Limonin was first reported (9) in 1938 as the cause of bitterness in Washington navel orange juice. It was not until 1961 that the complex chemical structure of limonin was finally worked out (10) and 1965 when Maier and Dryer (11) first reported the presence of limonin in commercial grapefruit juice. Limonin is a highly oxygenated triterpene derivative whose structural features include a furan ring, two lactone rings, a five-member ether ring, and an epoxide (Figure 1). Freshly squeezed citrus juices do not contain limonin. After extraction, limonin forms slowly over a period of several hours from a nonbitter precursor present in the fruit due to enzyme activity (12). Limonin forms rapidly if the juice is heated. The limonin precursor is limonoic acid A-ring lactone; being formed by closure of the D-ring as shown in Figure 1. An in-depth review of limonin and related structures has been presented by Maier et al. (13).
When Maier and Dryer (11) first discovered the presence of limonin in commercial grapefruit juice, thin-layer chromatography (TLC) was used. In 1970 Maier and Grant (14) developed a specific TLC assay for limonin. Scott (15), using an adaptation of their assay, reported limonin in many Florida citrus cultivars, including grapefruit. Tatum and Berry (16) in 1973 developed a quicker TLC method, which was adopted commercially.
Limonin today is generally assayed by high-performance liquid chromatography (HPLC), and a number of methods have been published. The real challenge with limonin determination by HPLC is one of detection. Although limonin is a major contributor to bitterness in citrus juices, it is only present at the low part per million level. Limonin does not contain a good UV absorbing chro-mophore, does not fluoresce, and is not easily reduced or oxidized using electrochemical detection. This limits LC methods to refractive index or UV detection at the lower wavelengths unless limonin is derivatized to enhance UV absorbance or fluorescence.
One of the first HPLC methods was developed by Fisher (17) and utilized refractive index detection. Subsequent HPLC methods (18-21) have utilized the more sensitive detection method of UV absorbance at lower wavelengths of 205 to 215 nm. Because limonin occurs in citrus juices in the low part per million range and most substances absorb in the UV below 220 nm, preparation of the sample
Limonoic acid A-ring lactone
Figure 1. Structures of limonin and limonoic acid A-ring lactone, the precursor to limonin.
to obtain an extract free of interferences upon analysis is demanding. Rouseff and Fisher (18) developed an accurate method using chloroform extraction to isolate limonin. Analysis with a cyano column by normal phase chromatography with hexane:isopropanol as the mobile phase and UV detection at 214 nm produced excellent results. Analysis of the same chloroform extracts under reversed phase conditions with acetonitrile:water on a cyano column resulted in a good separation but short analytical column life.
A number of reversed phase chromotography methods that utilize the more robust octyl (C8) and octydecyl (C18) analytical columns have also been published. Sample cleanup to remove interfering components was accomplished by solid phase extraction (19-21). Solid phase extraction (SPE) offers several advantages over solvent extraction. It is less time and labor intensive. Less solvent is required for preparation of each sample. Reversed phase chromatography is also generally preferred over normal phase chromatography as water comprises most of the mobile phase solvent and can be produced relatively inexpensively. For limonin analysis, one problem with methods that utilize C8 and C18 reversed phase columns is that the mobile phase conditions must be changed depending on the type of citrus juice being analyzed. Some grapefruit juice samples require different analysis conditions than those that provide a good separation of orange juice extracts. The mobile phases required also generally consist of mixtures of methanol:acetonitrile:water or methanol:tetrahydrofuran:water. Mobile phases containing methanol:acetonitrile:water give low broad peaks reducing detectability. Tetrahydrofuran tends to form epoxides that absorb strongly in the low UV requiring diligent use of fresh solvents.
In 1991 Widmer (22) developed a general citrus juice analysis method with sample preparation by SPE, analysis on a cyano column under reversed phase conditions using acetonitrile:water, and detection by UV at 214 nm. Unlike chloroform extracts of citrus juices, the SPE extract preparation was optimized so integrity of the cyano analytical column under reverse phase conditions was maintained, resulting in less cost per sample analysis. Limonin recovery using SPE from spiked grapefruit and model juices ranged from 95 to 108%. Results from 25 samples analyzed by the new method and the method of Rouseff and Fisher (18) also showed good agreement between the two methods with differences in the range of 5 to 13%. The method has since been modified for analysis of citrus juices and citrus peel extracts by direct injection with an in-line automated sample cleanup (23). Both limonin and naringin are determined simultaneously with an analysis time of 20 min. Sample preparation for citrus juice requires only heating and (1:1) dilution with 40% aqueous acetonitrile. Preparation of peel extracts is accomplished by sonication of the peel in aqueous acidified acetonitrile.
Other novel methods for limonin analysis that have been developed include the rapid radioimmunoassay (RIA) developed by Mansell and Weiler (24) and the enzyme linked immunoassay (EIA) developed by Jourdan et al. (25). The EIA method was commercialized and available as a rapid test kit (26) for a number of years. Carter and coworkers (27) in 1985 compared the RIA and EIA methods to HPLC methods available at the time. Samples analyzed by EIA tended to have limonin values slightly higher than those analyzed by HPLC. Widmer and Rouseff (28) reported on a collaborative study done to assess the commercial EIA method. Problems with reproducibility in citrus quality assurance labs (private communications), sample analysis costs that were higher than anticipated, and subsequent development of rapid and reliable HPLC methods likely resulted in the loss of interest by the citrus industry for the commercial EIA method.
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