Analysis of Oxidative Carbonyl Compounds by UPLC-High Resolution

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New Analytical Methods

Analysis of Oxidative Carbonyl Compounds by UPLCHigh Resolution Mass Spectrometry in Milk Powder Zhen Rohfritsch, Olivier Schafer, and Francesca Giuffrida J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00674 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 1, 2019

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Journal of Agricultural and Food Chemistry

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Analysis of Oxidative Carbonyl Compounds by UPLC-High Resolution Mass Spectrometry in Milk Powder Zhen Rohfritsch*, Olivier Schafer, Francesca Giuffrida Nestlé Research, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland *Corresponding author at: Nestlé Research, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland Email

addresses:

[email protected],

[email protected]

ACS Paragon Plus Environment

[email protected],


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ABSTRACT

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Long-chain polyunsaturated fatty acids are highly susceptible to lipid oxidation which

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causes undesirable odors and flavors in food. We present the development, validation and

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application of a semi-quantitative screening method to monitor volatile and non-volatile

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carbonyl compounds generated from lipids oxidation after 7-(diethylamino)-2-oxochromene-

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3-carbohydrazide (CHH) derivatization using liquid chromatography high-resolution mass

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spectrometry. An inclusion list containing eligible compounds was used in full scan mode to

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identify potential oxidative markers. In an antioxidants study using lecithin and tocopherols,

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the proposed method was successfully used to monitor the Docosahexaenoic acid (DHA)

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specific oxidative markers in a model milk powder system enriched with fish oils. The results

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showed that lecithin inhibits oxidation by reducing the peroxidation rate, while δ-tocopherol

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delays the oxidation with distinct induction periods. Here, we explore the optimum

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concentration of soy lecithin and δ-tocopherol needed to limit lipid oxidation in a complex

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food matrix such as milk powder.

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Keywords: lipid

oxidation, carbonyl compounds, off-flavor, DHA, milk powder,

antioxidation, lecithin, δ-tocopherol.


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INTRODUCTION

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Dietary lipids, both naturally occurring in raw food materials and added during food

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processing, play an important role in food nutrition and taste. Long-chain polyunsaturated

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fatty acids (LC-PUFAs), especially ω-3 fatty acids such as α-linolenic acid (ALA, 18:3),

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eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) provide

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important health benefits.1 These include preventing blood clotting and inflammation,

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promoting the functionality of the retina,2 developing brain and nervous tissue in infants,3

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and reducing cardiovascular disease risk. In the industrialized world, the intake of EPA and

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DHA, which mainly comes from marine organisms and fatty fish, is lower than recommended

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by the Food and Agriculture Organization (FAO). Thus, they are often added to foods such

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as infant formula, milk drinks, mayonnaise, and even pet food. However, LC-PUFAs are

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highly susceptible to oxidation, especially in the presence of catalytic systems i.e. light, heat,

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metals, and microorganisms. They oxidize to primary oxidation products (hydroperoxides)

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which further degrade into secondary oxidation products including epoxides, carbonyl

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compounds (aldehydes and ketones). The latter contribute to rancid, fishy, metallic and food

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oxidative off-flavors.3 As reactive oxygen species, carbonyl compounds could also introduce

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reversible and irreversible oxidative modifications on proteins, lipids, nucleic acids, causing

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the loss of essential nutrients such as amino acids, fat-soluble vitamins, and other bioactive

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compounds.4, 5

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Numerous analytical methods have been used to analyze carbonyl secondary oxidation

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products. Spectrophotometric methods are nonspecific. For instance, the thiobarbituric acid

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reactive substrates method is often used to detect malondialdehyde and other aldehydes in

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biological samples. P-Anisidine Value can measure aldehydes, especially the non-volatile

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unsaturated ones found in animal fats and vegetable oils. Spectroscopic methods such as

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infrared and nuclear magnetic resonance spectroscopy are non-destructive and can detect ACS Paragon Plus Environment


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primary and secondary oxidation products simultaneously. However, they suffer from low

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sensitivity, especially in complex food matrices.

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Chromatographic methods are widely used because of improved specificity. GC combined

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with headspace techniques are extensively applied to detect low molecular weight and

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volatile compounds such as aldehydes, ketones, alcohols, furan-, nitrogen- and phenolic

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derivatives, short carboxylic acids and hydrocarbons. The biggest advantage of GC

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headspace techniques is its ability for automation, however, it has some uncertainties in

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quantitation in liquid samples due to the gas-liquid phase partition of the volatiles.6,

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Headspace techniques include static headspace, headspace-solid phase micro-extraction

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and dynamic purge-and trap headspace. Static headspace is suitable for analyzing very light

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volatile compounds but it suffers from low sensitivity. It cannot detect higher boiling point

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volatiles and semi-volatiles due to their low partition in the gas headspace. Headspace-solid

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phase micro-extraction is more sensitive, it uses adsorption/desorption technique through a

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fiber coated with an adsorbent material to extract analytes from headspace gas. It has been

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mostly used for the detection of sensory defects in oxidized sunflower oil,8 fish oil emulsions

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and olive oil.9, 10 The main drawbacks of Headspace-solid phase micro-extraction include

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rapid fiber degradation, frequent fiber contamination or replacement, and the competitive

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adsorption on the adsorbent (fiber) when complex volatiles are present in foods.11 Dynamic

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headspace doesn’t rely on establishing an equilibrium between volatiles in the sample and

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the headspace and is more sensitive. For example, with purge-and-trap sampling, volatiles

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are continually removed by a stream of inert gas blown from the headspace of the sample

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and adsorbed onto a trap material. Later, the compounds are desorbed by either a high

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temperature during GC analysis or by a solvent during liquid chromatography (LC)

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analysis.12 The main disadvantages of this technique are the complexity of the method


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development and the number of different parameters which need to be optimized depending

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on the compounds to be analyzed.

