HPLC and UPLC methods for the determination of zearalenone in noodles, cereal snacks and infant formula

Accepted Manuscript HPLC and UPLC methods for the determination of zearalenone in noodles, cereal snacks and infant formula Hyun Ee Ok, Sung-Wook Choi, Meehye Kim, Hyang Sook Chun PII: DOI: Reference:

S0308-8146(14)00684-0 http://dx.doi.org/10.1016/j.foodchem.2014.04.111 FOCH 15773

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

12 December 2012 20 April 2014 29 April 2014

Please cite this article as: Ok, H.E., Choi, S-W., Kim, M., Chun, H.S., HPLC and UPLC methods for the determination of zearalenone in noodles, cereal snacks and infant formula, Food Chemistry (2014), doi: http:// dx.doi.org/10.1016/j.foodchem.2014.04.111

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Manuscript to be submitted for publication in:

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Food Chemistry

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HPLC and UPLC methods for the determination of zearalenone in

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noodles, cereal snacks and infant formula

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Hyun Ee Oka, Sung-Wook Choib, Meehye Kimc, Hyang Sook Chuna

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a

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Republic of Korea

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b

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Republic of Korea

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c

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Osong 363-700, Republic of Korea

School of Food Science and Technology, Chung-Ang University, Anseong 456-756,

Food Safety Research Division, Korea Food Research Institute, Sungnam 463–746,

Food Contaminants Team, National Institute of Food and Drug Safety Evaluation,

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* Corresponding author. Tel.: +82 31 670 3290; fax: +82 31 675 3108. E-mail addresses: [email protected] (HYANG SOOK CHUN)

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Running title: HPLC and UPLC methods for the determination of zearalenone in

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processed foods 1

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ABSTRACT

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High-performance liquid chromatography (HPLC) and ultra-performance liquid

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chromatography (UPLC) were compared to validate a method for determination of

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zearalenone (ZON) in noodles, cereal snacks, and infant formulas. The limits of

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detection and quantification in HPLC and UPLC were found to be 4.0 and 13.0 µg

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kg−1and 2.5 and 8.3 µg kg−1, respectively. The average recoveries of ZON by HPLC and

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UPLC ranged from 79.1% to 105.3% and from 85.1% to 114.5%, respectively. The

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measurement uncertainties of the two methods for ZON determination were within the

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maximum standard uncertainty. The two methods showed that the levels of ZON in 163

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naturally contaminated samples ranged from 4.3 to 8.3 µg kg−1 by HPLC and 3.1 to 17.6

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µg kg−1 by UPLC. These findings indicate that either method is suitable for the

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determination of ZON in noodles, cereal snacks, and infant formulas, but UPLC gives

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faster results with better sensitivity.

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Key words: Zearalenone, HPLC, UPLC, noodles, cereal snacks, infant formulas

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2

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1. Introduction

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Zearalenone [ZON; 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-β-resorcyclic acid

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lactone] is a nonsteroidal oestrogenic mycotoxin produced by Fusarium graminearum

46

and other Fusarium species, which are plant pathogenic fungi that infect a wide variety

47

of cereals (Saeger, Sibanda, & Peteghem, 2003; Visconti & Pascale, 1998). ZON

48

production has been reported in grains in the field, during harvest, commercial grain

49

processing, and storage (Saeger et al., 2003; Visconti & Pascale, 1998; Zinedine,

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Soriano, Molto, & Manes, 2007).

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ZON is a strongly estrogenic compound that causes reproductive problems in specific

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animal species including cattle, swine, and poultry, and possibly in humans (Saeger et

53

al., 2003). Fertility problems have been observed in animals such as swine and sheep

54

(Krska, Petterson, Josephs, Lemmens, MacDonald, & Welzig, 2003). ZON may be an

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important etiologic agent causing intoxication of infants or fetuses exposed to this

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mycotoxin, resulting in premature thelarche, pubarche, and breast enlargement (Council

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for Agricultural Science and Technology, 2003). Risk assessment of ZON performed by

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the Scientific Committee on Food (SCF) concluded that a temporary tolerable daily

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intake (t-TDI) is 0.2 µg kg−1 body weight (Scientific Committee on Food, 2000),

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whereas a provisional maximum tolerable daily intake of 0.5 µg kg−1 body weight was

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established by the Joint FAO/WHO Expert Committee on Food Additives (World

62

Health Organization, 2000). Recently, the Panel on Contaminants in the Food Chain

63

established 0.25 µg kg−1 body weight as a TDI for ZON (European Food Safety

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Authority, 2011).

