Kinetics of Forming Aldehydes in Frying Oils and Their Distribution in

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Kinetics of Forming Aldehydes in Frying Oils and Their Distribution in French Fries Revealed by LC-MS-based Chemometrics Lei Wang, A. Saari Csallany, Brian J. Kerr, Gerald C. Shurson, and Chi Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01127 • Publication Date (Web): 29 Apr 2016 Downloaded from http://pubs.acs.org on May 4, 2016

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Journal of Agricultural and Food Chemistry

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Kinetics of Forming Aldehydes in Frying Oils and Their Distribution in French Fries

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Revealed by LC-MS-based Chemometrics

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Lei Wang†, A. Saari Csallany†, Brian J. Kerr‡, Gerald C. Shurson§, and Chi Chen†*

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†

Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN 55108

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‡

National Laboratory for Agriculture and the Environment, USDA, Ames, IA 50011

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§

Department of Animal Science, University of Minnesota, St. Paul, MN 55108

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*To whom correspondence should be addressed.

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Running head: Aldehyde markers in frying oils and French fries

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Corresponding author: Dr. Chi Chen

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Department of Food Science and Nutrition

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University of Minnesota

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1334 Eckles Avenue, 225 FScN

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St. Paul, MN 55108

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Email: [email protected]

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Tel.: 612-624-7704, Fax: 612-625-5272

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ACS Paragon Plus Environment

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ABSTRACT. In this study, the kinetics of aldehyde formation in heated frying oils was

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characterized by 2-hydrazinoquinoline derivatization, liquid chromatography-mass spectrometry

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(LC-MS) analysis, principal component analysis (PCA), and hierarchical cluster analysis (HCA).

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The aldehydes contributing to time-dependent separation of heated soybean oil (HSO) in a PCA

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model were grouped by the HCA into three clusters (A1, A2, and B) based on their kinetics and

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fatty acid precursors. The increases of 4-hydroxynonenal (4-HNE) and A2/B ratio in HSO were

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well correlated with the duration of thermal stress. Chemometric and quantitative analysis of

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three frying oils (soybean, corn, and canola oils) and French fry extracts further supported the

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associations between aldehyde profiles and fatty acid precursors, and also revealed that the

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concentrations of pentanal, hexanal, acrolein, and the A2/B ratio in French fry extracts were

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more comparable to their values in the frying oils than other unsaturated aldehydes. All these

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results suggest the roles of specific aldehydes or aldehyde clusters as novel markers of lipid

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oxidation status for frying oils or fried foods.

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KEYWORDS: aldehydes, chemometrics, LC-MS, frying oil, French fries

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INTRODUCTION

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During deep frying of foods, thermal stress can extensively transform the chemical composition

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of frying oils, especially polyunsaturated fatty acids (PUFA)-enriched vegetable oils, and alter

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their nutritional value and toxicological profile.1, 2 Diverse reactions and chemical events, such as

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hydrolysis, radical formation, peroxidation, cyclization, polymerization, and homolytic and

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heterolytic cleavage occur in this process to convert triacylglycerols in frying oils to free fatty

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acids, mono- and di-acylglycerols, hydroperoxide primary lipid oxidation products (LOPs), and

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diverse secondary LOPs, such as cyclic fatty acids, polymeric products, aldehydes, ketones,

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alcohols, dienes, and acids.3 Among these LOPs, aldehydes, due to their reactivity with proteins

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and DNA, have been defined as major contributors to the adverse effects induced by heated

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frying oils in animal and cell-based toxicity studies.4,

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aldehyde species possess organoleptic properties, contributing to the flavor of fried foods.6, 7

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Besides their bioreactivity, many

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Because the reactivity and toxic effects of aldehydes are closely associated with their structures

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and concentrations, both qualitative (such as structural identification) and quantitative analyses

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of aldehydes are commonly performed to evaluate the oxidative status and toxic profile of frying

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oils and fried foods. In fact, among the numerous assays that have been developed and

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established to characterize the chemical properties of dietary lipids, aldehydes are the main

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targets of anisidine value, total oxidation (TOTOX) value, thiobarbituric acid reactive substances

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(TBARS), hexanal, and 2,4-decadienal assays.8,

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determining the presence of aldehydes, how to effectively monitor diverse aldehydes and their

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formation in frying oils and fried food remains to be a challenge. This situation can be largely

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attributed to: (1) under thermal stress, aldehyde production originates from multiple precursors

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Despite the availability of these assays for


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and multiple degradation routes, resulting in unstable composition of aldehydes in frying oils;

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and (2) in contrast to the complexity of aldehyde production, these established targeted assays

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are incapable of detecting and analyzing multiple aldehyde species simultaneously. At present,

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the kinetics of aldehyde production in frying oils as well as the correlations between their levels

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in frying oils and fried foods are generally undefined.

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In the study herein, a platform combining chemical derivatization, high-resolution liquid

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chromatography-mass spectrometry (LC-MS) analysis, and multivariate chemometric analysis

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was adopted to characterize the kinetics of aldehyde formation in vegetable oils heated at frying

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temperature and their distribution in French fries. The values of specific aldehydes and aldehyde

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clusters as the markers of thermal stress and oxidative degradation were evaluated based on their

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kinetic profiles and distribution.

