World Journal of Analytical Chemistry

Current Issue» Volume 2, Number 2 (2014)


Design of Experiment Assisted UV-Visible Spectrophotometric and RP-HPLC Method Development for Ambrisentan Estimation in Bulk and Formulations

1Department of Pharmaceutical Analysis, Nirmala College of Pharmacy, Guntur, India

2School of Medical Science & Technology, Indian Institute of Technology, Kharaghpur, India

3Faculty of Pharmacy, University of Zawia, Az Zawiyah, Libya

4Division of Biotechnology, Netaji Subhas Institute of Technology, New Delhi, India

World Journal of Analytical Chemistry. 2014, 2(2), 23-30
DOI: 10.12691/wjac-2-2-2
Copyright © 2014 Science and Education Publishing

Cite this paper:
Shanta K. Adiki, Prashanti M, Baishakhi Dey, Prakash Katakam, Fathi H. Assaleh, Nagiat T. Hwisa, Rajeev K. Singla, Babu R. Chandu. Design of Experiment Assisted UV-Visible Spectrophotometric and RP-HPLC Method Development for Ambrisentan Estimation in Bulk and Formulations. World Journal of Analytical Chemistry. 2014; 2(2):23-30. doi: 10.12691/wjac-2-2-2.

Correspondence to: Prakash  Katakam, Faculty of Pharmacy, University of Zawia, Az Zawiyah, Libya. Email:


Design of experiment (DOE) approach reinforces the robustness of the method being developed. This was employed for UV-visible (200-400 nm and 400-800 nm respectively) and RP-HPLC method development using C18 inertsil column and optimization of variables for ambrisentan (ABN) estimation in bulk and formulations. A two-level full factorial design assisted development of a visible spectroscopic method based on the principle of oxidation and coupling reaction of ABN with 3-methyl-2-benzothiazolinone hydrazone (MBTH reagent) in presence of FeCl3 to form bluish-green chromogen which is detectable in the visible range. second method estimates ABN in bulk and tablet dosage forms in the UV range using pH 7.4 phosphate buffer and the third one is a rapid, simple, stability indicating RP-HPLC method using a degassed mixture of orthophosphoric acid (OPA, 0.05M): acetonitrile (40:60) as mobile phase with water: acetonitrile (30:70) as diluent using PDA detector set at 264 nm for routine estimation of ABN in bulk and pharmaceutical dosage forms. Experimental design, data analysis and contour plots were developed using Minitab® 16.2.4 trial version (Minitab Inc). The percent assay of ABN detected by visible spectroscopy was 100.58±101.37% with RSD value 0.38%, by UV spectroscopy it was 99.1±99.8% with 0.5% RSD value and by RP-HPLC it was found 100.9%. The values of method validation parameters of three methodologies were found within the acceptance limits. The three methodologies could be regarded as specific, accurate, precise, with significant stability and robustness for routine estimation of ABN in bulk and tablet dosage forms.



