Nao Wu

Stéphane Balayssac

Gaëtan Assemat

Saïda Danoun

Sébastien Dejean

Myriam Malet-Martino

Véronique Gilard
email: gilard@chimie.ups-tlse.fr

Sébastien Déjean

Véronique Pr

Gilard

Evaluation of low-field versus high-field proton NMR spectroscopy for quality control of cinnamon samples

Keywords: cinnamon, NMR, compact NMR, flavor, dietary supplement, nutraceuticals

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Cinnamon is a well-known spice widely used for cooking in many countries around the world. Recent studies reviewed the use of cinnamon in human health with its beneficial as well as its adverse effects [START_REF] Silva | Beneficial Uses of Cinnamon in Health and Diseases[END_REF][START_REF] Hajimonfarednejad | Cinnamon: A systematic review of adverse events[END_REF]. Medicinal properties against diabetes, inflammations, gastrointestinal disorders, urinary infections and as a neuroprotective or antimicrobial agent were reported [START_REF] Nabavi | Evaluation of a Benchtop Cryogen-Free Low-Field H-1 NMR Spectrometer for the Analysis of Sexual Enhancement and Weight Loss Dietary Supplements Adulterated with Pharmaceutical Substances[END_REF][START_REF] Silva | Beneficial Uses of Cinnamon in Health and Diseases[END_REF]. Because of its actual or perceived medicinal properties, many health and cosmetic products take nowadays advantage of cinnamon.

Two main varieties of cinnamon are commonly available, "true cinnamon" or Ceylon cinnamon which corresponds to the Cinnamomum verum species, and cassia cinnamon which has several origins. The most common is Chinese cassia (Cinnamomum cassia Blume, syn.

Cinnamomum aromaticum Nees) but there are also Saigon cinnamon (Cinnamomum loureirii Nees) from Vietnam and Indonesian cassia (Cinnamomum burmanini Blume), mainly from Indonesia and the Philippines [START_REF] Avula | Authentication of true cinnamon (Cinnamon verum) utilising direct analysis in real time (DART)-QToF-MS[END_REF][START_REF] Woehrlin | Quantification of flavoring constituents in cinnamon: high variation of coumarin in cassia bark from the German retail market and in authentic samples from indonesia[END_REF]. "True cinnamon" has a characteristic taste different from cassia one because of a different chemical composition. Ceylon cinnamon has low levels of coumarin (0.004% w/w) by comparison to cassia cinnamon that contains until 1% [START_REF] Avula | Authentication of true cinnamon (Cinnamon verum) utilising direct analysis in real time (DART)-QToF-MS[END_REF]. Coumarin is known to be hepatotoxic and a tolerable daily intake of 0.1 mg/kg body weight has been defined in Europe to insure safety of consumers [START_REF] Efsa | Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) related to Coumarin[END_REF]. As cassia cinnamon is cheaper, it is more and more widely available in the European market; it is thus relevant to explore new quality control methods for the analysis of cinnamon samples.

The aim of this study was to evaluate the ability of low-field (LF) NMR as a method for quality control of cinnamon samples. With the recent introduction of compact NMR spectrometers that use permanent magnets giving rise to proton NMR frequencies between 40 and 80 MHz, new areas of applications have been opened [START_REF] Blumich | Low-field and benchtop NMR[END_REF][START_REF] Blumich | Desktop NMR and Its Applications From Materials Science To Organic Chemistry[END_REF]. In the field of food chemistry, analysis of meat [START_REF] Jakes | Authentication of beef versus horse meat using 60 MHz 1H NMR spectroscopy[END_REF], authenticity of Arabica coffee [START_REF] Gunning | 16-Omethylcafestol is present in ground roast Arabica coffees: Implications for authenticity testing[END_REF], analysis of edible oils [START_REF] Mcdowell | Low vs high field 1 H NMR spectroscopy for the detection of adulteration of cold pressed rapeseed oil with refined oils[END_REF]Parker, Limer, Watson, Defernez, Williamson, & Kemsley, 2014) and adulteration of dietary supplements with added active pharmaceutical ingredients (Pages, Gerdova, Williamson, Gilard, Martino, & Malet-Martino, 2014) were described.

The present work is the first application of LF NMR to cinnamon analysis for both food spices and dietary supplements. The analyses were performed using high-field (HF) NMR as the reference method and the LF capabilities and limits were assessed and discussed.

At last, a statistical treatment of LF NMR data was explored to model LF data and predict quantification.

Experimental

Materials

(E)-cinnamaldehyde (Acros Organic, 99%), coumarin (Acros Organic, 99%), cinnamyl alcohol (Acros Organic, 99%), dimethyl maleate (Sigma-Aldrich, 99%), o-methoxycinnamaldehyde (Sigma-Aldrich, 98%), (E)-cinnamic acid (Acros Organic, 98%), eugenol (Sigma-Aldrich, 99%), 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt (TSP, Acros Organic, 99.8% D), tetramethylsilane (TMS, Acros Organic, 99% D), Eu(fod)3 (Sigma-Aldrich, 99%), EuCl3 (Sigma-Aldrich, 99.9%), Eu(NO3)3.5H2O (Sigma-Aldrich, 99.9%), dimethyl sulfone (Sigma-Aldrich, 98%) were used as received without any further purification unless otherwise described. All deuterated solvents (99.80% D) were supplied from Eurisotop. An authentic sample of Cinnamomum verum was obtained for Extrasynthese (Lyon, France).

