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Selective 1D TOCSY NMR Experiments for a Rapid Identification of Minor Components in the Lipid Fraction of Milk and Dairy Products: Towards Spin-Chromatography? Christina Papaemmanouil, Constantinos G. Tsiafoulis, Dimitrios Alivertis, Ouranios Tzamaloukas, Despoina Miltiadou, Andreas Tzakos, and Ioannis P. Gerothanassis J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01335 • Publication Date (Web): 19 May 2015 Downloaded from http://pubs.acs.org on May 26, 2015

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Journal of Agricultural and Food Chemistry

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Selective 1D TOCSY NMR Experiments for a Rapid Identification of

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Minor Components in the Lipid Fraction of Milk and Dairy

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Products: Towards Spin-Chromatography?

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Christina Papaemmanouil, Constantinos G. Tsiafoulis*§‡, Dimitrios Alivertis#,

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Ouranios Tzamaloukas┴, Despoina Miltiadou┴, Andreas G. Tzakos, and Ioannis P.

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Gerothanassis*

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Section of Organic Chemistry and Biochemistry, Department of Chemistry, §NMR

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Center, ‡ Laboratory of Analytical Chemistry, Department of Chemistry, #Department

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of Biological Applications and Technology; University of Ioannina, Ioannina GR-451

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10, Greece

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┴

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University of Technology, P. O. Box 50329, Limassol 3603, Cyprus

Department of Agricultural Sciences, Biotechnology and Food Sciences, Cyprus

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1 ACS Paragon Plus Environment


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ABSTRACT: We report a rapid, direct and unequivocal spin-chromatographic

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separation and identification of minor components in the lipid fraction of milk and

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common dairy products with the use of selective 1D TOCSY NMR experiments. The

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method allows the complete backbone spin-coupling network to be elucidated even in

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strongly overlapped regions and in the presence of major components with 4x102 to

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3x103 stronger NMR signal intensities. The proposed spin chromatography method

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does not require any derivatization steps for the lipid fraction, is selective with

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excellent resolution, is sensitive with quantitation capability and compares favorably

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with 2D TOCSY and GC-MS methods of analysis. The results of the present study

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demonstrated that the 1D TOCSY NMR spin-chromatography method can become a

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procedure of primary interest in food analysis and generally in complex mixture

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analysis.

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KEYWORDS: lipid fraction, totally correlated NMR spectroscopy (TOCSY), spin-

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chromatography, dairy products

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INTRODUCTION

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Separation and identification of components from complex mixtures has occupied a

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central role in the field of natural products and food chemistry research. The classical

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protocol for the investigation of complex mixtures has been to apply various

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chromatographic techniques, to isolate a certain amount of a pure product and to

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identify its structure using various spectroscopic techniques1 and/or the use of

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specialized hyphenated spectroscopic techniques.2,3 On the other hand, NMR

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spectroscopy is increasingly used as an analytical tool for identification and

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quantification of low molecular weight metabolites on unfractionized biological

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fluids, natural product extracts and food samples.1,4-12

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Milk lipids are very important as they confer distinctive textural, nutritional and

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organoleptic properties on dairy products. Recent studies have reported that the

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consumption of saturated fatty acids has been linked to increased risk of

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cardiovascular disease, whereas the consumption of milk conjugated linoleic acids

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(CLA) has beneficial effects and this issue is still subject to numerous studies.13

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Accurate analysis, therefore, of minor lipids is important for determining the nutritive

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value and preparing nutritional labeling materials for particular function or

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application. The analysis of minor lipids, however, is extremely challenging and

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complex since it can be very slow and laborious and may require various preparation

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and analysis steps.14

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The selective 1D TOCSY experiment has become an important NMR technique for

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establishing 1H-1H connectivity via scalar coupling in small and medium-size

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molecules15,16 and in the analysis of mixtures of related compounds.17 However, the

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method has found limited application in food matrices; to the best of our knowledge



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few studies have, so far, been published, among them in the metabolomics analysis of

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amino acids in honey18 and in mango juice.19 In the present study we report, for the

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first time, the direct identification of six minor species: (9-cis, 11-trans) 18:2 and (9-

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trans, 11-trans) 18:2 conjugated linoleic acid (CLA) isomers, caproleic acid, glycerol

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in 1,2 diglyceride (1,2 DAG), in 1 monoglyceride (1-MAG), and in 2 monoglyceride

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(2-MAG) in the lipid fraction of milk and halloumi cheese, without any derivatization

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steps, with the use of a spin- chromatography procedure based on the spin diffusion

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process of selective 1D TOCSY experiment.

