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Quantitation of Minor Components in Mango Juice with Band-Selective Excitation NMR Spectroscopy Shoraku Ryu, Masanori Koda, Takuya Miyakawa, and Masaru Tanokura J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03336 • Publication Date (Web): 04 Oct 2017 Downloaded from http://pubs.acs.org on October 8, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Quantitation of Minor Components in Mango Juice with Band-Selective

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Excitation NMR Spectroscopy

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Shoraku Ryu, Masanori Koda, Takuya Miyakawa and Masaru Tanokura*

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Laboratory of Basic Science on Healthy Longevity, Department of Applied Biological

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Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo,1-1-1

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Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

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*Corresponding

author

(Tel:

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[email protected])

+81-3-5841-5165;

Fax:

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1

ACS Paragon Plus Environment

+81-3-5841-8023;

E-mail:


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ABSTRACT: 1H NMR-based metabolic analysis of foods has been widely applied. However,

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dynamic range problems frequently impede its application because foodstuffs are composed of

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various organic compounds in a wide range of concentrations. Band-selective excitation 1H

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NMR spectroscopy has been found to be a useful tool for observing the minor components in

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foods. Since quantitative information is important for metabolic analysis of foods and for

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complete metabolome data, quantitation with the band-selective excitation 1H NMR method

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was carefully investigated in the present study. As a result, the concentrations of minor

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components in mango juice of the ‘Carabao’ cultivar were successfully quantitated by

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band-selective excitation 1H NMR using standard curves that exhibited good linearity. The

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band-selective excitation 1H NMR techniques was therefore effective for determining the

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concentrations of minor components in foods.

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KEYWORDS: NMR, band-selective excitation, quantitation, mango juice

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INTRODUCTION

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During the past several decades, metabolic profiling has become important in food science for

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identifying and quantitating a number of metabolites, belonging to various classes of compounds.

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For metabolic profiling, various methods of component analysis are employed, such as liquid

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chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry

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(GC-MS), capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF-MS), and

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nuclear magnetic resonance (NMR) spectroscopy. Among them, 1H NMR spectroscopy is

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perceived as one of the most robust methods for metabolic profiling due to its multiple

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advantages, such as it being non-destructive, comprehensive, quantitative, and fast.1-3 In the past

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ten years, this technique has become an efficient method for combining statistical analysis with

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origin authentication, varietal differentiation and quality evaluation of foods without a separation

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procedure.4-11

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Foodstuffs are composed of various organic compounds in a wide range of concentrations.

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Minor components at relatively low concentrations in foods frequently contribute to the food

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characterization, such as geographic origins, botanical origins, and food processing. For example,

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minor components in mango juice are useful for discriminating the cultivars.7 In honey and olive

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oil minor components are used for identifying botanical and geographic origins, respectively.12–

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Despite the importance of minor components in foods, they may not be detected in 1H NMR

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spectra if they are in the baseline or below the lowest dynamic range of the A/D converter

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because the major components (e.g., sugars, lipids, ethanol, and acetic acid) existing at relatively

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high concentrations define the maximum of the dynamic range. This problem frequently

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impedes the application of NMR in metabolic profiling of foods. In a previous study, we

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investigated the band-selective excitation of 1H NMR as an efficient approach to overcoming the

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dynamic range problem in NMR,17 and found it to be a useful tool for providing information

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about minor components in mango juice and used it to successfully discriminate the cultivars of

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mango fruits.7 In addition, there are several reports for the band-selective applications in the field

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of NMR food analysis.12,18–22

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A thorough metabolic profiling analysis of foods requires both the identification and

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quantitation of the organic compounds because the concentration information is important for a

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complete metabolome of foods. However, in a band-selective excitation 1H NMR spectrum, the

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ratio between the signal intensities may not directly reflect the concentration ratio because of the

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influence of relaxation times and coupling constants.21,23 There are very few studies focusing on

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quantitative characteristics for band-selective excitation.22 For this reason, it is still unclear

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whether band-selective excitation 1H NMR is suitable for quantitation of the minor components

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in foods.

