Authentication of saffron using 60 MHz 1H NMR spectroscopy

60 MHz proton NMR spectroscopy was used to analyse extracts from saffron spice and a range of potential adulterants and mixtures. Using a simple extraction procedure, good quality spectra were obtained which contain peaks from the characteristic metabolites picrocrocin and crocins, fatty acids and kaempferol. The spectra of samples from trusted suppliers were used to train one-class classification models by SIMCA, nearest neighbour and isolation forest methods. Applying these to spectra of saffron samples purchased from the online marketplace, it was found that 7 out of 33 samples were highly anomalous. From comparison with the spectra of known mixtures and confirmatory spectral analysis using 600 MHz NMR, it is probable that these contain considerable amounts of undisclosed foreign matter

Quantitative authenticity testing of buffalo mozzarella via alpha(s1)-Casein using multiple reaction monitoring mass spectrometry

We address the detection and quantitation of bovine milk in 'buffalo' mozzarella cheese using multiple reaction monitoring (MRM) mass spectrometry (MS). Focussing on the abundant protein alpha(s1)-casein, present in both species but with 10 amino acid sequence differences, we extract a list of marker peptides specific to each species. 'Identical' peptides, exactly the same in both species, are used for relative quantitation of alpha(s1)-casein in each milk type, whereas 'similar' peptides, present in both species but differing typically by one amino acid, are used to demonstrate relative quantitation in binary cheese mixtures. In addition, we report a pilot survey of UK supermarket and restaurant products labelled as 'buffalo mozzarella', finding that 2/3 of restaurant meals and supermarket pizzas are either mislabelled or adulterated

Low vs high field 1h NMR spectroscopy for the detection of adulteration of cold pressed rapeseed oil with refined oils

Cold pressed rapeseed oil (CPRO) is a relatively recent development in rapeseed processing, which produces a quality product with a high market value. High field NMR (400 MHz) is a well-established tool in food analysis, while low-field NMR (60 MHz) is much less studied. This study aims to establish the effectiveness of both techniques in identifying binary adulteration in CPRO. Three adulteration scenarios were investigated: a) CPRO and refined rapeseed oil (RRO); b) CPRO and refined sunflower oil (RSO); and c) CPRO and RRO or RSO. A range of classification techniques were trialled as well as partial least squares regression to gauge predictive quantification performance. The 400 MHz NMR achieved classification rates of 100% in the scenarios with a single adulterant, and 93% in the multiple adulterant scenario. The 60 MHz NMR produced lower but still encouraging classification rates (RSO 92%; RRO 85%; both RRO and RSO 87%)

Using induced chlorophyll production to monitor the physiological state of stored potatoes (Solanum tuberosum L.)

A Visible/Near-infrared (Vis/NIR) spectrometer equipped with a fibre-optic probe was used to stimulate and measure chlorophyll production in potato tubers, at low levels that produce no visible greening in the skin. Subtle responses to changes in the light stimulus were also tracked. When used with a static experimental setup, these measurements are precise. However, the technique is very sensitive to the exact geometry of the tuber-probe arrangement, and careful positioning of the probe is crucial. Complementary studies established that tissue under the apical buds (‘eyes’) has greater capacity to produce chlorophyll than other locations on the tuber surface. A long-term study of multiple tubers suggested that different cultivars behave differently in terms of the rate of chlorophyll production. These behavioural differences may be related to the batch dormancy status; validating this potential relationship is the focus of ongoing work

Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture

Low-field H-1 NMR spectroscopy for distinguishing between arabica and robusta ground roast coffees

