Determination of Ephedra Alkaloids and Synephrine in Dietary Supplements via Strong Cation-Exchange SPE and LC–MS/MS Detection

Ephedra alkaloids are phenethylamines that occur naturally in plants, including the herb Ma Huang used in traditional Chinese medicine. The aim of this study was to develop a multi-analyte procedure for the extraction, cleanup, and quantification of the ephedra alkaloids in functional foods and natural products. High capacity strong cation-exchange SPE cartridges were used for the isolation of the phenethylamines from dietary supplements. HPLC separation, including separation of the stereoisomers, was carried out using a UCT Selectra® PFPP column prior to detection by LC–MS/MS.

Procedure

1. Sample Pretreatment

Weigh 1 ± 0.1 g of dietary supplement in question into a 15 mL polypropylene centrifuge tube. Add 10 mL of 1% formic acid to each sample. Shake or vortex sample for 15 min to fully extract the ephedra alkaloids*. Ensure samples are fully dissolved. Centrifuge the sample for 10 min at ≥3000 × g and 4 °C.

* For this study, a SPEX® SamplePrep® GenoGrinder® was used to pulverize the tablets.

2. SPE Procedure

a) SPE CONDITIONING

1. Add 2 × 4 mL of methanol to CUBCX1HL56 SPE cartridge.

2. Add 4 mL of ultrapure water.

3. Add 4 mL of 1% formic acid.

Note: Do not let the cartridge go dry otherwise repeat steps 1 through 3.

b) SAMPLE LOADING

1. Load supernatant from sample pretreatment step.

2. Allow sample to percolate through the cartridge or apply a vacuum if necessary (adjust vacuum for flow of 1–3 mL per min).

c) WASH CARTRIDGE

1. Add 2 × 4 mL of 0.1% formic acid and slowly draw through.

2. Add 2 × 4 mL methanol and slowly draw through.

3. Dry under vacuum for ≈30 s to remove excess solvent.

d) ELUTION

1. Elute the ephedra alkaloids using 8 mL of methanol containing 2% ammonium hydroxide.

2. Evaporate off the methanol solvent at 40 °C under a gentle stream of nitrogen until it reaches a volume of ≈1 mL.

3. Add 1 mL of aqueous mobile phase (10 mM ammonium acetate).

4. Evaporate off any remaining methanol.

Instrumental

Results

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