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Quantitative 1H NMR analysis of egg yolk, alcohol and total sugar content in egg liqueurs Monika Hohmann, Verena Koospal, Claudia Bauer-Christoph, Norbert Christoph, Helmut Wachter, B. W.K. Diehl, and Ulrike Holzgrabe J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00940 • Publication Date (Web): 10 Apr 2015 Downloaded from http://pubs.acs.org on April 13, 2015

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Journal of Agricultural and Food Chemistry

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Quantitative 1H NMR analysis of egg yolk, alcohol and

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total sugar content in egg liqueurs

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Monika Hohmann1,2*, Verena Koospal2, Claudia Bauer-Christoph2, Norbert Christoph2, Helmut Wachter2,

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Bernd Diehl3, Ulrike Holzgrabe1

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Germany

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2

Bavarian Health and Food Safety Authority, Luitpoldstraße 1, 97082 Würzburg, Germany

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Spectral Service, Emil-Hoffmann-Str. 33, 50996 Köln, Germany

Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg,

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Corresponding author: Monika Hohmann

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Phone: +49 9131 68087159

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Fax: +49 9131 68087210

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Email: [email protected]

ACS Paragon Plus Environment


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Abstract

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Analyzing egg liqueurs for compliance with legal requirements means several different time-consuming

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preparations and analytical processes. In this paper, we describe the approach to use quantitative

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1

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rapid sample preparations for water-soluble or nonpolar ingredients. Fifteen egg liqueurs were analyzed

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for alcoholic strength, content of total sugar and egg yolk (estimated by cholesterol as marker substance)

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with both, classical methods and quantitative 1H NMR spectroscopy. The results of both methods

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showed excellent correlations for alcoholic strength (R = 0.996, p < 0.001), content of total sugar

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(R = 0.989, p < 0.001) and cholesterol (R = 0.995, p < 0.001). Besides, NMR spectra revealed further

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information: a signal of phosphatidylcholine at about δ= 3.20 ppm served a second marker for the egg

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yolk content and characteristic signals of lactose at δ=4.46 ppm and butyric acid at δ=0.97 ppm indicated

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the use of milk products that has to be declared for lactose-intolerant consumers.

H NMR spectroscopy as an accurate alternative technique. 1H NMR analysis comprised two different

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Keywords: quantitative 1H NMR, qNMR, egg liqueur, total sugar, alcoholic strength, egg yolk, cholesterol


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Introduction

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According to the Regulation (EC) No 110/20081, egg liqueur “is a spirit drink, (…) obtained from ethyl

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alcohol of agricultural origin, (…) quality egg yolk, egg white and sugar or honey”. Besides that, this

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legislation stipulates a content of at least 150 g sugar or honey (expressed as invert sugar) per litre,

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140 g/L pure egg yolk and 14 % alcoholic strength by volume for the final product. For liqueurs with egg,

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the minimum content of egg yolk must be 70 g/L and the minimum alcoholic strength by volume has to

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be 15%. Furthermore, a deviation of only ± 0.3% between the actual and the labelled alcoholic strength

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by volume is regarded as tolerable.2

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The food industries as well as food control authorities have the duty to verify egg liqueurs in compliance

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with legal requirements. Especially the egg yolk content is a crucial factor which may be underused with

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lower concentrations than prescribed by law, in the interest of maximizing benefits or due to improper

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manufacturing processes.

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Appropriate analytical methods are needed in order to control the officially required specifications of

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egg liqueurs. The alcoholic strength and total sugar content can be analyzed directly, while the content

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of egg yolk is calculated indirectly via the concentration of typical constituents of egg yolk. According to

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the reference method, alcoholic strength of highly viscous spirits is determined by the density of a

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distillate using pycnometry3. The content of total sugar can be analyzed chromatographically,

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enzymatically or by a redox titration (back titration after reaction with Cu2+ as oxidizing agent)4 and for

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the content of egg yolk, several characteristic indicator substances can be used, 5 like cholesterol, 6

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phosphorus compounds7 such as phospholipids or specific egg proteins8,9. Given its relative constant