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LC or LCMS methods allow better quantitation of volatile and non-volatile carbonyl

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compounds in liquid samples, but it requires an additional derivatization step and often uses

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hydrazine compounds (R-NH-NH2) to increase the sensitivity of detection. For example, 2,4-

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dinitrophenyl hydrazine (2,4-DNPH) has frequently been applied to environmental air,

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water,13-15 plasma, urine and other biological samples.16 However, the strong acidic

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condition required for this reaction may cause undesirable reactions (such as decomposition

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of carbohydrates) and interfere with the analysis.17 In addition, 2,4-DNPH reacts

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preferentially with apolar lipid-esterified carbonylated compounds rather than the low

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molecular weight ones. Recently, 7-(diethylamino)-2-oxochromene-3-carbohydrazide

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(CHH) has been reported as a derivatization agent for both lipid-esterified and small

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carbonyl compounds.5, 18

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In this study, we developed and validated a semi-quantitative method uses reversed

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phase liquid chromatography-electrospray-high resolution mass spectrometry (LC-ESI-

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HRMS) after CHH derivatization for studying lipid oxidation products, especially carbonyl

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compounds and other non-volatiles in food samples without lipid extraction. We used this

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method to analyze the oxidative status of new food recipes, in this case in fish oil enriched

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milk powder, and to select the optimal antioxidant system.

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MATERIALS AND METHODS

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CHEMICALS

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The following reference standards were purchased from Sigma-Aldrich (Buchs,

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Switzerland): acetaldehyde, propanal, 2-propenal (acrolein), butanal, pentanal, (Z)-4-

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pentenal, (E)-2-pentenal, 1-penten-3-one, pentan-2-one, pentan-3-one, hexanal, (E)-2-



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hexenal, (Z)-3-hexenal, hexan-2-one, hexan-3-one, octanal, (E)-2-octenal, octan-4-one, 1-

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octen-3-one, (E)-2-heptenal, (Z)-4-heptenal, heptanal, nonanal, nonan-2-one, (E)-2-

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nonenal, (E)-2-decenal, (Z)-4-decenal, (E)-2-undecenal, (E,E)-2,4-hexadienal, (E,E)-2,4-

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heptadienal, (E,E)-2,4-octadienal, (Z)-1,5-octadien-3-one, (E,Z)-3,5-octadien-2-one, (E,Z)-

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2,6-nonadienal, (Z,Z)-3,6-nonadienal, 7-(diethylamino)-2-oxochromene-3-carbohydrazide

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(7-(diethylamino)coumarin-3-carbohydrazide), and hexanal-d12. (E,E)-2,4-decadienal was

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bought from AromaLAB AG (Planegg, Germany). (E)-4-hydroxy-2-hexenal, (E)-4-hydroxy-

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2-nonenal, (E)-4,5-epoxy-(E)-2-decenal, (E)-4,5-epoxy-(E,Z)-2,7-decadienal were ordered

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from Cayman Chemical (distributed by Adipogen AG, Liestal, Switzerland). Ammonium

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formate (MS grade, ≥ 99.0%) was supplied from Sigma-Aldrich (Buchs, Switzerland). Formic

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acid (99%, ULC/MS grade), acetonitrile (ULC/MS grade, >99%) were acquired from Chemie

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Brunschwig AG (Basel, Switzerland), and water (ULC/MS grade) was from Biosolve Chemie

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(Valkenswaard, Netherland). The milk powder used for validation was purchased from a

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local super market. Milk powder used for accelerated oxidation was prepared in-house. The

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stock solutions (10 mg/mL, in acetonitrile) of the individual standard solution including

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internal standard (hexanal-d12) were prepared separately. For validation, a standard mixture

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(10 µg/mL, in acetonitrile) containing propanal, (E)-2-hexenal, hexan-3-one, hexanal, (E)-2-

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heptenal,

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decadienal, nonan-2-one, and (E)-2-decenal was mixed from stock solutions. An internal

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standard working solution (10 µg/mL) was diluted from the stock solution described above.

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(Z,Z)-3,6-nonadienal,

1-octen-3-one,

octanal,

(E)-2-nonenal,

(E,E)-2,4-

SAMPLE PREPARATION AND CHH DERIVATIZATION Milk Powder

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Milk powder (3.0 ± 0.03 g) was reconstituted in 10 mL ULC/MS grade water. An aliquot

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(100 µL, 0.3 mg milk powder/µL water) was transferred into a reaction vial (clear, PP, Treff


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AG), mixed with 5 µL internal standard solution (hexanal-d12, 10 µg/mL, in acetonitrile), 100

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µL CHH solution (8 mM, in acetonitrile/water (1/1, v/v)) and homogenized (Fast Prep® 24,

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MP Biomedicals) for 10s at 6.5 m/s.

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The derivatization reaction was performed at 37 °C for 1.5 hours at 1400 rpm in a

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thermomixer (Comfort, Eppendorf). After derivatization, a mixture of acetonitrile/water (7/3,

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v/v) was added up to a final volume of 500 µL. The sample was then mixed (Vortex Genie

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2, Bender & Hobein AG) and centrifuged at 4852 rpm (rcf 2500 ꓫ g) at 20 °C for 2 min

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(5417R, Eppendorf). The supernatant was eventually transferred into high-performance

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liquid chromatography (HPLC) injection vial for analysis.

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Fish oil

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0.2 g high DHA NIF® fish oil sample (Sofinol, Manno, Switzerland) was dissolved in 10 ml

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chloroform/methanol (1/2, v/v). The dispersion was shaken for 10 min at 2500 rpm by a

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mechanic shaker and centrifuged for 10 min at 2500 rpm. 100 μl of supernatant was

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transferred into an Eppendorf tube. The CHH derivatization was performed using chloroform

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instead of acetonitrile in the CHH solution. It was followed by a sample dilution up to 500 μl

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with acetonitrile, vortexing, and 2 min centrifugation at 4852 rpm (rcf 2500 ꓫ g, 20 °C) before

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LC-HRMS analysis.