The fourteen samples of cinnamon for cooking were bought from French malls or markets, and the fourteen dietary supplements were purchased online or in French specialized shops like organic groceries or dietetic stores (Table 1). All samples were analysed before their expiry date.

Preparation of samples for NMR analysis

Extraction methodology

All the preliminary extraction experiments described in this section were analysed with the HF NMR spectrometer. In order to choose the best solvent to extract commercial samples, solid-liquid extractions with deuterated methanol (MeOH-d4), deuterated chloroform (CDCl3), heavy water (D2O) and mixtures of solvents MeOH-d4:D2O (50%:50%; 80%:20%) were investigated. A liquid-liquid extraction of an aqueous suspension of cinnamon powder with CDCl3 was also tested.

Briefly, around 100 mg of the powdered commercial cinnamon sample 25 were weighed and mixed with 1 mL of the chosen solvent, shaked for 20 s with a vortexer, sonicated for 10 min in a stoppered tube and centrifuged at 5000 g for 5 min. For the liquidliquid extraction, 0.5 mL D2O were added to the powdered sample followed by 1 mL CDCl3.

The sample was then mixed, sonicated and centrifuged as described above. After centrifugation, the CDCl3 phase was submitted to NMR analysis. In the final step, 700 µL of supernatant were put into a 5 mm NMR tube and 60 µL of TSP were added (10 mM in MeOH-d4) for NMR analysis. For the samples extracted with CDCl3, 10 µL of a solution (120 mM) of dimethylsulfone as internal reference were mixed with 500 µL of supernatant.

Each extraction condition was tested in duplicate.

Experiments with successive extractions of the powdered samples were also implemented. For the first step, the extraction protocol was the same as previously described, i.e. around 100 mg of samples 10 (n=2) and 25 (n=2) were mixed with 1 mL MeOH-d4, shaked for 20 s with a vortexer, sonicated for 10 min and centrifuged. The supernatant was removed and 700 µL were mixed with 60 µL TSP (10 mM, MeOH-d4) for NMR analysis.

The residual powder was dried on a filter paper to absorb the residual liquid and then reextracted leading to the second extraction sample. This step was repeated once to obtain the third extraction sample. The supernatants from the second and third extractions were then analysed by mixing 700 µL of supernatant with 60 µL TSP (10 mM, MeOH-d4). The mean results of extraction provided a recovery of 88.2 ± 0.9% and 86.1 ± 1.8% for (E)cinnamaldehyde and coumarin respectively in the first extraction. Residual active compounds were measured at 10.6 ± 0.8% and 12.4 ± 1.6% in the second extraction and 1.2 ± 0.1% and 1.5 ± 0.2% in the third extraction. The same procedure of three successive extractions was repeated for the two samples 10 and 25 with only 15 mg of powder. The recoveries were 96.8 ± 0.9% and 97.5 ± 0.7% for (E)-cinnamaldehyde and coumarin respectively in the first extraction, 3.2 ± 0.9% and 2.5 ± 0.7 % in the second extraction and nothing in the third one.

Preparation of commercial samples for NMR analysis

One mL of MeOH-d4 was added to around 15 or 100 mg exactly weighed of each powdered commercial cinnamon sample placed in a stoppered tube. After 20 s of vortex mixing and 10 min of sonication, the mixture was centrifuged at 5000 g for 5 min. 700 µL of supernatant were then collected, mixed with 60 µL of TSP (10 mM) and transferred into a 5 mm NMR tube. The extracts from 100 mg of cinnamon powder were analysed with both HF and LF NMR and those from 15 mg only with HF NMR for the absolute quantification of (E)-cinnamaldehyde and coumarin. All samples were prepared and analysed in duplicate for both HF and LF NMR.

Experiments with chemical shift reagents

100 mg of sample 25 were extracted with MeOD-d4 as described in section 2.2.2. The supernatant (700 µL) was mixed with 20 µL of a solution of Eu(fod)3, EuCl3, or Eu(NO3)3 in MeOH-d4 at a concentration of 750 mM. TSP (60 µL, 10 mM) was added as an internal reference. The final concentrations for europium salts were 20 mM. For the blank sample, 20 µL of MeOD-d4 were used instead of the europium salt solution.

Standard samples for statistical analysis

Solutions of standard (E)-cinnamaldehyde and coumarin in MeOH-d4 at different concentrations were prepared as follows. Stock solutions (5 mg/mL) of each standard were diluted to obtain concentrations corresponding to real amounts in commercial samples, i.e. 3,5,8,10,12,15,17,20,25 and 30 mg/g for (E)-cinnamaldehyde and 2, 4 and 6 mg/g for coumarin. These values take into account a correction factor linked to the extraction yield of real samples (see section 3.2.1.) A blank MeOH-d4 solution was also analysed. A solution of TSP as internal reference was added for NMR analysis as previously described.