3

65

ZON is commonly found worldwide in corn, corn products, sorghum, and rye. In

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2003, the Food Safety Agency in the UK quantified ZON in 39 of 333 samples at levels

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ranging from 8.0 to 231.8 µg kg−1. The highest levels were found in samples of

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breakfast cereals and cereal-based snacks (Food Standards Agency, 2003). ZON was

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found in corn, barley, and unpolished rice harvested in Korea, and the mean levels of

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contamination were 42.8, 33.3, and 22.0 µg kg−1, respectively (Ok, Chang, Choi, Kim,

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Koo, & Chun, 2007).

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According to the Food and Agriculture Organization, six countries had regulated

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acceptable ZON levels by 1996, but by 2003, levels of ZON in foods and animal feeds

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were regulated by 16 countries. Acceptable limits for ZON in maize and other cereals

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currently vary from 50 to 1000 µg kg−1 (Food and Agriculture Organization, 2004). The

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European Commission has established maximum acceptable levels of ZON in bread,

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pastries, snacks, cereal snacks, and breakfast cereals at 50 µg kg−1, and 20 µg kg−1 in

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processed cereal-based foods and baby foods for infants and young children (European

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Commission, 2006a). Recently, to protect the health of infants and young children, a

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vulnerable group, South Korea has recently specified the maximum levels of ZON in

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snacks, and baby foods for infants and young children, at 50 and 20 µg kg−1,

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respectively (Korea Food and Drug Administration, 2011).

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Previous collaborative trial efforts have focused on cereal samples, and a few of the

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methods have been collaboratively tested with complex matrices such as processed food

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and baby food. Because it is necessary to determine rather low levels of ZON in baby

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food in support of legislation, validated methods, and preferably cost-effective methods

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with reduced analysis time, are needed for the analysis of these matrices.

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High-performance liquid chromatography (HPLC) is a common and well-established

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separation technique frequently used to determine ZON contamination in cereal samples.

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Currently, the new trend in analytical methods is fast LC. The most relevant papers that

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have been recently published regarding the new instrumental and column technology

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describe the use of new stationary phases, particularly monolith columns, high- and

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low-temperature separations, and ultra-performance liquid chromatography (UPLC)

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methods with sub-2 µm, and novel porous-shell particle-packed columns (Guiochon,

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2007). The present paper reports HPLC and UPLC methods with fluorescence detection,

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which are useful for routine determination of ZON in noodles, cereal snacks, and infant

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formulas. Both methods were validated by parameters including linearity, accuracy,

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precision, and measurement uncertainty, and applied to the analysis of ZON in naturally

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contaminated samples.

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2. Materials and methods

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2.1. Reagents

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ZON Standard (Z2125) with purity of >99% and Tween 20 were purchased from

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Sigma-Aldrich (St Louis, MO, USA). BCR 717 maize (low-level ZON) used as a

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certified reference material was purchased from the Institute for Reference Materials

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and Measurements (IRMM, Geel, Belgium). All solvents were suitable for LC analysis

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and were purchased from J.T. Baker (Phillipsburg, NJ, USA).

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2.2. Food materials

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The following noodles, snacks, and infant formulas were collected from grocery

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markets in South Korea: 35 noodles (dry), 30 instant noodles, 63 snacks (32 maize-

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based snacks, and 30 wheat-based snacks), and 36 infant formulas. To validate the

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procedure, dry noodles, instant noodles, corn-based snacks, and infant formulas found

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to be free of ZON were selected. The composition of the four food materials was wheat

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flour and salt for dry noodles; wheat flour, potato starch, modified starch, eggshell

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calcium, salt, vegetable extract, alkali additives, and mix additives for instant noodles;

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wheat flour, corn flour, palm oil, sugar, and salt for cereal snacks; and skim milk

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powder, whey protein, cow’s milk protein, breast-milk protein, rice, black rice, soybean

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oil, coconut oil, sunflower oil, vegetables, fruit, eggshell calcium, and functional breast-

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milk ingredients for infant formula. A minimum sample size of 2 kg was purchased, and

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the samples were stored under cool conditions (<8°C) until blending. All samples were

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finely ground using a food mill until the sample could pass through a 0.85 mm sieve,

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and were kept in airtight aluminum foil zipper bags in a refrigerator (4–8°C) before

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analysis.