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In the present study, the quantitative characteristics were statistically compared between

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band-selective excitation 1H NMR and non-selective 1H NMR. Moreover, the technique was

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successfully used to quantitate the minor components in mango juice of the ‘Carabao’ cultivar

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and provide concentration data for those components.

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MATERIALS AND METHODS

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Materials and Sample Preparation. Mangos of the ‘Carabao’ cultivar were purchased at

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a local market, and deuterium oxide (D2O, 99.9% D) was obtained from Shoko Co., Ltd. (Tokyo,

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Japan). All other chemicals were of spectroscopic grade and were purchased from Wako Pure

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Chemical Industries, Ltd. (Osaka, Japan) and Nacalai Tesque Inc. (Kyoto, Japan). Mango juice

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was prepared according to a previous report6 and was stored at −20 °C until it was used for

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NMR analyses. For the NMR analyses, 70 µL of D2O was added to 630 µL of mango juice, then

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the sample was transferred to a 5 mm NMR tube (Tokyo Chemical Industry Co., Ltd., Tokyo,

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Japan). A sealed capillary tube with 0.83 mM [2,2,3,3-d4] sodium 3-(trimethylsilyl) propanoic

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acid (TSP-d4), 0.72 mM hydroxymethylfurfural (HMF) and 5.5 mM MnCl2 was coaxially

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inserted into the NMR tube as an external reference for concentration determination. To

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investigate the quantitative properties of band-selective excitation, alanine was chosen since it is

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found in mango juice (the target sample in this study) and in many other foods. For quantitation

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of mango juice components, histidine, shikimic acid, trigonelline, uridine and adenosine were

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used as standard solutions dissolved in D2O at five concentrations (0.2, 0.5, 1, 2 and 5 mM).

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These standard solutions (700 µL) were transferred to a 5 mm NMR tube for NMR

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measurements.

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NMR Spectroscopy. All NMR spectra were obtained at 20 °C on a Unity INOVA-500

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spectrometer (Agilent Technologies, Santa Clara, CA). The water signal was suppressed by a

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presaturation method. The selective excitation was achieved using DPFGSE24 combined with

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Re-Burp.25 The pulse lengths of Re-Burp were 2.0 and 2.9 ms for covering 6.0−11.0 ppm and

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−0.2 to 3.1 ppm, respectively. The spin-lattice relaxation times (T1) were measured by inversion

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recovery experiments and the delay time (d1) was determined with the following equation.5

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d1 ≥ 5 × T1 − aq

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where aq is acquisition time. The acquisition parameters of the 1H NMR spectra were as

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follows: spectral width, 8 000 Hz; number of data points, 32 k; acquisition time, 2.048 s; number

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of scans, 128. The receiver gain was set at 18 dB for non-selective and 60 dB for band-selective

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excitation 1H NMR. The measurements for alanine were performed in triplicate.

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NMR Data Processing and Quantitation of Minor Components. Free induction

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decades (FIDs) were converted to spectra using uniform processing and phasing parameters

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(exponential apodization with 0.25 Hz line broadening). The baseline was carefully calibrated

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manually. The signal of alanine at 1.47 ppm was chosen as the reference for chemical shifts,

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which was the shift of the alanine signal when the signal of external TSP was set to 0 ppm in the

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absence of MnCl2. The signals of HMF and TSP were used as concentration standards for the

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low-field and high-field regions, respectively. Their integral values were set to 1 and the relative

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integral values were calculated for all other observed signals. The standard curves, which

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characterize the correlation between integral values and concentrations, were determined using

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the standard solutions. Experimental concentrations of mango juice components were

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determined by the integral value of each signal in the low-field region and the corresponding

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standard curve according to the following equation.