NMR analysis

HF NMR analysis

HF 1 H NMR experiments were performed on a Bruker Avance 400 spectrometer (Bruker Biospin AG, Fallanden, Switzerland) equipped with a TXO probe at 298 K. For qualitative experiments, HF 1 H NMR spectra were recorded with the following parameters: pulse width 13.5 µs (flip angle 90°), acquisition time 2.56 s, spectral width 16 ppm, 32K data points and relaxation delay 1.0 s. The number of scans was 32 or 128, corresponding to a recording time of ca. 2 or 8 min. Typical acquisition parameters for quantitative experiments were as follows: pulse width 4.53 µs (flip angle 30°), acquisition time 5.12 s, spectral width 16 ppm, 64K data points, and a relaxation delay of 10 s; the number of scans was 256 or 512 giving a recording time of ca. 65 or 130 min. A 30° pulse angle was selected instead of 90° to both ensure relaxation and save recording time because the relaxation delay required is then lower. The signal of TSP set at 0 ppm was used as an internal reference for chemical shift measurement and quantification.

Before quantitative analyses, the longitudinal relaxation times T1 of protons of (E)cinnamaldehyde, coumarin, and TSP in standard solutions and in the commercial samples 10 and 25 were measured by the inversion-recovery pulse sequence method with recovery delays from 0.001 to 50 s. The T1s found were less than 6 s (i.e. H1 (E)-cinnamaldehyde 5.0 s, H4 coumarin 5.8 s), whereas the TSP protons have a relaxation time of 3.7 s. All 1 H resonances were thus considered as fully relaxed since 99% of the signal intensity of the proton with the longest T1 (5.8 s) were recovered with a repetition time of 15.1 s.

NMR assignments of (E)-cinnamaldehyde, coumarin, (E)-cinnamic acid, o-methoxycinnamaldehyde, (E)-cinnamyl alcohol and eugenol were achieved by comparison with spectra of standard compounds and with 13 C and 2D (gCOSY, gHSQC, gHMBC) experiments to clear up ambiguities. Data are given in the supplementary section (Table S1).

1D 1 H NMR data were processed using the TOPSPIN 3.1 software with one level of zero-filling and Fourier transformation after multiplying FIDs by an exponential linebroadening function of 0.3 Hz, then phasing and baseline correction were applied. T1 values were calculated with the T1/T2 relaxation module included in the TOPSPIN software.

LF NMR analysis

Spectra were acquired on a Pulsar TM benchtop NMR spectrometer (Oxford Instruments, Abingdon, UK) operating at a frequency of 59.7 MHz for 1 H. The temperature inside the spectrometer was 310 K. The acquisition was performed with the SpinFlow 1.2.0.1 software (Oxford Instruments) and the processing with MNova 11.0 (Mestrelab Research, Santiago de Compostela, Spain). FIDs were recorded with a flip angle of 90° (12.0 µs), a spectral width of 5000 Hz, and 8K complex points (acquisition time of 1.64 s). The relaxation delay was set at 2 s, and 2000 transients were recorded leading to a total acquisition time of 2 h. For data processing, the FIDs with one level of zero-filling were apodized with an exponential (0.3 Hz) filter and automatic Whittaker smoother baseline correction was applied.

The T1s of standard (E)-cinnamaldehyde (H1), coumarin (left part of the H4 doublet) and TSP were measured (n=4) in MeOH-d4 by the inversion recovery pulse sequence method with 20 recovery delays from 0.001 to 40 s. Values were 3.8 ± 0.1 s, 4.3 ± 0.2 s and 3.1 ± 0.1 s, respectively. T1 measurements were also performed on the commercial samples 10 and 25 in duplicate. Values obtained were 3.5 ± 0.2 s, 3.9 ± 0.4 s and 3.1 ± 0.1 s for T1 of (E)cinnamaldehyde (H1), coumarin (H4) and TSP, respectively. From these T1 measurements, it was possible to calculate the recovery of the signals compared to TSP in our recording conditions. It was 90% for (E)-cinnamaldehyde and 82% for coumarin with RSD <2% for standard samples but reaching 5% for commercial samples for which variability is greater.

At last, the LOD and the apparent LOQ were determined by analyzing the diluted standard solutions of known concentration for signal-to-noise ratios (SNR) of 3 and 10, respectively. We mention here an "apparent" LOQ insofar as we will show later that the experimental conditions used were not fully quantitative. The SNR was estimated using the SNR peak calculator tool included in the MNova software. The LOD of the left signal of H4 of coumarin was 0.096 mg/mL and the apparent LOQ was 0.29 mg/mL for the same signal.

For the H1 of (E)-cinnamaldehyde, the LOD and apparent LOQ values were 0.048 mg/mL and 0.19 mg/mL respectively. The limits of apparent quantification thus corresponded to 1.9 mg/g and 2.9 mg/g for (E)-cinnamaldehyde and coumarin, respectively, in real samples.