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2.3. Extraction and purification

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Ground sample (25 g) was placed in a 200 mL beaker with 100 mL of acetonitrile:

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distilled water (75:25, v/v), 2.0 g of NaCl, and 1 mL Tween 20, and homogenized for 3

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min using a high-speed blender (Ultra Turrax, IKA, Staufen, Germany). After extraction,

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the sample was filtered through filter paper (Whatman No. 1) and a 10 mL of filtrate

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extract was diluted with 40 mL of distilled water. If the dilution solution was not clear,

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it was centrifuged at 14000 rpm (23700 ×g) for 15 min. After filtration through a GF/B

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filter, 25 mL of the filtrate was passed through an immunoaffinity column (IAC,

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ZearalaTest WB, Vicam, MA, USA) at a flow rate of one drop per second. The IAC was

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washed with 20 mL of distilled water and dried by rapidly passing air through it. The

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ZON was eluted into a 10 mL flask with 3 mL methanol. The eluent was evaporated in a

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water bath at 50°C. Dried residues were reconstituted with 1 mL of mobile phase

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(distilled water:ACN:methanol = 35:10:55, v/v/v) and filtered through a syringe filter

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(0.2 µm). Finally, 2 µL and 20 µL of this solution obtained from the same sample were

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injected into the UPLC and HPLC apparatus, respectively. ZON, its solutions and all

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food samples suspected of being contaminated with ZON were handled according to

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safety recommendations. Procedures including grinding, extraction and clean-up were

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performed with the use of gloves in the hood. Decontamination of all volumetric flasks

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and vials containing concentrated sample extract or reference standard solutions has

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been performed by 2% (v/v) sodium hypochlorite solution treatment.

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2.4. HPLC/UPLC

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The UPLC apparatus comprised a Waters Acquity UPLC system (Milford, MA,

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USA) equipped with a binary solvent manager, a sample manager, a column heater, and

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a fluorescence detector (FLD). The analytical column was an Acquity BEH C18 (2.1 ×

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100 mm, 1.7 µm) proceeded by an Acquity UPLC column in-line filter (0.2 µm). The

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flow rate of the mobile phase was 0.3 mL min-1. Data acquisition and instrument control

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were performed using Empower 2 software (Waters). The HPLC-FLD apparatus was a

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Jasco HPLC system (JASCO International, Tokyo, Japan) equipped with a binary pump

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and autosampler. The analytical column was a Synergi Hydro-RP 80Å column

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(Phenomenex, 4.6 × 250 mm, 4 µm, Torrance, CA, USA), proceeded by a Security

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Guard C18 cartridge (Phenomenex, 3.9 mm × 20 mm). The flow rate of the mobile phase

163

was 1.0 mL min−1. For HPLC and UPLC, the column was kept at a temperature of 40°C

164

and the detector was set for an excitation wavelength of 275 nm and emission

165

wavelength of 450 nm.

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2.5. LC/MS

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The LC/MS was performed using a Shiseido Model Nanospace SI-2 liquid

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chromatograph system (Shiseido, Kyoto, Japan) and an LCQ DECA XP mass

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spectrometer (Thermo Finnigan, San Jose, CA, USA) with electrospray ionization (ESI)

172

capabilities. Liquid chromatography separation was performed on a 150 mm × 1.0 mm

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i.d., 5 µm, Luna C18(2) column (Phenomenex, Torrance, CA). The LC mobile phase

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was a mixture of water (A, 0.1% formic acid, v/v) and ACN (B, 0.1% formic acid, v/v).

175

The initial gradient was 80% A and 20% B, and was equilibrated for 5 min.

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Subsequently, the solvent B was changed linearly to 80% in 20 min, and was held for

177

10 min. Solvent B was reduced to 20 % in 2 min and was then equilibrated for 28 min.