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content (on average 12.5 mg/g egg yolk10), cholesterol presents a frequently used compound to quantify

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the egg yolk content.11 It can for instance be analyzed enzymatically subsequent to saponification.6



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In general, the analysis of egg liqueurs with highly viscous consistency and sometimes instable emulsion

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is rather difficult with regard to precise sample handling.7 Since every single analyte requires its

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individual elaborative and time-consuming sample preparation and quantification method, efficient

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alternative screening methods would be preferable. Steam distillation has been described as a useful

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rapid alternative method to determine alcoholic strength and density,12 however, steam distillation does

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not enable the quantification of egg yolk content. The aim of this study was to develop a rapid and

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accurate alternative technique to analyze egg liqueurs for compliance with legal requirements. Hence,

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we investigated for the first time the use of quantitative 1H NMR spectroscopy (qNMR) for the

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quantitative and qualitative analysis of certain egg liqueur components.


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Materials and methods

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Chemicals and materials

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TMS (tetramethylsilane, 99.9%) and ethanol (99.8%) were purchased from Acros Chemicals (Geel,

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Belgium), caffeine, CDCl3 (99.8 atom% D), D2O (99.9 atom% D), NaN3, K4[Fe(CN)6]·3H2O (99%), NaOH (99-

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100%), starch and TSP-d4 (3-(trimethylsilyl)-propionic acid-D4 sodium salt, 98 atom% D) from Merck

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(Darmstadt, Germany), cholesterol (99%), fructose (99%), glucose (99.5%), KI (99.5%) and

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sucrose (99.5%) from Sigma (St. Louis, MO, USA), CuSO4·5H2O (99.5%), KOH (85%),

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KNaC4H4O6·4H2O (99%), Na2S2O3 (99.5%), ZnSO4 (99.5%) and MeOH-d4 (99.8 atom% D) from Carl Roth

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(Karlsruhe, Germany), MeOH (99,8%) from Fisher Scientific (Pittsburgh, PA, USA), H2SO4 (96%) from Fluka

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(Buchs, Switzerland) and isopropyl alcohol from VWR Scientific Products (New York, NY, USA). A ready kit

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for enzymatic determination of cholesterol was purchased from r-biopharm (Boehringer Mannheim/r-

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biopharm, Darmstadt, Germany) and sea sand from Merck (Darmstadt, Germany).

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TSP-d4 solution consisted of 7 mM TSP-d4 and 2 mM NaN3 in D2O, Carrez I solution of

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15% K4[Fe(CN)6]·3H2O in demineralized water, Carrez II solution of 30% ZnSO4 in demineralized water

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and CDCl3 solution of 7 mM TMS and 21.5 mM caffeine in CDCl3.

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Sample preparation for egg liqueurs

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Fifteen different egg liqueurs were purchased from supermarkets in Germany. For analysis of alcoholic

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strength and total sugar content, about 2 g of egg liqueur was diluted to 10 mL with demineralized water

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and 1000 µL of this dilution were mixed with 50 µL Carrez I solution, 50 µL Carrez II solution and 100 µL

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TSP-d4 solution; subsequently this mixture was centrifuged at 6260 g for 5 min and 600 µL of clear

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supernatant were transferred to 5 mm NMR tubes. For analyzing cholesterol, about 200 mg of egg

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liqueur were mixed with 500 µL MeOH-d4 and 500 µL CDCl3 solution. After shaking for 30 min, samples

H NMR analysis of egg liqueurs



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were centrifuged at 1565 g for 5 min and 600 µL of clear supernatant were transferred to 5 mm NMR

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tubes.

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Sample preparation for checking recovery rates

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The increase in concentration by addition of reference solutions (ethanol/ sucrose/ egg yolk mixture)

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was divided by the theoretical increase in concentration in order to yield the percentage of recovery

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rates.

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For checking recovery rates of ethanol and total sugar content, five different egg liqueurs were analyzed.