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METHOD OPTIMIZATION

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A liquid-liquid extraction (LLE) step after CHH derivatization was evaluated for recovery

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determination. An aliquot of reconstituted milk was submitted to a LLE using n-hexane to

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remove potential polar interferences stemming from food matrices. After CHH derivatization,

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the sample was transferred into a Pyrex glass tube and mixed with 4 mL n-hexane in a multi-

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Tube Vortexer (DVX-2500, VWR, Switzerland) at 2200 rpm for 15 min. Followed by

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centrifugation for 2 min at 2325 rpm (rcf 1100 ꓫ g, Sigma Laboratory Centrifuge 4K15C, ACS Paragon Plus Environment


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Germany). The n-hexane phase was transferred into another Pyrex tube, evaporated under

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nitrogen gas until dry and reconstituted with 1 mL acetonitrile/water (7/3, v/v) prior to

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analysis.

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METHOD VALIDATION

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The method was validated with selected available standards for linearity, precision,

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trueness, and limit of detection/limit of quantitation (LoD/LoQ). The linearity test was

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performed in water with concentrations ranging from 0 to 2.5 g/mL (0, 0.0025, 0.0125,

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0.0625, 0.25, 0.625, 1, 1.5, 2, and 2.5 g/mL), and in milk powder from 0 to 33.33 g/g (0,

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0.033, 0.17, 0.83, 3.33, 8.33, 13.33, 20, 26.67, and 33.33 g/g). An internal standard

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(hexanal-d12) was used for quantitation based on isotope dilution. To calculate precision,

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trueness and LoD/LoQ, aliquots of reconstituted milk were spiked at three levels: 0.17, 3.33

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and 33.33 µg/g, and analyzed in duplicates (k=2) by one operator over six different

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days/conditions (n=6). The spiking standards (10 µg/mL, in acetonitrile) contained propanal,

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(E)-2-hexenal, hexan-3-one, hexanal, (E)-2-heptenal, (Z,Z)-3,6-nonadienal, 1-octen-3-one,

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octanal, (E)-2-nonenal, (E,E)-2,4-decadienal, nonan-2-one, and (E)-2-decenal.

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LC-ESI-HRMS CONDITIONS

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Samples were analyzed by an ultra-performance liquid chromatography (UPLC) (Dionex

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UltiMate 3000 system, Thermo Scientific)–QExactive Plus (Thermo Scientific) system.

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Compounds were separated on a BEH C18 UPLC column (130 Å, 1.7 µm, 100 mm x 2.1

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mm i.d., Waters Milford, MA, USA) equipped with a BEH C18 (130 Å, 1.7 µm, 5 mm x 2.1

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mm i.d.) guard column. The mobile phase was a mixture of 10 mM ammonium formate in

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water solution (A) and 0.1 % formic acid in acetonitrile solution (B) eluting at a flow rate of

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0.40 mL/min. The gradient started with 40% mobile phase B, which increases to 98% over

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12 min, held for 5 min, then reduced to 40% within 1 min and held again for 2 min. The total ACS Paragon Plus Environment

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run time was 20 min. The temperature of the autosampler and the column oven were set at

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5 °C and 40 °C, respectively. The injection volume was 10 L.

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The compounds were detected in positive ionization mode by a heated electrospray

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source using a full scan MS (100-1000 Da) and a data-dependent MS2 of targeted

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compounds. The instrumental parameters were set as following: sheath gas flow rate 47,

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auxiliary gas flow rate 15, spray voltage 3.8 kV, capillary temperature 320 °C, S-lens RF

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level 80, the auxiliary gas heater temperature 425 °C.

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ACCELERATED OXIDATION OF FISH OIL AND MILK POWDER

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Storage studies and analysis of secondary oxidation products were conducted on both

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high DHA milk powder and pure fish oil (Table 3). Milk powders fortified with high DHA fish

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oil (about 3.65 g DHA/100 g milk powder) were prepared in-house. First, skimmed milk

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powder was dissolved in water. Then fish oil from tuna (Sofinol, Manno, Switzerland),

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rapeseed oil (8.23% on the dried matter), and soy lecithin (Topcithin NGM, Cargill

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Texturizing Solutions, Hamburg, Germany) were mixed together before being added to the

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milk powder aqueous phase. Afterward, samples were homogenized, freeze-dried, ground

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to powder, then packed in small aluminum bags and stored up to 30 days at 30 °C. At the

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time points of 0, 4, 8, 12, 15, 18, and 30 days, duplicate bags were removed from storage

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for further analysis. To monitor lipid secondary oxidation products, samples were prepared

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and analyzed by LC-HRMS. Two batches of fish oil containing mainly δ-tocopherol and its

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analogs (Table in Figure 4) were purchased. D-Delta-rich tocopherols concentrate (≥ 90%,

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Eisai Pharma-Chem Europe Ltd., London, UK) were used for δ-tocopherol fortification in the

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antioxidant study.

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Accelerated oxidation studies on pure fish oil were performed at controlled temperatures (38 °C) over a time period of 30 days and analyzed at different time points.



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DATA ANALYSIS

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The identification of the compounds was based on exact mass in full scan mode, retention

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times and MS/MS fragmentation by higher energy collision-induced dissociation. Q-Stat, a

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proprietary statistic calculation tool was used for validation. This software meets the

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recommendations of the European Commission Council directive 96/23/EC and ISO 11843

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concerning the performance of the analytical method and the interpretation of results.

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Software XcaliburTM (version 2.2) was employed for quantitative analysis, peak identification

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and quantitation. For untargeted screening, the Compounds Discoverer™ (version 2.1,

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Thermofisher) software was utilized for peak identification, integration and data analysis. A

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list of targeted masses (mainly aldehydes and ketones) based on accurate molecule masses

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and retention times were used for targeted screening.

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RESULTS AND DISCUSSION

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IDENTIFICATION OF CHH DERIVATIVES

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Reactive carbonyl compounds (aldehydes/ketones) react with the strong nucleophilic

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hydrazine group of CHH to form hydrazone carbonyl-CHH derivatives (Figure 1). Carbonyl-

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CHH derivatives were identified by exact mass as protonated adducts, their specific

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fragment ions, and retention times if standards were available. As an example, selected ion

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chromatograms of carbonyl-CHH compounds used for validation are shown in Figure 1.

