Preparative HPLC for large scale isolation, and salting-out assisted liquid-liquid extraction based method for HPLC-DAD determination of khat (Catha edulis Forsk) alkaloids

Abstract

Background: Khat (Catha edulis Forsk) is an evergreen shrub of the Celastraceae family. It is widely cultivated in Yemen and East Africa, where its fresh leaves are habitually chewed for their momentary pleasures and stimulation as amphetamine-like effects. The main psychostimulant constituents of khat are the phenylpropylamino alkaloids: cathinone, cathine and norephedrine.

Results: In this study, simple procedures based on preparative HPLC and salting-out assisted liquid-liquid extraction (SALLE) based methods were developed respectively for large scale isolation and the extraction of psychoactive phenylpropylamino alkaloids; cathinone, cathine and norephedrine, from khat (Catha edulis Forsk) chewing leaves, a stimulant and drug of abuse plant. The three khat alkaloids were directly isolated from the crude oxalate salt by preparative HPLC-DAD method with purity > 98%. In addition, a modified (SALLE) method has been developed and evaluated for the extraction efficiency of psychoactive phenylpropylamino alkaloids from khat (Catha edulis Forsk) chewing leaves. An in situ two steps extraction protocol was followed without dispersive SPE clean up. The method involves extraction of the samples with 1% HAc and QuEChERS salt (1.0 g of CH₃COONa and 6.0 g of MgSO₄) followed by subsequent in situ liquid-liquid partitioning by adding ethyl acetate and NaOH solution. The optimized method allowed recoveries of 80-86% for the three alkaloids from khat sample with relative standard deviation (RSD) values less than 15% and limits of detection (0.85-1.9 μg/mL).

Conclusion: The method was found to be simple, cost-effective and provides cleaner chromatogram with good selectivity and reproducibility. The SALLE based protocol provided as good results as the conventional extraction method (ultrasonic assisted extraction followed by solid phase extraction, UAE-SPE) and hence the method can be applicable in forensic and biomedical sectors.

Keywords: Alkaloids; Cathine; Cathinone; Khat; Norephedrine; Preparative HPLC; Salting-out assisted liquid–liquid extraction.

Fig. 1

The molecular structures of the khat alkaloids

Fig. 2

Preparative HPLC chromatogram of the a oxalate salt (right to left: cathinone, cathine, norephedrine and oxalate ion) and b borohydride reduced product (right to left: cathine and norephedrine)

Fig. 3

Superimposed HPLC–DAD chromatograms of fractions collected from prep HPLC and concentrated as oxalate salts. CA cathinone, NPE norpseudoephedrine or cathine, NE norephedrine

Fig. 4

a Effect of soaking time, b effect of shaking time and c effect of pH on the extraction efficiency of norephedrine, cathine and cathinone

Fig. 5

Superimposed HPLC–DAD chromatograms obtained for: a spiked and unspiked samples extracted with SALLE, and pure standards (60, 60, and 30 µg/mL respectively of norephedrine, cathine and cathinone) and b sample extracted with SALLE and UAE–SPE methods