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For ethanol, 5 g of egg liqueur was diluted to 50 mL with demineralized water and 5 g of the same egg

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liqueur was mixed with 0.8 g of pure ethanol and diluted to 50 mL with demineralized water. 1 mL of

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each dilution was mixed with 50 µL Carrez I solution, 50 µL Carrez II solution and 100 µL of TSP-d4

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solution and centrifuged at 6260g for 5 min. For total sugar content, 2 g of egg liqueur were diluted to

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10 mL with demineralized water. 1 mL of this dilution was mixed with 100 µL sucrose solution

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(containing 200 g/L sucrose) and 1 mL was mixed with 100 µL demineralized water. Subsequently, the

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samples were mixed with 50 µL Carrez I solution, 50 µL Carrez II solution, 100 µL of TSP-d4 solution and

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centrifuged at 6260 g for 5 min. Each 600 µL of clear supernatant were transferred to 5 mm NMR tubes

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and the ethanol or sucrose content was determined by qNMR.

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For checking cholesterol recovery rates, five egg liqueurs were prepared by adding different amounts

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(ranging between 6.6 and 25.9 g) of an egg yolk mixture (containing 335.3 g/L egg yolk, respectively

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4.2 g/L cholesterol on the assumption of 12.5 mg/g cholesterol in egg yolk10) to 50 g of an egg liqueur

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with known content of cholesterol (calculated as the average value of five qNMR results). 200 mg of this

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were mixed with 500 µL MeOH-d4 and 500 µL CDCl3 solution. After shaking for 30 min, samples were

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centrifuged at 1565 g for 5 min and 600 µL of clear supernatant were transferred to 5 mm NMR tubes.

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1

H NMR measurement


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Hardware and software equipment for 1H NMR measurement comprised a Bruker Avance 400

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spectrometer with a 5 mM SEI probe with Z-gradient coils, an automatic SampleChanger (SampleXpress),

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a BCU 05 temperature unit and TopSpin 3.0 software.

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Samples in aqueous solution were measured without rotation at 301.8 ± 0.1 K using 4 dummy scans prior

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to 16 scans. Acquisition parameters have been set as follows: size of FID = 64k,

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spectral width = 20.55 ppm, receiver gain = 16, acquisition time = 3.98 s, relaxation delay = 4 s, FID

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resolution = 0.25 Hz per point. Water suppression was achieved by an experiment using a 90o pulse with

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a NOESY-presaturation pulse sequence (Bruker 1D noesygppr1d) with irradiation of the water frequency

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during the recycle and mixing time delays. All spectra were automatically phased and manually baseline

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corrected.

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Samples in MeOH-d4/CDCl3 solution were measured without rotation at 300 ± 0.1 K using 2 dummy scans

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prior to 256 scans. Acquisition parameters have been set as follows: size of FID = 64k, spectral width =

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25.82 ppm, receiver gain was automatically set by rga command, acquisition time = 3.17 s, relaxation

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delay = 1 s, FID resolution = 0.32 Hz per point. A flip angle of 30° was used within the zg30 experiment.

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All spectra were manually phased and baseline corrected.

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Quantification

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Integration ranges are displayed in Table 1. Quantification was performed by external calibration with

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concentration ranges from 12.5 to 22.5% by volume for ethanol, 100 to 500 g/L for sucrose, 20 to 80 g/L

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for glucose and fructose and 1 to 3 g/L for cholesterol. For each calibration graph, linearity was verified

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by Mandel’s fitting test (F-values were lower than reported values in F-tables with a confidence level of

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95%). The limit of detection (LOD) / limit of quantification (LOQ) were determined according to

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DIN 3264516 (significance level 5%; uncertainty of result 33%) and amounted to 0.80 / 2.78 % by volume



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for ethanol, 6.79 / 26.11 g/L for sucrose, 0.56 / 2.23 g/L for glucose, 1.39 / 5.33 g/L for fructose and

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0.08 / 0.28 g/L for cholesterol, respectively.

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To consider the influence of the pulse length on signal intensities, the signal areas of ethanol, sucrose,

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glucose and fructose were converted into their theoretical values for an equal 90o pulse length of 11 µs.