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Total run time was 60 min. The flow rate was set at 0.3 mL min−1. Injection volume was

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2 µL. The following MS parameters were employed. The capillary voltage was set to

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−15 V, the spray voltage to 4 kV, the capillary temperature to 275°C, and the sheath gas

181

flow to 20 arbitrary units. ZON determination was conducted using the selected ion

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monitoring (isolation width 1.0 m/z) mode of each base ion peak at m/z 317.4 in the

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negative mode. 8

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2.6. Method validation

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The HPLC and UPLC methods for determination of ZON in noodles, snacks, and

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infant formulas were validated as in-house methods (International Union of Pure and

189

Applied Chemistry, 2002). A stock solution containing 250 µg mL-1 of ZON was

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prepared with ACN. Intermediate standard solution (5 µg mL-1) was prepared by

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diluting the stock solution with ACN and stored at -18°C. For the linearity test and for

192

the determination of LOD and LOQ, working standard solutions of ZON were prepared

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with mobile phase in five concentrations from 10 ng mL−1 to 500 ng mL−1, and

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UPLC/HPLC measurements were performed (10 ng mL−1 and 500 ng mL−1

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corresponding to 8.06 µg kg−1 and 403.2 µg kg−1 in samples, respectively). In the

196

assessment of linearity, calibration curve was plotted 10, 20, 50, 100, 200 and 500 µg

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kg-1 for ZON. Calibration curves were evaluated by the analysis of the distribution

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properties of the residuals. Selectivity was tested by adding ZON to positive samples in

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dried noodle, instant noodle, snack, and infant formula and then observing the increase

200

of ZON peak. Also, the retention time of peak was checked in the samples in order to

201

see if it corresponded with the retention time in the calibration samples. Recovery and

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repeatability were determined in five replicates after spiking the four food matrices that

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were found to be free of ZON at levels of 20, 50, and 200 µg kg−1. Aliquots of 25 g of

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homogenized food samples were spiked with adequate volumes of intermediate standard

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solution until the desired concentration was reached, and stayed for overnight in the

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fume food. These samples were extracted and analyzed pursuant to the method

207

described above. The recovery was determined in five replicates and expressed as a 9

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percentage by comparing the observed values to the spiked levels. The repeatability

209

(RSDr) and reproducibility (RSDR) were determined by performing 5 repeated

210

experiments on a single day and on five different days. The limit of detection (LOD) of

211

the chromatographic procedure was determined as the system limit of detection for the

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pure standard at a signal-to-noise ratio of 3:1. The limit of quantification (LOQ),

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defined as the lowest amount of analyte in a sample which can be quantitatively

214

determined, was experimentally assessed by analyzing spiked noodle, instant noodle,

215

snack, and infant formula samples in triplicate. Trueness was determined using CRMs

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(BCR 717) and was expressed as recovery of the accepted reference value. The

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concentrations of ZON in each food were not corrected for a recovery rate.

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2.6. Measurement uncertainty

220 221

Uncertainty of ZON determination in noodles, snacks, and infant formulas spiked

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with 200 µg kg−1 ZON was estimated using HPLC and UPLC. A metrology approach to

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measurement uncertainty was based on the following intralaboratory data: studies of

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precision, data for performance of the analytical process and quantification of ZON. We

225

considered sources of uncertainty including those arising from balances, volumetric

226

measuring devices, reference material, linear calibration curve interpolation, and

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instrumental factors. The measurement uncertainty (U), which is the expanded

228

uncertainty, was obtained by multiplying the combined standard uncertainty by a

229

coverage factor, k = 2, which gives a confidence level of approximately 95%. In

230

addition, a fitness-for-purpose approach that specifies maximum levels of uncertainty

231

was used to assess the acceptability of the analytical method to be used in the laboratory 10

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(European Commission, 2006b). The equation for maximum standard uncertainty (U݂,

233

µg kg−1) is as follows: Uƒ = ට(

234

୐୓ୈ ଶ ) ଶ

+ (α × C)ଶ

235

where C is the concentration of interest and α is a constant numeric factor dependent

236

on the value of C. In cases where the concentration ranges from 51 to 500 µg kg−1, α is

237

0.18.

238 239 240

3. Results and discussion

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3.1. System performance of HPLC and UPLC methods

242 243

The analysis and retention times were 20 and 6.4 min in the HPLC method,

244

respectively, and 10 and 2.7 min in the UPLC method, respectively (Table 1). The

245

linearity of the relationship was evaluated across a range of 10–500 ng mL−1. The slope

246

was slightly steeper for HPLC than UPLC, while the intercept for UPLC was near zero.