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Experimental Concentration =

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( )(

!" #"

!" #"

) × Theoretical Concentration''

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where Integral and Integralstandard represent the integral value of each signal and methyl proton

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signal of standard compounds (HMF or TSP), respectively, protons and protonsstandard are the

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number of protons constituting each signal and the methyl proton signal of the standard

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compounds (HMF or TSP), respectively, and Theoretical Concentrationstandard is 0.83 mM. The

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concentrations of the minor components appearing in the low field region were determined

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using the standard curves. The components observed in the high field region, such as choline,

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GABA, citric acid, quinic acid, and alanine, were quantitated by non-selective excitation 1H

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NMR spectra.

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Statistical Analysis. The linearity of standard curves was evaluated with the coefficient of 7



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determination R2, and the linear regression parameters (slope and y-intercept) were calculated.

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According to guidance from the International Conference on Harmonisation (ICH),26 the

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detection limit (DL) and quantitation limit (QL) were estimated using the following equations.

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QL = 10σ/S

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DL = 3.3σ/S

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where σ represents the standard deviation of the response calculated using regression analysis,

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and S is the slope of the calibration curve.

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RESULTS AND DISCUSSION

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Quantitation of Alanine as a Standard Sample. The standard curves for quantitation

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were generated with the alanine solution. The spectra and the integrals of the β-proton signals

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with band-selective excitation and non-selective excitation are shown in Figure 1. The relative

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integral value of β-proton signals (1.47 ppm) was calculated in comparison to the value of the

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TSP signal in 5.5 mM MnCl2 (0.355 ppm), which was set to 1 (Figure 1). MnCl2 was added to

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shorten the relaxation time and recycling delay. The half-width of the methyl proton signal of

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TSP-d4 increased by 11.5 times in the presence of 5.5 mM MnCl2. The standard curve for

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band-selective excitation 1H NMR exhibited good linearity based on the coefficient of

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determination, with R2 values of 0.9997, which is almost the same value as that of the

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non-selective 1H NMR (R2 = 0.9999) (Figure 1B). However, the ratio of integral value to

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concentration (Int/Con slope) for those measured with the non-selective and the band-selective

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1

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indicating an excellent consistency between the theoretical and the experimental concentrations.

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In contrast, the Exp Con/Theo Con slope was calculated as 1.41 indicating a worse consistency

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in quantitation with band-selective excitation 1H NMR. Because the intensity of 1H NMR

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signals should be proportional to the absolute quantity of hydrogen atoms, these results indicated

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that the signal intensity was attenuated during band-selective excitation, which leads to an

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inadequate spectrum for direct quantitative analysis. On the other hand, the high coefficient of

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determination (R2) showed that band-selective excitation 1H NMR can be reliably utilized to

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quantitate components by plotting standard curves for the individual protons.

H NMR were different. The Exp Con/Theo Con slope for the non-selective 1H NMR was 1.02,

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According to the ICH guidance, we calculated DL and QL using σ values and the slope of

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standard curve and compared the results between the non-selective and the band-selective

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excitation 1H NMR spectra. The σ values of the series of three measurements calculated with

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regression analysis were 0.028 and 0.020 mM for non-selective and band-selective excitation 1H

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NMR, respectively. The DL and QL values were 0.241 and 0.729 mM for non-selective 1H

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NMR spectra, respectively. On the other hand, the DL and QL values for band-selective

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excitation were 0.120 and 0.379 mM, respectively. These results showed that band-selective

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excitation 1H NMR could have 2-fold higher sensitivity, which indicates it could be used to

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detect and quantitate components that are half as concentrated as components non-selective 1H

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NMR could detect.

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The Influence of Relaxation Delay Time on the Quantitation. For precise quantitation

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with 1H NMR, a long relaxation delay times (d1) are usually used to allow the equilibrium

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magnetization state to be re-established before the next pulse cycle begins. Consequently, the

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experimental time is considerably lengthened. To decrease the time needed for a quantitative

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experiment, we examined whether the similar good linearity could be obtained with a short d1

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when using standard curves.

