Graphical exploration of 600- and 60-MHz proton NMR spectral datasets from ground roast coffee extracts.

This article uses a variety of graphical and mathematical approaches to analyse 600- and 60-MHz ('benchtop') proton NMR spectra acquired from lipophilic and hydrophilic extracts of roasted coffee beans. The collection of 40 authenticated samples comprised various coffee species, cultivars and hybrids. The spectral datasets were analysed by a combination of metabolomics approaches, cross-correlation and whole spectrum methods, assisted by visualisation and mathematical techniques not conventionally employed to treat NMR data. A large amount of information content was shared between the 600-MHz and benchtop datasets, including in its magnitude spectral form, suggesting the potential for a lower cost, lower tech route to conducting informative metabolomics studies.

Detection, discrimination and quantification of amphetamine, cathinone and nor-ephedrine regioisomers using benchtop 1 H and 19 F nuclear magnetic resonance spectroscopy.

Amphetamine and cathinone derivatives are abused recreationally due to the sense of euphoria they provide to the user. Methodologies for the rapid detection of the drug derivative present in a seized sample, or an indication of the drug class, are beneficial to law enforcement and healthcare providers. Identifying the drug class is prudent because derivatisation of these drugs, to produce regioisomers, for example, occurs frequently to circumvent global and local drug laws. Thus, newly encountered derivatives might not be present in a spectral library. Employment of benchtop nuclear magnetic resonance (NMR) could be used to provide rapid analysis of seized samples as well as identifying the class of drug present. Discrimination of individual amphetamine-, methcathinone-, N-ethylcathinone and nor-ephedrine-derived fluorinated and methylated regioisomers is achieved herein using qualitative automated 1 H NMR analysis and compared to gas chromatography-mass spectrometry (GC-MS) data. Two seized drug samples, SS1 and SS2, were identified to contain 4-fluoroamphetamine by 1 H NMR (match score median = 0.9933) and GC-MS (RRt = 5.42-5.43 min). The amount of 4-fluoroamphetamine present was 42.8%-43.4% w/w and 48.7%-49.2% w/w for SS1 and SS2, respectively, from quantitative 19 F NMR analysis, which is in agreement with the amount determined by GC-MS (39.9%-41.4% w/w and 49.0%-49.3% w/w). The total time for the qualitative 1 H NMR and quantitative 19 F NMR analysis is ~10 min. This contrasts to ~40 min for the GC-MS method. The NMR method also benefits from minimal sample preparation. Thus, benchtop NMR affords rapid, and discriminatory, analysis of the drug present in a seized sample.

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.

Comparative study of the analysis of seized samples by GC-MS, 1H NMR and FT-IR spectroscopy within a Night-Time Economy (NTE) setting.

Rapid analysis of surrendered or seized drug samples provides important intelligence for health (e.g. treatment or harm reduction), and custodial services. Herein, three in-situ techniques, GC-MS, 1H NMR and FT-IR spectroscopy, with searchable libraries, are used to analyse 318 samples qualitatively, using technique specific library-based searches, obtained over the period 24th - 29th August 2019. 259 samples were identified as consisting of a single component, of which cocaine was the most prevalent (n = 158). Median match scores for all three techniques were ≥ 0.84 and showed agreement except for metformin (n = 1), oxandrolone (identified as vitamin K by IR (n = 4)), diazepam (identified as zolpidem by FT-IR (n = 2)) and 2-Br-4,5-DMPEA (n = 1), a structural isomer of 2C-B identified as a polymer of cellulose (cardboard) by FT-IR. 51 samples were found to consist of two or more components, of which 49 were adulterated cocaine samples (45 binary and 4 tertiary samples). GC-MS identified all components present in the 49 adulterated cocaine samples, whereas IR identified only cocaine in 88 % of cases (adulterant only = 12 %). The breakdown for 1H NMR spectroscopy was all components identified (51 %), cocaine only (33 %), adulterant only (10 %), cocaine and one adulterant (tertiary mixtures only, 6 %).

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.