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MATERIALS AND METHODS

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Vegetable oils and chemicals. Refined soybean, corn, and canola oils were purchased from

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local

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hydrazinoquinoline (HQ) from Alfa Aesar (Ward Hill, MA); 2,2′-dipyridyl disulfide (DPDS)

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from MP Biomedicals (Santa Ana, CA); and LC-MS-grade water and acetonitrile from Fisher

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Scientific (Houston, TX). Aldehyde standards, including 2,4-decadienal, 2-undecenal, 2-decenal,

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2,4-undecadienal, 2-octenal, pentanal, 2,4-heptadienal, and 2-heptenal were acquired from

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Bedoukian Research (Danbury, CT); acrolein from Sigma-Aldrich (St. Louis, MO); 4-

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hydroxynonenal (4-HNE) from Cayman Chemical (Ann Arbor, MI); and acetone-d6 from Acros

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Organics (Morris Plains, NJ).

grocery stores

(St.

Paul,

MN);

hexanal,

triphenylphosphine (TPP),


and

2-

4

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Preparation of frying oil samples. Soybean oil (300 mL) was placed in a 500 mL round-bottom

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glass flask and then heated in an electric heating mantle with power controller. The temperature

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of soybean oil was gradually increased from room temperature (22oC) to 185oC in approximately

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1 h with bubbling air (50 mL/min). Heated soybean oil (HSO) samples were collected at 45oC,

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65oC, 85oC, 105oC, 125oC, 145oC, 165oC, and 185oC. Afterwards, the temperature was held

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constant at 185oC for 6 h with samples collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h

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of heating. Corn and canola oils were heated by the same procedure, but sampled only at 0 min,

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30 min, and 6 h of heating at 185oC. Control soybean oil (CSO) was stored at room temperature

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(22oC).

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Oil extraction from French fries. Ten French fry samples were purchased separately from five

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brands of fast food restaurants (abbreviated as A, B, K, M, W) in St. Paul, Minnesota,

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representing two samples from each brand. The oil contents in minced French fries were

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extracted twice by 2 volumes of dichloromethane (v/w). The extracts were filtered through a

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column packed with anhydrous sodium sulfate to remove water and insolubles. After evaporating

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the solvent by nitrogen, the samples were stored at -80oC prior to further analysis.

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Measurement of peroxide value (PV). The PV of oil samples were determined by a modified

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colorimetric assay.10 Briefly, 10 µL of 20 mM FeCl2 solution (in 200 mM HCl) and 1 µL of oil

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sample were added into 100 µL of 1 mM xylenol orange solution (in a 1:1 butanol-methanol

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mixture) in a 96-well plate. After 10 min of shaking and incubation at room temperature, the

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absorbance of reaction mix was measured at 560 nm using a Molecular Devices SpectraMax 250


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microplate reader (Sunnyvale, CA). The standard curve was prepared with FeCl3 with a linear

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range from 1 to 20 µg/mL.

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Measurement of TBARS value. The TBARS values of oil samples were measured using a

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modified method.11 Briefly, 20 µL of oil sample or malondialdehyde (MDA) standard was added

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to 200 µL of a solution containing 15% w/v trichloroacetic acid, 0.375% w/v thiobarbituric acid,

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and 0.25 M HCl in a 2 mL screw-top tube, and then heated at 100oC for 30 min. After cooling on

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ice, the reaction mixture was then centrifuged at 3,000 × g for 5 min. The aqueous phase was

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transferred to a 96-well plate and measured at 535 nm using a Molecular Devices SpectraMax

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250 microplate reader. The TBARS values were calculated using a MDA standard curve ranging

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from 2.5 to 200 µM.

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Derivatization of aldehydes in oil samples. Prior to the LC-MS analysis, aldehydes in oil

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samples were first derivatized by HQ using a modified method.12 Briefly, 4 µL of oil sample was

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added into a 200 µL of freshly-prepared acetonitrile solution containing 1 mM DPDS, 1 mM

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TPP, 1 mM HQ, and 100 µM acetone-d6 (internal standard). The reaction mixture was incubated

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at 60°C for 30 min, chilled on ice, and then centrifuged at 18,000 × g for 10 min. A 100 µL of

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supernatant was mixed with 100 µL of ice-cold H2O. After centrifugation at 18,000 × g for 10

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min, the supernatant was transferred into a HPLC vial for LC-MS analysis.

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LC-MS analysis. A 5 µL sample aliquot was injected into an Acquity ultra-performance liquid

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chromatography (UPLC) system (Waters, Milford, MA) and separated in a BEH C18 column

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(Waters) using a mobile phase (A: H2O containing 0.05 % acetic acid, v:v, and 2 mM


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ammonium acetate; B: H2O:acetonitrile=5:95, v:v, containing 0.05 % acetic acid, v:v, and 2 mM

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ammonium acetate). The LC eluant was introduced into a Xevo-G2-S quadrupole time-of-flight

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mass spectrometer (QTOFMS, Waters) for accurate mass measurement and ion counting.

