Development and Validation of Stability-indicating RP-HPLC Method for Coumarin Assay in Bulk Drugs and Pharmaceutical Products



  • S. Lakshmana Prabu
  • S Thiyagarajan
  • P Balan
  • T N K Suriyaprakash
  • Sharavanan S P


A simple, economic, selective, precise, rapid and reproducible stability indicating HPLC method for the analysis of coumarin (CMN) in bulk drugs and from pharmaceutical formulation has been developed and validated. Separation was achieved on a symmetry C18 column using a mobile phase consisting of a mixture of methanol:water in the ratio of (70:30%v/v) at a flow rate of 1ml/min with detection at 276 nm. Linear regression analysis data for the calibration revealed a good linear relationship between response and concentration in the range 2 μgml-1 to 14 μg ml1 of CMN; the correlation coefficient was 0.999. The LOD and LOQ were 30 and 100 ng ml-1 respectively. The method was validated for accuracy, precision, recovery, reproducibility, specificity, robustness and degradation studies. The statistical analysis showed the method to be precise, reproducible, selective, specific and accurate for analysis and estimation of coumarin in bulk drugs and pharmaceutical dosage forms. 


Download data is not yet available.


Metrics Loading ...


Coumarin, RP-HPLC, method validation, pharmaceutical dosage form




How to Cite

Prabu SL, Thiyagarajan S, Balan P, Suriyaprakash TNK, S P S. Development and Validation of Stability-indicating RP-HPLC Method for Coumarin Assay in Bulk Drugs and Pharmaceutical Products. Scopus Indexed [Internet]. 2013 Nov. 30 [cited 2024 May 18];6(3):2187-92. Available from:



Research Articles


Bogan DP and O’Kennedy R (1996). Simultaneous determination of coumarin, 7-hydroxycoumarin and 7-hydroxycoumarin glucuronide in human serum and plasma by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 686: 267-273.

Celeghini RMS, Vilegas JHM and Lancas FM, (2001). Extraction and Quantitative HPLC Analysis of Coumarin in Hydroalcoholic Extracts of Mikania lomerata Spreng: ("guaco") Leaves. J Braz Chem Soc 12: 706-709.

Chen J and Hu G (1999). The determination of coumarin in foods by high performance liquid chromatography (HPLC). Se Pu 17: 203-205.

Chen Y, Fan GR, Chen B, Xie Y, Wu HL and Wu YT(2006). Separation and quantitative analysis of coumarin compounds from Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook. f by pressurized capillary electrochromatography. J Pharm Biomed Anal 41: 105-116.

Christakopoulos K, Feldhusen H, Norin A, Palmqvist L and Wahlberg I (1992). Determination of natural levels of coumarin in different types of tobacco using a mass fragmentographic method. J Agric Food Chem 40: 1358-1361.

Cok I, Aygun Kocabas N, Cholerton S, Karakaya AE and Sardas S (2001). Determination of coumarin metabolism in Turkish population. Hum Exp Toxicol 20: 179-184.

Dolenc A, Kristl J, Baumgartner S, and Planinsek O (2009). Advantages of celecoxib nanosuspension formulation and transformation into tablets. Int J Pharm 376: 204–212

Egan DA and O'Kennedy R (1992). Rapid and sensitive determination of coumarin and 7-hydroxycoumarin and its glucuronide conjugate in urine and plasma by high-performance liquid chromatography. Chromatogr 582: 137-143.

Feng C, Ruan JL and Cai YL (2010). Simultaneous determination and pharmacokinetics of four coumarins in rat plasma after oral administration of traditional chinese medicine "YIGONG" capsule by SPE-HPLC. J Braz Chem Soc 21: 2272-2277.

Hawryl MA, Soczewinski E and Dzido TH (2000). Separation of coumarins from Archangelica officinalis in high-performance liquid chromatography and thin-layer chromatography systems. J Chromatogr A 886: 75-81.

Hoult JR and Paya M (1996). Pharmacological and biochemical actions of simple coumarins: Natural products with therapeutic potential. Gen Pharmacol 27: 713-722.

Hu XR, Ho JB, Wang Y, Zhu YW and Tian JJ (2011). Oxidative spectroelectrochemistry of two representative coumarins. Electrochimica Acta 56: 2919-2925.