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MATERIALS AND METHODS

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Materials. Conjugated (9-cis, 11-trans) 18:2 linoleic acid, purity ≥ 96% (HPLC),

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conjugated (9-trans, 11-trans) 18:2 linoleic acid, purity ≥ 98% (HPLC), were

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purchased from Fluka. Caproleic acid, purity ≥ 96%, was purchased from Sigma -

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Aldrich, chloroform and methanol (analytical grade) were obtained from Fisher

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Scientific (U.K.), CDCl3 (99.8%) from Deutero (Kastellaun, Germany) and the 37

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FAME standard mix from Sigma-Aldrich.

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Sample Preparations. The lipid fractions of milk samples were prepared as

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previously described.20 The lipid fractions of halloumi cheese were prepared as

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follows: cheese samples were frozen in liquid N2 and pulverized in a ceramic mortar.

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After lyophilization for two days, 300 mg of cheese was used for the extraction of the

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lipid phase, using the Bligh and Dyer method, as previously described.20

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NMR Instrumentation. NMR experiments were performed on a Bruker AV500

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spectrometer (Bruker Biospin, Rheinstetten, Germany) using the Topsin 2.1 suite. The


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1D TOCSY experiments were carried out using standard Bruker pulse program

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(selmlgp). A shaped pulse length of 20 ms for selective excitation was used followed

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either by a MLEV-17 TOCSY spin lock18 or by applying the DIPSI-2 pulse train and

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by incorporating a z-filter before acquisition21 for the suppression of artifacts. The

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spin-lock was adjusted to 7.1 KHz, corresponding to a low power 90o pulse of 35 µs;

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this allows safe operation without problems of significant heating of the samples with

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spin-lock times up to 400 ms.

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GC-MS Analysis. Fatty acid methyl esters (FAME’s) were prepared by trans-

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esterification with methanolic potassium hydroxide according to the ISO 15884:2002

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method22 as previously described.23 Fatty acid profiles were generated by analyzing

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the FAME samples on a GC-MS-QP 2010 Plus Gas Chromatography Mass

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Spectrometer (Shimadzu, Duisburg, Germany) equipped with a HT 280 T auto

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sampler (HTA, Brescia, Italy). Details of the GC-MS analysis are given

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elsewhere.20,23

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RESULTS AND DISCUSSION

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1D TOCSY of Model Compounds – Effects of Mixing Time

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Figure 1 illustrates a series of selective 1D TOCSY spectra of the model (9-cis, 11-

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trans) 18:2 CLA isomer where the H11 olefinic proton (δ= 6.27 ppm)20,24,25 has been

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selected for excitation using a range of mixing times and, thus, affecting the number

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of transfers within the spin system.26 For a mixing time τm = 33 ms, the selective 1D

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TOCSY transfers magnetization to its J-coupled partners within the conjugated

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olefinic protons and the C(13)H2 protons (Figure 1(b)). Using longer mixing times,



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the magnetization is transferred throughout the full spin system and, for τm values of

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200 and 400 ms, resulted in the complete analysis and structure elucidation of the

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compound (Figure 1(d), (e)). Figure S1 illustrates a similar series of selective 1D

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TOCSY spectra of the (9-trans, 11-trans) 18:2 CLA isomer where the H10, H11 (δ=

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5.97 ppm) protons have been selected. Again, the 1D TOCSY experiment allows

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structural information to be extracted in a time-efficient manner and with high

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spectral resolution.

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Method Application in the Lipid Fraction of Milk and Dairy Products

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An important application of the 1D experiment is the selective excitation of a

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proton sub spectrum belonging to a single chemical component contained in a

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complex mixture, thus providing a form of spin-chromatography. The selective

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excitation of a suitable “target” resonance of the compound of interest can then reveal

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the whole spin system, even if the 1D 1H NMR spectrum is heavily overlapped in the

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region of the other peaks in the selected spin coupling network. Figure 2(a) illustrates

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a typical 500 MHz 1H NMR experiment of the lipid fraction of a lyophilized halloumi

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cheese sample in CDCl3. Selective TOCSY excitation, with 400 ms mixing time, of

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the H11 proton of the (9-cis, 11-trans) 18:2 CLA isomer (Figure 2(b)), illustrates

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effective magnetization transfer from H11 to H2 and H11 to H18. Therefore, its

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structure becomes amenable to detailed analysis although signals from H2 to H8 and

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H12 to H18 protons are completely hidden in a conventional 1D 1H NMR spectrum

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under the resonances of the abundant components with 4x102 to 3x103 stronger

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intensities.