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Capillary voltage and cone voltage for electrospray ionization was maintained at 3 kV and 30 V

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for positive-mode detection. Source temperature and desolvation temperature were set at 120°C

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and 350°C, respectively. Nitrogen was used as both cone gas (50 L/h) and desolvation gas (600

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L/h), and argon was used as collision gas. For accurate mass measurement, the mass

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spectrometer was calibrated with sodium formate solution with mass-to-charge ratio (m/z) of 50-

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1,000 and monitored by the intermittent injection of the lock mass leucine enkephalin ([M+H]+ =

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m/z 556.2771) in real time. Mass chromatograms and mass spectral data were acquired and

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processed by MassLynxTM software (Waters) in centroided format. The chemical identities of

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compounds of interest were determined by accurate mass measurement, elemental composition

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analysis, MSMS fragmentation, and comparisons with authentic standards if available.

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Individual compound concentrations were determined by calculating the ratio between the peak

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area of compound and the peak area of internal standard and fitting with a standard curve using

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QuanLynxTM software (Waters).

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Chemometric analysis and data visualization. After data acquisition in the UPLC-QTOFMS

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system, the chromatographic and spectral data of samples were deconvoluted by MarkerLynxTM

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software (Waters). A multivariate data matrix containing information on sample identity, ion

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identity (retention time and m/z), and ion abundance was generated through centroiding,

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deisotoping, filtering, peak recognition and integration. The intensity of each ion was calculated

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by normalizing the single ion counts (SIC) versus the total ion counts (TIC) in the whole


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chromatogram. The data matrix was exported into SIMCA-P+TM software (Umetrics, Kinnelon,

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NJ) and transformed by Pareto scaling. Principal component analysis (PCA) was then performed

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to model the data matrix and define the correlations among the samples. The compounds

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contributing to the sample separation in the model were identified in the loadings plot of the

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model. After Z score transformation, the relative abundance of identified compounds in samples

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were presented in heat maps generated by the R program (http://www.R-project.org), and

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correlations among these compounds were defined by hierarchical cluster analysis (HCA).13

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Statistics. The statistical significances among frying oils were analyzed by one-way ANOVA

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and Tukey’s multiple comparison tests using PROC GLM procedure of SAS version 9.1 (SAS

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Institute, Cary, NC). A P value of < 0.05 was considered as statistically significant.

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RESULTS

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Lipid oxidation status in HSO based on kinetic changes of PV and TBARS concentrations.

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PV and TBARS value are two widely used indicators of the presence of primary and secondary

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LOPs in frying oils, respectively. In this study, soybean oil was sampled multiple times at

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different temperatures to determine changes in PV and TBARS values. The PV of soybean oil

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started to increase from 145oC, peaked when heating temperature reached 185oC, and decreased

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rapidly thereafter (Figure 1A). This transient increase of PV in the early time points of heating is

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consistent with other PV profile results reported at various frying conditions,14, 15 suggesting that

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hydroperoxide primary LOPs are highly unstable within this temperature range. Compared to PV,

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the TBARS value, which reflects the concentration of MDA, a secondary LOP of PUFA, also

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increased at 145oC and began to plateau within the first hour of heating at 185°C (Figure 1B).


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Overall, even though PV and TBARS values partially reflect heating-induced chemical changes

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in soybean oil, the transient increase of PV and the plateauing of TBARS value in their kinetic

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profiles do not allow an accurate assessment of the duration of thermal stress on frying oils or

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enable the establishment of dose-response relationships in toxicological evaluation of heated oils.

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LC-MS-based chemometric analysis of thermal stress-induced production of aldehydes in

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soybean oil.

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Considering that reactive aldehydes are major secondary LOPs responsible for the adverse

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biological effects associated with frying oil consumption, the kinetics of forming aldehydes in

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HSO was examined by LC-MS-based chemometrics to determine whether individual aldehydes

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or the aldehyde profile may serve as better indicators of thermal stress exposure in frying oils.

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Because direct detection of aldehydes in LC-MS analysis is hampered by their poor retention in

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common C18 reverse-phase columns, and their insufficient ionization efficiency in electrospray

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MS system, HQ was adopted as the derivatization agent to facilitate chromatographic separation

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and MS detection of aldehydes.12 As shown in the scores plot (Figure 2A), heating-induced

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changes in soybean oil are reflected by the distribution pattern of HQ-derivatized samples in the

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PCA model, and can be summarized as follows: (1) Increasing the temperature from 45ºC to

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165ºC did not significantly increase aldehyde production because the samples collected in this

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temperature range were clustered together with CSO in the model (Figure 2A). The increase of

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PV began at 145oC (Figure 1A), which suggests that aldehyde production lagged behind the

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formation of hydroperoxides, which is consistent to the sequence of forming primary and

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secondary LOPs;1 (2) Increasing the temperature from 165ºC to 185ºC triggered dramatic

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changes of aldehyde profile in HSO, which was reflected by the clear separation of samples


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collected at these two temperatures in the model (Figure 2A); (3) Time-dependent chemical

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changes in HSO occurred from 0 min to 6 h when HSO was held at 185ºC (Figure 2A).

