Determination of Bixin and Norbixin

Determination of Bixin and Norbixin in Human Blood Plasma by GC (26)

Bixin is a naturally occurring carotenoid and has a unique structure among alkaloids as it contains two carboxylic groups one of which is a methyl ester.

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Courtesy of the Journal of Chromatography

Figure 44. Chromatogrm of Carbonyl Compounds Contained in Hexane Extract of Urine Monitored as Pentafluoro-phenylhydrazine

Bixin occurs in the seeds of the Annatto plant Bixa Orellna together with the water soluble hydrolyzed derivative norbixin and is used as Annatto Food colors. Annatto has been the normal part of the Latin American diet for many centuries. The normal intake of bixin or nor bixin is relatively low (less than 4 mg per day) but its long-term effect on human metabolism should be known. The assay uses liquid-liquid extraction followed by LC analysis and sample preparation is carried out in three stages. An internal standard (Sudan 1) is added to the plasma, the serum proteins are denatured by the addition of alcohol and the bixin, norbixin and standard are extracted into chloroform. The chloroform extract is evaporated to dryness and the residue taken up in methanol. A sample of the methanol solution is injected onto the column and the amount of bixin and norbixin estimated by comparing the peaks to that of the standard. Details of the procedure are as follows. 10 ml of blood was centrifuged and the plasma isolated. 3 ml of the plasma and then treated with 6 ml of alcohol and allowed to stand for 30 min. 3 ml of chloroform was then added and the mixture shaken in a separating funnel and the lower layer discarded.