[1]  Higuchi, T., Brochmann-Hansen, E, Pharmaceutical Analysis, CBS publications, New Delhi, 1997.
[2]  Douglas, A., Skoog, F., Holler, J, Fundamentals of Analytical Chemistry, Broks/Cole publications, Stamford, 2009.
[3]  Willard, H.H., Merritt, L.L., Dean, J.A., Settle, F.A, Instrumental Methods of Analysis, CBS Publishers, New Delhi, 1986.
[4]  The Merck Index. An encyclopedia of chemicals drugs and biologics. 14th edition, Merck and Co., New Jersey, 2006.
[5]  Martindale. The Complete Drug Reference, 33rd edition, Edited by Sweetman S.C, Pharmaceutical Press, London, 2002.
Show More References
[6]  Nirogi, R., Kandikere, V., Komarneni, P., Aleti, R., Padala, N, “Kalaikadhiban I: LC-ESI-MS/MS method for quantification of ambrisentan in plasma and application to rat pharmacokinetic study,” Biomedical Chromatography, 26 (10). 1150-1156, Oct. 2012.
[7]  Dousa, M., Gibala, P, “Rapid determination of ambrisentan enantiomers by enantioselective liquid chromatography using cellulose-based chiral stationary phase in reverse phase mode,” Journal of Separation Science, 35 (7), 798-803, Apr. 2012.
[8]  Kumar, N.S., Avula, P.R., Telu, V., Sekaran, C.B, “Extractive spectrophotometric determination of ambrisentan,” Advanced Pharmaceutical Bulletin, 3 (1). 231-237. Feb. 2013.
[9]  Seshamamba, B.S.V., Satyanarayana, P.V.V., Sekaran, C.B, “Spectrophotometric methods for the determination of ambrisentan using charge transfer reagents,” Journal of Applied Chemical Reserach, 7 (3). 7-14. Jul. 2013.
[10]  Prathyusha, V., Siddartha, B., Sowmya, B., Madhavi, C., Kalyani, N, “A validated UV spectrophotometric method for the estimation of ambrisentan in pure and tablet dosage forms,” Inventi Rapid: Pharm Analysis & Quality Assurance 2014. Article ID Inventi: ppaqa/1266/13.
[11]  Nanjappan, S.K., Gandham, N.K, “A stability indicating reversed phase high performance liquid chromatography method for ambrisentan: an endothelin receptor antagonist,” Journal of Chromatographic Science, in press. 2013.
[12]  Narayana, M.B.V., Chandrasekhar, K.B., Rao, B.M, “A validated specific stability indicating RPHPLC assay method for ambrisentan and its related substances,” Journal of Chromatographic Science, in press. 2013.
[13]  Ramalingam, S., Rajappan, M., Valliappan, K, “HPLC method for the simultaneous determination of levocetirizine, ambroxol and montelukast in human plasma employing response surface methodology,” International Journal of Drug Development and Research, 4 (3). 173-185. Jul. 2012.
[14]  Wani, Y.B., Patil, D.D, “An experimental design approach for optimization of spectrophotometric method for estimation of cefixime trihydrate using ninhydrin as derivatizing reagent in bulk and pharmaceutical formulation,” Journal of Saudi Chemical Society, 2013. Online.
[15]  Gohel, N.R., Patel, B.K., Parmar, V.K, “Chemometrics assisted UV spectrophotometric and RP-HPLC methods for the simultaneous determination of tolperisone hydrochloride and diclofenac sodium in their combined pharmaceutical formulation,” Scientia Pharmaceutica, 81 (4), 983-1001. Dec. 2013.
[16]  Vadeya, C.M., Yadav, D.S., Jogia, H.A., Chauhan, P.L, “Design of experiment (DOE) utilization to develop a simple and robust reversed phase HPLC technique for related substances estimation of omeprazole formulations,” Scientia Pharmaceutica, 81 (4), 1043-1056. Dec. 2013.
[17]  Balakrishna, M., Aziz, U., Prabhakar, R., Suma, C.H, “RP-HPLC-PDA method for the analysis of ambrisentan in bulk drugs and pharmaceutical dosage forms,” Int J Chem Pharm Sci, 4 (4), 45-50. Dec. 2013.
[18]  Yanmandru, V.K., Murali, D., Rambabu, C., “New visible spectrophotometric methods for determination of ambrisentan,” Bull. Pharm Res Poster presentation, Proceedings of APP 1st Annual National Convention, 2012.
[19]  Sreenivasulu, J., Nagaraju, C.H.V.S., Rajan, S.T., Eswaraiah, S., Ramana, P.V., “Development and validation of novel stability indicating RP-HPLC method for estimation of related substances and degradation products in Ambrisentan.,” Anal Chem , 14 (1) 2014.
[20]  Nageswara Rao, R., Ramakrishna, K., “LC-MS/MS characterization of forced degradation products of ambrisentan: development and validation of a stability-indicating RP-HPLC method” New J Chem Mar. 2014.
[21]  Yoshinari, Y., Miho, T., Hideki, H., Keita, H., Yasuo, O., Yuto, Y., Kenichiro, T., Toshimasa, T., Hiroshi, Y., Kunihiko, I., “Simultaneous microdetermination of bosentan, ambrisentan, sildenafil, and tadalafil in plasma using liquid chromatography/tandem mass spectrometry for pediatric patients with pulmonary arterial hypertension,” J Pharm Biomed Anal, 89, 227-232. Feb. 2014.
[22]  Intenational Conference on Hormonization (ICH) Harmonized Tripartate Guideline. Validation of analytical procedures: Text and Methodology, Q2 (R1). Switzerland: 2005, 1-13.
Show Less References


A New Simple and Rapid Method for the Determination of Sodium Hyaluronate in Active Pharmaceutical Ingredient and Ophthalmic Formulations by DP5 Photorode

1Department of Pharmaceutical Technology, Anna University of Technology, Thiruchirapalli, Trichy, Tamilnadu, India

2Department of Analytical Research & Development, Jamjoom Pharmaceuticals, Jeddah, Saudi Arabia

World Journal of Analytical Chemistry. 2014, 2(2), 15-22
DOI: 10.12691/wjac-2-2-1
Copyright © 2014 Science and Education Publishing

Cite this paper:
K. Ruckmani, Saleem Z. Shaikh, Pavne Khalil, Aamer Khatri, Javed Akmal. A New Simple and Rapid Method for the Determination of Sodium Hyaluronate in Active Pharmaceutical Ingredient and Ophthalmic Formulations by DP5 Photorode. World Journal of Analytical Chemistry. 2014; 2(2):15-22. doi: 10.12691/wjac-2-2-1.