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Subsequently, a group of aldehydes were identified as the major contributors to the separation of

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CSO and different HSO samples in a loadings plot (Figure 2B). Their structural identities were

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further determined as 2,4-decadienal (I), 2-undecenal (II), 2-decenal (III), 2,4-undecadienal (IV),

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4-HNE (V), 2-octenal (VI), pentanal (VII), hexanal (VIII), acrolein (IX), 2,4-heptadienal (X),

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and 2-heptenal (XI) after comparing their retention time and MSMS fragmentograms with

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authentic standards (Table S1 and Figure S1). In addition to confirming the structures of these

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aldehydes, other markers of thermal stress were also identified in the model. For example,

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compound XII was tentatively defined as a 4-oxononanal, based on its molecular formula of

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C9H16O2 and MSMS fragmentogram, but was not confirmed due to the lack of a standard.

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Moreover, the distribution pattern of these aldehydes in the loading plot indicated that individual

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aldehydes had different contributions to principal components 1 and 2 of the PCA model, which

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underlie the temperature- and time-dependent sample separation. To further define the kinetics of

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aldehyde formation and thermal stress-induced changes, a HCA was performed on these

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aldehyde markers together with PV and TBARS. Twelve aldehydes were divided into three

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clusters, named as A1, A2, and B based on the branching of dendrogram and their kinetic

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patterns in the heat map (Figure 2C). The TBARS was grouped with cluster A1 aldehydes, while

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PV was separated from others in the dendrogram due to its distinctive pattern of change (Figure

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2C).

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Quantitative analysis of these aldehyde markers further confirmed the conclusions on the kinetics

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of individual aldehydes from the PCA and HCA models (Figure 3A-L), and the results can be


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summarized as follows: (1) 2,4-Decadienal (I) was the most abundant among all identified

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aldehydes (Figure 3A); (2) Concentrations of cluster B aldehydes, including 2,4-heptadienal (X),

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2-heptenal (XI), and acrolein (IX), peaked at 30 min of heating at 185ºC and decreased thereafter

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(Figure 3I-K); (3) In contrast to cluster B aldehydes, concentrations of aldehydes belonging to

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clusters A1 and A2 (I-VIII) increased continuously at 185ºC (Figure 3A-H). The increases of

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cluster A1 aldehydes, including hexanal (VIII), pentanal (VII), 2,4-decadienal (I), and 2-octenal

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(VI), were attenuated after 1 h of heating at 185ºC while the increases of cluster A2 aldehydes,

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including 2,4-undecadienal (IV), 2-decenal (III), 2-undecenal (II), and 4-HNE (V), were more

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sustained (Figure 2C and 3A-H); (4) Different from other aldehydes, pentanal (VII) and hexanal

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(VIII) existed in detectable amounts in CSO (Figure 3G-H), while 4-HNE (V) was formed at a

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relatively constant rate (r2 = 0.99) during heating at 185ºC (Figure 3E). Overall, the total

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aldehyde concentrations in HSO, which is the sum of all eleven identified aldehydes, had a

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similar kinetic profile with cluster A1 aldehydes (Figure 3L). More importantly, analyzing the

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relative abundance of three aldehyde clusters in the total aldehydes indicated that the percentage

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of cluster A1 was relatively stable across 6 h of heating while the increase of cluster A2

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aldehydes as a proportion of total aldehydes was accompanied with a decrease of cluster B

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aldehydes (Figure 3M). The opposite trends of changes between cluster A2 and B aldehydes

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were further reflected by the values of A2/B ratio, which were in a good linear correlation (r2 =

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0.96) with the duration of thermal stress (Figure 3N).

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LC-MS-based chemometric analysis of thermal stress-induced production of aldehydes in

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frying oils and their distribution in French fries.


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Soybean, corn and canola oils are three widely used vegetable oils for preparing fried food. To

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understand the distribution of aldehydes in these frying oils and fried foods, soybean, corn and

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canola oil samples collected before and during heating (0, 30, and 360 min at 185ºC) and ten

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French fry oil extract samples were derivatized by HQ and then compared by LC-MS-based

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chemometric analysis. The distribution of control oils, HSO, heated corn oil (HCO) and heated

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canola oil (HCAO) samples in the scores plot of a PCA model indicated that progressive and

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time-dependent changes in the chemical composition occurred in all of these oils (Figure 4A).

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More importantly, changes in HCAO were greatly different from that in the other two oils, while

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the changes in HCO and HSO were relatively comparable (Figure 4A). The aldehydes

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contributing to the separation of three frying oils in the PCA model were identified as two groups

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of compounds in the loadings plot (Figure 4B), in which 2-undecenal (II), 2-decenal (III), and

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2,4-heptadienal (X) were better correlated to HCAO while 2,4-decadienal (I), hexanal (VIII), and

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2-heptenal (XI) were better correlated to HSO and HCO (Figure 4B). Another prominent feature

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of this PCA model is the clear separation of French fry extracts from frying oils (Figure 4A). The

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markers contributing to this separation were also identified in the loadings plot (Figure 4B) and

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likely contained oxo fatty acids such as 8-oxo-octanoic acid based on elemental composition

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analysis, MSMS, and database search (data not shown). This observation suggests that the

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chemical composition of French fry extracts was greatly different from that of heated oils.