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For this, each signal area was multiplied by the factual 90o pulse length (in µs) and divided by 11. To

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quantify cholesterol, the ratio of the signal area of cholesterol compared to the signal area of caffeine

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was used for examination of egg liqueurs as well as for calibration references.

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Conventional analytics of egg liqueurs

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Alcoholic strength was determined pycnometrically after distillation,3 total sugar content as reducing

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sugars by a redox titration after reaction with Cu2+ as oxidizing agent (Luff-Schorl method)4 and

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cholesterol was analyzed enzymatically subsequent to saponification (for saponification: 10 mL of 1 M

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KOH solution were added to 2 g egg liqueur and 1 g sea sand and heated under reflux for 25 min, the

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supernatant and two amounts of each 6 mL isopropyl alcohol (used for washing solid residues) were

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diluted to 25.0 mL with isopropyl alcohol and in case of turbid solutions, filtration was performed using a

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folded filter). Enzymatic determination of cholesterol was performed according to the instructions of a

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ready kit (purchased from Boehringer Mannheim/r-biopharm, Darmstadt, Germany).

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Results and discussion

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Sample preparation for 1H NMR measurement

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Foremost, optimized conditions for sample preparation were developed. Considering the high viscosity

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of egg liqueurs, direct 1H NMR measurements are not applicable. Firstly, it is difficult to accurately fill

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viscous egg liqueurs into NMR tubes and secondly, poor spectral resolution is obtained (Figure 1). By

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precipitation of proteins the viscosity of liqueurs is reduced, which increases spectral resolutions. Figure

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1 shows a 1H NMR spectrum of an egg liqueur (1:2 diluted with water) without further preparation steps ACS Paragon Plus Environment

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and of an egg liqueur (1:5 diluted) after Carrez-precipitation. 1H NMR spectra of the supernatant,

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obtained after Carrez-precipitation of diluted egg liqueurs, revealed distinct resonance signals of

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ethanol, sucrose, glucose and fructose (Figure 2) and the respective signal areas enabled the

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determination of alcoholic strength (as ethanol) and total sugar content (sum of sucrose, glucose and

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fructose).

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As cholesterol is difficult to dissolve in water, it was not detectable by use of this preparation process.

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Therefore MeOH-d4 and CDCl3 (50:50, v:v) were added to a small amount of liqueur and this solution was

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subsequently mixed for the extraction of cholesterol. Although this mixture contained nonpolar solvents

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and water (originating from liqueurs) at the same time, no layer separation occurred during extraction.

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Thus, it can be excluded that extraction losses of cholesterol are caused by partially dissolved cholesterol

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in different solvent phases. The appearance of a homogeneous solution can be attributed to the

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emulsifying effect of phospholipids out of egg yolk. Aside from that, due to the precipitation of proteins

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by MeOH-d4, a clear and non-viscous solution was available after centrifugation and the supernatant was

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used for qNMR analysis of cholesterol (Figure 3).

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Capabilities and requirements of quantitative 1H NMR

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As quantitative 1H NMR spectroscopy (qNMR) presents a primary method,17 the concentration of

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substances can be directly derived from the signal areas without the application of response factors or

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reference substances.18 One major advantage of qNMR is the possibility for straightforward sample

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preparations, since every step of sample pre-treatment implies a potential source of error. Furthermore,

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in contrast to analytical methods that are restricted in applicability by the selectivity of detectors,

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1

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conditions for multi-method approaches.