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Collision energy ranging from 10 to 30 eV was optimized for propanal, (Z,Z)-3,6-

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nonadienal, nonan-2-one and (E,E)-2,4-octadienal (Figure 2). Collision energy of 25 eV has

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been selected for method setup. Alkanals, such as propanal-CHH derivative (m/z 316.16)

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was identified by detection of fragment ions at m/z 244.09, 262.10 and 218.11. The fragment

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ion at m/z 244.09 corresponds to the neutral loss of hydrazine (-N2H4).5 Aldehyde specific

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fragments could not be generated for propanal due to the absence of preferential


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fragmentation sites, e.g. double bounds or functional groups along the aliphatic chain.19 In

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addition to m/z 244.09 and 262.10, the cleavage of the double bond between C-3 and C-4

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of unsaturated C9 aldehyde (Z,Z)-3,6-nonadienal-CHH derivative (m/z 396.22) gave a

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fragment ion at m/z 316.16. Similarly, a fragment ion at m/z 384.05 corresponding to the

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neutral loss of a methyl group (–CH3: 15.02 Da) was observed for ketone nonan-2-one-CHH

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derivative (m/z 400.26). For alkadienal (E,E)-2,4-octadienal-CHH derivative (m/z 382.21), a

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fragment ion at m/z 316.16 was generated by the split between C-3 and C-4.

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METHOD OPTIMIZATION AND VALIDATION

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In food, lipids and polar components often cause signal suppression and column damage

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in reversed LC conditions. Therefore, depending on the polarity or functional groups of the

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compounds of interests, a cleanup step is normally required for complex food matrices. The

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loss of the analytes caused by liquid-liquid extraction was investigated on 9 volatile

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compounds spiked in milk powders (6.7 µg/g sample) by comparing their concentrations

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after LLE with the concentrations measured in non-LLE treated samples. In the latter case,

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the concentration of added standards was considered as 100%. The peak areas in the

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sample blank were subtracted for calculating their recoveries (Figure 3). The relative

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standard deviation values (n=3) ranged between 1-4% for samples without LLE and 9-22%

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for those with liquid-liquid extraction. Sample recoveries after LLE ranged from 55 to 77%,

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showing that not all carbonyl-CHH derivatives could be extracted by n-hexane. The

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extractability of carbonyl-CHH derivatives could be affected by their polarity represented by

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the logPow value (octanol-water partition coefficient). It is suspected that most of the

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carbonyl-CHH derivatives are moderate polar. Some of them may be amphiphilic due to a

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moderate polar CHH (logPow=1.262) and either polar or nonpolar carbonyl compounds

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(logPow=0.3-3.4 for aldehydes and ketones). The relative low recovery may be explained by



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the nonpolar character of n-hexane (logP=3.9). Due to the broad range of the carbonyl

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compounds considered in this untargeted screening study, any cleanup procedure may

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cause potential loss of signal. Therefore no sample cleanup was used for LLE.

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CHH assay reaction time (1, 1.5 and 2 hours) and CHH concentration (1 and 10 mM) were

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evaluated in triplicates (data not shown). Except for pentanal and hexanal, the peak intensity

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of selected carbonyl-CHH increased proportionally with derivatization time and CHH

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concentration. Small chain alkanals (≤C6) such as pentanal and hexanal may react more

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efficiently with CHH and reach the equilibrium faster than long chain alkenals (>C6) like 2,4-

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heptadienal or 2,4-nonadienal. Based on signal intensity and convenience of manipulation,

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a CHH assay time of 1.5 hr. and a concentration of 8 mM were selected as final experimental

244

conditions.

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For method validation, strong linearity was obtained in both water and milk powder with

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R2 coefficients higher than 0.98 in water and 0.95 in milk powder (Table 1). The linearity of

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(E)-2-heptenal was determined in water because of the high interfering background signal

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present in the milk powder used. The matrix effect in milk powder was observed for some of

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the selected compounds (data not shown). Comparing their responses in water, (E)-2-

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nonenal, (E)-2-decenal, (E,E)-2,4-decadienal, propanal shown signal enhancement, while

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signal suppression was observed for (Z,Z)-3,6-nonadienal and octanal. Nonan-2-one, 1-

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octen-3-one, (E)-2-hexenal, hexan-3-one and hexanal did not show significant matrix effect.

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Therefore, to avoid over- or under estimation, matrix-matched calibration was used for

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absolute quantitation.

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Linearity, precision and recovery (Table 1) were determined at three different levels,

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although only the results at 3.33 µg/g are discussed here. This concentration corresponds

257

to the LoQ level at which the targeted statistic criteria (CV(r) < 15%, CV(iR) < 20%) are

258

met.20 At this concentration, the results showed satisfactory repeatability CV(r) < 15% and


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slightly worse intermediate reproducibility CV(iR) < 20% for all compounds except (Z,Z)- 3,6-

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nonadienal. Recovery values were not significantly different from 100% apart from (E)-2-

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nonenal (77%). The complete set of results is given as supplementary material (S1). Since

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each compound has a specific MS response, the LoQ determined at unified concentration

263

is not compound specific. Therefore, a decision limit/detection capability (CCα/CCβ)

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approach was used for more specific calculation.20 CCα, equivalent to LoD, is the limit at

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and above which it can be concluded that a sample is positive with an error probability of α

266

(1%). CCβ, equivalent to LoQ, is the smallest content of the substance that may be detected,

267

identified and/or quantified in a sample with an error probability of β (5%). With CCα/CCβ

268

calculation, the LoD/LoQ could be lowered. Due to the high amount of (E)-2-heptenal

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present in the milk powder, its LoD or LoQ was determined as the lowest concentration

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during the calibration at which the analyte has a response 3 or 10 times higher than the one

271

in the blank.21 This corresponded to 2.5 /12.5 ng/mL in the solvent, equivalent to 0.04/0.21

272

µg/g in milk powder.

