Abstract
Two chromatographic techniques are described for the separation and quantitative determination of azadirachtin A and B, salannin, and nimbin present in the crude extract of neem seed kernels and commercial neem based formulations. The high performance thin-layer chromatography (HPTLC) separation of markers was carried out on Merck TLC aluminium sheets of silica gel 60 F254 using ethylacetate-benzene (7.0: 3.0, v/v) as mobile phase. The other technique was based on extended length packed column supercritical fluid chromatographic (PC-SFC) separation of the markers using Cyano column (250 mm × 4.6 mm I.D, S-5.0 μ) and Kromasil 100 NH2 column (250 mm × 4.6 mm I.D, 5.0 μ) connected in series. The detection was carried out using photodiode array detector at 338 K using methanol-modified carbon dioxide (10%) as the mobile phase at flow rate of 2.0 mL min−1. The current study assesses the effect of extending column length during PC-SFC experiment to obtain maximum resolution between a number of unknown components and known markers present in neem seed extracts. Both the chromatographic methods were validated in terms of precision, robustness, recovery, limits of detection and quantitation. The analysis of variance (ANOVA) and Student’s t-test were applied to correlate the results of quantitative determination of markers by means of HPTLC and PC-SFC method.
Similar content being viewed by others
References
Butterworth JH, Morgan ED (1968) J Chem Soc Chem Commun 23–24
Butterworth JH, Morgan ED (1972) J Insect Physiol 17:969–977
Broughton HB, Ley SV, Slawin AMZ, Williams DJ, Morgan ED (1986) J Chem Soc Chem Commun 46–50
Kraus W, Bokel M, Klenk A, Pohnl H (1985) Tetrahedron Lett 26:6435–6436
Jacobson M, Green MB, Hedin PA (1986) ACS Symposium Series, American Chemical Society, Washington, DC, 269:220–222
Mordue AJ, Blackwell A (1993) J Insect Physiol 39:903–924
Schmutterer H (1995) Insecta, Insects in the Neem Tree (H. Schmutterer, ed.) VCH, Weinheim, 167–365
Harris M, Henderson R, McCrindle M, Overton KH, D. W. Turner (1968) Tetrahedron 24:1517–1523
Warthen JD, Uebel EC, Dutky SR, Lusby WR, Feingold H (1978) Agric Res Results, (North eastern Ser.) ARR-NE-2
Warthen JD, Stokes JB, Jacobson M, Kozempel MF (1984) J Liq Chromatogr 7:591–595
Yamasaki RB, Klocke JA, Lee SM, Stone GA, Darlington MV (1986) J Chromatogr 356:220–226
Thejavathi R, Shirish R, Yakkundi S, Ravindranath B (1995) J Chromatogr A 705:374–379
Kaushik N (2002) Anal Bioanal Chem 374:1199–1204
Barrek S, Paisse O, Grenier-Loustalot MF (2004) Anal Bioanal Chem 378:753–763
Schaaf O, Jarvis AP, van der Esch SA, Giagnacovo G, Oldham NJ (2000) J Chromatogr A 886:89–97
Vuorela P, Rahko EL, Hiltunen R, Vuorela H (1994) J Chromatogr A 670:191–198
Ferenczi-Fodor K, Vigh Z, Nagy-Turak A, Renger B, Zeller M (2001) J AOAC Int 84:1265–1276
Ferenczi-Fodor K, Vigh Z (2001) Planar Chromatography-A Retrospective View from the Third Millennium (Sz. Nyiredy, ed.), Springer Scientific Publisher, Budapest, 336–352
European Pharmacopoeia (1999) Council of Europe, Strasbourg, 3rd edn 1998, Suppl., pp. 5–7
Thin layer chromatography (2002) Monograph 2.2.27, European Pharmacopoeia, Published by Council of Europe 4.5:3638–3642
Mantovani G, Vaccari G, Dosi E, Lodi G (1998) Carbohydr Polym 37:263–269
Doner LW (2001) Chromatographia 53:579–583
Sherma J, Fried B (1996) Handbook of Thin-Layer Chromatography, Marcel Dekker, New York, 2nd edn pp. 129-148 and pp. 273–306