HF NMR quantification

Concentrations were measured by comparing the signal areas of targeted protons [i.e. H1 (δ 9.66 ppm, doublet (d)) of (E)-cinnamaldehyde, H4 (δ 7.96 ppm, d) of coumarin, H2 (δ 6.47 ppm, d) of (E)-cinnamic acid, right part of the H1 d at 9.62 ppm of o-methoxycinnamaldehyde, H2 (δ 6.36 ppm, doublet triplet) of (E)-cinnamyl alcohol, and -O-CH3 (δ 3.82 ppm, singlet) of eugenol (Table S1)] to the area of the reference signal, the area of each NMR peak being directly proportional to the number of protons giving rise to it.

The amount of each compound expressed in mg/g of sample was determined using the following general equation: The standard deviations (SD) (Table 1) were obtained from the amounts calculated from n=2 or n=3 experiments depending on samples.

( / ) = × × × × !" (1)

Chemometric analysis of LF spectra

Fourier transform, phasing and manual multipoint baseline correction were performed on each spectrum. Then the data matrix was obtained by an automatic binning approach (bin width of 0.01 ppm) and buckets were normalized by dividing their areas by that of the internal reference TSP for standard solutions and also by the correction factor linked to the extraction yield (see section 3.2.1.) for commercial samples. Principal Component Analyses (PCA) with Pareto scaling were performed on the LF NMR spectra of all commercial samples (56 spectra). Partial least squares regressions (PLS1) were built with the areas of specific LF NMR signals of standard (E)-cinnamaldehyde (22 spectra) and coumarin (8 spectra) solutions as the set of independent variables (X) and their known concentrations as the set of dependent variables (Y). These PLS1 models were validated using cross-validation method (Q 2 ) and CV-ANOVA. The quantification of (E)-cinnamaldehyde and coumarin in commercial samples was then predicted according to their respective PLS1 models. PCAs and PLS1s were performed with the SIMCA-P+ 13.0 software (Umetrics, Umea, Sweden). As the coupling constants are independent of the magnetic field, the signals appear obviously much more spread with more overlaps in the spectrum at LF. For instance the H2 doublet of doublet of (E)-cinnamaldehyde becomes a second order system with a slight "roof" effect at LF. Likewise, H3 and H4 of coumarin give two doublets at HF (Δν/J >60) but tend to an AB system at LF with Δν/J=9.4. Besides, some signals overlap e.g. H7, 8, 9 and 10 of coumarin co-resonate as a broad multiplet that spreads from 7.1 to 7.7 ppm at LF.

Results and discussion

HF and LF NMR assignment of signals

NMR assignments of spectra recorded at 400 MHz are reported in Table S1. cinnamic acid and eugenol were observed whereas those of coumarin could not be detected.

Implementation of the experimental conditions

Extraction tests and solvent choice

To select the best extraction procedure, different extraction tests were performed on the commercial dietary supplement 25 which contains significant amounts of (E)cinnamaldehyde and coumarin as shown later.

In the literature different solvents have been proposed for the quantification of active compounds of cinnamon. The most common are methanol [START_REF] Archer | Determination of Cinnamaldehyde, Coumarin and Cinnamyl Alcohol in Cinnamon and Cassia by High-Performance Liquid-Chromatography[END_REF][START_REF] Farag | NMR approach for the authentication of 10 cinnamon spice accessions analyzed via chemometric tools[END_REF][START_REF] Wang | Cassia Cinnamon as a Source of Coumarin in Cinnamon-Flavored Food and Food Supplements in the United States[END_REF] and methanol/water or ethanol/water mixtures [START_REF] Ballin | Coumarin content in cinnamon containing food products on the Danish market[END_REF][START_REF] Blahova | Assessment of coumarin levels in ground cinnamon available in the Czech retail market[END_REF][START_REF] Ding | Discrimination of cinnamon bark and cinnamon twig samples sourced from various countries using HPLC-based fingerprint analysis[END_REF][START_REF] Lungarini | Coumarin and cinnamaldehyde in cinnamon marketed in Italy: a natural chemical hazard?[END_REF][START_REF] Sproll | HPLC analysis and safety assessment of coumarin in foods[END_REF]. Only one study carried out by NMR used CDCl3 as extracting solvent [START_REF] Killday | Cinnamon: an Important Natural Health Product with Quantification of Key Components using NMR Spectroscopy[END_REF].

A first set of experiments was thus dedicated to the screening of different solvents. In order to establish the extraction procedure, three successive extractions were performed on 100 mg or 15 mg of samples 10 and 25. From these preliminary experiments (see results in section 2.2.1), we chose to analyse all the commercial samples after a single extraction in MeOH-d4. HF NMR analyses were done from both 15 and 100 mg of powder.