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The concentrations of aldehydes (I-XI) in three heated oils and ten French fry extracts were

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further quantified. Consistent to the results of PCA modeling, the formation and distribution of

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aldehydes differed greatly among the three heated oils, in which HCO had higher levels (P soybean > corn) (Figure 5I-J). As for 2-heptenal, it can be


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generated from both linoleic acid and α-linolenic acid.6, 29 However, the results from this study

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suggest that it shares a similar kinetic profile with acrolein and 2,4-heptadienal in HSO (Figure

340

3K).

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Overall, combining the existing knowledge on the oxidative degradation of unsaturated fatty

343

acids, the observed kinetic profiles of aldehydes suggests strong correlations between individual

344

aldehydes and fatty acid precursors, and further supports the use of specific aldehydes and

345

aldehyde profiles as more accurate markers of thermal stress than PV and TBARS.

346 347

Potentials of using 4-HNE and aldehyde profiles as markers of thermal stress for heated

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oils and fried foods. Chemometric modeling and quantitative analysis utilized in this study

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offers an opportunity to examine the benefits and limitations of some existing oxidation

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evaluation methods by comparing their kinetic profiles with the profiles of other aldehydes in

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heated oils. Four common lipid oxidation markers associated with aldehydes, including PV,

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TBARS, hexanal, and 2,4-decadienal,were evaluated. The kinetic profile of PV reflected the

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transient increase of lipid hydroperoxides in soybean oil, but it differed from the profiles of

354

aldehyde LOPs greatly (Figure 1A and 2C). Thus, PV does not represent the actual oxidative

355

status of heated oil, especially after prolonged thermal stress. The plateauing of the TBARS

356

curve after 1 h of frying temperature also does not accurately reflect the actual status of forming

357

LOPs in HSO (Figure 1B and 2C). The kinetic profiles of hexanal and 2,4-decadienal were

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similar to each other and also to the profiles of other linoleic acid-derived aldehydes in HSO

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(Figure 2C), but differed from the profiles of aldehydes derived from oleic acid and α-linolenic

360

acid. Overall, these four aldehyde-associated markers, like many other established assays, only


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target the oxidation products from specific degradation pathways, and thus cannot reflect general

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oxidative status of heated oils.30, 31 Furthermore, the lack of a linear correlation (time vs level) in

363

the kinetic curves of these markers also diminishes their value as accurate indicators of the

364

length of time that oils have been subjected to thermal-stress conditions. It should be noted,

365

besides these aldehyde-associated markers, other markers, including total polar materials (TPMs)

366

and polymerized triglycerides (PTGs), are also commonly used to monitor the quality of frying

367

oils.8 Even though those markers were not evaluated in current study, a similar analysis of their

368

kinetic profiles in future studies will likely provide more insights on how well they can reflect

369

the progress of lipid oxidation in frying oils.

370 371

Novel information obtained from profiling aldehydes in this study provide some insights on how

372

to overcome the drawbacks of using traditional markers (i.e. PV, anisidine value, TOTOX value,

373

TBARS, hexanal, and 2, 4-decadienal assays). Among all examined aldehydes, 4-HNE is an

374

established marker of in vivo lipid peroxidation,20 and was only recently identified as a

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secondary LOP in heated oil.32, 33 Compared to other identified aldehydes in this study, including

376

2,4-decadienal and hexanal, the concentration of 4-HNE in HSO had a greater linear correlation

377

with the duration of thermal stress (Figure 3E), suggesting that 4-HNE may be a more accurate

378

aldehyde marker to use when monitoring the oxidation status of soybean oil, than 2,4-decadienal

379

and hexanal. Moreover, 4-HNE was also detected in HCO and HCAO (Figure 5E), and its levels

380

in both oils increased progressively at three sampling time points (0, 30, 360 min when heated at

381

185ºC). Besides 4-HNE, the ratio between cluster A2 aldehydes and cluster B aldehydes (A2/B

382

ratio), also had a sound linear correlation with the duration of thermal stress in HSO (Figure 3N),

383

and had its value increased progressively at three sampling time points (0, 30, 360 min heating at


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185 ºC) in HCO and HCAO (Figure 5N). Considering oleic acid and α-linolenic acid are major

385

contributors to A2 and B aldehyde clusters, respectively, the A2/B ratio reflects the sensitivities

386

of oleic acid and α-linolenic acid to oxidative degradation. Overall, these observations warrant

387

future studies on whether 4-HNE and A2/B ratio could function as general markers to assess

388

oxidative stress for diverse frying oils.