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

Rapid Identification of Novel Psychoactive and Other Controlled Substances Using Low-Field 1H NMR Spectroscopy.

An automated approach to the collection of 1H NMR (nuclear magnetic resonance) spectra using a benchtop NMR spectrometer and the subsequent analysis, processing, and elucidation of components present in seized drug samples are reported. An algorithm is developed to compare spectral data to a reference library of over 300 1H NMR spectra, ranking matches by a correlation-based score. A threshold for identification was set at 0.838, below which identification of the component present was deemed unreliable. Using this system, 432 samples were surveyed and validated against contemporaneously acquired GC-MS (gas chromatography-mass spectrometry) data. Following removal of samples which possessed no peaks in the GC-MS trace or in both the 1H NMR spectrum and GC-MS trace, the remaining 416 samples matched in 93% of cases. Thirteen of these samples were binary mixtures. A partial match (one component not identified) was obtained for 6% of samples surveyed whilst only 1% of samples did not match at all.

Correction: Metabolite quantification of faecal extracts from colorectal cancer patients and healthy controls.

[This corrects the article DOI: 10.18632/oncotarget.26022.].

Metabolite quantification of faecal extracts from colorectal cancer patients and healthy controls.

Colorectal cancer (CRC), a primary cause of morbidity and mortality worldwide is expected to rise in the coming years. A better understanding of the metabolic changes taking place during the disease progression is needed for effective improvements of screening strategies and treatments. In the present study, Nuclear Magnetic Resonance (NMR) metabolomics was used to quantify the absolute concentrations of metabolites in faecal extracts from two cohorts of CRC patients and healthy controls. The quantification of over 80 compounds revealed that patients with CRC had increased faecal concentrations of branched chain fatty acids (BCFA), isovalerate and isobutyrate plus valerate and phenylacetate but diminished concentrations of amino acids, sugars, methanol and bile acids (deoxycholate, lithodeoxycholate and cholate). These results suggest that alterations in microbial activity and composition could have triggered an increase in utilisation of host intestinal slough cells and mucins and led to an increase in BCFA, valerate and phenylacetate. Concurrently, a general reduction in the microbial metabolic function may have led to reduced levels of other components (amino acids, sugars and bile acids) normally produced under healthy conditions. This study provides a thorough listing of the most abundant compounds found in human faecal waters and presents a template for absolute quantification of metabolites. The production of BCFA and phenylacetate in colonic carcinogenesis warrants further investigations.

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.

Acute Consumption of Flavan-3-ol-Enriched Dark Chocolate Affects Human Endogenous Metabolism.

Flavan-3-ols and methylxanthines have potential beneficial effects on human health including reducing cardiovascular risk. We performed a randomized controlled crossover intervention trial to assess the acute effects of consumption of flavan-3-ol-enriched dark chocolate, compared with standard dark chocolate and white chocolate, on the human metabolome. We assessed the metabolome in urine and blood plasma samples collected before and at 2 and 6 h after consumption of chocolates in 42 healthy volunteers using a nontargeted metabolomics approach. Plasma samples were assessed and showed differentiation between time points with no further separation among the three chocolate treatments. Multivariate statistics applied to urine samples could readily separate the postprandial time points and distinguish between the treatments. Most of the markers responsible for the multivariate discrimination between the chocolates were of dietary origin. Interestingly, small but significant level changes were also observed for a subset of endogenous metabolites. 1H NMR revealed that flavan-3-ol-enriched dark chocolate and standard dark chocolate reduced urinary levels of creatinine, lactate, some amino acids, and related degradation products and increased the levels of pyruvate and 4-hydroxyphenylacetate, a phenolic compound of bacterial origin. This study demonstrates that an acute chocolate intervention can significantly affect human metabolism.

Low-field (1)H 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 (60MHz) bench-top (1)H 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 60MHz 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.

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.

Meat Authentication via Multiple Reaction Monitoring Mass Spectrometry of Myoglobin Peptides.