MeOH-d4, mixtures

The 15 mg samples were considered for the assay of (E)-cinnamaldehyde and coumarin as we showed that a lower amount of powder ensures a better extraction yield, and the 100 mg samples for the assay of the other chemicals (eugenol, o-methoxy-cinnamaldehyde, (E)cinnamyl alcohol, (E)-cinnamic acid) whose concentrations are much lower. The LF capabilities were assessed with 100 mg of powder only (discussed in 3.2.3. section).

LF NMR exploratory experiments with Chemical Shift Reagents (CSR)

CSR have been explored extensively in the years 1960-70 when NMR spectrometers operated with 1 H frequencies around or under 100 MHz. Recently, the use of CSR was reported for the new generation of compact LF NMR spectrometers to increase the frequency dispersion of the targeted NMR signals [START_REF] Singh | Compact NMR Spectroscopy with Shift Reagents[END_REF]. In most experiments with CSR, the best solvent for improving spectral resolution is CDCl3. In the context of cinnamon analysis, we showed above that CDCl3 did not provide a good extraction yield and that MeOH-d4 was the best solvent. Although the interaction of the paramagnetic ion with the oxygen atom of MeOH-d4 reduces its availability for interacting with other species, we nevertheless decided to test CSR in our experimental conditions. Our aim was to spread the signals and particularly to downfield slightly (≈20 Hz) the H4 signal of coumarin thus making its area easier to measure. Three CSR (Eu(fod)3, Eu(NO3)3 and EuCl3) were tested (Figure S3). For a TSP calibrated at 0 ppm, the chemical shift of the H4 signal did not shift significantly. Indeed, the left part of the doublet moves from 8.

LF quantitative analysis: attempt to implement and limitations

Quantitative HF NMR is nowadays a recognized method for the analysis of complex samples [START_REF] Bharti | Quantitative 1H NMR spectroscopy[END_REF][START_REF] Monakhova | Monitoring daily routine performance in quantitative NMR (qNMR) spectroscopy: Is the system suitability test necessary?[END_REF][START_REF] Simmler | Universal quantitative NMR analysis of complex natural samples[END_REF] and was taken as the standard reference method in this study. However, the implementation of LF quantitative analysis remains a challenging task [START_REF] Matviychuk | A field-invariant method for quantitative analysis with benchtop NMR[END_REF]. In this section, we discuss the attempt to set up and encountered limitations for cinnamon analysis.

The first option was to quantify the samples prepared with a correct extraction yield, i.e. using the same solution for both HF and LF NMR quantifications. For HF 1 H NMR quantitative analysis, 15 mg of commercial sample were sufficient to measure accurate concentrations in a reasonable recording time of 1 h with a LOQ of 0.09 mg/g and 0.15 mg/g for (E)-cinnamaldehyde and coumarin respectively. With the same parameters and recording time on the LF spectrometer, the LOQ was estimated at 6 mg/g and 10 mg/g due to the decrease of the SNR per unit time by a factor of ≈ 60. This means that if we wanted to obtain a correct SNR ratio for coumarin quantification, the experimental time would have been superior to 100 h for each analysis of commercial samples. Even with extracts from 100 mg of commercial powder, at least 17 h were necessary to obtain a SNR of ≈10 for samples containing ≈3 mg/g of coumarin (Figure S4). It thus appeared that it was not possible to implement correct quantitative measurements of coumarin with both a full relaxation of the signals and under a reasonable recording time compatible with routine analyses.

Another way to proceed was to run LF NMR analysis using concentrated samples, i.e.

samples extracted from 100 mg, recorded in a reasonable experiment time of 2 h in non-fully relaxed conditions, and to consider several correction factors: (i) a correction factor linked to the extraction yield, (ii) a second integrating the fact that signals were not fully relaxed in 2 h of recording [START_REF] Bharti | Improved quantification from 1H-NMR spectra using reduced repetition times[END_REF], (iii) a third applied to coumarin only, linked to the roof effect of the second order system of H4 and H3 protons.

Moreover, the LF quantification was also altered by the automatic baseline correction procedure that introduces experimental errors related to the SNR of the considered signal.

Taking into account all the correction factors described above, the global resulting RSD for quantification would be >15%. We thus concluded that cinnamon samples could not be accurately quantified at LF in our experimental conditions. Nevertheless, we will further investigate in section 3.3.2. a statistical treatment of LF NMR spectra obtained from commercial samples recorded for 2 h after extraction of 100 mg samples.

