Origin Determination of Walnuts (Juglans regia L.) on a Worldwide and Regional Level by Inductively Coupled Plasma Mass Spectrometry and Chemometrics

“To counteract food fraud, this study aimed at the differentiation of walnuts on a global and regional level using an isotopolomics approach. Thus, the multi-elemental profiles of 237 walnut samples from ten countries and three years of harvest were analyzed with inductively coupled plasma mass spectrometry (ICP-MS), and the resulting element profiles were evaluated with chemometrics. Using support vector machine (SVM) for classification, validated by stratified nested cross validation, a prediction accuracy of 73% could be achieved. Leave-one-out cross validation was also applied for comparison and led to less satisfactory results because of the higher variations in sensitivity for distinct classes. Prediction was still possible using only elemental ratios instead of the absolute element concentrations; consequently, a drying step is not mandatory. In addition, the isotopolomics approach provided the classification of walnut samples on a regional level in France, Germany, and Italy, with accuracies of 91%, 77%, and 94%, respectively. The ratio of the model’s accuracy to a random sample distribution was calculated, providing a new parameter with which to evaluate and compare the performance of classification models. The walnut cultivar and harvest year had no observable influence on the origin differentiation. Our results show the high potential of element profiling for the origin authentication of walnuts.”

2.1. Reagents and Materials

The elemental analyses of walnuts were based on a previous study [13]. In Table S1 (supporting information), the reagents and materials used in this study are listed.

2.2. Sample Preparation

A total of 237 reference samples of relevant, market-available walnuts from three years of harvest (2017, 2018, and 2019) were collected and analyzed in this study. The walnut samples originated from ten countries and were purchased as shelled or in-shell goods. Thanks to the cooperation with regional producers and project partners who work according to our internal guidelines to ensure the authenticity of the reference material (e.g., by applying the HACCP guidelines, FSSC 22,000, or providing the structure meta date), authentic walnut samples could be acquired. See Table S2 (Supporting Information) for detailed information and Figure 1 for a visual illustration. On arrival, walnut samples were frozen and stored at −80 °C until further processing could take place. Element patterns are less influenced by storage than other profiling levels, such as the metabolome [27]. At the applied storage conditions of −80 °C, enzyme activities are inhibited—i.e., cell lysis is also inhibited [28]. One German walnut sample (harvest year 2018, Hesse) was selected as a quality control (QC) sample.
Figure 1. Overview of the 237 walnut samples with regard to their origin and harvest year.

2.3. Sample Preparation and Digestion

For each walnut sample, 100 g of walnut kernels were milled using a knife mill (Grindomix GM 300, Retsch, Haan, Germany) with the addition of dry ice. If necessary, in-shell walnuts were shelled before. Homogenized samples were freeze-dried for 48 h (Beta 1–8 LDplus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany), including a stirring step after 24 h.
The sample digestion of 500 mg of homogenized and lyophilized walnut material was performed using an Ethos.lab microwave (MLS GmbH, Leutkirch, Germany), as described in reference [13] and in Table S3 (Supporting Information). For each digestion run, one vessel was selected for the QC sample and one vessel for a blank. The QC sample was later used for quality assurance and the calculation of the method’s precision (see Section 3.2).

2.4. Analytical Procedure and Instrumentation

Multi-elemental analyses were performed on an HR-ICP-MS Element2 (ThermoFisher Inc., Waltham, MA, USA) coupled with an SC-E4 Autosampler (Elemental Scientific Inc., Omaha, NE, USA), following a method validated in a previous study [13].
The multi-element method included 47 isotopes: Li, Be, B, Na, Mg, Al, K, Ca, Sc, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Y, Mo, Ag, Cd, Te, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Tl, Pb, Bi, Th, U. The calculated limit of detection and limit of quantitation are given in Table S4 (Supporting Information). Here, also the respective element used for internal standardization is given.
Quantitation was conducted by external calibration. Instrument optimization, mass calibration, and mass offset were performed daily. For further instrumental conditions and method validation, refer to Table S5 (Supplementary Materials) and reference no. [13]. Tubes and pipette tips were pre-cleaned by soaking in 3% (v/v) nitric acid overnight and subsequently rinsed with ultrapure water and dried.

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