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Similar experiments were performed with caproleic acid (Figure 2(c)). Selective

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TOCSY excitation of the H10a protons at 4.97 ppm with τm = 400 ms resulted in the

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effective magnetization transfer throughout the complete proton spin system, although

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the signals from H8 to H2 are completely hidden in the conventional 1D 1H NMR

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spectrum. Selective TOCSY excitation of the doublet at 3.72 ppm reveals the

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complete spin system of the glycerol moiety in 1,2 DAG at 5.20 ppm (2’-CHOCO),

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4.28 ppm and 4.13 ppm (1’b, 1’a-CH2OCO, respectively) (Figure 2(d)).

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Figure S2 illustrates a similar series of selective 1D TOCSY spectra of the lipid

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fraction of lyophilized milk sample. Again the 1D TOCSY allows the analysis and

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structure elucidation of minor compounds in a time-efficient manner and with high

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spectral resolution.

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Comparison of the 1D TOCSY spectrum of the model (9-cis, 11-trans) 18:2 CLA

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isomer with the one obtained from the lipid fraction of the lyophilized halloumi

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cheese sample illustrates a significant difference of the C(2)H2 spin system (Figure 3).

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In the model compound it appears as a triplet (δ= 2.33 ppm, 3J= 7.2 Hz) due to

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coupling with the C(3)H2 protons while in the spectrum of the extract it appears at δ=

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2.30 ppm with a complex multiplet pattern. This clearly demonstrates that the (9-cis,

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11-trans) 18:2 CLA isomer in the extract exists predominantly as an ester and not as a

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free acid. Due to the asymmetry of the sn2 carbon of the glycerol moiety the C(3)H2

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protons become magnetically non equivalent, thus, resulting in a multiplet spin

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pattern. This result is very important in investigating the hydrolysis products of the

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ester linkage between an acyl group and the glycerol backbone. The resulting free

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fatty acids and their catabolic products have been found to be among the primary

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factors for the aroma of hard cheese where lipolysis reaches high levels.24



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Figure 4 illustrates the great potential of the 1D TOCSY experiments in the case of

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minor species with resonances in overcrowded spectral regions of the 1D 1H NMR

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spectrum. The spin systems of the glycerol moieties in both 2-MAG (Figure 4(b1),

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(b2)) and 1-MAG (Figure 4(c1), (c2)) were clearly resolved and provided an

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unequivocal assignment of both species although the 1-MAG resonances are strongly

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overlapped in the region of 3.75 to 3.55 ppm (Figure 4(a)).

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The excellent selectivity of the 1D TOCSY is demonstrated also in the case of

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strongly overlapped resonances of the 18:2 CLA geometric isomers. Figure S3

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illustrates a selected 1H NMR region of the (9, 11) 18:2 CLA resonances of the lipid

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fraction of a lyophilized halloumi cheese sample. The apparent triplet at 5.92 ppm

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(3J= 10.9 Hz) has been assigned to the H10 olefinic proton of the (9-cis, 11-trans)

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18:2 CLA isomer and the strongly overlapped minor peak at 5.97 ppm to the

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composite signal of the H10 and H11 protons of the (9-trans, 11-trans) 18:2 CLA

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isomer. Selective 1D TOCSY of the resonance at 5.97 ppm clearly demonstrates

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magnetization transfer to H9 and H12 resonances at 5.54 ppm which are not

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overlapped with other resonances of the lipid fraction (Figure S3(c)) and, thus, can be

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used for the assignment of the (9-trans, 11-trans) 18:2 CLA isomer. A limitation of

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the method, however, is that since the identification is based on chemical shifts, the

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length of the fatty acid and the position of the unsaturation remains ambiguous. For

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instance the 1H NMR spectrum of (9-cis, 11-trans) 18:2 has almost identical chemical

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shifts and J-couplings as that from (9-cis, 11-trans) 20:2 and (11-cis, 13-trans) 20:2.