Analysis of commercial samples

HF NMR analysis: from profiling to quantitative analysis

Amounts of (E)-cinnamaldehyde, coumarin, eugenol, o-methoxy-cinnamaldehyde, cinnamyl alcohol and (E)-cinnamic acid in cinnamon commercial samples measured from HF 1 H NMR spectra are reported in Table 1. These data allow distinguishing several categories of samples. In four dietary supplements (16, 19, 21 and 24), neither (E)-cinnamaldehyde nor coumarin was detected but only signals of fatty acids and/or sugars. None of the typical chemicals of cinnamon being detected, not even (E)-cinnamaldehyde, we assess that these dietary supplements did not contain cinnamon powder (or at trace level) contrary to what was announced on the label. Eugenol which was previously reported as a marker of Cinnamomum verum [START_REF] Farag | NMR approach for the authentication of 10 cinnamon spice accessions analyzed via chemometric tools[END_REF][START_REF] Wang | Cassia Cinnamon as a Source of Coumarin in Cinnamon-Flavored Food and Food Supplements in the United States[END_REF] was observed and quantified in samples 5, 6, 9, 17 and 18. Moreover, coumarin was not detected in these samples which could be thus classified as "true cinnamon" (Cinnamomum verum) samples. Indeed, the amount of coumarin in Cinnamomum verum (0.04 mg/g) is under the LOQ of HF NMR for this compound (0.15 mg/g). Two samples of food cinnamon, 2 and 11, contained the highest amounts of both (E)-cinnamaldehyde and coumarin in agreement with the Vietnamese origin claimed by the producers and previously reported data on Vietnamese cinnamon (Cinnamomum loureirii) [START_REF] Wang | Cassia Cinnamon as a Source of Coumarin in Cinnamon-Flavored Food and Food Supplements in the United States[END_REF]. Samples containing detectable levels of coumarin could be classified as cassia cinnamon (samples 1-4, 8, 10-15, 20, 25-28) without a more precise classification according to geographical origin or species (burmanii, zeylanicum…). Classification of samples 7, 22

and 23 could not be assessed from NMR data insofar as only (E)-cinnamaldehyde was detected. The proposed classification of samples from HF NMR data is reported in Table 1.

Moreover, we can observe that the two samples 22 and 23 contain high levels of (E)-cinnamic acid with 4.1 and 3.7 mg/g respectively, which can be due to a longer storage of these samples that leads to the oxidation of (E)-cinnamaldehyde [START_REF] Woehrlin | Quantification of flavoring constituents in cinnamon: high variation of coumarin in cassia bark from the German retail market and in authentic samples from indonesia[END_REF].

The toxicity of cinnamon samples is related to their coumarin content and the hepatotoxic level of 0.1 mg/kg body weight must be considered. Consumption of cinnamon in food is very variable and linked to dietary habits of each country. For instance, it was reported that in Germany the consumption of cinnamon present in various desserts can reach 17 g over the ten days of the Christmas period, so a mean value of 1.7 g/day [START_REF] Abraham | Toxicology and risk assessment of coumarin: focus on human data[END_REF]. In food cinnamon samples reported in Table 1, the highest concentration of coumarin found was 6.1 mg/g. This kind of spice sample may thus present a risk for the health of a big consumer of cinnamon cookies or other Christmas desserts.

The issue is different for dietary supplements for which a dosage is recommended by the manufacturer. Indeed, the coumarin content of dietary supplements can be discussed considering the daily amount ingested according to the recommended dosage indicated on the packaging. For samples 15, 20, 25-28, it was calculated, from HF NMR data, between 0.65 and 8.43 mg (mean 4.6 mg). For the two dietary supplements 27 and 28, the daily coumarin intake, 7.72 and 8.43 mg respectively, is over the hepatotoxic level of 0.1 mg/kg considering an average body weight of 70 kg. Therefore, their daily ingestion can be at risk for consumers.

LF NMR analysis: from chemometric approach to quality control

As shown above (see section 3.2.3.), the LF quantification of cinnamon samples could not be done correctly. We nevertheless chose to extract 100 mg of powder from commercial samples, to record the LF NMR spectra in non-fully relaxed conditions and then to apply a statistical treatment.

First, an unsupervised PCA of all samples was performed as it provides an informative first look at the dataset structure and indicates trends of clustering. The PCA was built from the LF NMR spectra limited to the region of olefinic and aromatic protons (5.7-11 ppm) of all samples (Figure 3A). Other parts of NMR spectra were excluded in order to remove (i) areas affected by the residual signals of water and methanol at 3.8-5.7 ppm and 2.8-3.8 ppm respectively, (ii) signals of fatty acids (0.5-2.5 ppm) and (iii) noise (2.5-2.8 ppm) (Figure 2B).

Based on the quantitative HF analysis, three classes of samples were colored on the PCA score plot : samples which contain neither (E)-cinnamaldehyde nor coumarin (16,19,21,24) in red, samples in agreement with Ceylan cinnamon i.e. without coumarin but containing eugenol (5, 6, 9, 17, 18) in blue, and all the remaining samples in green. Two compact clusters were identified: the first one corresponds to the dietary supplements which contain no cinnamon (in red) and the second to samples containing true cinnamon (Cinnamomum verum)

(in blue). The wide dispersion of the last group (in green) is related to the NMR signals at 6.4-7.0 ppm and 7.3-7.7 ppm corresponding to all the protons (except H1) of (E)-cinnamaldehyde (Figure 1) which is present in varying amounts (0.2 to 36 mg/g) (Table 1). The effect of (E)cinnamaldehyde in the samples dispersion is shown by the loading plot illustrated in Figure S5. For coumarin, in order to check if the whole doublet can be considered for the next step, a PCA score plot based on parts of the H3 (right component) and H4 (left component) was performed. It showed a clear grouping of samples where coumarin was not detected (red and blue in Figure 3B). Samples 2 and 11 with the highest levels of coumarin (6.1 and 5.4 mg/g respectively, Table 1) are located in the lower right part of the score plot. The two samples 22

and 23 seem atypical because they contain significant amounts of (E)-cinnamic acid (Table 1) whose H2 has the same chemical shift than the right resonance of the H3 of coumarin at LF (Figure S6). In the same way, the broad signal around 8 ppm is only detected in sample 22 and to a lesser extent in sample 23. For the next steps, the two formulations 22 and 23 were no longer considered and the analysis was restricted to the left resonance of the H4 of coumarin.