Correspondence to: Saleem  Z. Shaikh, Department of Pharmaceutical Technology, Anna University of Technology, Thiruchirapalli, Trichy, Tamilnadu, India. Email:


A new, simple, rapid and reliable turbidimetric routine method for the determination of sodium hyaluronate in active pharmaceutical ingredient and ophthalmic solution is described by using DP5 Phototrode. The turbidity of an aqueous ophthalmic sample solution was measured using a DP5 Phototrode™ for photometric indicated titration. Near the equivalence point, a precipitate between titrant and analyte is formed, and the solution becomes turbid. The method involves measurement of the equivalence point at minimum light transmission through the sample, containing cationic quaternary ammonium compound, cetylpyridinium chloride (CPC) solution as a dispersing agent, is measured at 520 nm. The results obtained for in-house prepared formulation and other marketed ophthalmic solution are compared with those obtained by the published HPLC method. A calibration curve was obtained from 0.08 to 0.122 mg mL–1 (r > 0.9998). Within-day % RSD was 1.08 and between-day % RSD was 1.10. Specificity/ selectivity experiments revealed the absence of interference from excipients, recovery from spiked samples for sodium hyaluronate was 98.3–100.9%. The developed method was applied to the determination of sodium hyaluronate in pharmaceutical drug substance and drug product.