389 390

Human exposure to aldehyde LOPs is mainly through consuming fried foods which comprise

391

LOPs from both frying oils and inherent lipid content. To explore whether individual aldehydes

392

or aldehyde profiles could serve as effective markers of oxidative status in fried foods, the

393

concentrations and composition of aldehydes in the extracts of commercial French fry samples

394

were examined. The majority of aldehydes had their concentrations in French fry extracts far

395

below their concentrations in the heated oils prepared under current experimental conditions

396

(Figure 5A-F and J-K). Two events might contribute to this observation. One possibility is that

397

the solvent evaporation in preparation of French fry oil extracts, such as the use of nitrogen in

398

current study, might decrease the concentrations of volatile aldehydes. More importantly, the

399

concentration of aldehyde can be decreased by the reactions between unsaturated aldehydes in

400

frying oil and the chemical components of the potatoes, such as primary amines.34-36 Therefore,

401

the concentrations of these aldehydes (I-VI and X-XI) in French fries might not reflect the actual

402

oxidation status of oils used in cooking. However, the concentrations of pentanal (VII), hexanal

403

(VIII), acrolein (IX), and A2/B ratio were within the range of their values in the three heated

404

vegetable oils (Figure 5G-I and 5N). This selective retention of pentanal and hexanal in French

405

fries may be explained by the fact that these two saturated alkanals are much less reactive than

406

other unsaturated alkenals, alkadienals, and hydroxyalkenal in heated oils,20 while significant


18

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407

presence of acrolein in French fries may be attributed to its multiple sources, because acrolein

408

can be formed from carbohydrates, fatty acids, and amino acids.37 As for the A2/B ratio, even

409

though the levels of aldehydes comprising A2 and B clusters differed greatly between heated oils

410

and French fry extracts (Figure 3 and 5), its values in French fry extracts were still of the same

411

magnitude as the ones in heated oils. The use of the A2/B ratio as an accurate marker for

412

evaluating frying oil residuals in fried foods needs to be further evaluated using more defined

413

experimental settings, such as measuring the kinetic profile of aldehydes in fried foods.

414 415

In summary, the kinetics of forming aldehydes in frying oils and the distribution of aldehydes in

416

French fries were defined by high-resolution LC-MS analysis and multivariate chemometrics in

417

this study. The results demonstrated the capacity of LC-MS-based chemometrics in both

418

comprehensive profiling and specific characterization of these LOPs, making it a robust platform

419

for monitoring lipid oxidation of heated oils and identifying effective markers. The observations

420

of temperature-sensitive and time-dependent aldehyde formation in frying oils indicated the

421

limitation of several traditional lipid oxidation assays for the lack of strong correlation with the

422

duration of thermal stress, and also suggested the potential of 4-HNE and aldehyde clusters

423

(A2/B ratio) as the markers of thermal stress for frying oils and fried foods.

424 425

AUTHOR INFORMATION

426 427

Corresponding Author *(C.C.) Tel.: 612-624-7704. Fax: 612-625-5272. Email: [email protected]

428 429 430

Funding This research was partially supported by the Agricultural Experiment Station project MIN‐18‐

431

092 (C. C.) from the United States Department of Agriculture (USDA).


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432 433

Notes The authors declare no competing financial interest.

434

ACKNOWLEDGMENTS

435

We specially thank Bedoukian Research for providing the aldehyde standards for structural

436

confirmation.

