Anthocyanin Profiling Method Based on Isocratic Elution for Comparable Speed, Reproducibility, and Quantitation with Gradient Elution
2017
Singh, Jashbir | Sharma, Khushbu | Walia, Suresh | Saha, Supradip
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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