A rapid multiple reaction monitoring (MRM) mass spectrometric method for the detection and relative quantitation of the adulteration of meat with that of an undeclared species is presented. Our approach uses corresponding proteins from the different species under investigation and corresponding peptides from those proteins, or CPCP. Selected peptide markers can be used for species detection. The use of ratios of MRM transition peak areas for corresponding peptides is proposed for relative quantitation. The approach is introduced by use of myoglobin from four meats: beef, pork, horse and lamb. Focusing in the present work on species identification, by use of predictive tools, we determine peptide markers that allow the identification of all four meats and detection of one meat added to another at levels of 1% (w/w). Candidate corresponding peptide pairs to be used for the relative quantification of one meat added to another have been observed. Preliminary quantitation data presented here are encouraging.

60 MHz 1H NMR spectroscopy for the analysis of edible oils.

We report the first results from a new 60 MHz 1H nuclear magnetic resonance (NMR) bench-top spectrometer, Pulsar, in a study simulating the adulteration of olive oil with hazelnut oil. There were qualitative differences between spectra from the two oil types. A single internal ratio of two isolated groups of peaks could detect hazelnut oil in olive oil at the level of ∼13%w/w, whereas a whole-spectrum chemometric approach brought the limit of detection down to 11.2%w/w for a set of independent test samples. The Pulsar's performance was compared to that of Fourier transform infrared (FTIR) spectroscopy. The Pulsar delivered comparable sensitivity and improved specificity, making it a superior screening tool. We also mapped NMR onto FTIR spectra using a correlation-matrix approach. Interpretation of this heat-map combined with the established annotations of the NMR spectra suggested a hitherto undocumented feature in the IR spectrum at ∼1130 cm-1, attributable to a double-bond vibration.

Metabolomics of fecal extracts detects altered metabolic activity of gut microbiota in ulcerative colitis and irritable bowel syndrome.

(1)H NMR spectroscopy of aqueous fecal extracts has been used to investigate differences in metabolic activity of gut microbiota in patients with ulcerative colitis (UC) (n = 13), irritable bowel syndrome (IBS) (n = 10), and healthy controls (C) (n = 22). Up to four samples per individual were collected over 2 years giving a total of 124 samples. Multivariate discriminant analysis, based on NMR data from all three groups, was able to predict UC and C group membership with good sensitivity and specificity; classification of IBS samples was less successful and could not be used for diagnosis. Trends were detected toward increased taurine and cadaverine levels in UC with increased bile acid and decreased branched chain fatty acids in IBS relative to controls; changes in short chain fatty acids and amino acids were not significant. Previous PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the same fecal material had shown alterations of the gut microbiota when comparing UC and IBS groups with controls. Hierarchical cluster analysis showed that DGGE profiles from the same individual were stable over time, but NMR spectra were more variable; canonical correlation analysis of NMR and DGGE data partly separated the three groups and revealed a correlation between the gut microbiota profile and metabolite composition.

Prediction of variability in CYP3A4 induction using a combined 1H NMR metabonomics and targeted UPLC-MS approach.

The activity of Cytochrome P450 3A4 (CYP3A4) enzyme is associated with many adverse or poor therapeutic responses to drugs. We used (1)H NMR-based metabonomics to identify a metabolic signature associated with variation in induced CYP3A4 activity. A total of 301 female twins, aged 45--84, participated in this study. Each volunteer was administered a potent inducer of CYP3A4 (St. John's Wort) for 14 days and the activity of CYP3A4 was quantified through the metabolism of the exogenously administered probe drug quinine sulfate (300 mg). Pre- and postintervention fasting urine samples were used to obtain metabolite profiles, using (1)H NMR spectroscopy, and were analyzed using UPLC--MS to obtain a marker for CYP3A4 induction, via the ratio of 3-hydroxyquinine to quinine (3OH-Q:Q). Multiple linear regression was used to build a predictive model for 3OH-Q:Q values based on the preintervention metabolite profiles. A combination of seven metabolites and seven covariates showed a strong (r = 0.62) relationship with log(3OH-Q:Q). This regression model demonstrated significant (p < 0.00001) predictive ability when applied to an independent validation set. Our results highlight the promise of metabonomics for predicting CYP3A4-mediated drug response.