Figure 4 shows that a quantification based on the LF NMR data focused on typical signals of (E)-cinnamaldehyde (H1) or coumarin (left component of the H4 doublet) can be predicted. For this approach, spectra of standard and commercial samples were acquired and processed under the same conditions. Only the correction factor linked to the extraction yields of commercial samples was included in the normalization of the data. PLS1 models were first built with the LF NMR spectra of solutions of standard (E)-cinnamaldehyde and coumarin of known concentrations corresponding to real amounts in commercial samples. Each model was validated (Q 2 > 0.99 and CV-ANOVA<10 -8 ) and used to predict concentrations of these compounds in commercial samples. The first scatter plot (Figure 4A) shows the good correlation (R 2 = 0.98) between the predicted concentrations of (E)-cinnamaldehyde and the real concentrations measured with HF NMR for the three classes of samples (red, blue and green) from 0 to ≈40 mg/g for the more concentrated samples 2 and 11. The same result was obtained for coumarin in food samples (Figure 4B). Figure 4C shows the bar plot of the approximate concentrations of coumarin predicted by the same PLS1 but restricted to dietary supplements (without samples 22 and 23). Here, the maximal recommended daily dosage was considered for data treatment. This graph highlights that the two samples 27 and 28 containing ≈6-8 mg of coumarin may lead to the ingestion of daily high levels of coumarin by the consumer. They can thus pose a health risk because they are located in the hepatotoxic range for a body weight of about 70 kg.

This chemometric approach demonstrates that key information on the quality of commercial cinnamon samples can be reached from LF NMR data obtained with a quick and easy sample preparation step and 2 h of NMR spectra recording. Moreover, it allows predicting quantification of (E)-cinnamaldehyde and coumarin levels in commercial samples.

Conclusion

The aim of the present study was to evaluate the pros and cons of LF NMR, a technique reported here for the first time for quality control of commercial cinnamon samples.

Although LF NMR spectra of cinnamon samples are crowded and assignments tricky due to signal overlaps, characteristic resonances of (E)-cinnamaldehyde and coumarin can nevertheless be detected. LF absolute quantification cannot be proposed as a routine control method for cinnamon analysis but the treatment of LF NMR data with a chemometric approach is a promising way to enhance the power of LF NMR and thus to consider it as a full analytical technique. These encouraging results must lead to continuing the evaluation of benchtop LF NMR spectrometers for quality control applications in food analysis.

Captions for figures

[

  REF] is the concentration of the internal reference (TSP), Ax and AREF the integrated signal areas of the targeted compound and TSP, respectively, Nx and NREF the number of protons giving rise to the signal considered, V the volume of solution, MWx the molecular weight of each compound, Wsample the mass of cinnamon sample used for the NMR assay.

Figure 1

 1 Figure 1 compares the spectra of the same NMR tube containing a solution of standard (E)-cinnamaldehyde (A and B) or coumarin (C and D) recorded at 400 (A and C) and 60 (B and D) MHz. As the coupling constants are independent of the magnetic field, the signals

Figure 2

 2 Figure2illustrates the 1 H NMR profiles of the cinnamon food sample 2. The HF spectrum shows, in addition to signals of coumarin and (E)-cinnamaldehyde, some typical resonances of o-methoxy-cinnamaldehyde, cinnamyl alcool and (E)-cinnamic acid that were assigned by comparison to previous work[START_REF] Farag | NMR approach for the authentication of 10 cinnamon spice accessions analyzed via chemometric tools[END_REF] and by addition of standard compounds in the medium. NMR assignments of spectra of standard compounds recorded at 400 MHz are reported in TableS1. Another flavouring compound, eugenol, was detected in HF spectra of some samples (not present in the spectrum shown Figure2A) thanks to its characteristic singlet at 3.82 ppm (O-CH3)[START_REF] Farag | NMR approach for the authentication of 10 cinnamon spice accessions analyzed via chemometric tools[END_REF] and other signals from its aromatic ring (δ (ppm) 6.72, d 2 Hz; 6.69, d 8.0 Hz; 6.58, dd 8.0, 2.0 Hz). Only (E)-cinnamaldehyde (H1, δ 9.66 ppm and H2, δ 6.73 ppm) and coumarin (H3, δ 6.42 ppm and H4, δ 7.94 ppm) signals are readily observed in the LF spectrum of the cinnamon food sample 2 (Figure2B). To complete the samples screening, the HF 1 H NMR spectrum of an additional sample of an authentic standard of Cinnamomum verum is reported in FigureS1and compared with sample 2. In this spectrum, signals of (E)-cinnamaldehyde, o-methoxy-cinnamaldehyde, cinnamyl alcool, (E)-