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Figure S4 illustrates a comparison of the 1D TOCSY spectrum of Figure 2(b) with

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the respective extracted column and row in the 2D TOCSY experiment. It is evident

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that the 1D TOCSY: (i) increases significantly the digital resolution since in the 2D

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TOCSY it is limited by the number of points taken in the indirect dimension, (ii) 8 ACS Paragon Plus Environment

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removes dynamic range issues from the spectrum; (iii) the whole spin system can be

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excited even in the region of strong signal overlapping with major components with

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4x102 to 3x103 stronger NMR signal intensities and (iv) decreases the experimental

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time.

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Quantification using 1D TOCSY – Comparison with 1D 1H NMR and GC-MS

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Method of Analysis

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The excellent sensitivity and digital resolution of the 1D TOCSY experiment

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allows the quantification of minor components. The quantitative analysis was based

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on the standard addition method. Known amounts of caproleic acid were added to the

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sample and the respective spin chromatograms were recorded with the selective

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excitation of the H9 proton of the caproleic acid. The pulse repetition time

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(acquisition time + relaxation delay) was set at 5xT1 (~16 s) of the long relaxation

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time of the H9 and H10 protons (~3.2 s) of the caproleic acid. The results were

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compared with those obtained by the use of 1D 1H NMR quantitation.

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The effect of the mixing time on the quantitation results was studied in detail.

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Several mixing times were used, more specific 33, 100 and 200 ms (τm= 33 ms

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corresponds to the value of ½ 3J, where 3J is the coupling of the H9 and H10 protons).

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The sensitivity of the 1D TOCSY method was higher using smaller mixing time

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values but at the expense of the quantitation accuracy. Longer mixing times resulted

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in a moderate decrease of sensitivity, approx. by a factor of two, however, a

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significantly improved accuracy of the method was achieved (the relative error was

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calculated 31 % for τm= 33 ms, due to twisted lineshapes,27 whereas only 2 % for τm=

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200 ms). Moreover for τm= 200 ms the sensitivity and accuracy of the method was 9 ACS Paragon Plus Environment


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almost equal using either the H9 or the H10 signals (data not shown). It should be

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pointed out that one of the advantages of the spin chromatographic procedure is the

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ability to quantify the analyte of interest either using the excited proton or by using a

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nearby proton where the magnetization was transferred.

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1D TOCSY quantitation was achieved following the procedure described by

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Sandusky et al.28 Table 1 provides the quantification data obtained for the milk

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samples using the 1D TOCSY procedure. A concentration value (in tube) of

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1.02±0.03 mmol L-1 was determined using the 1D TOCSY and the H9 proton

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integration (Figure 5 and Figure S5) compared to 0.97±0.02 mmol L-1 as determined

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by using the conventional 1D 1H NMR with internal standard. For the second milk

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sample, the respective results were 1.92±0.06 and 1.84±0.06 mmol L-1. The results

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demonstrate the excellent agreement between the 1D TOCSY method with the

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conventional 1D 1H NMR method.

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The limit of detection (LD) for the spin chromatographic quantitation of the

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caproleic acid was calculated by the use of two different methods, based on the

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calibration function29 or the SNR method.30,31 By means of the calibration function,29

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a calibration curve of the standard addition method was used; the LD was found 0.07

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± 0.01 mmol L-1 (in tube) or 0.01 mg g-1 for the milk sample. Using the SNR method

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the LD was calculated by applying the 3xC/(S/N) equation in a standard sample of

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caproleic acid, where C represents the concentration of the analyte as determined by

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1

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the signal-to-noise value of the respective 1D TOCSY spectra was calculated. For the

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concentration levels of 0.97, 2.04 and 2.98 mmol L-1 of caproleic acid (in tube) the

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3xC/(S/N) value was calculated 0.10, 0.11 and 0.08 mmol L-1 using the 1D TOCSY

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spectra, and 0.004, 0.007 and 0.010 mmol L-1 using the 1D 1H NMR spectra,

H NMR (data were treated without using a line broadening exponential function) and


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respectively; thus, the LD value of caproleic acid is 0.10 mmol L-1 (in tube) or 0.01 ±

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0.001 mg g-1 for the milk sample and 0.010 mmol L-1 (in tube) or 0.001 ± 0.0001 mg

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g-1 for 1D TOCSY and 1D 1H NMR methods, respectively.