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Coefficient of determination values (r2) for both methods were 0.9999. For HPLC and

248

UPLC, LOD were 4.0 and 2.5 µg kg−1, respectively. The LOQ for HPLC were 10.0 µg

249

kg−1 for noodle and instant noodle, 8.1 µg kg−1 for snacks, and 8.3 µg kg−1 for infant

250

formula, respectively. For UPLC, LOQ were 10.0 µg kg−1 for noodle, 9.2 µg kg−1 for

251

instant noodle, 8.3 µg kg−1 for snacks, and 8.0 µg kg−1 for infant formula, respectively.

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Based on these findings, major advantages of the UPLC-FLD method with respect to

253

the HPLC-FLD methods are reductions in the chromatographic run time and the

254

consumption of organic solvents. These were in agreement with other previously

255

published papers (Pascale, Panzarini, & Visconti, 2011; Wu, Wang, Wang, Xiao, Ma, & 11

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Zhang, 2008). In addition, the sensitivity of the UPLC-FLD method is suitable for

257

quantitative determination of ZON below the maximum admissible levels in South

258

Korea for noodles, cereal snacks, and infant formula.

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The standard ZON chromatograms obtained using the HPLC and UPLC methods are

260

shown in Fig. 1. Chromatograms from naturally contaminated cereal snacks and infant

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formulas were similar to those of the standard. There was no interference peak near the

262

ZON peak. The presence of ZON in the standard and naturally contaminated samples

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was confirmed by LC-MS with negative electrospray ionization (−ESI). ESI in the

264

negative ion mode was found to be well adapted for the analysis of ZON. Both

265

polarities were used for ionization of ZON, and sensitivity was greater in the negative

266

ion mode. ZON appeared as an [M−1]− ion at m/z 317.4.

267 268

3.2. Accuracy and precision of the HPLC and UPLC methods

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Results of the ZON recovery study are summarized in Table 2. For all test samples,

271

the average recoveries of ZON added at 20–500 µg kg−1 ranged from 79.1% to 105.3%

272

in HPLC, and from 85.1% to 114.5% in UPLC, respectively. The standard deviation

273

(SD) and relative standard deviation (RSDr) of ZON ranged from 0.3 to 14.6 µg kg−1

274

and 1.6% to 11.1%, respectively, in HPLC, and from 0.4 to 11.9 µg kg−1 and 1.6% to

275

11.1%, respectively, in UPLC. BCR 717 (maize, 83 ± 9 µg kg-1) was also tested for

276

ZON as a certified reference material. The average recovery, SD, and RSDr of BCR 717

277

were 90.5%, 13.6 µg kg−1, and 18.1% for HPLC, respectively. The average recovery, SD,

278

and RSDr of BCR 717 were 94.2%, 5.5 µg kg−1, and 7.0% for UPLC, respectively. All

279

results obtained for the certified reference material were within the acceptable range. 12

280

The performance criteria established by Commission regulation 401/2006/EC as

281

applicable for ZON in the ≤ 50 µg kg−1 level are 60%–120% for recovery, ≤ 40% for

282

RSDr, and ≤ 50% for RSDR, and in the > 50 µg kg−1 level are 70 – 120% for recovery,

283

≤ 25% for RSDr, and ≤ 40% for RSDR (European Commission, 2006b).

284 285

3.3. Measurement uncertainty of HPLC and UPLC methods

286 287

To assess the suitability of the analytical method to be used in the laboratory,

288

uncertainty of ZON measurement in noodles, snacks, and infant formulas spiked with

289

200 µg kg−1 ZON was estimated using HPLC and UPLC (Table 3). After estimation of

290

all uncertainty contributions, they were joined according to their laws of combination

291

(EURACHEM/CITAC, 2000) obtaining a combined standard uncertainty: the final U,

292

for a level of confidence of approximately 95% and considering a coverage factor of 2,

293

was found to be 15%−31% for HPLC and 14%−22% for UPLC. In the European Union,

294

where the analytical recovery is 100%, the U for the analysis will be of the order ±32%

295

of the analytical result at the concentration of 1 mg kg−1 (European Commission, 2004).

