu, India. His research area interest includes Pharmaceutical Analysis, Electro Chemical Sensors, Optical Sensors and Ionic liquids.

Naga Gayatri Sambhani is the full time Research Scholar. Working under the supervision of Dr. Valsala Madhavan Nair Biju.


1 Sweetman, S. and Martindale, C.: “The complete drug reference.” 36th edition, Pharmaceutical press, Lambeth High Street, London, (2009), p: 742. Search in Google Scholar

2 Aronson, J. K. and Oxford, UK. MEYLER'S side Effects of Drugs. The International Encyclopedia of Adverse Drug Reactions and Interactions. 15th Edition. (2005), P: 2250. Search in Google Scholar

3 Silvestro, L., Viano, I., Baiocchi, C., Saini, G., Marmont, F. and Ferro, R.: Quantitation of melphalan in plasma of patients by reverse-phase HPLC with electrochemical detection. Journal of Chromatography B: Biomedical Sciences and Applications. 2 (1991) 443450. 10.1016/0378-4347(91)80054-G Search in Google Scholar

4 Taha Ibtisam, A. K., Raad Ahmad, A. J. and Rogers, J. H.: melphalan estimation by quantitative thin-layer chromatography. Cancer Chemother Pharmaco. 5 (1981) 181184. PMid:7296753; 10.1007/BF00258477 Search in Google Scholar

5 Krishnaveni, G., Sailaja, O. and Lakshmareddy, K.: Simple, Sensitive, Specific and Validated Calorimetric Method Development for the Quantitative Estimation of melphalan Hydrochloride in Bulk and Pharmaceutical Dosage forms. SPJPBS. 2 (2014) 110117. Google Scholar. Search in Google Scholar

6 Brightman, K., Finlay, G., Jarvis, I., Knowlton, T. and Manktelow, C. T.: A Stability Indicating method for Determination of melphalan and Related Impurity Content by Gradient HPLC. J Pharm Biomed Ana. 3(1999) 439447. 10.1016/S0731-7085(99)00011-4 Search in Google Scholar

7 Edler, M., Jakubowski, N. and Linscheid, M.: Quantitative Determination of melphalan DNA adducts using HPLC – ICPMS. Journal of Mass Spectrometry. 4 (2006) 507516. PMid:16541389; 10.1002/jms.1009 Search in Google Scholar

8 Kato, Y., Kaneko, H., Matsushita, T., Inamori, K., Egi, S. and Togawa: Direct Injection analysis of melphalan is plasma using column-switching HPLC. Ther Drug Monit. 14 (1992) 6671. PMid:1546392; 10.1097/00007691-199202000-00011 Search in Google Scholar

9 Bosanquet, A. G. and Gilby, E. D.: Measurement of plasma melphalan at Therapeutic Concentrations using Isocratic HPLC. J Chromatogr. 232 (1982) 345354. PMid:7153282; 10.1016/S0378-4347(00)84174-2 Search in Google Scholar

10 Mirkou, A., Vignal, B., Cohn, S., Guillaumont, M., Glehen, O. and Guitton, J.: Assays for the Quantification of melphalan and its Hydrolysis Products in Human Plasma by Liquid Chromatography – Tandem Mass Spectrometry. J Chromatogr B. 877 (2009) 30893096. PMid:19674945; 10.1016/j.jchromb.2009.07.033 Search in Google Scholar

11 Sparidans, W. R., Martens, I., Valkenburg, B. J. L., Van Lersel, den Hartigh, J., Schellens, H. and Bejinn, H. J.: Liquid Chromatography Tandem Mass Spectometric Assay for the PARP-1 Inhibitor Olaparib in Combination with Nitrogen Mustard melphalan in Human Plasma. J Chromatogr B. 879 (2011) 18511856. PMid:21622034; 10.1016/j.jchromb.2011.05.003 Search in Google Scholar

12 Pinguet, F., Joulia, J. M., Martel, P., Grosse, P. Y., Astre, C. and Bressolle, F.: High Performance Liquid Chromatographic Assay of melphalan in Human Plasma: Application to Pharmacokinetic Studies. J Chromatogr B. 686(1996) 4349. 10.1016/S0378-4347(96)00184-3 Search in Google Scholar

13 Zhen-Yu, W., Thompson, M. J., Roberts, M. S., Addison, R. S., Cannell, G. R., Grabs, A. J. and Smithers, B. M.: High-Performance Liquid Chromatographic assay of Measurement of melphalan and its Hydrolysis Products in Perfusate and Plasma and melphalan in Tissues from Human and Rat Isolated Limb Perfusions. J Chromatogr B. 673 (1995) 267279. 10.1016/0378-4347(95)00277-5 Search in Google Scholar

14 Poujol, S., Pinguet, F., Mougenot, P., Malosse, F., Fabbro, M., Culine, S., Astre, C. and Bressolle, F.: Quantitation of melphalan in Human Plasma by Liquid Chromatography and Solid Phase Extraction: Application to Pharmacokinetic Study. J LIQ CHROMATOGR R T., 27 (2004) 301313. 10.1081/JLC-120027101 Search in Google Scholar