This work reports a new screening protocol for addressing issues of coffee authenticity using low-field (60 MHz) bench-top H-1 NMR spectroscopy. Using a simple chloroform-based extraction, useful spectra were obtained from the lipophilic fraction of ground roast coffees. It was found that 16-O-methylcafestol (16-OMC, a recognized marker compound for robusta beans) gives rise to an isolated peak in the 60 MHz spectrum, which can be used as an indicator of the presence of robusta beans in the sample. A total of 81 extracts from authenticated coffees and mixtures were analysed, from which the detection limit of robusta in arabica was estimated to be between 10% and 20% w/w. Using the established protocol, a surveillance exercise was conducted of 27 retail samples of ground roast coffees which were labelled as "100% arabica". None were found to contain undeclared robusta content above the estimated detection limit. (C) 2016 Published by Elsevier Ltd

High-throughput screening of argan oil composition and authenticity using benchtop 1H NMR

We use 60-MHz benchtop nuclear magnetic resonance (NMR) to acquire(1)H spectra from argan oils of assured origin. We show that the low-field NMR spectrum of neat oil contains sufficient information to make estimates of compositional parameters and to inform on the presence of minor compounds. A screening method for quality and authenticity is presented based on nearest-neighbour outlier detection. A variety of oil types are used to challenge the method. In a survey of retail-purchased oils, several instances of fraud were found

Mitigating instrument effects in 60 MHz 1H NMR spectroscopy for authenticity screening of edible oils

Low field (60 MHz) 1H NMR spectroscopy was used to analyse a large (n = 410) collection of edible oils, including olive and argan, in an authenticity screening scenario. Experimental work was carried out on multiple spectrometers at two different laboratories, aiming to explore multivariate model stability and transfer between instruments. Three modelling methods were employed: Partial Least Squares Discriminant Analysis, Random Forests, and a One Class Classification approach. Clear inter-instrument differences were observed between replicated data collections, sufficient to compromise effective transfer of models based on raw data between instruments. As mitigations to this issue, various data pre-treatments were investigated: Piecewise Direct Standardisation, Standard Normal Variates, and Rank Transformation. Datasets comprised both phase corrected and magnitude spectra, and it was found that that the latter spectral form may offer some advantages in the context of pattern recognition and classification modelling, particularly when used in combination with the Rank Transformation pre-treatment

Quantification of MDMA in seized tablets using benchtop 1H NMR spectroscopy in the absence of internal standards

Recreational MDMA use is a worldwide problem. Tablet dosage varies, thus entailing a requirement for quantitative analysis. The quantification of MDMA in tablets using benchtop 1H NMR spectroscopy via either linear regression (‘manual’ method) or partial least square regression (‘automated’ method) approaches are reported, without the need for an internal standard, and compared against contemporaneously obtained GC–MS data. Twenty samples were evaluated of which 15 were proven to contain MDMA, via qualitative NMR (hit score ≥ 0.97) and GC–MS (Rt = 5.6 min) analysis. Quantitative NMR analysis showed that the mean value of MDMA content was 42.6% w/w by the manual method and 45.9% w/w by the automated method. The mean value obtained from GC analysis was 44.0% w/w. A substantial proportion (n = 9) of the tablets tested possessed > 190 mg of MDMA (range 133–223 mg, average of all techniques’ calculations for each tablet). This value is higher than the reported average MDMA content of tablets by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), which was ca. 125 mg of MDMA per tablet in 2016

16-O-methylcafestol is present in ground roast Arabica coffees: Implications for authenticity testing

High-field and low-field proton NMR spectroscopy were used to analyse lipophilic extracts from ground roast coffees. Using a sample preparation method that produced concentrated extracts, a small marker peak at 3.16 ppm was observed in 30 Arabica coffees of assured origin. This signal has previously been believed absent from Arabicas, and has been used as a marker for detecting adulteration with robusta. Via 2D 600 MHz NMR and LC-MS, 16-O-methylcafestol and 16-O-methylkahweol were detected for the first time in Arabica roast coffee and shown to be responsible for the marker peak. Using low-field NMR, robusta in Arabica could be detected at levels of the order of 1-2% w/w. A surveillance study of retail purchased "100% Arabica" coffees found that 6 out of 60 samples displayed the 3.16 ppm marker signal to a degree commensurate with adulteration at levels of 3-30% w/w