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OXIDATIVE MARKERS IN FISH OIL AND MILK POWDER

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Both fish oil and high DHA milk powder were stored at 38/30 °C up to 30 days and

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analyzed for lipid oxidative markers at time points (0, 4, 8, 12, 15, 18, 25 and 30 days) by

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the LC-HRMS method. Compounds increasing over time including aldehydes, ketones, and

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epoxy aldehydes were tentatively identified as potential markers responsible for oxidative

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off-flavors in fish oil and high DHA milk powders (Table 2).

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The high DHA milk powders contained rapeseed oil, which is rich in oleic, linoleic and

280

ALA, while fish oil is rich in DHA and EPA (Table 4). Therefore, lipid oxidation products from

281

ω-3 (DHA, EPA and ALA), ω-6 (linoleic acid) and ω-9 (oleic acid) FAs were expected. As

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ω-3 PUFA degradation products, 2,4,7,10,13-hexadecapentaenal, 2,4,7-decatrienal



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(E,Z,Z/E,E,Z) isomers, (E,E)-2,4-heptadienal, (Z)-4-heptenal, (E,Z)-3,5-octadien-2-one, E-

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4,5-epoxy-(E,Z)-2,7-decadienal,

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hexenal, propanal, and 1-penten-3-one were detected. Among them, 2,4,7-decatrienal

286

(E,Z,Z/E,E,Z) isomers, originating from DHA 13-OOH/EPA 11-OOH/ALA 9-OOH, are some

287

of the most important contributors to fishy flavors.17, 22, 23 2,4-Heptadienal stemming from

288

DHA 16-OOH/EPA 14-OOH/ALA 12-OOH is characterized by a distinct grainy, straw-like

289

flavor at high concentration (>1 mg/kg) and contributes to the general oxidized flavor of fish

290

oils.22 (E,E)-2,4-heptadienal has been described as fatty and fishy flavor.24 (Z)-4-heptenal is

291

another characteristic volatile compound responsible for a stale burnt flavor,17, 25 and it acts

292

as a modifier to contribute to the overall burnt/fishy flavors in oxidized fish oils and other oils

293

containing PUFAs.22,

294

bonded hydration and retro-aldol condensation of (E,Z)-2,6-nonadienal, which originates

295

from the EPA 12-OOH/DHA 14-OOH pathway.27 (E,Z)-3,5-octadien-2-one is derived from

296

autoxidation of EPA and strongly related to green, fruity and fatty, as well as plastic and

297

synthetic odors.28, 29 Compounds that contribute to metallic and pungent flavors are (E)-4,5-

298

epoxy-(E,Z)-2,7-decadienal (originating from EPA 15-OOH) and (E)-4,5-epoxy-(E)-2-

299

decenal.23 Other volatile compounds formed from ω-3 PUFA peroxidation and associated

300

with fishy flavors are (Z,Z)-3,6-nonadienal, 3-hexenal, propanal and 1-penten-3-one.

301

Coming from the degradation of DHA 14-OOH/EPA 12-OOH/ALA 10-OOH, (Z,Z)-3,6-

302

nonadienal is responsible for fatty, soapy and cucumber flavors. 3-hexenal is a degradation

303

product of DHA 17-OOH/EPA 15-OOH/ALA 13-OOH and has a cucumber-like aroma.

304

Propanal comes from DHA 20-OOH/EPA 18-OOH/ALA 16-OOH degradation and has an

305

earthy odor. 1-penten-3-one stems from EPA 15-OOH and has been linked with pungent

306

green odor.9 Additionally detected aldehydes including 2,4,7,10,13-hexadecapentaenal,

26

(E)-4,5-epoxy-(E)-2-decenal,

(Z,Z)-3,6-nonadienal,

3-

(Z)-4-heptenal has been proposed to form through α/β double-


Page 15 of 34


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307

specific DHA degradation product through the 7-hydroperoxide pathway,30 and 2,4,7,10-

308

tridecatetraenal, originates through DHA 10-OOH/EPA 8-OOH.

309

Hexanal (grassy and fishy) and 2,4-decadienal (fried and fatty) were identified as

310

indicators of ω-6 fatty acids oxidation. 2,4-decadienal contributes to general oxidized

311

flavor,17, 22 while its (E,E)-2,4-decadienal isomer has been reported to correlate with rancid,

312

fishy flavors in fish oil enriched mayonnaise.29 Both hexanal and 2,4-decadienal were found

313

in oxidized cod liver oil and menhaden fish oil.22, 27 (E)-2-decenal (fatty and waxy) resulting

314

from ω-9 FAs oxidation was also detected.

315

ANTIOXIDANT EFFECTS OF LECITHIN AND δ-TOCOPHEROL

316

Phospholipids, especially lecithin are widely used as natural emulsifiers in the food industry

317

and have been gaining interest as natural antioxidants to control lipid oxidation. Tocopherols

318

can also behave as antioxidants and interrupt lipid autoxidation by interfering with either the

319

chain propagation or the decomposition processes. Many studies in the literature have

320

shown that tocopherols at high concentration have a pro-oxidant effect.31, 32 δ-tocopherol

321

was chosen for the study because it is highly effective at inhibiting the decomposition of

322

hydroperoxides,27 and also because the fish oil used in the milk powders studied here

323

contained the high concentration of it.

324

Among many detected volatiles, the antioxidant effect of lecithin and tocopherols in high

325

DHA milk powder was demonstrated by two representative fish oil degradation products

326

(E,E)-2,4-heptadienal and 2,4,7-decatrienal (Figure 4). The quantitation of (E,E)-2,4-

327

heptadienal was based on the matrix-matched standard calibration with an internal standard.

328

Duo to the lack of an available standard for 2,4,7-decatrienal, its relative formation was

329

calculated using the ratio between its peak area and an internal standard.



