Gas Chromatographic Identification of Umonin in Citrus Juice

A method for analyzing citrus juice for Limonin is presented. The method begins by isolating and concentrating the Limonin in the test sample. Limonin is extracted with chloro form and a non-Limonin containing portion re moved by partitioning between acetonitrile and hexane. The acetonitrile portion is evaporated and 0.250 ml of acetonitrile added quantitatively. An aliquot, depending upon the conceneration of Limonin, is injected in a 5% SE-30 column and Limonin determined quantitatively by comparison to a standard curve. This method is quite specific for Limonin, with a sensitivity of 0.25 ppm. Limonin, (Fig. 1), the main bitter constituent of grapefruit and orange juice, is a triterpenoid oxidation product containing two lactone rings, one ketone group, and one furan ring (1). Limonin has been quantitatively analyzed by many methods, (3, 4, 7, 9), each of which suffers to some degree in length of analysis and sensi tivity. This absence of a relatively quick, re producible method for quantitative determination Fig. 1. Limonin molecule. KRUGER, COLTER: IDENTIFICATION OF LIMONIN 207 has hampered investigation of acceptable levels of Limonin in citrus juice. The method currently in use (7) involves direct TLC of an acetonitrile extract containing Limonin, spraying with Ehrlich's Reagent, and treatment with gaseous hydrogen chloride to re veal the Limonin spots. Quantitation is made by visual comparison with Limonin of known con centration. Although this method suffers from reproducibility, since the color intensities of the spots are so difficult to reproduce, it is quite adequate where lower limits of Limonin have been set (e.g. below 5 ppm). A new approach to quantitation was therefore sought. Fales and Luukkainen (5) had found that steroids with high melting points (up to 254°C) could be successfully analyzed by GC on a 1% SE-30 column at 223°C. Fales and Pisano (6) also found that physiologically important amines that were highly polar and only slightly volatile could be chromatographed on a 4% SE-30 column at 205°C. Martin et al. (8) did a study using a 5% SE-30 column at 270 °C on the relationship between the log of the retention time and the boiling point of high boiling phenothiazine drugs. He was able to elute the compounds having high boiling points of over 430°C in a time span of 10 min. Limonin has a melting point of 296°C and was, therefore, analyzed without derivative formation. The sample extraction in the proposed method follows to some extent the Maier and Grant (7) TCL procedure, with some minor deviations. It was found that 2 x 10-ml chloroform does not extract all the Limonin from the sample. This was remedied by using 3 x 10-ml chloroform and adding 3 g of magnesium suJfate as a salting out and de-emulsifying agent. Also, to aid in recovery of the Limonin in the final step of the proposed method, a 50-ml round bottom flask was used. This proposed method has several advantages. Eight juice samples can be analyzed in a day and its sensitivity and precision are at the 0.25 ppm