Liquid chromatographic preparative method for isolating ergot alkaloids, using a particle-loaded membrane extracting disk

A liquid chromatographic method is described for the determination of ergot alkaloids in wheat. Ergonovine, ergotamine, ergocornine, alpha -ergocrypti ne, and ergocristine are extracted from wheat with methanol-0.25% concentra ted H3PO4 (40 + 60) pH 2.2, cleaned up by using a solid-phase extraction (S PE) disk, and separated by reversed-phase liquid chromatography with fluore scence detection. Ergot alkaloids are basic compounds that form water-solub le salts in acidic aqueous solution. Because ergot alkaloid salts are posit ively charged, they can be easily and selectively trapped on a negatively c harged strong cation-exchange SPE disk. A strong wash solvent, methanol-0.2 5% concentrated H3PO4 (40 + 60) was used to remove matrix Interferences not bonded by ionic interactions with the cation-exchange column, The ergot al kaloids were eluted from the ion-exchange column by adjusting the pH of the elution solvents to slightly basic conditions (pH 9). The SPE disk concent rated and cleanly separated the ergot alkaloids from matrix interferences. Standard calibration curves for ergot alkaloids for the concentration range 0.1-2.0 mug/mL were linear. The SPE disk had a column capacity equivalent to about 1 g extracted wheat. At spiking levels of 2.3-46 ng/g for ergonovi ne and 20-400 ng/g for ergotamine, ergocornine, alpha -ergocryptine, and er gocristine, the mean recovery was 88.1% with a coefficient of variation (CV ) of 5.33%. The recovery data ranged from 79.1 to 95.9%. Ergonovine had the lowest overall recovery and the largest CV. The method has an estimated re liable limit of detection and limit of quantitation of <5 and <20 ng/g, res pectively, for each ergot alkaloid tested.