Page 16 of 34

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330

The results showed that lecithin acted as a retarder of oxidation by reducing the oxidation

331

rate in the high DHA milk powder (containing 1500 mg/kg δ-tocopherol stemming from fish

332

oils). The lecithin predominantly inhibited the formation kinetic of (E,E)-2,4-heptadienal

333

(Figure 4a) and relative formation of 2,4,7-decatrienal (Figure 4c) when different ratios of

334

lecithin/fish oil were added (0.5/1 and 1/1, Figure 4a and 4c, left axes) compare to the one

335

without lecithin (0/1, Figure 4a and 4c, right axes). 1/1 lecithin/fish oil provided the best

336

inhibition for (E,E)-2,4-heptadienal and 2,4,7-decatrienal, the latter having also an induction

337

period of 8 days.

338

Possible explanations can be drawn by the fact that lecithin consists mainly of phospholipids

339

(40-50%),

340

phosphatidylcholine (12-18%) and phosphatidylethanolamine (10-15%) (Table 3).

341

Phospholipids behave as antioxidants in food by binding to pro-oxidants such as metals and

342

providing a protective barrier against the penetration of radicals.27 Phospholipids can also

343

produce antioxidative compounds through Maillard reactions during lipid oxidation that

344

synergize with phenolic antioxidants such as tocopherols by regenerating them.33, 34 They

345

also decompose hydroperoxides into non-radical compounds such as fatty acid alcohols.35

346

However, it has been reported that sometimes phospholipids do not show antioxidant

347

activity, or even acted as pro-oxidants in bulk oil and low pH oil-in-water emulsions.35-37

348

Δ-tocopherol showed concentration-dependent inhibition of lipid oxidation by either

349

decreasing the oxidation rate of (E,E)-2,4-heptadienal, or inducing the induction periods for

350

2,4,7-decatrienal. Because the fish oil used for the milk powder system examined here

351

already contained 1500 mg/kg δ-tocopherol (Table 4), D-Delta-rich tocopherols concentrate

352

were added to make up around 1500, 3000, 4000, 5500 and 7000 mg/kg δ-tocopherol in

353

milk powder (with 1/1 lecithin/fish oil added) to better study its effects (Figure 4b, 4d). As the

354

reference, 1500 mg/kg δ-tocopherol without lecithin was used. The induction period of 2,4,7-

and

the

soy

lecithin

used

in

this

study


contained

predominantly

Page 17 of 34


17

355

decatrienal increased with increasing concentration of δ-tocopherol until 4000 mg/kg (an

356

induction period of 15 days) except at 3000 mg/kg (induction period was zero), continue

357

increasing concentration led to the decrease of the induction period. The same trend was

358

found for the oxidation rate of (E,E)-2,4-heptadienal, at 4000 mg/kg, which was the lowest.

359

Our finding confirmed that δ-tocopherol still exhibits optimal concentration at higher levels,

360

unlike α-tocopherol which is more effective at low concentration. Their effectiveness can

361

increase with concentration, up to a certain point where further addition of antioxidants does

362

not further decrease lipid oxidation.38 Based on the literature, tocopherols work either as

363

natural chain-breaking antioxidants which exhibit a very distinct induction period by losing

364

hydrogen radical to LOO thus halting radical oxidation propagation,39 or inhibiting

365

hydroperoxide decomposition.40 It is reported that the antioxidant activity of tocopherols

366

depends on their homolog, concentration, and matrix. The relative antioxidant activity of

367

tocopherols depends on the tocopherol concentration, the type of lipid substrates and other

368

factors.40 For example, the order in fats and oils is δ > γ ≈ β > α, while in vivo is in the

369

reversed order.

370

Synergistic effects between lecithin and tocopherols were also reported with regard to

371

lecithin’s ability to regenerate tocopherols from their oxidized form.37 Such effects have been

372

partially observed in our study. The combination of lecithin (lecithin/fish oil 1/1) and 1500

373

mg/kg δ-tocopherol demonstrated a better inhibition of oxidation than without lecithin. The

374

effect of lecithin (lecithin/fish oil 1/1) only could not be observed because of the presence of

375

tocopherols in the fish oil. Ideally stripped fish oil without tocopherols should be used in the

376

future. This synergistic effect depends on the type of phospholipid and tocopherol analogs

377

used in food. Decker et al. found that phosphatidylethanolamine exhibits a better antioxidant

378

effect in synergy with δ-tocopherols than with α-tocopherols in some oil-in-water

379

emulsions.41 This study shows that the combination of δ-tocopherol and soy lecithin



Page 18 of 34

18

380

produces antioxidant activity in a high DHA milk powder system. This could provide the basis

381

for a clean label strategy using natural antioxidants.

382

LC-MS detection of carbonyl-CHH derivatives, especially volatile aldehydes and ketones,

383

has proven a useful alternative method to GC-headspace technique for the detection of

384

oxidative markers responsible for fishy off-flavors in milk powder and fish oil. By studying

385

the formation kinetics or relative comparison of the selected markers, this method can be

386

used to evaluate the effectivity of antioxidants. It can be used in different complex food

387

matrices with slight modifications (procedures not covered here) without lipid extraction. This

388

method was developed for research purposes to screen lipid oxidation products and

389

understand potential oxidation mechanisms. Due to the broad range of lipid oxidation

390

products such as carbonyl compounds, standards are not always available for quantitation,

391

therefore qualitative screening was often used to monitor oxidative markers. However, there

392

is room for some improvements. Data treatment and interpretation using a full scan

393

screening method remain a time-consuming task. The significant investment for a LC-HRMS

394

instrument, the lack of harmonized validation procedure and the time dedicated to train

395

skilled users are the main points to be addressed for cost-effective implementation in the

396

routine laboratory. However, an adaptation using LC, LC-MS or quick tests for suitable

397

markers discovered with this method can be developed later for regular application. To

398

better use the full scan data, studies using fingerprint approach and chemometric data

399

treatment are ongoing. Furthermore, lipid oxidation involves different mechanisms and

400

pathways, the full extent of lipid oxidation should be determined more accurately. Future

401

work includes LCMS detection of other lipid oxidation products, such as hydroperoxides,

402

epoxides, and their co-oxidation products to better describe the interactions of lipid oxidation

403

products with other components in foods, for example, protein-lipid co-oxidation products.42-

404

44


Page 19 of 34


19

405

ACKNOWLEDGMENTS

406

The authors would like to thank Caroline Fradin for making available the data generated

407

during her internship at Nestlé. We also thank Aidan Makwana and Maria-Belén Sanchez

408

Bridge for their dedication in the redaction of this manuscript.