   and CDCl3 were tested with a direct extraction of 100 mg of powder. Moreover, a liquid-liquid extraction of a D2O suspension of powder by CDCl3 was also explored. The extraction yields were evaluated by integration of the H1 signal of (E)-cinnamaldehyde and H3 and H4 signals of coumarin on the HF 1 H NMR spectra. Results are reported in FigureS2. The solvents that extracted the highest quantities of (E)-cinnamaldehyde and coumarin were MeOH-d4 and the mixture MeOH-d4:D2O in the 80:20 ratio. This better extraction yield with methanol is in agreement with most published studies[START_REF] Ballin | Coumarin content in cinnamon containing food products on the Danish market[END_REF][START_REF] Blahova | Assessment of coumarin levels in ground cinnamon available in the Czech retail market[END_REF][START_REF] Farag | NMR approach for the authentication of 10 cinnamon spice accessions analyzed via chemometric tools[END_REF]. The extraction yields found in CDCl3 relatively to MeOH-d4 were poor (10.6 and 9.5% for (E)-cinnamaldehyde and coumarin, respectively) demonstrating that it was not possible to implement a quantitative analysis of cinnamon samples in this solvent unlike previously reported[START_REF] Killday | Cinnamon: an Important Natural Health Product with Quantification of Key Components using NMR Spectroscopy[END_REF]. For the next steps of the study, since our objective was to propose a method on a LF NMR spectrometer, we chose to use MeOH-d4 and not a MeOH-d4:D2O mixture as extraction solvent. Indeed a single solvent facilitates the implementation of the LF NMR experiments because the lock procedure is more convenient.

  02 ppm without CSR to 8.03 (Δδ = 0.5 Hz), 8.09 (Δδ = 4.4 Hz) and 8.18 ppm (Δδ = 9.7 Hz) with addition of Eu(fod)3, Eu(NO3)3 and EuCl3 respectively. Moreover, for EuCl3 which induces the highest chemical shift modification of the coumarin resonance, a broadening of the signals, as well as a sharp decrease of the (E)-cinnamaldehyde signal, were observed because EuCl3 induced strongest interaction with the chemicals. From these experiments, it appears that the use of CSR for the analysis of cinnamon extracts under our experimental conditions did not constitute an interesting tool for better discrimination of signals. Experiments with CSR were not thus deeply investigated due to the restricted solvent choice imposed by the extraction step.

Figure 1 .

 1 Figure 1. 1 H NMR spectra of solutions of standard (E)-cinnamaldehyde (A and B) and coumarin (C and D) in MeOH-d4. (A) and (C) spectra are recorded at HF and (B) and (D) at LF. Recording times were 2 min at HF and 2 h at LF. See Figure 2 for chemical structures and proton numbering of (E)-cinnamaldehyde and coumarin.

Figure 2 .

 2 Figure 2. (A) HF and (B) LF 1 H NMR spectra of cinnamon food (sample 2) highlighting typical signals of the main components. Recording times were 8 min at HF and 2 h at LF.

Figure 3 .

 3 Figure 3. (A) Three-dimensional score plot of a PCA built from the LF NMR data of all the samples using signals in the range 5.7 to 11 ppm. (B) Score plot of a PCA built from the LF NMR data focusing on the signals of H3 (right component at 6.34 ppm) and H4 (left component at 8.03 ppm) of coumarin. Samples without cinnamon (16, 19, 21, 24) are colored in red, samples with true cinnamon (5, 6, 9, 17, 18) in blue and other samples in green.

Figure 4 .

 4 Figure 4. Chemometric analysis of LF NMR data. Representation of predicted values obtained from PLS1 models built with LF NMR data versus HF NMR quantitative data for (A) (E)-cinnamaldehyde in all samples and (B) coumarin in food samples. (C) Bar plot of predicted contents of coumarin in dietary supplement samples according to their recommended dosage.

  Figure 1

Table 1 .

 1 Quantitative data of cinnamon commercial samples from HF NMR analysis (n=2) a and comparison of labelled information to the proposed classification from HF NMR data.

	Sample	(E)-cinnamaldehyde	Coumarin	Eugenol	o-methoxy-cinnamaldehyde	Cinnamyl alcohol	(E)-cinnamic acid

Acknowledgments

The authors thank the Chinese Scholarship Council (CSC) for Ph.D. funding and Dr Chantal Galaup for helpful discussions on CSR reagents.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 8.4 a Some samples were extracted in triplicate. b Calculated from the HF NMR quantification according to the recommended dosage on the dietary supplement label. c For these 3 samples, the commercial information does not fit with the NMR analysis; Ceylon cinnamon was replaced by cassia cinnamon.