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The quantitative results of the spin chromatography and 1H NMR were compared

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with those obtained with the use of the GC-MS method of analysis (ISO

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15884:2002).22 The results are in accordance with the GC-MS method of analysis;

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caproleic acid was found to be 0.28 % and 0.68% of the total lipids when calculated

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by GC-MS and was measured 0.26 % and 0.74 % of the total lipids when calculated

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by 1D 1H NMR for the first and second milk sample, respectively.

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The proposed selective 1D TOCSY spin-chromatographic separation procedure,

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therefore, is an excellent technique in mixture analysis of minor components, through

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a careful selection of the spin system to be excited. The method might become of

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primary interest in food research including targeting metabolomics32 since: (i) it is

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rapid, selective and nondestructive, (ii) it allows the chemical identification of minor

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components even in strongly overlapped spectral regions, (iii) does not require

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derivatization steps and (iv) allows the quantification of analytes of interest.

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AUTHOR INFORMATION

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Corresponding Authors

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*(I.P.G.) E-mail: [email protected]; Phone: +30 26510 08389

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*(C.G.T) E-mail: [email protected]; Phone: +30 26510 08315

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Notes



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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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This work was supported by the Cyprus Research Promotion Foundation, European

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Regional Development Fund and Charalambides- Christies Ltd dairy industry. Thanks

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are given to the anonymous reviewers for critical and constructive criticisms.

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Supporting Information Available

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Figure S1: 500 MHz 1H NMR and 1D TOCSY spectra for several mixing times of (9-

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trans, 11-trans) 18:2 conjugated linoleic acid; Figure S2: 500 MHz 1H NMR and 1D-

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TOCSY spectra of the lipid fraction of a lyophilized milk sample; Figure S3: Selected

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regions of 500 MHz 1H NMR and 1D TOCSY spectra for (9-cis, 11-trans) and (9-

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trans, 11-trans) 18:2 CLAs of the lipid fraction of a lyophilized cheese sample; Figure

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S4: Selected regions of 500 MHz 2D TOCSY and 1D TOCSY spectra of the lipid

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fraction of a lyophilized cheese sample; Figure S5: 1D TOCSY quantitation

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procedure displaying the 1D TOCSY spectra for successive addition of caproleic acid

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in a milk sample.

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This information is available free of charge via the Internet at http: //pubs.acs.org


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Rapid determination of total conjugated linoleic acid content in select Canadian

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cheeses by 1H NMR spectroscopy. J. Agric. Food Chem. 2013, 61, 9915–9921.

339

(26) Sachleben, J. R.; Yi, R.; Volden, P. A.; Conzen, S.D. Aliphatic chain length by

340

isotropic mixing (ALCHIM): determining composition of complex lipid samples

341

by 1H NMR spectroscopy. J. Biomol. NMR 2014, 59, 161-173.

342

(27) An effective elimination of the twisted lineshapes (anti-phase components, ZQ

343

artefacts) for short τm values can be achieved with the use of the ZQ-filter

344

experiment (Thrippleton, J.M.; Keeler, J. Elimination of zero-quantum

345

interference in two-dimensional NMR spectra, Angew. Chem., Int. Ed. 2003, 42,

346

3938 –3941).

347

(28) Sandusky, P.; Amponsah, E.A.; Raftery, D. Use of optimized 1D TOCSY NMR

348

for improved quantitation and metabolomic analysis of biofluids, J. Biomol. NMR

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2011, 49, 281-290.

350 351

(29) Danzer, K. Analytical Chemistry, Theoretical and Metrological Fundamentals; Springer- Verlag: Berlin, 2007; pp.74, 147.


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(30) Tsiafoulis. C.G.; Exarchou, V.; Tziova, P.P.; Bairaktari, E.; Gerothanassis, I.P.;

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Troganis, A.N. A new method for the determination of free L-carnitine in serum

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samples based on high field single quantum coherence filtering 1H-NMR

355

spectroscopy. Anal. Bioanal. Chem. 2011, 399, 2285-2294.

356

(31) Maniara, G.; Rajamoorthi, K.; Rajan, S.; Stockton, G. Method performance and 1

validation for quantitative analysis by

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Applications to analytical standards and agricultural chemicals. Anal. Chem.