409

CONFLICT OF INTEREST

410 411 412

The authors declare that no conflict of interest exists. SUPPORTING INFORMATION DESCRIPTION S1-Validation results



Page 20 of 34

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413

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25

FIGURE CAPTIONS Figure 1: Selected ion chromatograms of 7-(diethylamino)-2-oxochromene-3-carbohydrazide (CHH) derivatives in standard solution. 1: propanal (316.16), 2: (E)-2-hexenal (356.19), 3: ISTD hexanal-d12 (370.28), 4: hexanal/hexan-3-one (358.21), 5: (E)-2-heptenal (370.21), 6: (Z,Z)-3,6-nonadienal (396.22), 7: 1-octen-3-one (384.22), 8: octanal (386.24), 9: (E)-2-nonenal (398.24), 10: (E,E)-2,4-decadienal (410.24), 11: nonan-2-one (400.26), 12: (E)-2-decenal (412.26). Peak at RT=5.72 is a suspected isomer impurity from standard hexan-3-one. It was originally found in the individual standard solution of hexan-3-one and was detected in all standard mixture solutions Figure 2: MS2 spectra of propanal-CHH (a), (Z,Z)-3,6-nonadienal-CHH (b), nonan-2-one-CHH (c) and (E,E)-2,4octadienal-CHH (d). Figure 3: The recovery of liquid-liquid-extraction for selected carbonyl-CHH derivatives in milk powder. Figure 4: The effect of lecithin and δ-tocopherol on the formation of (E,E)-2,4-heptadienal (quantitation) and 2,4,7decatrienal (relative response) at selected storage time during accelerated oxidation of high DHA milk powders fortified with fish oil. a, c: The antioxidant effect of lecithin concentration (lecithin to fish oil ratio at 0/1, 0.5/1 and 1/1) with the presence of approximate 1500 mg/kg δ-tocopherol. b, d: the concentration-dependent antioxidant effect of δ-tocopherol (1500 (A), 3000 (A), 4000 (B), 5500 (B) and 7000 (B) mg/kg) with the presence of lecithin (1/1 lecithin to fish oil ratio). A and B indicate the two different batches of fish oils used for milk powder fortification (tocopherols composition refers to the table). Because of the difference in scale and for better readability, 1500 mg/kg δ-tocopherol without lecithin added represented by the blue curve follows right axes, the other curves follow left axes.



Page 26 of 34

26

FIGURES Figure 1:


Page 27 of 34


27

Figure 2:



Page 28 of 34

28

Figure 3:


Page 29 of 34


29

Figure 4:



Page 30 of 34

30

TABLES Table 1: Validation results for compounds with their retention time, linearity, repeatability, intermediate reproducibility, limit of detection, limit of quantitation, and recovery. Compound

Retention Linearity Linearity in Repeatability (%) Reproducibility (%) time range solvent/matrix 3.3 (µg/g) 3.3 (µg/g) (min) (µg/g) Propanal 3.29 0.03-33.3 0.98/098 12.8 11.9 (E)-2-hexenal 5.98 0.03-33.3 0.99/0.99 6.9 6.9 Hexan-3-one/hexanal 6.36 0.03-33.3 0.99/0.99 3.6 10.4 (Z,Z)-3,6-nonadienal 7.63 0.03-33.3 0.98/0.99 7.4 25.6 (E)-2-heptenal 6.99 0.03-33.3 0.99/1-octen-3-one 7.92 0.03-33.3 0.99/0.99 6.3 15.9 Octanal 8.29 0.03-33.3 0.99/0.99 10.3 17.1 Nonan-2-one 8.75 0.03-33.3 0.98/0.98 3.1 19.3 (E)-2-nonenal 8.84 0.03-33.3 0.99/0.99 9.0 12.3 (E,E)-2,4-decadienal 9.06 0.03-33.3 0.98/0.98 7.8 13.3 (E)-2-decenal 9.70 0.03-33.3 0.99/0.95 8.9 11.4 *: based on limit of detection/limit of quantitation determined from standard calibration.


Limit of detection (µg/g) 0.565 0.392 1.154 0.396 0.042* 0.458 1.468 0.464 0.395 0.293 0.333

Limit of quantitation (µg/g) 0.940 0.630 1.892 0.712 0.208* 0.784 2.410 0.744 0.690 0.543 0.574

Recovery (%) 3.3 (µg/g) 99.4 97.3 112.4 103.2 92.5 109.3 95.9 77.1 102.3 104.5

Page 31 of 34


31

Table 2: Carbonyl-CHH derivatives tentatively identified in oxidized fish oil (in bold) and high DHA milk powder. The formulae, molecule weight, mass accuracy, retention times and flavor description are given.

Compounds Acetaldehydea 2-propenal (acrolein)a Propanala Butadienalb Malondialdehydeb Butanala 4-pentenala/1-penten-3-onea (E)-2-pentenala Pentan-2-onea/pentan-3-onea 3-methyl-2-butenalb 3-methylbutanalb 3-hydroxybutan-2-oneb (E,E)-2,4-hexadienala (Z)-3-hexenala (E)-2-hexenala Pentane-2,3-dioneb Hexanala/hexan-2-onea/hexan-3-onea 4-hydroxy-2-methyl-butanalb (E,E)-2,4-heptadienala (E)-2-heptenala (Z)-2-heptenala (E)-4-hydroxy-2-hexenala Heptanala (E,Z)-3,5-octadien-2-onea (E,Z)-2,4-octadienalb (E,E)-2,4-octadienala (E)-3-octen-2-oneb (E)-2-octenala Octanala/octan-4-onea (E,Z)-2,6-nonadienala (Z,Z)-3,6-nonadienala (Z)-3-nonenalb (E)-2-nonenala Nonan-4-oneb Nonanala/nonan-2-onea 2,4,7-decatrienalb (E,E)-2,4-decadienala (E)-2-decenala (Z)-4-decenala (E)-4-hydroxy-2-nonenala (E)-4,5-epoxy-(E,Z)-2,7-decadienala (E,E)-2,4-undecadienalb (E)-4,5-epoxy-(E)-2-decenala (E)-2-undecenala 3,6,9-dodecatrienalb 2,4-dodecadienalb 2,4,7,10-tridecatetraenalb 2,4,7-tridecatrienalb 3,6,9,12-pentadecatetraenalb 2,4,7,10,13-hexadecapentaenalb

