Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/15/2003
Publication Date: 5/23/2003
Citation: Manners, G.D., Schoch, T.K., Hidalgo, M. 2003. Analysis of bitter limonoids in citrus juices by atmospheric pressure chemical ionization liquid and electrospray ionization liquid chromatography-mass spectrometry. Journal of Agricultural and Food Chemistry. Vol 51, No. 13, p. 3709-3714. Interpretive Summary: Limonin is a bitter compound that occurs naturally in citrus. When present in amounts greater than 6 ppm, the juice becomes unacceptable to consumers. Orange juice producers continue to look for new methods to quickly and accurately determine the amount of limonin in citrus juice. Here we describe a method for analyzing limonin and related bitter compounds using liquid chromatography (LC) coupled with mass spectrometry (MS). The method utilizes the ability of LC to separate compounds in a mixture, and the ability of MS to detect and selectively quantify unique electronically charged fragments formed from the limonin and related compounds in the presence of other non-bitter substances. The charged fragments are unique for each bitter limonoid present in citrus juice and the ability of the MS to measure the charged fragments makes it 40 times more sensitive than current limonin analysis methods. These characteristics allow highly selective measurements of very small amounts of bitter compounds in citrus juice without interference. The specificity, coupled with sensitivity, make the method more accurate and widely applicable. These factors will allow juice producers to determine a "total bitterness factor" for juice which represents a sum of limonin and related compounds which could not be previously measured accurately.
Technical Abstract: Normal phase chromatography utilizing 3u spherical silica in a microbore column coupled to a mass spectrometer operating in a positive ion atmospheric pressure chemical ionization (APCI) mode was found to be a rapid, selective and highly sensitive method for the analysis of both the bitter limonoids (limonin and nomilin) and the non-bitter limonoids (deacetylnomilin and obacunone) in citrus juices. Analysis was performed on the chloroform soluble materials obtained by a simplified extraction and processing procedure applied to clarified citrus juices. The method includes an internal standard, is capable of detecting limonoids down to the low picogram range and is capable of quantifying accurately "total limonoid bitterness" in citrus juices as represented by the combined occurrence of limonin and nomilin.