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To gain benefit from these advantages, general prerequisites need to be fulfilled in order to yield

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accurate quantification results. In general, appropriate spectrometer configuration, measuring

H NMR spectroscopy enables simultaneous detection of all 1H atoms and thus, implements optimum



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conditions as well as suitable processing parameters must be considered. Furthermore, distinct and well

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separated resonance signals as well as sufficiently high concentrations are needed for proper integration

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and the relaxation delay between consecutive pulses must be set adequate to ensure complete

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relaxation of the substances to be quantified.19 Another effect to be considered is that the NMR signal

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strength is affected by the 90o pulse length that is inversely proportional to the signal intensity.20, 21

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When sample preparations were applied as previously described, distinct signals for ethanol, sucrose,

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glucose, fructose and cholesterol were achieved (Figures 2 and 3) and integration ranges for qNMR were

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set adequate to eliminate nearby resonance signals from other components. With the exception of

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fructose and glucose that were not contained in each egg liqueur, all concentrations were high above the

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respective limit of quantification. After integration, quantification was performed by external calibration

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with individual calibration graphs for each substance (ethanol, sucrose, glucose, fructose and

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cholesterol). The sample preparation for qNMR of cholesterol included caffeine as internal standard in

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the extraction solvents in order to avoid quantification errors caused by evaporation of volatile solvents.

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Thus, instead of the signal area of cholesterol, its ratio to the signal area of caffeine was used for

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examination.

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To consider the influence of the 90o pulse length on signal intensities, the signal areas for ethanol,

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sucrose, glucose and fructose were converted into their respective value for an equal 90o pulse length

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(11 µs; materials and methods). Since all resonance signals were affected by variations of the 90o pulse

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length, the signal areas of cholesterol and caffeine as internal standard were likewise influenced. Thus,

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the impact of the pulse length was already eliminated during examination of the cholesterol content,

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since the cholesterol signal areas were considered as their respective ratio to the signal areas of caffeine

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as internal standard.


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The correctness and precision of qNMR was investigated by recovery rates (n = 5) and replicate

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measurements (n = 5). Accurate results were achieved for all quantified components (Table 2). Thus, the

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conditions for quantification were set adequately.

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Comparison of the results of qNMR with conventional analysis

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The direct comparison with the results of established conventional analysis is an important step in order

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to verify applicability and validity of qNMR as a rapid alternative method. Even if no factual

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concentrations for the egg liqueur samples were available, the methods accordance between established

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classical methods and qNMR gives a first impression of the suitability of NMR analysis. For comparison

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with qNMR, the following conventional analytical methods were applied: pycnometry after distillation

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for alcoholic strength, redox titration for sugar and enzymatic analysis (after saponification of lipids) for

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cholesterol. However, the respective results are subject to individual uncertainties of measurement and

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thus, they do not reflect real values but rather trusted estimators.

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Correlations between data of qNMR and conventional methods were calculated for their comparability.

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Since correlation coefficients do not accommodate systematic differences, too optimistic results may be

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delivered and thus, we additionally used the Bland-Altman-plot to factor in eventual biases.22 It is

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performed by plotting the differences between two methods against the respective mean value of both

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methods for each sample. For a better overview, 3 parallel lines for the mean deviation and mean

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deviation ± 1.96fold standard deviation are indicated in the plot. This diagram offers information about

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systematic deviations between two methods, the variance around the mean deviation and eventual

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dependencies between deviations and concentration range.23 We analyzed overall fifteen egg liqueurs by

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both, quantitative 1H NMR analysis and classical analytical techniques. The results are displayed in Table

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3.



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When qNMR results were plotted against results of customary analysis techniques, high correlations

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were achieved for alcoholic strength (R = 0.996, p < 0.001), content of total sugar (R = 0.989, p < 0.001)

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and content of cholesterol (R = 0.995 p < 0.001).

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Regarding alcoholic strength, the absolute deviation between the methods averaged 0.2 ± 0.3% by

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volume and the relative deviation 1.0 ± 1.5% (with an average alcoholic strength of 19.0% by volume,

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determined by results of pycnometry). Thus, the results showed a tendency for marginally higher values

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for qNMR analysis. Beside a possible systematic error of qNMR, this may be due to a bias of the classical

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method (for instance caused by incomplete distillation processes prior to pycnometric analysis). The

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Bland-Altman plot shows no dependency between the concentration range and the deviation between

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the methods (Figure 4). The deviations were rather symmetrically distributed between mean deviation

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± 1.96fold standard deviation (0.2 ± 0.6% by volume).