296

Recently, the EU also proposed that for evaluating the acceptability of the analytical

297

method (the fitness for purpose), the uncertainty function approach, specifying the

298

maximum acceptable uncertainty, may be used (European Commission, 2006b).

299

According to the equation for the maximum standard uncertainty established in the EU,

300

the Uf level at 200 µg kg−1 concentration of ZON was 36 µg kg−1(equivalent to 18%) in

301

this study. Thus, U using a coverage factor of 2 was calculated to be 36% (European 13

302

Commission, 2006b). Therefore, in this study, HPLC and UPLC methods for ZON in

303

noodles, snacks, and infant formulas produced results within the maximum standard

304

uncertainty. In the case of ZON results in other matrices, the U was determined to be

305

±34% at a level of 226 µg kg−1 ZON in wheat (Scudamore, Guy, Kelleher, &

306

MacDonald, 2008) and ±25% at a range of 20−300 µg kg−1 ZON in wheat, maize, and

307

rice (Sebaei, Gomaa, Mohamed, & El-Dien, 2012).

308 309

3.4. Occurrence of ZON in cereal products and infant formulas

310 311

To demonstrate the effective application of the established method on real samples,

312

examples of noodles, snacks (wheat based and maize based), and infant formulas were

313

analyzed in duplicate for their ZON content. Results are shown in Table 4. Using HPLC

314

analysis, only four samples out of 163 were found to contain ZON at levels greater than

315

the LOD, and the contamination level ranged from 4.3 to 8.3 µg kg−1. Using UPLC, 16

316

samples were contaminated with ZON at levels greater than the LOD, and the

317

contamination level was 3.1–17.6 µg kg−1. Of all the sample matrices, infant formulas

318

showed a slightly higher frequency (6% and 17% for HPLC and UPLC, respectively) of

319

contamination levels than other samples, although the level was low. In an EFSA report

320

(CONTAM) published in 2011, the group ‘infant formulae, powder’ (n = 19) occurred in

321

5% sample (>LOD) and the mean and maximum concentrations were 0.3 and 5.0 µg

322

kg−1, respectively (European Food Safety Authority, 2011). ZON was not detected in

323

any of the 77 baby products for infants and young children in the UK (Food Standard

324

Agency, 2011). In samples of noodles, ZON was not detected by the HPLC method and

325

was detected in three samples at a level below 10 µg kg−1 using the UPLC method. In 14

326

the case of wheat milling products, the frequency of ZON was 14% (>LOD), and the

327

mean and maximum concentrations were 4.9 and 507 µg kg−1, respectively (European

328

Food Safety Authority, 2011). The difference between wheat milling products and

329

noodle study results may have been the result of a processing effect. The production of

330

instant noodles with the addition 1% potassium carbonate resulted in a 48%–62%

331

reduction of ZON (Matsuura et al., 1981). ZON in wheat- and maize-based snacks was

332

detected only in one sample using the HPLC method. By contrast, using the UPLC

333

method, ZON was detected in seven samples, but the level of contamination was low.

334

Higher detection by the UPLC method can be explained by its lower LOD, which meant

335

higher sensitivity, as compared to that of HPLC method. UPLC, using small particles

336

(<2 µm) in short columns (5 cm) can drastically decrease the analysis time, and gain the

337

efficiency. Comparing to the conventional HPLC method, UPLC showed many

338

advantages, including reduced run time, less solvent consumption and increased peak

339

capacities (Núñez, Gallart-Ayala, Martins, & Lucci, 2012). In the EFSA report, ZON

340

contaminated 16% (>LOD) of 121 samples of snacks. Where the level below LOD was

341

estimated as zero, the mean concentration was 2.6 µg kg−1, and it can be related to the

342

high corn content of certain snack foods and also to the addition of corn germ oil in this

343

type of food (European Food Safety Authority, 2011). The results of the EFSA report for

344

snacks and noodles were slightly higher than those of this study in terms of the

345

frequency and mean level obtained by the UPLC method. In Korea, ZON was detected

346

in 38 samples among 432 samples (8.8% incidence), including 3 snacks, 2 biscuits and

347

33 other cereal products. The ZON contamination levels were in the range of 6.0 – 11.8

348

µg kg-1 for snack, 14.8 – 17.8 µg kg-1 for biscuit. Dried noodle and fried noodle was not

349

contaminated in all samples (Jang et al., 2011). These results were very similar to those 15

350

of this study, and suggest that the exposure to ZON through intake of the selected food

351

in Korea is not considered as serious.