15 Małsecki, F. and Crawhall, J. C.: Determination of melphalan and Chlorambucil in Tablet Dosage form Using High-Performance Liquid Chromatography and Amperometric Detection. Analytical Letters. 23(1990) 16851693. 10.1080/00032719008052518 Search in Google Scholar

16 Nath, C. E., Zeng, L., Eslick, A., Trotman, J. and Earl, J. W.: An Isocratic UV HPLC Assay for Analysis of Total and Free melphalan Concentrations in Human Plasma. Acta Chromatographica. 20 (2008) 383398. 10.1556/AChrom.20.2008.3.6 Search in Google Scholar

17 Chang, S. Y., Alberts, D. S., Melnick, L. R., Walson, P. D. and Salmon, S. E.: High Pressure Liquid Chromatographic Analysis of Melphaian in Plasma. J Pharm Sci Exp Pharmacol67 (1978) 679682. PMid:641809; 10.1002/jps.2600670529 Search in Google Scholar

18 Mohamed, A.-M. I., Omar, M. A., Hammad, M. A. and Mohamed, A. A.: Development and Validation of highly Sensitive Stability Indicating Spectrofluorimetric Method for Determination of Amlodipine in Pharmaceutical Preparations and Human Plasma. J Fluoresc26 (2016) 21412149. PMid:27566613; 10.1007/s10895-016-1910-4 Search in Google Scholar

19 Belal, F., Ibrahim, F., Sheribah, Z. and Alaa, H.: Spectrofluorimetric Determination of Clonazepam: Application to Content Uniformity Testing and Human Plasma. Luminescence (2017) 18. PMid:28516499; 10.1002/bio.3325 Search in Google Scholar

20 Ibrahim, F., El-Enany, N., Shalan, S. and Elsharawy, R.: Validated Spectrofluorimetric Determination of Clonazepam in Pharmaceutical Preparations. Luminescence31 (2016) 682. PMid:26335592; 10.1002/bio.3011 Search in Google Scholar

21 Mandal, B., Balabathula, P., Mittal, N. and Mittal, G. C.: Development and Validation of a Spectrofluorimetric Method for the Determination of Erlotinib in Spiked Human Plasma. J Fluoresc22 (2012) 14251429. PMid:22875639; 10.1007/s10895-012-1103-8 Search in Google Scholar

22 Walash, M. I., Ibrahim, F., Manal, I., Eid, S. and Abass, A.: Stability-Indicating Spectrofluorimetric Method for Determination of Itopride Hydrochloride in Raw Material and Pharmaceutical Formulations. J Fluoresc23(2013) 1293. PMid:23852162; 10.1007/s10895-013-1263-1 Search in Google Scholar

23 Sambhani, N. G. and Biju, V. M. N.: J Fluoresc: A Micelle Enhanced Spectrofluorimetric Determination of Pirfenidone: Application to Content Uniformity Testing and Human Urine. 28 (2018) 951. PMid:29974315; 10.1007/s10895-018-2258-8 Search in Google Scholar

24 Rasul, M. J., Shah, J., Jved, M. A. and Yousaf, N.: Micelle Based Spectrofluorimetric Determination of Chloroquine Phosphate in Commercial Formulation and Human Plasma. 51 (2014) 491. 10.3139/113.110333 Search in Google Scholar

25 Zidan, D. W., Hassan, W. S., Elmasry, M. S. and Shalaby, A. A.: A novel spectrofluorimetric method for determination of imatinib in pure, pharmaceutical preparation, human plasma, and human urine. Luminescence. 33(2018) 232242. PMid:29094459; 10.1002/bio.3406 Search in Google Scholar

26 ICH. Harmonized tripartite guideline, validation of analytical procedures: text and methodology, Q2 (r1), current step 4 version, parent guidelines on methodology. Dated November 6 1996, Incorporated in November2005. Search in Google Scholar

27 US Pharmacopoeial Convention. The United States Pharmacopeia 30 The National Formulary 25. Rockville, MD, USA: US Pharmacopeial Convention, electronic version, 2007. Search in Google Scholar

28 Hinze, W. L., Singh, H. N., Baba, Y. and Harvey, N. G.: Micellar enhanced analytical fluorimetry. TrAC Trends Anal Chem. 3 (1984) 193199. 10.1016/0165-9936(84)85006-2 Search in Google Scholar

29 Miller, J. N. and Miller, J. C.: Statistics and chemo metrics for analytical chemistry. 5th ed.Harlow, UK: Pearson Education, (2005) 3973, 107–49, 256. Search in Google Scholar

30 Kiran, K. K.: (Published Doctoral Thesis) Development and Validation of New Visible Spectrophotometric and RP-HPLC Assay Methods for Selected Drugs in Pure and Dosage Formulations. (2014) Acharya Nagarjuna University, Andhra Pradesh, India; Search in Google Scholar

Received: 2018-09-13
Accepted: 2019-03-22
Published Online: 2020-01-23
Published in Print: 2020-01-21

© 2020, Carl Hanser Publisher, Munich