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For total sugar, the absolute mean deviation between the methods amounted to -15 ± 13g/L and the

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respective relative mean deviation to -4.1 ± 3.7% (with an average sugar content of 357 g/L, determined

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by results of titrimetry). Thus, the results of qNMR were found to be on average lower than the results of

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titrimetric analysis. The integral used for qNMR of total sugar derived from the same 1H NMR spectrum

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that was used for quantification of ethanol. Since qNMR data of ethanol complied well with values of

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classical techniques for alcoholic strength, it is unlikely that the sample preparation of qNMR is

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responsible for the deviation between the methods in terms of total sugar content. Besides, lower

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results of qNMR may be due to the fact that only sucrose, fructose and glucose were taken into account

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for examination, while titration involved all reducing sugars. For instance, egg liqueurs occasionally

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contain cream or condensed milk and an addition of milk products implies an addition of lactose (on

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average 3.3 g/100 g cream or 10.2 g/100 g condensed milk24), which is analyzed as reducing sugar during

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titrimetric analysis but not considered for qNMR. Thus, the average deviation between the methods was

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higher for egg liqueurs with lactose (-19 g/L; averaged by the results of nine egg liqueurs) than for egg


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liqueurs without lactose (-8 g/L; averaged by the results of six egg liqueurs). As the legal requirement for

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total sugar in egg liqueurs is referred to invert sugar1 which is defined as sum of sucrose, glucose and

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fructose, this rather supports the approach of qNMR. Furthermore, performing classical titrimetry

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involved elaborative sample preparation as well as imprecise endpoints of titration, which can cause

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measurement errors and possibly resulted in deviations from qNMR data. The Bland-Altman plot

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demonstrates that the deviations between the methods tended to grow with increasing concentration

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(Figure 4). As titration of sugars was performed by means of a back titration, high sugar concentrations

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resulted in small needs of titrant, which in turn means that the measurement error potentially increased

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with growing content of total sugar. This represents a possible explanation for the dependencies of

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deviations between the methods and the concentration range of total sugar. By all means, the aim of

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analyzing the total sugar content is simply to ensure that the minimum of 150 g/L 1 is exceeded. For this

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purpose qNMR is definitely suitable, especially as the absolute mean deviation between qNMR and

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classical analysis only amounted to -5 ± 10 g/L for egg liqueurs with sugar contents of less than 350 g/L.

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The absolute mean deviation between qNMR and enzymatic analysis of cholesterol amounted

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to -0.2 ± 0.1 g/L and the relative mean deviation to -9.7 ± 4.2% (with an average cholesterol content of

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2.3 g/L, determined by results of enzymatic analysis), showing on average lower values for qNMR than

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for enzymatic investigations. As the Bland-Altman plot demonstrates (Figure 4), the deviations between

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the methods were independent from the measuring range. An overestimation of enzymatic results due

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to insufficient specificity of cholesterol oxidase25 is unlikely, since egg liqueurs do not contain vegetable

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oils and structurally similar sterols are potentially also included in qNMR results. Comparing the sample

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preparations for both techniques, enzymatic determination is in general more elaborative and

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consequently more prone to errors than qNMR. However, since no factual data of cholesterol contents

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were available for the analyzed liqueurs, the reason for the observed deviations cannot certainly be

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identified. Similar to the legislative background for total sugar, the purpose of cholesterol quantification



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is to control the egg yolk content with respect to the given minimum of 140 g/L1 (respectively 1.75 g/L

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cholesterol, on the assumption of 12.5 mg/g cholesterol in egg yolk.10 As a screening method for this,

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qNMR achieved absolutely appropriate results. In addition, since the deviation between enzymatic

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determination and qNMR was systematically, correction factors can be applied to adjust qNMR values to

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respective data of enzymatic analysis by use of the linear correlation between the methods results

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(cholesterolqNMR (g/L) = 0,978*cholesterolenzymatic analysis(g/L) - 0,176 g/L).