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1998, 70, 4921-4928.

360 361

H and

31

357

P NMR spectroscopy.

(32) Sundekilde, U. K.; Larsen, L. B.; Bertram, H. C. NMR-based milk metabolomics. Metabolites 2013, 3, 204–222.

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FIGURE CAPTIONS

365

Figure 1. (a) 500 MHz 1D NMR spectrum of 20 mM solution of the (9-cis, 11-trans)

366

18:2 conjugated linoleic acid in CDCl3 (T= 298 K, acquisition time= 4.3 s, relaxation

367

delay= 5 s, number of scans= 256, experimental time ~25 min); (b)-(e) selective 1D

368

TOCSY spectra of the above solution using a mixing time of τm= 33 ms (b), 70 ms

369

(c), 200 ms (d), and 400 ms (e). The asterisk denotes the selected H11 resonance

370

which was excited. For (b)-(e) the magnetization transfer network is illustrated.

371

Figure 2. Spin chromatogram of the lipid fraction of a lyophilized cheese sample in

372

CDCl3: (a) 500 MHz 1H NMR spectrum of the lipid fraction of a lyophilized cheese

373

sample in CDCl3 (298 K, number of scans= 256, acquisition time= 4.3 s, relaxation

374

delay= 5 s, total experiment time ~25 min). The major lipid resonances are denoted

375

(sn1, sn2 and sn3 indicate the stereospecific numbering of esterified glycerol). The

376

insert shows x512 magnification of the spectrum in order to display resonances from

377

the 18:2 CLA and other minor species. (b)-(d) 1D TOCSY spectra of 2(a) with τm=

378

400 ms (ns= 256, total experiment time ~25 min). The asterisks denote the resonances

379

which were excited by the use of a selective pulse.

380

Figure 3. Selected regions of: (a) Figure 2(a); (b) 1D TOCSY spectrum of Figure

381

2(b); (c) 1D TOCSY of the model (9-cis, 11-trans) 18:2 CLA isomer. In (b) and (c)

382

the selective excitation pulse was set on the H11 proton (δ= 4.92 ppm). The insert in

383

(b) shows x16 magnification of the spectrum in order to display the C(2)H2COOR

384

resonances.

385

Figure 4. Selected regions of: (a) Figure 2(a); (b) 1D TOCSY spectrum which

386

demonstrates the spin system of the glycerol moiety in 2-MAG (in (b1) and (b2) the

387

selective excitation pulse was set on the 1’,3’ CH2OH (δ= 3.82 ppm) and 18 ACS Paragon Plus Environment

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2’ CHOCOR (δ= 4.92 ppm) peaks in 2-MAG, respectively); (c) 1D TOCSY spectrum

389

of the spin system of the glycerol moiety in 1-MAG (in (c1) and (c2) the selective

390

excitation pulse was set on the 3’ CH2OH (δ= 3.67 ppm) and 2’ CH2OH (δ= 3.92

391

ppm) peaks in 1-MAG, respectively).

392

Figure 5. Quantification of caproleic acid (CA) in the lipid fraction of a lyophilized

393

milk using the 1D TOCSY method (see also Figure S5).

394



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Table 1: Results obtained using 1D TOCSY NMR, conventional 1D 1H NMR and GC-MS methods for the determination of the caproleic acid in two milk samples. Sample

Analyte / Units

1D

1

H NMRa

TOCSYa 1

Caproleic acid/mM

1.02±0.03

Caproleic acid / %

Relativeb

GC-MS

deviation (%) 0.97±0.02a

Relativec deviation (%)

5.15

0.26±0.01

0.28

-7.14

0.68

8.82

of the lipid fraction 2

Caproleic acid/mM % of the lipid

1.92±0.06

1.83±0.06a

1.75

0.74±0.01

fraction a

Results are expressed as mM of caproleic acid (in tube; standard deviation (n=3)). bResults are

expressed as 100 x [ (1D TOCSYvalue)- (1D 1H NMRvalue)] /(1D 1H NMRvalue). cResults are expressed as 100 x [(1D 1H NMRvalue) -(GC-MSvalue)] /(GC-MSvalue).


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Figure 1



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Figure 2


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Figure 3



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Figure 4


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Figure 5



TOC Graphic

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Selective One-Dimensional Total Correlation ... - ACS Publications

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