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The ε-proton signal of histidine (Hε) at 8.64 ppm was chosen for its long T1 value (7.75 s).

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First, we plotted the standard curves with band-selective excitation with d1 value of 38 s

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(>5×T1−aq); a 128-scan experiment with d1 = 38 s takes 113 minutes to run. The curve

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displayed a linearity with the R2 value of 0.9993. Next, we compared that curve with the

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standard curve obtained when the d1 value was set to 2 s, which is the typical measurement

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condition for 1H NMR. A 128-scan experiment with a d1 = 2 s takes only 36 min to run. In the

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standard curve with a 2 s delay time, the ratio of integral to concentration was obviously lower

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than the ratio in the experiment with a long enough delay time (≥5×T1−aq) (Figure 1C) because

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2 s is an insufficient relaxation time during scans. Despite the attenuation of the signal intensities,

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a good linearity (R2 = 0.9953) was also seen in the standard curve in band-selective excitation 1H

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NMR with a 2 s relaxation time. These results demonstrated that the shorter relaxation delay is

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also applicable for quantitation with a specific standard curve. This method using shorter delay

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times would help reduce the experimental time in the case of long T1 if the S/N ratios are

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sufficiently high.

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Quantitation of Minor Components of Mango Juice. Figure 2A shows the expanded 1H

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NMR spectra of the mango juice from 5.8 to 10.0 ppm. Only a few signals were observed in this

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region even with 1024 scans. When using band-selective excitation, the receiver gain could be

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increased and many signals were then detected with just 256 scans (Figure 2B). In our previous

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study, the minor components of mango juice were assigned with band-selective excitation 1D

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and 2D NMR spectra.7 In the present study, five mango fruits of the ‘Carabao’ cultivar were

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measured with band-selective excitation 1H NMR spectra and non-selective 1H NMR spectra,

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which contained signals of many major components such as amino acids (histidine, alanine,

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γ-aminobutyric acid (GABA), etc.), organic acids (citric acid, shikimic acid, and quinic acid),

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alkaloids (trigonelline), nucleosides (adenosine and uridine), and others (choline, etc.) (Figure 3).

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We quantitated the minor components of mango juice with band-selective excitation 1H NMR

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spectra and other minor components with non-selective 1H NMR spectra. Considering their low

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concentration in mango juice, the delay time was set to be > 5×T1−aq in all the experiments,

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where T1 was the longitudinal relaxation time of the histidine Hε proton, which has the

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longest T1 observed in the spectrum. Although some amine protons from compounds such

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as glutathione and glutamine were clearly observed in the low-field region, it was difficult

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to quantitate those chemicals because of the exchangeability of their protons.27,28

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HMF was used as the external standard for the down field region (6.0−11.0 ppm). The

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standard curves were prepared for five standard chemicals (trigonelline, histidine, uridine,

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shikimic acid, and adenosine). All chemicals showed good linearity for their standard curves

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(Figure 5 and Table 1). The concentrations of the minor components were determined using the

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standard curves. Because the signals of citric acid overlapped with other signals, we applied

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curve fitting to calculate the signal intensity. The results of the quantitation are summarized in

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Figure 5 and Table 2. Because the signals of shikimic acid were observed in both the down-field

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(6.59 ppm) and up-field regions (2.76 ppm), the concentrations of shikimic acid calculated from

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both the non-selective and band-selective excitation 1H NMR spectra of mango juice were

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compared. The quantitative values were 6.1 ± 0.4 mM and 5.6 ± 0.6 mM (mean ± standard

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deviation) for non-selective and band-selective excitation 1H NMR, respectively (Table 2 and

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Figure 5), indicating that the results of quantitation using standard curves by band-selective

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excitation 1H NMR are consistent with the results using non-selective 1H NMR spectra.