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Taken the results together, the comparison of qNMR and classical data confirms NMR spectroscopy to be

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a suitable method to verify accordance of egg liqueurs with the given legal limits. qNMR can be used as a

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screening tool and in case of suspicious results the respective samples undergo additional analysis by

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reference methods. This enables risk orientated sample management and high sample throughputs due

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to rapid determinations by qNMR. For the future, final assessment of the qNMR method should be

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achieved by analyzing egg liqueurs with known content of alcoholic strength, total sugar and cholesterol,

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for instance by participating in inter-laboratory ring tests.

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1

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Aside from the opportunity to use qNMR as a powerful innovative and economizing analytical tool for

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simultaneous quantitative analysis of several components, 1H NMR also provides further comprehensive

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information on egg liqueur composition, since each spectrum provides a fingerprint of all organic

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components.

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For instance, a signal of phosphatidylcholine (δ≈3.20 ppm, s, 9, choline-N(CH3)3; assignment according to

289

SDBS spectral database13) was detected in 1H NMR spectra after extraction with MeOH-d4/CDCl3 that is

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performed during cholesterol analysis (Figure 3). It is often claimed that the natural deviation of

291

cholesterol in egg yolk is very high5 (for instance due to breed26 and age of hens27) and its validity as

H NMR spectroscopic fingerprint of the basic egg liqueur composition


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indicator for the content of egg yolk accordingly low. Thus, simultaneous analysis of phosphatidylcholine

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as a second marker substance of egg yolk ensures the results for the egg yolk content via cholesterol. As

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the chemical shift of this signal depends on the concentration level of phosphatidylcholine, integration

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ranges were adapted at 3 Hz left and right sided from the signal top for each spectrum. A comparison of

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the signal integrals for cholesterol (Chol) and phosphatidylcholine (PC) showed a very high correlation

297

with R = 0.991 (p < 0.001). The ratio of the integrals (signal areaPC/signal areachol) averaged a value of

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8.94 ± 0.78. Hence, it can serve as value for the plausibility of the calculated egg yolk content by

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cholesterol quantification.

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Additionally, a significant signal of lactose (δ≈4.46 ppm; d, 1, C1H; assignment according to Bruker

301

BBIOREFCODE Database28) in the 1H NMR spectrum after Carrez-precipitation (Figure 2) as well as a

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characteristic signal of butyric acid (δ≈0.97 ppm; t, 3, C4H3; assignment according to SDBS spectral

303

database13) in the 1H NMR spectrum after extraction with MeOH-d4/CDCl3 (Figure 3) indicated an

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addition of milk products that has to be labelled to inform lactose-intolerant consumers 2. The

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qualification of lactose or butyric acid by 1H NMR spectroscopy compensates an additional classical

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analysis of lactose (e.g. by enzymatic analysis), to control if milk products were added despite missing

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labelling.

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Finally, 1H NMR spectra generally indicate striking deviations from the typical composition of egg

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liqueurs. The example of melamine in milk powder29 clearly demonstrated that it is of advantage to use

310

analytical methods which also detect not suspected adulterations. Thus, an increased use of methods

311

like qNMR (with non-selective detection and wide information) is in favour of improved consumer

312

protection.

313

Acknowledgement

314

Special thanks are given to Carolin Psotta for performing classical analysis of egg liqueurs.



315

Funding

316

This research was funded by the Bavarian State Ministry of the Environment and Consumer Protection.


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317

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Figure Captions Figure 1. 1

H NMR spectrum of an egg liqueur without further preparation (1:2 diluted with demineralized water;

dotted line) and after Carrez-precipitation (1:5 diluted with demineralized water; constant line) at the spectral range from δ=3.1 to 4.3 ppm, measured with a 400 MHz spectrometer. Figure 2. 1

H NMR spectrum of an egg liqueur after Carrez-precipitation (1:5 diluted with demineralized water) at

the spectral range from δ=0.2 to 5.7 ppm with an enlarged view on the spectral range from δ=4.1 to 5.6 ppm, measured with a 400 MHz spectrometer. Figure 3. 1

H NMR spectrum of an egg liqueur extract (MeOH-d4:CDCl3 = 50:50 (v/v)) at the spectral range from δ=0

to 8.0 ppm with an enlarged view on the spectral range from δ=7.4 to 8.0 ppm, δ=3.1 to 3.3 ppm and δ=0.7 to 1.1 ppm, measured with a 400 MHz spectrometer. Figure 4. Bland-Altman plot for a comparison of the results of qNMR and classical analysis methods for the quantification of alcoholic strength, total sugar and cholesterol in fifteen different egg liqueurs; the mean deviation between the methods is plotted as constant line and the mean deviation ± 1.96fold standard deviation as dotted line.