352 353

4. Conclusions

354 355

Because ZON contamination of cereal and cereal products is widespread, reliable

356

analytical techniques are required for quality and safety assurance of food products. Our

357

findings indicate that both methodologies, HPLC and UPLC, are suitable for the

358

determination of ZON in noodles, snacks and infant formulas, and can be implemented

359

for their routine analysis. However UPLC method gives faster results with better

360

sensitivity.

361 362

Acknowledgements

363 364

This research was supported by a grant (10162NIFDSE009) from the National

365

Institute of Food and Drug Safety Evaluation in 2010, and by the IT R&D program of

366

MoTIE/MISP/KEIT (10044580), Republic of Korea.

367 368

References

369 370

Council for Agricultural Science and Technology (CAST) (2003). Potential economic

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costs of mycotoxins in the United States. In Mycotoxins: risks in plant, animals,

372

and human systems. Task Force Report No. 139. January 2003, ISBN 1-887383-

373

22-0.

16

374

EFSA Panel on Contaminants in the Food Chain (CONTAM) (2011). Scientific opinion

375

on the risks for public health related to the presence of zearalenone in food.

376

European Food Safety Authority (EFSA) Journal, 9, 2197−2321.

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EURACHEM. (2000). Quantifying uncertainty in analytical measurement 2 nd. London: EURACHEM. European Commission (EC) (2004). Report on the relationship between analytical

380

results, measurement uncertainty, recovery factors and the provisions of EU food

381

and feed legislation, with particular reference to community legislation concerning.

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trichothecenes and zearalenone (Number 35/03). Food Survey Information

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Sheets available at URL

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http://www.food.gov.uk/science/research/surveillance/fsis2003/35cereal.

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Guiochon, G. (2007). Monolithic columns in high-performance liquid chromatography. Journal of Chromatography A, 1168, 101 −108. International Union of Pure and Applied Chemistry (IUPAC) (2002). Harmonized

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Korean Journal of Food Science and Technology, 43, 224-229.

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(2003). Zearalenone in maize: stability testing and matrix characterization of a

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Matsuura, Y., Yoshizawa, T., & Morooka, N. (1981). Effect of food additives and

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occurrence of deoxynivalenol and zearalenone in cereals and their products.

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photodiode array detection. Talanta, 89, 231-236.

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Saeger, S. D., Sibanda, L., & Peteghem, C. V. (2003). Analysis of zearalenone and α-

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Scientific Committee on Food (SCF) (2000). Opinion of the Scientific Committee on Food on Fusarium toxins. Part 2: Zearalenone (ZEA). 12 pp. Scudamore, K., Guy, R. C. E., Kelleher, B., & MacDonald, S. J. (2008). Fate of the

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of wholemeal wheat grain. Food Additives and Contaminants, 25(3), 331 − 337.

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450

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452

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453

oestrogenic mycotoxin. Food Chemical and Toxicology, 45, 1−18.

454

20

455

Figure caption

456 457

Fig. 1. Chromatograms of ZON standard obtained by using HPLC(a), UPLC(b) and

458

LC/MS(c) in standard solution (250 ng mL−1), snack, and infant formula. STD; standard,

459

EU; emission unit value.

460 461 462

21

463

Table 1.

464

Comparison of HPLC and UPLC system performance for ZON determination. Parameter

HPLC

UPLC

LOD, µg kg-1

4.0

2.5

LOQ, µg kg-1

8.1 – 10.0

8.0 – 9.2

Range, ng mL-1

10 – 500

Curve equation

y = 420.6x+589.3

y = 358.9x+75.3

r2

0.9999

0.9999

Slope confident interval (p = 95%, n=10)

± 1.67

± 1.0

Intercept confident interval (p = 95%, n=10)

± 97.63

± 74.5

0.44

0.06

1163.3

9587.6

Peak resolution

1.56

2.08

Peak asymmetry

1.32

1.06

Retention time (min)

6.4

2.7

Peak width at half height Theoretical plates

465 466 467 468 469 470 471

22

472

Table 2.