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(19) Gutteridge, J. M. C. The HPTLC separation of malondialdehyde from peroxidised linoleic acid. J. High Resolut. Chromatogr. 1978, 1, 311-312. (20) Esterbauer, H.; Schaur, R. J.; Zollner, H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 1991, 11, 81-128. (21) Esterbauer, H.; Cheeseman, K. H. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol. 1990, 186, 407-421. (22) Ho, C. T.; Chen, Q. Lipids in food flavors. An overview. In Lipids in Food Flavors; ACS Symposium Seres 558, Ho, C. T.; Hartman, T. G., Eds.; American Chemical Society: Washington, DC, 1994; pp 2-14. (23) Bax, S.; Hakka, M. H.; Glaude, P. A.; Herbinet, O.; Battin-Leclerc, F. Experimental study of the oxidation of methyl oleate in a jet-stirred reactor. Combust. Flame. 2010, 157, 1220-1229. (24) Schneider, C.; Boeglin, W. E.; Yin, H. Y.; Stec, D. F.; Hachey, D. L.; Porter, N. A.; Brash, A. R. Synthesis of dihydroperoxides of linoleic and linolenic acids and studies on their transformation to 4-hydroperoxynonenal. Lipids. 2005, 40, 1155-1162. (25) Schneider, C.; Porter, N. A.; Brash, A. R. Autoxidative transformation of chiral omega6 hydroxy linoleic and arachidonic acids to chiral 4-hydroxy-2E-nonenal. Chem. Res. Toxicol. 2004, 17, 937-941. (26) Schneider, C.; Tallman, K. A.; Porter, N. A.; Brash, A. R. Two distinct pathways of formation of 4-hydroxynonenal - mechanisms of nonenzymatic transformation of the 9-and 13hydroperoxides of linoleic acid to 4-hydroxyalkenals. J. Biol. Chem. 2001, 276, 20831-20838. (27) Endo, Y.; Hayashi, C.; Yamanaka, T.; Takayose, K.; Yamaoka, M.; Tsuno, T.; Nakajima, S. Linolenic acid as the main source of acrolein formed during heating of vegetable oils. J. Am. Oil Chem. Soc. 2013, 90, 959-964. (28) Nielsen, G. S.; Larsen, L. M.; Poll, L. Formation of volatile compounds in model experiments with crude leek (Allium ampeloprasum Var. Lancelot) enzyme extract and linoleic acid or linolenic acid. J. Agric. Food Chem. 2004, 52, 2315-2321. (29) Neff, W. E.; Mounts, T. L.; Rinsch, W. M.; Konishi, H. Photooxidation of soybean oils as affected by triacylglycerol composition and structure. J. Am. Oil Chem. Soc. 1993, 70, 163-168. (30) Shurson, G. C.; Kerr, B. J.; Hanson, A. R. Evaluating the quality of feed fats and oils and their effects on pig growth performance. J. Anim. Sci. Biotechno. 2015, 6, 10. (31) Liu, P.; Kerr, B. J.; Chen, C.; Weber, T. E.; Johnston, L. J.; Shurson, G. C. Methods to create thermally oxidized lipids and comparison of analytical procedures to characterize peroxidation. J. Anim. Sci. 2014, 92, 2950-2959. (32) Seppanen, C. M.; Csallany, A. S. Formation of 4-hydroxynonenal, a toxic aldehyde, in soybean oil at frying temperature. J. Am. Oil Chem. Soc. 2002, 79, 1033-1038. (33) Csallany, A. S.; Han, I.; Shoeman, D. W.; Chen, C.; Yuan, J. Y. 4-Hydroxynonenal (HNE), a toxic aldehyde in French fries from fast food restaurants. J. Am. Oil Chem. Soc. 2015, 92, 1413-1419. (34) Kim, Y. S.; Ho, C. T. Formation of pyridines from thermal interaction of glutamine or glutamic acid with a mixture of alkadienals in aqueous and oil media. J. Food Lipids. 1998, 5, 173-182. (35) Kim, Y. S.; Hartman, T. G.; Ho, C. T. Formation of 2-pentylpyridine from the thermal interaction of amino acids and 2,4-decadienal. J. Agric. Food Chem. 1996, 44, 3906-3908. (36) Uchida, K. Histidine and lysine as targets of oxidative modification. Amino Acids. 2003, 25, 249-257.


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(37) Stevens, J. F.; Maier, C. S. Acrolein: sources, metabolism, and biomolecular interactions relevant to human health and disease. Mol. Nutr. Food Res. 2008, 52, 7-25.

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532

FIGURE LEGENDS

533

Figure 1. Kinetics of established lipid oxidation markers. (A) PV of HSO samples. (B) TBARS

534

value of HSO samples.

535 536

Figure 2. LC-MS-based chemometric analysis of aldehyde production in HSO. Aldehydes in

537

CSO and HSO samples were derivatized by HQ prior to the LC-MS analysis. (A) Scores plot of

538

a PCA model on CSO and HSO sample. The t[1] and t[2] values represent the scores of each

539

sample in the principal components (PC) 1 and 2, respectively. The identities of examined

540

samples are labeled. The HSO samples from 45ºC to 165ºC and the samples collected at 185ºC

541

are encircled. (B) Loadings plot of the ions detected by HQ derivatization and LC-MS analysis.

542

Major aldehyde ions (I-XII) correlated to the HSO samples are labeled. The p[1] and p[2] values

543

represent the contributing weights of each ion to the PC 1 and 2 of the PCA model, respectively.

544

(C) Heat map and dendrogram of aldehyde markers of thermal stress. The markers are

545

hierarchically clustered as clusters A1, A2, and B. The relative abundance of each marker in all

546

samples were compared by their Z scores and presented in the inlaid color key.

547 548

Figure 3. Kinetics of individual aldehyde markers (I-XI) and aldehyde clusters (A1, A2, and B).

549

The concentrations of aldehyde markers in HSO were quantified by LC-MS analysis. (A) 2,4-

550

Decadienal. (B) 2-Undecenal. (C) 2-Decenal. (D) 2,4-Undecadienal. (E) 4-HNE. (F) 2-Octenal.

551

(G) Pentanal. (H) Hexanal. (I) Acrolein. (J) 2,4-Heptadienal. (K) 2-Heptenal. (L) Total

552

aldehydes. The value is the sum of eleven aldehyde markers (I-XI). (M) Kinetics of aldehyde

553

clusters A1, A2, and B in HSO samples. Each cluster is expressed by its percentage in total

554

aldehydes. (N) Kinetics of A2/B ratio.


24

Page 25 of 31


555 556 557

Figure 4. LC-MS-based chemometric analysis of aldehydes in three frying oils (soybean, corn,

558

and canola oils) and French fry oil extracts. (A) Scores plot of a PCA model on frying oils and

559

French fry extracts. Each type of oil has unheated controls (●), 0 min (■), 30 min (♦), and 360

560

min (▼) samples collected at 185ºC frying temperature. French fry oil extracts are labeled with

561

their sources (A, B, K, M, W). (B) Loadings plot of the ions detected by HQ derivatization and

562

LC-MS analysis. Major aldehyde markers associated with the sample groups are labeled.

