Chromatography Limonoids

Manners GD, Breksa AP III, Schoch TK, Hidalgo MB. Analysis of bitter limonoids in citrus juices by atmospheric pressure chemical ionization and electrospray ionization liquid chromatography-mass spectrometry. J Agric Food Chem 2003 51(13) 3709-3714. 

In the past ten years thinlayer chromatography (TLC) has proven useful for semi-quantitative measurements of fruit and juice samples, however this method is by its very nature 1) rather insensitive (pg range) 2) slow (1 hour or more for sample application and solvent development) 3) requires pre-purification steps (liquid-liquid extraction or precipitation) 4) detection is difficult (especially true for limonoids) and 5) sample throughput is normally less than 50 per person per day. 

A variety of methods have been proposed for the detection and quantification of bitter limonoids in citrus. Most methods were developed specifically for the quantification of limonin in citrus juices. The early techniques included colorimetry, fluorimetry, and gas chromatography. 

The colorimetric method (Wilson and Crutchfield 1968) and the fluorometric method (Fisher 1973) involve the chemical derivation of the limonoids. However, the reactions are not specific for limonin, and these techniques depend on isolating limonin from interfering compounds. Kruger and Colter (1972) developed a derivitization procedure to allow for the non-volatile limonin in citrus juices to be analyzed by gas chromatography. 

In the food industry, high-performance liquid chromatography (HPLC) is the most widely used technique for the determination of limonin levels in citrus juices, since it is accurate and reliable. Most of the HPLC methods developed are for the analysis of limonin in citrus juices. These methods may not work for other bitter limonoid aglycones and/or tissues. 

Liu Y, Alford AR, Rajab MS, Bentley MD (1990) Effects and modes of action of citrus limonoids against Leptinotarsa decemlineata. Physiol Entomol 15 37-45 Maier VP, Beverly GD (1968) Limonin monolactone, the nonbitter precursor responsible for delayed bitterness in certain citrus juices. J Food Sci 33 488-492 Maier VP, Grant ER (1970) Specific thin-layer chromatography assay of limonin, a citrus bitter principle. J Agric Food Chem 18 250-252... 

Rouseff RL, Fisher JF (1980) Determination of limonin and related limonoids in citrus juices by high performance liquid chromatography. Anal Chem 52 1228-1233 Rouseff RL, Mansell RL (1982) Comparison of high performance liquid chromatographic and radioimmunoassay limonin values from commercial grapefruit juice. Proc Fla State Hortic Soc 95 249-252... 

Raman, G., Cho, M., Brodbelt, J.S., and Patil, B.S. 2005. Isolation and purification of closely related Citrus limonoid glucosides by flash chromatography. Phytochemical Analysis, 16, (3), 155-160. 

Schoch T.K., Manners G.D., Hasegawa S. Analysis of limonoid glu-cosides from citrus by electrospray ionization liquid chromatography-mass spectrometry. Journal of Agricultural and Food Chemistry, 49 1102 1108 (2001). 

Certain compounds of the prieurianin group were reported to be separable by t.l.c. in Glasgow, but to isomerise on attempted separation in Durban (35, 136). This was eventually traced as an effect caused by the thickness of the absorbent layer. Thin plates, which run rapidly, separate the mixtures while thicker plates, which run more slowly, isomerise them. Not all commercially-available plates are suitable for these separations. Particular difficulty was experienced with the limonoids of Trichilia emetica. These proved inseparable by conventional methods 200) but were separated by a special technique of high pressure liquid chromatography 138). Because of this, some compounds which have been isolated may be artefacts a case in point is the limonoids of Aphanamixispolystacha (55) which were isolated only after repeated column chromatography. 

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