Abstract
The present work aims at developing a simple and rapid method for the quantification of anthocyanins irrespective of acylation present or not. The proposed analytical approach is based on isocratic elution mode in order to avoid the complexity of gradient elution system. The optimal conditions for the separation of anthocyanins were obtained as 14 % acetonitrile in water containing 2 % trifluroacetic acid with a flow rate of 0.6 ml min−1 when analyzed in Syzygium cumini (Jamun) anthocyanins. The method was successfully separated eight anthocyanins present in Jamun using C18 column as a stationary phase. Anthocyanins characterization was done by using electrospray ionization mass spectrometry (ESI-MS). LOD and LOQ value was calculated based on signal-to-noise ratio (S/N ratio) and ordinary least square regression (OLSR) approaches. LOD were obtained as 0.9, 0.4, and 0.3 μg ml−1 by S/N ratio method and 0.27, 1.2, and 1.0 μg ml−1unit by OLSR method for M3-G, C3-G and D3-G, respectively. In the isocratic elution mode, critical pair resolution was 1.95 whereas it was 0.85 under the gradient elution mode. Interestingly, ratio of peak area and peak height in gradient and isocratic elution was virtually same for almost all the evaluated peaks. More importantly, the method was validated with other three sources of anthocyanins (black carrot, red cabbage, and berry) and found variable resolution and response. After analyzing all the data, it may be concluded that the proposed method is useful for separation and identification of anthocyanin irrespective of acylation present in it when the number of anthocyanin is ≤10.
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References
Aguilar MI, Hodder AN, Hearn MTW (1991) High-performance ion-exchange chromatography of proteins. In: Hearn MTW (ed) HPLC proteins, peptides polynucleotides. VCH, New York, p 199
Berry VV, Shansky RE (1984) Comparison of two novel techniques for eliminating liquid chromatography gradient ghost peaks. J Liq Chromatogr 7:943–952
Castaneda-Ovando A, Pacheco-Hernandez MDL, Paez-Hernandez ME, Rodriguez JA, Galan-Vidal CA (2009) Chemical studies of anthocyanins: a review. Food Chem 113:859–871
De Pascual-Teresa S, Sanchez-Ballesta MT (2008) Anthocyanins: from plant to health. Phytochem Rev 7:281–299
Dolan JW, Snyder LR, Djordjevic NM, Hill DW, Waeghe TJ (1999) Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time: I. Peak capacity limitations. J Chromatgr A 857:1–20
Faria F, Marques MC, Mercadante AZ (2011) Identification of bioactive compounds from jambolão (Syzygium cumini) and antioxidant capacity evaluation in different pH conditions. Food Chem 126:1571–1578
Hou DX (2003) Potential mechanisms of cancer chemoprevention by anthocyanins. Curr Mol Med 3:149–159
Ichiyanagi T, Hatano Y, Matsugo S, Konishi T (2004) Structural dependence of HPLC separation pattern of anthocyanins from bilberry (Vaccinium myrtillus L). Chem Pharm Bull 52:628–630
Jandera P (2000) Comparison of various modes and phase systems for analytical HPLC, Handbook of Analytical Separations 1: 1
Li L, Adams LS, Chen S, Killian C, Ahmed A, Seeram NP (2009a) Eugenia jambolana Lam. berry extract inhibits growth and induces apoptosis of human breast cancer but not non-tumorigenic breast cells. J Agric Food Chem 57:826–831
Li L, Zhang YJ, Seeram NP (2009b) Structure of anthocyanins from Eugenia jambolana fruit. Nat Prod Commun 4:217–219
Matsui T, Ebuchi S, Kobayashi M, Fukui K, Sugita K, Terahara N (2002) Anti-hyperglycemic effect of diacylated anthocyanin derived from Ipomoea batatas cultivar Ayamurasaki can be achieved through the α-glucosidase inhibitory action. J Agric Food Chem 50:7244–7248
Patterson SD (1992) Use of solvent selectivity optimization procedures for high-performance liquid chromatographic method development. J Chromatgr A 592:43–49
Schellinger AP, Carr PW (2006) Isocratic and gradient elution chromatography: a comparison in terms of speed, retention reproducibility and quantitation. J Chromatgr A 1109:253–266
Schott RPW (1993) Silica gel and bonded phases. Their production, properties and use in LC. Wiley, New York, p 261
Snyder LR, Kirkland JJ, Glajch JL (2012) Practical HPLC method development. Wiley, New York
Unger KK (1990) Chromatographic science series 47. Marcel Dekker, New York, p 840
Vial J, Jardy A (1999) Experimental comparison of the different approaches to estimate LOD and LOQ of an HPLC method. Anal Chem 71:2672–2677
Williams S (2014) Ghost peaks in reversed-phase gradient HPLC: a review and update. J Chromatogr A 1052:1–11
Woodward G, Kroon P, Cassidy A, Kay C (2009) Anthocyanin stability and recovery: implications for the analysis of clinical and experimental samples. J Sci Food Agric 57:5271–5278
Acknowledgments
Present work was funded by National Agricultural Innovation Project (ICAR). The authors would like to thank Head, Division of Agricultural Chemicals, Indian Agricultural Research Institute for providing all facilities.
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Jashbir Singh declares that he has no conflict of interest. Khushbu Sharma declares that she has no conflict of interest. Suresh Walia declares that he has no conflict of interest. Supradip Saha declares that he has no conflict of interest.
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Singh, J., Sharma, K., Walia, S. et al. Anthocyanin Profiling Method Based on Isocratic Elution for Comparable Speed, Reproducibility, and Quantitation with Gradient Elution. Food Anal. Methods 10, 118–128 (2017). https://doi.org/10.1007/s12161-016-0561-z
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DOI: https://doi.org/10.1007/s12161-016-0561-z