Honey flavonoids: Detected by micellar liquid chromatography

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  • Published: Oct 4, 2010
  • Author: Steve Down
  • Channels: HPLC
thumbnail image: Honey flavonoids: Detected by micellar liquid chromatography

Honey antioxidants

Of the many components in honey, the natural antioxidants have attracted much attention. They comprise series of phenolic compounds such as flavonoids, cinnamic acids and substituted benzoic acids and impart health benefits to the honeys. They have also been shown to be useful markers of the floral source or geographical origin of the product.

For instance, citrus honeys are characterised by the presence of hesperetin which is generally absent from honeys of diverse floral origin such as rosemary, lavender, almond, clover and rhododendron. Similarly, quercetin is a biomarker for sunflower honey. Geographically, European honeys are characterised by the presence of pinocembrin, pinobanksin and chrysin.

Traditionally, flavonoids are detected and measured in honey (and many other food products) by HPLC using aqueous-organic mobile phases. However, two Iranian scientists have proposed that a less common mode, micellar liquid chromatography (MLC) might be a better option.


Micellar liquid chromatography

In MLC, the mobile phase consists of an aqueous solution of surfactant above the critical micellar concentration, often modified by an organic compound which reduces peak broadening for a more efficient separation of the analytes. Separation relies on competitive binding between the micelles in the mobile phase and the stationary phase, which is often the typical reversed-phase alkyl-bonded silica.

MLC has the acknowledged advantages of low volatility, low cost, reduced toxicity and the ability to separate ionic and non-ionic compounds in the absence of gradient elution.

Mohammad Reza Hadjmohammadi and Saman Nazari from the University of Mazandran, Babolsar, Iran, employed MLC for the separation of the three key citrus honey flavonoids quercetin, hesperetin and chrysin. The honey was collected directly from the hive in a citrus garden at Babol, north of Iran, and diluted with water. The flavonoids were extracted by SPE using a C18 cartridge and eluting with methanol.


Optimisation of flavonoid separation by experimental design

There are four key variables involved in MLC and their effects on the retention times can be interdependent and nonlinear, so the researchers adopted a systematic approach to their optimisation. A two-level full factorial was used to determine the significance of the variables followed by central composite design (CCD) using the Pareto optimal method to establish the optimum values.

A C18 column was employed with a UV detector operating at 269 nm. The concentration of the surfactant sodium dodecyl sulphate (SDS), the alkyl chain length (N) of the alcohol used as organic modifier, and the volume percentages of added modifier and added acetic acid were investigated. An acidic mobile phase was required due to the nature of the flavonoids and acetic acid was preferred over phosphate or acetate.

The most important factor was N, with increasing values lowering the retention time. The remaining factors also had an effect, reducing the retention time as their values were increased.

Due to interactions between the separate variables, CCD was used to derive several models of the retention time. The best model was selected on the basis of the balance between resolution and retention time, leading to optimum condition of 0.124 M SDS, 7.8 vol.% ethanol as the organic modifier and 5.0 vol.% acetic acid.


MLC of citrus honey flavonoids

Under the optimised conditions, the detection limits of hesperetin, quercetin and chrysin were 0.0126, 0.0091 and 0.0079 mg/L, respectively, with linear calibration ranges of 0.05-50.0 mg/L. The peak area repeatabilities and retention time repeatabilities were excellent at 1.42-2.99% and 0.28-0.87%, respectively. Recoveries were all greater than 92%.

The flavonoid contents of the Iranian citrus honey were found to be 1.24, 0.31 and 0.48 mg/kg for hesperetin, quercetin and chrysin, respectively.

Although separation by MLC requires a little work on optimisation using experimental design, the optimised method separated the three flavonoids within 25 min, free from interfering peaks and with symmetrical peak shape, providing a viable alternative to conventional reversed-phase HPLC.


The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

 
 
 
Three of the principle honey flavonoids have been separated and quantified by micellar liquid chromatography using full factorial design to optimise the separation conditions for symmetrical peak shapes and good resolution

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