563 564

Figure 5. Quantification of aldehyde markers in HSO, HCO, HCAO and French fry extracts

565

through LC-MS analysis. Each type of oil has unheated controls (●), 0 min (■), 30 min (▲), and

566

360 min (▼) samples collected at 185ºC frying temperature (two samples per time point per oil

567

and duplicate measurement for each sample). The time point with the highest level of individual

568

or total aldehydes in three frying oils was selected for statistic analysis. Different letter labels (a,

569

b, or c) indicate significant differences from each other while the same letter no differences. (A)

570

2,4-Decadienal. (B) 2-Undecenal. (C) 2-Decenal. (D) 2,4-Undecadienal. (E) 4-HNE. (F) 2-

571

Octenal. (G) Pentanal. (H) Hexanal. (I) Acrolein. (J) 2,4-Heptadienal. (K) 2-Heptenal. (L) Total

572

aldehydes. (M) Relative abundances of aldehyde clusters A1, A2, and B in HSO, HCO, and

573

HCAO samples. Each cluster is expressed by its percentage in total aldehydes. (N) The ratios of

574

A2/B in HSO, HCO, HCAO, and French fry extracts.

575 576


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577

Page 26 of 31

TABLE OF CONTENTS

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Page 27 of 31


Figure 1. Kinetics of established lipid oxidation markers. (A) PV of HSO samples. (B) TBARS value of HSO samples. 57x18mm (600 x 600 DPI)



Figure 2. LC-MS-based chemometric analysis of aldehyde production in HSO. Aldehydes in CSO and HSO samples were derivatized by HQ prior to the LC-MS analysis. (A) Scores plot of a PCA model on CSO and HSO sample. The t[1] and t[2] values represent the scores of each sample in the principal components (PC) 1 and 2, respectively. The identities of examined samples are labeled. The HSO samples from 45ºC to 165ºC and the samples collected at 185ºC are encircled. (B) Loadings plot of the ions detected by HQ derivatization and LC-MS analysis. Major aldehyde ions (I-XII) correlated to the HSO samples are labeled. The p[1] and p[2] values represent the contributing weights of each ion to the PC 1 and 2 of the PCA model, respectively. (C) Heat map and dendrogram of aldehyde markers of thermal stress. The markers are hierarchically grouped as clusters A1, A2, and B. The relative abundance of each marker in all samples were compared by their Z scores and presented in the inlaid color key. 127x127mm (600 x 600 DPI)


Page 28 of 31

Page 29 of 31


Figure 3. Kinetics of individual aldehyde markers (I-XI) and aldehyde clusters (A1, A2, and B). The concentrations of aldehyde markers in HSO were quantified by LC-MS analysis. (A) 2,4-Decadienal. (B) 2Undecenal. (C) 2-Decenal. (D) 2,4-Undecadienal. (E) 4-HNE. (F) 2-Octenal. (G) Pentanal. (H) Hexanal. (I) Acrolein. (J) 2,4-Heptadienal. (K) 2-Heptenal. (L) Total aldehydes. The value is the sum of eleven aldehyde markers (I-XI). (M) Kinetics of aldehyde clusters A1, A2, and B in HSO samples. Each cluster is expressed by its percentage in total aldehydes. (N) Kinetics of A2/B ratio. 152x130mm (600 x 600 DPI)



Figure 4. LC-MS-based chemometric analysis of aldehydes in three frying oils (soybean, corn, and canola oils) and French fry oil extracts. (A) Scores plot of a PCA model on frying oils and French fry extracts. Each type of oil has unheated controls (●), 0 min (■), 30 min (♦), and 360 min (▼) samples collected at 185ºC frying temperature. French fry oil extracts are labeled with their sources (A, B, K, M, W). (B) Loadings plot of the ions detected by HQ derivatization and LC-MS analysis. Major aldehyde markers associated with the sample groups are labeled. 69x27mm (600 x 600 DPI)


Page 30 of 31

Page 31 of 31


Figure 5. Quantification of aldehyde markers in HSO, HCO, HCAO and French fry extracts through LC-MS analysis. Each type of oil has unheated controls (●), 0 min (■), 30 min (▲), and 360 min (▼) samples collected at 185ºC frying temperature (two samples per time point per oil and duplicate measurement for each sample). The time point with the highest level of individual or total aldehydes in three frying oils was selected for statistic analysis. Different letter labels (a, b, or c) indicate significant differences from each other while the same letter no differences. (A) 2,4-Decadienal. (B) 2-Undecenal. (C) 2-Decenal. (D) 2,4Undecadienal. (E) 4-HNE. (F) 2-Octenal. (G) Pentanal. (H) Hexanal. (I) Acrolein. (J) 2,4-Heptadienal. (K) 2Heptenal. (L) Total aldehydes. (M) Relative abundances of aldehyde clusters A1, A2, and B in HSO, HCO, and HCAO samples. Each cluster is expressed by its percentage in total aldehydes. (N) The ratios of A2/B in HSO, HCO, HCAO, and French fry extracts. 177x177mm (600 x 600 DPI)


Kinetics of Forming Aldehydes in Frying Oils and Their Distribution in

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