Renger B (1993) J AOAC Int 76:7–14
Abdelrahman AN, Karim EIA, Ibrahim KEE (1994) J Pharm Biomed Anal 12:205–208
Kaniou I, Zachariadis G, Kalligas G, Tsoukali H, Stratis J (1994) J Liq Chromatogr 17:1385–1398
Ponder GW, Stewart JT (1994) J Chromatogr A 659:177–183
Weins C, Hauck HE (1996) LC GC 14:456–464
McCarthy KE, Wang Q, Tsai EW, Gilbert RE, Brooks MA (1998) J Pharm Biomed Anal 17:671–677
Jovanovic SE, Agbaba D, Zivanov-Stakic D, Vladimirov S (1998) J Pharm Biomed Anal 18:893–898
WHO, Guidelines for the Assessment of Herbal Medicine, Munich, World Health Organization, 1991
Küppers S, Lorenschaft B, Schmitz FP, Klesper E (1989) J Chromatogr 475:85–94
Schmitz FP, Klesper E (1990) J Supercrit Fluids 3:29–34
van Puyvelde F, van Rompay P, Chimowitz EH (1992) J Supercrit Fluids 5:227–232
Linnemann KH, Wilsch A, Schneider GM (1986) J Chromatogr 369:39–42
Gyllenhaal O, Vessman J (1993) J Chromatogr 628:275–280
Petersson P, Lundell N, Markides KE (1992) J Chromatogr 623:129–134
Jenkins TJ, Kaplan M, Simmonds MR (1991) Analyst 116:1305–1308
Bhoir IC, Sundaresan M, Bhagwat AM (1997) Anal Chim Acta 354:123–128
Patel YP, Sundaresan M, Bhagwat AM, Raman B (1997) Indian J Pharm Sci 59:3–8
Patil ST, Sundaresan M, Bhoir IC, Bhagwat AM (1998) Talanta 47:3–10
Bhoir SI, Sundaresan M, Bhoir IC, Bhagwat AM (2001) J Chromatogr B 757:39–47
Bari VR, Dhorda UJ, Sundaresan M (1997) Talanta 45:297–304
Bhoir IC, Sundaresan M, Bhagwat AM, Raman B (1998) J Pharm Biomed Anal 17:539–546
Bari VR, Sundaresan M, Dhorda UJ (1998) Anal Chim Acta 376:221–225
Patil ST, Sundaresan M, Bhoir IC (1999) Talanta 48:1179–1189
Bhoir IC, Raman B, Sundaresan M, Bhagwat AM (1997) Anal Chim Acta 354:123–132
Patil ST, Sundaresan M, Bhoir IC (1999) Anal Chim Acta 384:143–150
Simmons BR, Jagota NK, Stewart JT (1994) J Pharm Biomed Anal 12:1317–1323
Smith MS, Oxford J, Evans MB (1994) J Chromatogr 683:402–406
Strode JTB, Taylor LT, Howard AL, Brooks MA (1994) J Pharm Biomed Anal 12:1003–1009
Mount DL, Todd GD, Navaratnam V (1995) J Chromatogr 666:183–187
Patel YP, Sundaresan M, Dhorda UJ (1998) Talanta 47:625–630
Berger TA, Wilson WH (1993) Anal Chem 65:1451–1455
ICH, Q2A Validation of Analytical Procedure: Methodology, International Conference on Harmonization, Geneva, October, 1994
ICH, Q2B Validation of Analytical Procedure: Methodology, International Conference on Harmonization, Geneva, March, 1996
ICH Guidance on Analytical Method Validation, International Convention on Quality for the Pharmaceutical Industry, Toronto, Canada, September 2002
McCarthy KE, Wang Q, Tsai EW, Gilbert RE, Brooks MA (1998) J Pharm Biomed Anal 17:671–677
Fried B, Sherma J (1994) Thin-Layer Chromatography, Techniques and Applications, third edn Marcel Dekker, New York
Sethi PD (1996) High Performance Thin Layer Chromatography, Quantitative Analysis of Pharmaceutical Formulations, CBS Publishers and Distributors, New Delhi
Miller JC, Miller JN (1992) Statistics for Analytical Chemistry, second ed., Ellis Horwood, New York
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Agrawal, H., Kaul, N., Paradkar, A.R. et al. Standardization of Crude Extract of Neem Seed Kernels (Azadirachta indica A. Juss) and Commercial Neem Based Formulations Using HPTLC and Extended Length Packed-Columns SFC Method. Chroma 62, 183–195 (2005). https://doi.org/10.1365/s10337-005-0588-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1365/s10337-005-0588-6