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Table 1. Resonance assignments and integration ranges for egg liqueurs and calibration references of 1

H NMR spectra (400 MHz, with MeOH-d4:CDCl3 (50:50, v:v) as solvent for caffeine and cholesterol and

H2O: D2O (90:10, v:v) for ethanol, fructose, glucose and sucrose).

resonance assignment with δ in ppm

structural formula

integration ranges (δ in ppm) for calibration egg liqueurs references

caffeine13: δ ≈ 7.8 (s, 1, C8H)

7.83 - 7.75

7.77 - 7.69

cholesterol13 δ ≈ 0.7 (s, 3, C18H3)

0.72 - 0.67

0.72 - 0.67

1.25 - 1.10

1.06 - 0.99

α-D-fructofuranose/ β-D-fructofuranose14 δ ≈ 4.1 (m, 1, C3H)/ δ ≈ 4.1 (m, 1, C3H ), δ ≈ 4.1 (m, 1, C4H )

4.12 - 4.09

4.13 - 4.10

α-D-glucopyranose15 δ ≈ 5.2 (d, 1, C1H)

5.25 - 5.21

5.26 - 5.22

ethanol13 δ ≈ 1.1 (t, 3, C2H3)

sucrose13 δ ≈ 5.4 (d, 1, glucopyranosyl-C1H)

+/- 5 Hz from signal middle at about 5.40


Page 23 of 29


Table 2. Precision and recovery rates for qNMR of ethanol, total sugar and cholesterol (n = 5).

component ethanol total sugar cholesterol

precision 0.5% 3.2% 2.3%

recovery rate 97.6 ± 2.0% 96.1 ± 7.6% 106.6 ± 8.1%



Table 3. Results of qNMR and respective classical analysis methods for quantification of alcoholic strength (pynometry after distillation as classical method), total sugar (redox titration as classical method) and cholesterol (enzymatic analysis as classical method) in fifteen different egg liqueurs.

Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

alcoholic strength in vol% NMR classical Δ 15.8 15.7 0.1 18.3 17.9 0.4 17.7 17.3 0.3 17.9 18.3 -0.4 19.9 19.8 0.1 17.9 17.7 0.2 26.2 25.9 0.3 19.9 20.2 -0.3 20.6 20.4 0.1 20.7 20.3 0.4 14.2 13.4 0.8 20.2 19.9 0.4 20.0 19.8 0.2 22.4 22.4 0.0 16.2 15.9 0.4 Ø19.0

c(total sugar) in g/L NMR classical Δ 369 394 -26 374 395 -22 458 485 -27 363 390 -27 370 395 -25 372 408 -37 316 329 -13 259 268 -10 347 352 -4 311 300 11 327 340 -13 301 297 5 330 341 -11 401 420 -19 233 234 -1 Ø357


c(cholesterol) in g/L NMR classical Δ 1.5 1.6 -0.2 1.0 1.2 -0.2 2.5 2.6 -0.2 1.0 1.2 -0.2 1.3 1.6 -0.3 0.9 1.1 -0.2 4.0 4.2 -0.2 3.0 3.4 -0.4 2.6 2.9 -0.3 2.7 2.8 -0.1 1.1 1.4 -0.4 2.4 2.8 -0.3 1.7 1.8 -0.1 3.5 3.7 -0.3 2.5 2.7 -0.2 Ø2.3

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Figure 1.



Figure 2.


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Figure 3.



Figure 4.


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TOC graphic.


Quantitative 1H NMR Analysis of Egg Yolk, Alcohol ... - ACS Publications

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