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In conclusion, the present study has indicated that band-selective excitation 1H NMR

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spectroscopy is an efficient technique for determining the concentrations of minor components

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in complex mixtures using standard curves. Even in the case of a short delay time (for example,

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2 s), the quantitative analysis can be carried out if the S/N ratio is sufficient. A previous report on

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the metabolic profiling of mango juices suggested that the concentration variations of minor

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components might cause the differences between groups rather than certain marker

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components;7 however, it was unclear whether signal intensity had any correlation with actual

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concentration of each component. The present study has elucidated the signal intensity is

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linearly correlated with the concentration, which has revealed that band-selective excitation 1H

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NMR spectra exactly provides quantitative information for metabolic profiling.

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ASSOCIATED CONTENT

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Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website.

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Calibration data for working curves of alanine; and calibration data for standard curves

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of the Hε Proton of Histidine (8.64 ppm) (PDF)

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AUTHOR INFORMATION

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Corresponding Author

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*Tel: +81-3-5841-5165; Fax: +81-3-5841-8023; E-mail: [email protected]

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ORCD

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Masaru Tanokura: 0000-0001-5072-2480

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Notes

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The authors declare no competing financial interest.

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FIGURE CAPTIONS

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Figure 1. (A) The spectra and (B) standard curves of alanine for non-selective (black line, ▲)

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and band-selective excitation (red line, ♦) 1H NMR spectra. (C) The influence of relaxation

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delay (d1) on the quantitative nature of the integral of the signal at 8.64 ppm (Hε proton) of

318

histidine in standard samples. The inset circle indicates the 1H position used for quantitation. The

319

relaxation delay was 2 s (■) and 38 s (♦)

320

321

Figure 2. Expanded 1H NMR spectra of mango juice (5.8̶− ̶ 10 ppm) observed with (A)

322

non-selective excitation (number of scans, 1024; receiver gain, 18 dB) and (B) band-selective

323

excitation (number of scans, 256; receiver gain, 60 dB)

324

325

Figure 3. (A) Low-field region of the band-selective excitation 1H NMR spectra of mango juice

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from five different fruits (‘Carabao’ cultivar). (B) High-field region of the non-selective 1H

327

NMR spectra of mango juice from five different fruits (‘Carabao’ cultivar).

328

329

Figure 4. Standard curves for minor components in the low-field region (6.0−11.0 ppm): ○,

330

adenosine; □, trigonelline; ∆, histidine; +, uridine; and *, shikimic acid.

331

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332

333

Figure 5. The concentrations of minor components in mango juice (A) at concentrations lower

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than 5 mM and (B) at concentrations higher than 5 mM. For shikimic acid. The labels “nonsel”

335

and “sel” represent the quantitative values determined by the signals of non-selective and

336

band-selective excitation 1H NMR spectra, respectively.

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Table 1. Calibration Data for Minor Components in the Low-Field Region (6.0−11.0

ppm). Analyte

Int/Con slope Int/Con intercept

R2

T1

trigonelline (8.83 ppm)

3.03

0.032

1.0000

4.59

histidine (8.64 ppm) uridine (7.86 ppm)

1.42 1.65

0.075 0.047

0.9994 0.9997

7.75 1.45

shikimic acid (6.59 ppm) adenosine (6.08 ppm)

1.51 1.61

0.035 0.035

0.9996 0.9998

3.77 2.87

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Table 2. Concentrations of Minor Components in Mango Juice.

Component

Average concentration (SD*) (mM)

trigonelline

0.08 (0.02)

histidine

0.12 (0.03)

uridine

0.09 (0.01)

adenosine choline GABA

0.12 (0.01) 0.29 (0.04) 1.12 (0.22)

quinic acid alanine citric acid shikimic acid (nonsel) shikimic acid (sel)

2.63 (0.38) 0.92 (0.44) 36.5 (6.08) 6.11 (0.37) 5.63 (0.62)

*

SD stands for standard deviation.

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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

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Table of Contents Graphic

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Quantitation of Minor Components in Mango Juice ... - ACS Publications

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