STANDARDIZATION OF A RAPID SPECTROPHOTOMETRIC METHOD FOR LYCOPENE ANALYSIS

Health professionals and consumers are becoming increasingly aware of the benefits of lycopene consumption; tomatoes provide the greatest source of this important phytonutrient. Our laboratory has developed a standard method for the extraction and determination of lycopene in tomato products. This is a sensitive, rapid and reproducible assay utilizing a mixture of hexane/ethanol/acetone for extraction. Following incubation for 1 hour, the hexane layer is removed using a Drummond pipettor and lycopene is determined by measuring absorbance at 503 nm. This method will be useful for breeding programs and processors who are interested in evaluation of a large number of samples in a rapid fashion. In this manuscript we describe the method and how it was validated. INTRODUCTION There is currently no single standardized protocol for the routine determination of lycopene in tomatoes. In 2003, the California League of Food Processors requested that we evaluate published procedures for determination of lycopene, as well as develop a rapid method if such a procedure did not exist already. Based on our review of the many publications where lycopene content of tomatoes was determined, we concluded that a method based on solvent extraction followed by the direct spectrophotometric determination of lycopene will have the best combination of speed, simplicity, and analytical precision. Our focus was to examine and refine methods of this type with the aim of developing a single simplified protocol. We also include some data on the possible use of a reflectance colorimeter to estimate lycopene in tomato juice. SOLVENT EXTRACTION OF LYCOPENE While many solvents and solvent combinations have been used to extract lycopene from tomatoes, none appears to offer an advantage over a mixture of hexane:ethanol:acetone at 2:1:1 (hereafter referred to as HEA), first used by Sadler et al. (1990). This original procedure called for 1 gram of tomato juice and 100 ml of the mixed solvents. Fish et al. (2002) later showed that these amounts could be reduced to 0.2 grams of juice and 20 ml of solvent. In either case an aliquot of tomato homogenate is weighed into a vial or flask, the solvent is added, and the lycopene is allowed to extract over the next 15 to 30 min. Water is added (15 ml per 100 ml of solvent) to cause a phase separation. The upper hexane phase contains all the lycopene, which can then be quantified spectrophotometrically or by High Performance Liquid Chromatography (HPLC). This solvent system has been used by many other investigators (e.g. Sharma and LeMaguer, 1996; Arias et al., 2000) and by the California League of Food Processors; and we believe the method should be adopted by the World Processing Tomato Council as the standard chemical method for all lycopene determinations. We have examined some of the details involved with using these solvents for extraction. Total Solvent Volume If many samples are to be analyzed it is desirable to minimize the amount of solvent used per sample to reduce the cost of the assay. These costs include not only the Proc. X IS on the Processing Tomato Eds.: A. B’Chir and S. Colvine Acta Hort. 758, ISHS 2007 111 purchase of the solvents but also the disposal costs. For the spectrophotometric quantification of extracted lycopene, only 3 ml of extract is actually needed. HPLC determinations require even less. Since the volume of the upper hexane layer is about half that of the total solvent volume, there is in theory no need to use more than 6 ml of the mixed solvents for the extraction. However, as a practical matter, to easily remove 3 ml of the upper layer without disturbing the lower layer, we have found that it is better if the total volume of solvent is slightly larger. For most of the work described here we have used 8 ml of the HEA solvent. To dispense this solvent we generally use a glass repipetter. Once properly calibrated we have found that this type of pipetter gives reproducible results. If only a few samples are to be analyzed it is generally not worth the trouble to set up this type of repeating pipetter. Instead a total solvent volume of 10 ml can be used, dispensed with a volumetric pipette. One problem with using a reduced volume of organic solvent for extraction is that the amount of tomato that can be added to the assay without overloading it becomes very small. To get reasonable optical densities with 8 ml of extracting solvent, only about 100 mg, or about three drops, of juice from a typical tomato is needed. If added by weight, this requires weighing the sample to the nearest milligram or two. This can be a tedious and time consuming process when large numbers of samples are to be analyzed. As an alternative, we have found that adding the sample by volume, using a positive displacement pipetter, is a very accurate and more practical approach for handling these small amounts of liquid. Since the final result needs to be expressed as the amount of lycopene per weight of tomato, the weight of tomato juice dispensed by a 100 μL Drummond pipetter with various tomato products was examined (Table 1). In most cases the weight is very close to 100 mg, as would be expected since tomato juice is mostly water. Two exceptions are catsup, which has a density greater than one, due to the presence of added sugars; and raw tomato juice prepared in an ordinary blender, which had a density less than one, due to the large amount of air in the solution. The reproducibility of these pipetters is excellent, in most cases ±1%. For the routine determination of lycopene this is a very acceptable level of precision. The advantages of dispensing the sample by volume are that it is rapid, and each assay receives the same sample size, eliminating the need to record the exact weight for each individual sample. This method of dispensing the sample is particularly appropriate for situations where large numbers of identically prepared samples are to be analyzed. We routinely use this procedure for analyzing samples of microwave hot break juice from our annual tomato variety evaluation trial. Before analyzing any large number of samples of a given type, it is a good idea to verify the precision and accuracy of the specific pipetter to be used with that type of sample by obtaining data like that in given in Table 1. Despite all the advantages of reducing the amount of solvent used and dispensing the sample with a positive displacement pipetter, there are still cases where the original Sadler procedure using 100 ml of solvent and 1 gram of sample may be preferable. For instance, if the material is too viscous or contains too many large particles to be pipetted it may be necessary to add the sample by weight. The larger sample size is more easily weighed to the required accuracy. Small heterogeneities in the sample are also less likely to be a problem if a larger sample is used. The larger volume of solvent can be added using a simple graduated cylinder rather than a repipetter. For lab situations where delicate precision items like a repipetter or a Drummond pipetter might not be used correctly or survive at all, the simpler large volume procedure might be more appropriate. Time Course of Extraction Extraction of lycopene with the HEA solvent system is rapid. Samples of tomato juice (100 μL) were incubated in 8 ml of solvent in a screw cap tube for various times before water was added to separate the phases and the lycopene absorbance of the upper layer determined. Almost no change in absorbance was observed over the time course, indicating that extraction was nearly complete after only 2 minutes, the earliest time point we analyzed (Fig. 1, left). A recommendation for a minimum extraction time of 10