Hunter K (1983). Determination of coumarin anticoagulant rodenticide residues in animal tissue by high-performance liquid chromatography: II.Fluorescence detection using ion-pair chromatography. J Chromatogr A 270: 277-283.

ICH, International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Q2B(R1), Guideline on Validation of Analytical Procedure: Text and Methodology, Geneve, 2005.

Izquierdo MEF, Granados JQ, Mir MV and Martinez MCL (2000). Comparison of methods for determining coumarins in distilled beverages. Food Che 70: 251-258.

Jager L, Perfetti GA and Diachenko GW (2007). Determination of coumarin, vanillin, and ethyl vanillin in vanilla extract products: liquid chromatography mass spectrometry method development and validation studies. J Chromatogr A 1145: 83-88.

Jimenez M, Mateo JJ and Mateo R (2000). Determination of type A trichothecenes by high-performance liquid chromatography with coumarin-3-carbonyl chloride derivatisation and fluorescence detection. J Chromatogr A 870: 473-481.

Kollroser M and Schober C (2002). Determination of coumarin-type anticoagulants in human plasma by HPLC-electrospray ionization tandem mass spectrometry with an ion trap detector. Clin Chem 48: 84-91.

Maggi F, Barboni L, Caprioli G, Papa F, Ricciutelli M and Sagratini G (2011). HPLC quantification of coumarin in bastard balm (Melittis melissophyllum L., Lamiaceae). Fitoterapia 82: 1215-1221.

Mundy DE and Machin AF (1982). The multi-residue determination of coumarin-based anticoagulant rodenticides in animal materials by high-performance liquid chromatography. J Chromatogr 234: 427-435.

Orsi DD, Gagliardi L, Bolasco A and Tonelli D (2000). HPLC determination of 6-methylcoumarin and 3-pyridine methanol in toiletries for oral hygiene. Anal Sci 16: 1341-43.

Park AY, Park SY, Lee J, Jung M, Kim J and Kang SS (2009). Simultaneous determination of five coumarins in Angelicae dahuricaeRadix by HPLC/UV and LC-ESI-MS/MS. Biomed Chromatogr 23: 1034-1043.

Polzin GM, Stanfill SB, Brown CR, Ashley Dl and Watson CH (2007). Determination of eugenol, anethole, and coumarin in the mainstream cigarette smoke of Indonesian clove cigarettes. Food Chem Toxicol 45: 1948-1953.

Rahim AA, Saad B, Osman H, Hashim N, Yahya S and Talib KM (2011). Simultaneous determination of diethylene glycol, diethylene glycol monoethylether, coumarin and caffeine in food items by gas chromatography. Food Chem 126: 1412-16.

Scotter MJ, Roberts DPT and Rees GO (2011). Development and single-laboratory validation of an HPLC method for the determination of coumarin in foodstuffs using internal standardization and solid-phase extraction cleanup. Anal Methods 3: 414-419.

Sherma J, Schafer SL and Morris K (1987). Determination of coumarin in vanilla flavorings by quantitative high performance thin layerchromatography. J Chromatogr 10: 3585-3593.

Sproll C, Ruge W, Andlauer C, Godelmann R and Lachenmeier DW (2008). HPLC Analysis and Safety Assessment of Coumarin in Foods. Food Chem 109: 462-469.

Tan HIS, Ritschel WA and Sanders PR (1976). Determination of coumarin and umbelliferone mixtures in whole blood by spectrophotofluorometry. J Pharm Sci 65: 30-33.

Tsvetkova TM (1977). Thin-layer chromatographic determination of coumarin derivatives. Vet Med Nauki 14: 42-46.

Vogl S, Zehl M, Picker P, Urban E, Wawrosch C and Reznicek G (2011). Identification and quantification of coumarins in Peucedanum ostruthium (L.) Koch by HPLC-DAD and HPLC-DAD-MS. J Agric Food Chem 59: 4371-4377.

Wisneski HH (2001) Determination of coumarin in fragrance products by capillary gas chromatography with electron capture detection. J AOAC Int 84: 689-692.

Xu J, Zhao WM, Qian Z, Guan J and Li SP (2010). Fast determination of five components of coumarin, alkaloids and bibenzyls in Dendrobium spp. using pressurized liquid extraction and ultra-performance liquid chromatography. J Sep Sci 33: 1580-1586.