[1]  Puhl, W., Scharf P, “Intra-articular hyaluronan treatment for osteoarthritis”, Annals of the rheumatic diseases, 56(7). 441. Jul. 1997.
[2]  Karlsson, J., Sjögren, L.S., Lohmander, L.S, “Comparison of two hyaluronan drugs and placebo in patients with knee osteoarthritis. A controlled, randomized, double-blind, parallel-design multicentre study”, Rheumatology (Oxford, England), 41(11). 1240-8. Nov. 2002.
[3]  Jubb, R.W., Piva, S., Beinat, L., Dacre, J., Gishen, P.A, “one-year, randomised, placebo (saline) controlled clinical trial of 500-730 kDa sodium hyaluronate (Hyalgan) on the radiological change in osteoarthritis of the knee”, International journal of clinical practice,57(6): 467-74.
[4]  Kotz, R., Kolarz, G, “Intra-articular hyaluronic acid: Duration of effect and results of repeated treatment cycles”, American journal of orthopedics (Belle Mead, N.J.), 28(11 Suppl). 5-7. Nov.1999.
[5]  Bannuru, R.R., Natov, N.S., Dasi, U.R., Schmid, C.H., McAlindon, T.E, “Therapeutic trajectory following intra-articular hyaluronic acid injection in knee osteoarthritis – meta-analysis”, Osteoarthritis and Cartilage, 19(6). 611-9. Jun.2011
Show More References
[6]  Salk, R.S., Chang, T.J., D'Costa, W.F., Soomekh, D.J., Grogan. K.A, “Sodium Hyaluronate in the Treatment of Osteoarthritis of the Ankle: A Controlled, Randomized, Double-Blind Pilot Study”, The Journal of Bone and Joint Surgery 88(2). 295-302. Feb.2006.
[7]  Beasley, K.L., Weiss, M.A., Weiss, R.A, “Hyaluronic Acid Fillers: A Comprehensive Review”, Facial Plastic Surgery, 25(2). 86-94. May. 2009.
[8]  Shimmura, S., Ono. M., Shinozaki, K., Toda, I., Takamura, E., Mashima, Y., Tsubota K, “Sodium hyaluronate eyedrops in the treatment of dry eyes”, The British journal of ophthalmology, 79(11). 1007-11. Nov.1995.
[9]  Sato, T., Sacramento, O., Danka, W., Yoshida. K., and Urishibata, O., “Clinical Effects of dietary hyaluronic acid on dry, rough skin”. J. Aesthetic Dermatology. (12).109-120. 2002.
[10]  Kajimoto, O., Odanaka, W., Sakamoto, W., Yoshida. K., Takahashi, T., “Clinical Effects of Hyaluronic acid diet for dry skin”. J. New Rem & Clin: 90-102. 2001.
[11]  The British Pharmacopoeia 2013.
[12]  Meyers, R.A. Eds.John, Size-exclusion chromatography of polymers, Encyclopedia of Analytical Chemistry, John Wiley & Sons. Sep. 2006, PP 1-26.
[13]  Andrei Medvedovici, Alexandru Farca, and Victor David, “Derivatization reactions in liquid chromatography for drug assaying in biological fluids”, CRC Press, 2009, 47, 283-314.
[14]  Volpi, N., “On-line Turbidimetric/ESI-MS separation and characterization of hyaluronan oligosaccharides from 2-mers to 40- mers” Anal. Chem. 79(16).6390-7. Aug. 2007.
[15]  Kakehi, K., Kinoshita, M., Yasueda, S, “Hyaluronic acid: Separation and biological implications” J. Chromatogr. BAnalyt. Technol, Biomed.Life Sci., 25; 797(1-2). 347-55. Nov.2003.
[16]  Bao, L.J., Yang, J.C., He, Z.H., Yang, X.Y., Liang, W.D, “Zymohydrolysis with chondroitinase ABC and high performance liquid chromatography used for th eDetermination of sodium hyaluronatein shark fin”, Se Pu, 20(6).557-9. Nov. 2002.
[17]  Koshiishi, I., Takenouchi, M., Hasegawa, T., Imanari, T, “Enzymatic method for the simultaneous determination of of hyaluronan and chondroitin sulfates using high-performance liquid chromatography”, Anal. Biochem. 265(1). 49-54. Dec. 1998.
[18]  Midura, R.J., Salustri, A., Calabro, A., Yanagishita, M., Hascall, V.C,”High-resolution separation of disaccharide and oligosaccharide alditols from chondroitin sulphate, dermatan sulphate and hyaluronan using CarboPac PA1 chromatography” Glycobiology, 4(3).333-42. Jun. 1994.
[19]  Nurminen, M., Dejmek, A., Mårtensson, G., Thylen, A., Hjerpe, A, “Clinical utility of liquid-chromatographic analysis of effusions for hyaluronate content”, Clin. Chem. 40(5). 777-80. May.1994.
[20]  Agren, U.M., Tammi, R., Tammi, M, “A dot-blot assay of metabolically radiolabeled Hyaluronan”, Anal. Biochem. 217(2). 311-5. Mar. 1994.
[21]  Akiyama, H., Shidawara, S., Mada, A., Toyoda, H., Toida, T., Imanari, T, “Chemiluminescence high-performance liquid chromatography for the determination of hyaluronic acid, chondroitin sulphate and dermatan sulphate”, J.Chromatogr.Sep. 579(2). 203-7. Sep. 1992.
[22]  Saari, H., Tulamo, R.M., Konttinen, Y.T., Sorsa, T, “Methyl prednisolone acetate induced release of cartilage proteoglycans: determination by high performance liquid chromatography”, Ann. Rheum.Dis,. 51(2). 214-9. Feb.1992.
[23]  Orviský, E., Kéry, V., Stanciková, M, “Specific high performance liquid chromatographic determination of the molecular weight and concentration of Sodium hyaluronatein complex mixtures by labeled hyaluronate binding proteins”, Biomed.Chromatogr. 5(6).251-5. Nov. 1991.
[24]  Akiyama, H., Toyoda, H., Yamanashi. S., Sagehashi, Y., Toida, T., Imanari, T, “Microdetermination of sodium hyaluronatein human urine by high performance liquid chromatography”, Biomed. Chromatogr. 5(5).189-92. Sep.1991.
[25]  Zebrower, M., Kieras, F.J., Heaney-Kieras, J, “High pressure liquid chromatographic identification of sodium hyaluronateand chondroitin sulphate disaccharides”, Glycobiology. 1(3). 271-6. Jun. 1991.
[26]  Payan, E., Jouzeau, J.Y., Lapicque, F., Muller, N., Payan, J.P., Gegout, P., Bertin, P., Netter. P, “Assay of synovial fluid sodium hyaluronateusing high-performance liquid chromatography of hyaluronidase digests”, J.Chromatogr. 566(1).9-18. May. 1991.
[27]  Whitfield, D.M., Stojkovski, S., Pang, H., Baptista, J., Sarkar, B, “Diagnostic methods For the determination of iduronic acid in oligosaccharides”, Anal. Biochem. 194(2). 259-67. May.1991.
[28]  Fedarko, N.S., Termine, J.D., Robey, P.G., “High-performance liquid chromatographic separation of hyaluronan and four proteoglycans produced by human bone cell cultures”, Anal Biochem. 188(2). 398-407. Aug. 1990.
[29]  Motohashi, N., Mori, I, “Quantitation of sodium hyaluronateand chondroitin sulphates in rabbit synovial fluid by high-performance liquid chromatography of oligosaccharides enzymatically derived thereof”, Chem.Pharm.Bull.(TOKYO), 38(3). 769-73. Mar. 1990.
[30]  Gherezghiher, T., Koss, M.C., Nordquist, R.E., Wilkinson, C.P, “Rapid and sensitive method for measurement of sodium hyaluronateand isomeric chondroitin sulfates using high- performance liquid chromatography”, J. Chromatogr. 413.9-15. Jan. 1987.
[31]  Murphy, D., Pennock, C.A., London, K.J, “Gas-liquid chromatographic measurement of glucosamine and galactosamine content of urinary glycosaminoglycans”, Clin. Chim.Acta., 53(2). 145-52. Jun. 1974.
Show Less References