Separation solved by cyclodextrin in solvent

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  • Published: Nov 8, 2018
  • Author: Ryan De Vooght-Johnson
  • Copyright: Image: BlackJack3D/iStock.com
  • Channels: HPLC

Inexpensive methods needed to check enantiomeric purity


Credit: Andrew Brookes/Getty Images.

Many drug compounds exist as pairs of enantiomers, which often vary greatly in their activity, giving rise to a need to check the enantiomeric purity of these drugs. Typically, chiral HPLC is used for such analyses, but this technique is relatively expensive and involves the use of large quantities of solvent when multiple samples are present.

High-performance thin layer chromatography (HPTLC) is generally not as accurate as HPLC, but is less expensive and uses much less organic solvent, making it more environmentally friendly. The Alexandria University scientists used chiral β-cyclodextrin as a solvent additive to separate three β-blocker compounds: betaxolol, carvedilol and sotalol. The separation conditions were optimised using a Box-Behnken experimental design with three variables. This design has three levels for each variable; the experimental points can be considered as the centre point of a cube and 12 points in the mid-points of each edge of the cube.

Box-Behnken design used to optimise HPTLC conditions

HPTLC was carried out using Fluka aluminium plates coated with F254 fluorescent silica gel 60. A 100-µL microsyringe and a Linomat IV applicator (both Camag) were for sample application. After development in a solvent tank, the plate was scanned at 220 nm (for betaxolol and sotalol) and 245 nm (for carvedilol) using a Camag TLC Scanner III. Chiral resolution factors (the ratio of the Rf values of the two enantiomers) were calculated for each run.

The runs for the Box-Behnken design were devised by StatSoft Statistica 10 software, with the levels being suggested by preliminary data. The solvent was a mixture of acetonitrile, methanol, acetic acid and water, with a ratio of seven parts acetonitrile plus methanol to one part water. Three variables were considered: the concentration of β-cyclodextrin in the mobile phase (0.1, 0.55 or 1.1 mM), the amount of acetonitrile in the organic phase (30, 50 or 70%) and the volume of acetic acid added to the mobile phase (0.1, 0.2 or 0.3 mL). 15 runs were carried out, three of which were centre point duplicates, while the other 12 were mid-points of the cube edges.

The results for the three variables were found to be quadratic rather than linear, having curved response surfaces. The optimum conditions for enantiomeric separation were shown to be fairly similar for each of the three drugs. So the means of the optimum conditions for each drug were recommended as a standard method suitable for all three, giving 0.57 mM β-cyclodextrin, 48.85% acetonitrile (in the organic phase, not counting the water) and 0.18 mL acetic acid added. These conditions gave good separation (Rf difference greater than 0.2) for all three pairs of enantiomers. The method was shown to give good linearity, accuracy and precision. The limits of detection (LODs) ranged from 0.13 to 0.3 µg/band.

The method was used to quantify the amount of the inactive (+)-betaxolol in samples of active (-)-betaxolol. The active enantiomer was spiked with the inactive isomer at the 1% level; this contamination was successfully measured by the HPTLC method. The method was also applied to commercial racemic betaxolol tablets and eye drops, with equal amounts of the two enantiomers being found in both formulations as expected.

HTPLC can be used to determine the enantiomeric purity of β-blockers

The powerful experimental design enabled a practical HPTLC method to be devised to measure the enantiomeric purity of β-blockers. For laboratories with a high throughput of samples, HPTLC can be a less expensive and greener option than HPLC owing to its low solvent usage, while also giving higher throughput. Often it is only lack of expertise in the field that prevents laboratories using this technique for some of their routine analyses.

Related Links

Chirality, 30, 1195-1205. El-Kimary et al.: "Box‐Behnken response surface modeling assisted enantiomeric resolution of some racemic β‐blockers using HPTLC and β‐cyclodextrin as chiral mobile phase additive: Application to check the enantiomeric purity of betaxolol."

Wikipedia: Box-Behnken design

Journal of Pharmaceutical and Biomedical Analysis 2014, 98, 52-95. Loescher et al.: "High performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC) for the qualitative and quantitative analysis of Calendula officinalis—Advantages and limitations."

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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