Box–Behnken design makes rhubarb extracts fine

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  • Published: Dec 1, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Laboratory Informatics / Chemometrics & Informatics
thumbnail image: Box–Behnken design makes rhubarb extracts fine

Better assay methods needed for medicinal rhubarb

Rhubarb is used in traditional Chinese medicine, having laxative and anti-inflammatory properties. The species used, Rheum palmatum or Chinese rhubarb, is not the same as the culinary rhubarb, Rheum rhabarbarum, There are a number of active compounds in Chinese rhubarb, but current analytical methods only involve assays on a few of these, thus not giving an overall picture of the activity. However, measuring more of the active compounds using conventional analytical standards is not practicable for routine assays because such standards are relatively expensive to buy. Current methods of extracting active compounds from rhubarb for assay are also rather cumbersome.

The Tianjin researchers looked at 12 active compounds in Chinese rhubarb, optimising their ultrasonic-assisted extraction (UAE). They aimed to devise an assay method using quantitative analysis of multi-components with a single marker (QAMS), where only one analytical standard is required.

Rhubarb extraction optimised using a designed experiment

Rhubarb was extracted with aqueous ethanol in a 40 kHz ultrasonic bath. Four variables were optimised for the extraction of the 12 active compounds using a three-level Box–Behnken design (Stat-Ease Design-Expert 8.0.5 software). The variables examined were: the proportion of ethanol to water, the ratio of solvent to solid, the extraction time and the extraction temperature. In all, 30 runs were carried out in a random order, including six replicates at the centre of the design. A suitable quadratic polynomial model was derived from the results. The model gave a high correlation coefficient, showing good agreement between the predicted and experimental results (r = 0.978). The optimised conditions used a 24.1:1 ratio of solvent to solid, a solvent composition of 74.4% ethanol and 25.6% water, with an extraction time of 51 minutes at 64 °C. It is interesting to note that yields gradually decreased as the temperature was raised above 64 °C, possibly due to compound instability.

HPLC for quantitative determination of the compounds was carried out using a Shimadzu i-Series instrument fitted with a Kromosil C18 column. Gradient elution was employed, with the aqueous mobile phase being 50:1 water:formic acid and the organic phase being acetonitrile. The proportion of the latter was increased from 3 to 55% in a series of gradients. The total run time was 140 minutes. A PDA (DAD) detector was used with a detection wavelength of 280 nm. The method clearly separated all 12 compounds of interest and the internal standard (methyl 2,4-dihydroxybenzoate).

The identities of the compounds were confirmed by LC-MS/MS using an Agilent 1200 system connected to an Agilent 6310 ion trap mass spectrometer. The same column and mobile phase were used as with the PDA runs. Electrospray ionisation (ESI) was carried out in both positive and negative ion modes, the former being best for the internal standard, while the latter was best for the 12 rhubarb compounds.

The PDA method was shown to give good linearity and precision. Relative calibration factors (RCFs) were calculated for the 12 compounds with respect to the internal standard. Results using the RCFs were compared with those obtained by using external standards for each of the 12 compounds. The two methods were found to give similar results, meaning that the RCF method can be deployed for routine determination of these bioactive rhubarb compounds.

New method gives easy determination of bioactive rhubarb compounds

This paper is a good example of careful extraction optimisation using experimental design. The optimised ultrasonic extraction method combined with the use of RCFs for HPLC assays allows for the simple assay of the 12 rhubarb compounds, without the use of expensive standards. The only drawback is the relatively long HPLC run time. It remains to be seen how widely the new protocol will be adopted.

Related Links

Journal of Separation Science, 2017, 40, 3792-3800. Sun et al. Quantitative studies of rhubarb using quantitative analysis of multicomponents by single marker and response surface methodology.

Lipids, 2004, 39, 273-284. Malone et al. Determining the relative amounts of positional isomers in complex mixtures of triglycerides using reversed-phase high-performance liquid chromatography-tandem mass spectrometry.

Wikipedia, Box–Behnken Design

Article by Ryan De Vooght-Johnson

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

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