Formula C16 H19 N3 O3 C17 H19 N3 O3 C17 H21 N3 O3 C18 H19 N3 O3 C17 H19 N3 O4 C18 H23 N3 O3 C19 H23 N3 O3 C19 H23 N3 O3 C19 H23 N3 O3 C19 H23 N3 O3 C19 H25 N3 O3 C18 H23 N3 O4 C20 H23 N3 O3 C20 H25 N3 O3 C20 H25 N3 O3 C20 H25 N3 O3 C20 H27 N3 O3 C19 H25 N3 O4 C21 H25 N3 O3 C21 H27 N3 O3 C21 H27 N3 O3 C20 H25 N3 O4 C21 H29 N3 O3 C22 H27 N3 O3 C22 H27 N3 O3 C22 H27 N3 O3 C22 H29 N3 O3 C22 H29 N3 O3 C22 H31 N3 O3 C23 H29 N3 O3 C23 H29 N3 O3 C23 H31 N3 O3 C23 H31 N3 O3 C23 H33 N3 O3 C23 H33 N3 O3 C24 H29 N3 O3 C24 H31 N3 O3 C24 H33 N3 O3 C24 H33 N3 O3 C23 H31 N3 O4 C24 H29 N3 O4 C25 H33 N3 O3 C24 H31 N3 O4 C25 H35 N3 O3 C26 H33 N3 O3 C26 H35 N3 O3 C27 H33 N3 O3 C27 H35 N3 O3 C29 H37 N3 O3 C30 H37 N3 O3

Molecule ∆ Mass[ppm] Weight 301.14234 -0.99 313.14228 -1.15 315.15792 -1.17 325.1426 -0.01 329.13722 1.01 329.13367 1.02 341.17361 1.01 341.17361 -0.99 341.17361 -0.96 341.17376 -0.54 343.18914 -1.31 345.16845 -1.16 353.17364 -0.87 355.18922 -1.04 355.18923 -1.01 357.16843 -1.19 357.20476 -1.34 359.18416 -1.27 367.18928 -0.85 369.20488 -0.99 369.20492 -0.88 371.18417 -0.91 371.22053 -0.97 381.20479 -1.17 381.20488 -0.96 381.20488 -0.96 383.22048 -1.08 383.22048 -1.07 385.2358 -1.9 395.22057 -0.8 395.22059 -0.76 397.23615 0.79 397.23615 -0.98 399.25089 3.27 399.25089 0.91 407.22053 -0.88 409.23658 0.09 411.25183 -0.88 411.25183 -0.88 413.23084 1.4 423.21545 -0.85 423.25186 -0.79 425.23105 -0.97 425.2674 -1.03 435.2519 -0.67 437.26695 -2.04 447.25181 -0.85 449.26752 -0.73 475.28196 3.17 487.28311 -0.79

Identified based on reference materials. Tentatively identified. Compounds in bold are found both in oxidized fish oil and high DHA milk powder. a b


Retention Time [min] 2.33 3.25 3.32 6.98/4.72 3.42 4.27 4.64 4.99 4.71 7.57 6.75 2.41 5.39 5.80 6.03 2.19 6.45 3.00 6.38 7.02 6.51 3.01 7.44 6.93 7.09 7.33 7.48 7.96 8.37 7.85 7.67 8.59 8.85 9.95 9.24 8.18/8.29 9.07 9.69 9.15 5.83 4.86 9.08 7.40 10.52 9.33 10.09 9.59 10.51 10.42 10.77


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Table 3: Composition of soy lecithin used in milk powder oxidation study.

Lecithin composition

Unit

Phosphatidylcholine Phosphatidylethanolamine Phosphatidylinositol Phosphatidic acid C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 Acetone insolubles Toluene insolubles Hexane insolubles Acid value Peroxide value Moisture Gardner color Viscosity

12-18% 10-15% 8-14% 1-5% 12-18% 4-6% 10-20% 54-64% 5-9% min. 60% max. 0.3% max. 0.1% max. 28 mg KOH/g max. 5 meq O2/kg max. 0.65% max. 11 max. 12.5 Pa·s, 25°C


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Table 4: Composition of fish oil used in milk powder oxidation study.

Fish oil composition

Unit

C12:0 C14:0 C14:1 C15:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 Oleic C18:2 Linoleic C18:3 gamma C18:3 alpha C18:4 C20:0 C20:1 C20:2 C20:3 (n-6) C20:4 (n-6) C20:5 (EPA) C22:0 C22:1 C22:5 (n-6) C22:5 (n-3) C22:6 (DHA) C24:0 C24:1 Other fatty acids Acid value Peroxide value Delta-rich tocopherol concentrate Sensory evaluation: IN/OUT Induction period (Rancimat)

0.04% 3.35% 0.07% 1.12% 18.76% 4.23% 1.26% 0.15% 4.97% 11.57% 1.3% 0.14% 0.36% 0.76% 0.40% 0.56% 0.19% 0.11% 1.80% 4.72% 0.26% 0.16% 1.83% 0.83% 24.48% 0.25% 0.42% 8.62% < 0.2 mg/KOH/g oil < 0.5 meq O2/kg oil 1500 mg/kg Just IN 8.8 hours at 80°C



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TOC graphic


Analysis of Oxidative Carbonyl Compounds by UPLC-High Resolution

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