473

Average recovery a, RSDr and RSDR values obtained for ZON addition at 20, 50 and 200 µg kg-1 using HPLC and UPLC methods. a

b

RSDr, %

Recovery , %

Matrix

RSDRc, %

Recovery , %

20

50

200

20

50

200

20

50

200

20

50

200

101.2

105.3

102.7

1.6

6.2

3.0

98.2

106.1

105.4

7.8

10.6

6.1

79.1

85.6

85.0

9.5

11.1

8.6

71.9

77.8

81.0

20.7

16.7

9.8

114.4

88.6

89.8

4.7

3.3

2.8

119.9

86.7

87.1

7.4

4.5

4.9

96.2

97.7

99.9

9.6

3.3

3.1

96.2

96.9

99.0

9.2

4.5

3.5

97.7

94.3

94.4

6.4

6.0

4.4

96.6

91.9

92.9

11.3

9.1

6.1

HPLC Noodle, dry Instant noodle Snacks Infant formula Avg. of all matrixes BCR 717 (83 ± 9 µg kg-1)

90.5

18.1

-

-

UPLC Noodle, dry

104.1

104.0

100.4

11.1

7.0

5.9

106.8

100.2

99.9

12.6

11.7

8.6

96.5

85.1

87.6

4.3

8.3

3.4

91.2

83.9

83.9

10.3

10.7

6.7

Snacks

101.6

99.8

100.6

3.5

0.7

2.6

104.2

100.1

99.3

5.5

2.2

5.2

Infant formula

114.5

107.2

101.5

1.6

2.7

1.9

114.5

107.2

101.0

2.0

2.6

2.0

Instant noodle

474 475

Avg. of all matrixes 104.2 99.0 97.5 5.1 4.7 3.5 104.2 97.9 96.0 7.6 6.8 6.9 -1 BCR 717 (83 ± 9 µg kg ) 94.2 7.0 a % recovery = {(amount found in blank control spiked)/amount added} × 100; ZON in controls were not detected; recovery for repeatability test (n=5). b,c

Recovery and relative standard deviation for reproducibility (n=25).

23

476

Table 3.

477

Expanded uncertaintya of ZON levels using HPLC and UPLC at 200 µg kg−1 in noodles, snacks, and infant formulas. HPLC Measurand, µg kg-1

Expanded uncertainty, µg kg-1

Relative expanded uncertainty, %

Measurand, µg kg-1

Expanded uncertainty, µg kg-1

Relative expanded uncertainty, %

Noodle, dry

202.3

29.6

15

200.7

29.6

15

Instant noodle

164.3

48.9

31

175.2

39.0

22

Snacks

179.7

35.9

20

201.2

29.0

14

Infant formula

199.5

29.3

15

201.0

28.9

14

Sample

478

a

UPLC

k = 2, 95% confidence level.

479

24

480

Table 4.

481

Determination of ZON levels using HPLC and UPLC in naturally contaminated noodles, snacks, and infant formulas.

Commodity

482

a

No. of sample analyzed

HPLCa

UPLCa

No. of positive sample >LOD -
≥LOQ,

Mean, µg kg-1

µg kg-1

Range, µg kg-1

No. of positive sample >LOD -
≥LOQ,

Mean, µg kg-1

Range, µg kg-1

µg kg-1

Noodle, dry

35

0

0

0

-

1

0

0.2

8.1

Instant noodle

30

0

0

0

-

2

0

0.2

3.1-3.6

Snack (maize-based)

32

1

0

0.2

5.7

4

2

1.2

3.8-10.3

Snack (wheat-based)

30

0

0

0

-

2

1

0.5

4.0-8.3

Infant formula

36

1

1

0.4

4.3-8.3

5

4

2.0

3.3-17.6

Total

163

3

0

15

7

Samples
483 484 485 486 487 25

488 489

Fig. 1.

26

490 491 492 493 494 495

Highlight

496 497


HPLC and UPLC were compared to determine zearalenone in cereal products and infant formulas.

498


All performance criteria and the measurement uncertainties were within the acceptable range.

499


The two methods were applied to determine the levels of zearalenone in 163 naturally contaminated samples.

500


HPLC and UPLC methods are suitable for the determination of zearalenone in cereal products and infant formulas.

501 502

27