Discrimination between Orange Juice and Pulp Wash by 1 H Nuclear Magnetic Resonance Spectroscopy: Identification of Marker Compounds Gwe ´nae ¨lle Le Gall, Max Puaud, and Ian J. Colquhoun* Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom The potential of NMR spectroscopy and multivariate analysis methods to detect the adulteration of orange juice with pulp wash is demonstrated. Principal component analysis has been applied to 1 H NMR spectra of >300 orange and pulp wash juices, and stepwise linear discriminant analysis was used to classify the samples. A model with six principal components gave a high success rate of classification (94%) for both training and validation sets. An important principal component loading showed that dimethylproline played a key role in the discrimination between the two types of juice, with higher levels in pulp wash. Dimethylproline was not previously known as a marker compound for orange juice adulteration. An ANOVA test revealed at least 21 other NMR signals that differed significantly between the authentic and pulp wash groups. The compounds they represent could be seen as potential marker compounds in addition to dimethylproline. This makes NMR with chemometrics an attractive screening tool with advantages in terms of rapidity, simplicity, and diversity of information provided. Keywords: NMR; orange juice; pulp wash; authenticity; adulteration; multivariate analysis INTRODUCTION Orange juice is a high-value product traded on a massive scale (>20 L per capita was consumed in the United States in 1996). World demand has been growing during the 1990s, and expectations are that it will continue to grow for the foreseeable future. According to U.S. Food and Drug Administration (FDA) investiga- tions, some companies are known to have made millions of dollars selling fraudulent orange juice (1). Adultera- tion of orange juice may be done by the addition of water, sugars, pulp wash, or other citrus fruit juices such as grapefruit. Pulp wash is a second extract obtained by washing the separated pulp with water after the first pressing of the orange juice. Its chemical composition is similar in many respects to that of orange juice but it is paler, more bitter, and regarded as a lower quality product. Addition of “in-line” or “off-line” pulp wash to orange juice is at present forbidden in the EU, although the regulations are to change following adop- tion of a new Fruit Juice Directive. According to U.S. federal regulations frozen concentrated orange juice may contain “in line” pulp wash from the same oranges used to make the juice concentrate. “In line” addition of pulp wash to fresh and pasteurized juices is not permitted, nor is addition of “off line” pulp wash to any category of orange juice. The adulteration can also be quite elabo- rate, with the addition of citric acid, amino acids, or trace metals as well as sugars to mimic the chemical profile of the authentic orange juice. Many techniques have been investigated to tackle the problem of orange juice adulteration. Several reviews (2, 3) have listed the major techniques employed for this purpose, such as isotopic and chromatographic analyses. Most of the chromatographic methods are based on the study of one specific family of compounds (e.g., sugars). Instead of considering only one type of compound, spectroscopic methods such as NMR spectroscopy have the potential to monitor a wide range of chemicals (sugars, organic and amino acids, phenolics, etc.) in a single spectrum. Those methods also offer advantages in terms of rapidity and simplicity of sample prepara- tion. The richness of information, however, makes the spectra too complex to be analyzed or compared by eye. Multivariate analysis (MVA) is therefore applied di- rectly to the spectral data to extract the useful informa- tion. A few papers have previously been published concerning the application of MVA to chemical data for orange juice authentication (4-6). The present work shows the potential of NMR to discriminate orange juice from pulp wash using prin- cipal component analysis (PCA) followed by linear discriminant analysis (LDA) (7, 8). One of the advan- tages of NMR is that the chemical origins of the discrimination may be interpretable through an ANOVA test or through comparison of the principal component loadings with the spectra. This permits identification of compounds involved in the differentiation of orange juice and pulp wash and may uncover novel marker compounds. The ultimate aim would obviously be to distinguish authentic samples from ones adulterated by pulp wash and to measure the amount of pulp wash added. Because the two orange products are chem- ically very similar and pulp wash adulteration is dif- ficult to detect, the present work was aimed at detecting differences between the two “pure” products. The sample set is intended to be sufficiently large to cover the range of natural variability likely to be encountered in prac- tice. * Corresponding author (telephone +44 1603 255353; fax +44 1603 507723; e-mail ian.colquhoun@bbsrc.ac.uk). 580 J. Agric. Food Chem. 2001, 49, 580-588 10.1021/jf001046e CCC: $20.00 © 2001 American Chemical Society Published on Web 01/13/2001