Food Control 26 (2012) 300e304

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Food Control
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Occurrence of zearalenone in fresh corn and corn products collected from local
Hispanic markets in San Diego County, CA
Trieste C. Hewitt, Carrie L. Flack, Julia K. Kolodziejczyk, Ana M. Chacon, Kathleen L. D’Ovidio*
School of Exercise and Nutritional Sciences, San Diego State University, San Diego, CA, USA

a r t i c l e i n f o a b s t r a c t

Article history: Estrogenic mycotoxins such as zearalenone (ZON) produced by several species of Fusarium fungi can be
Received 17 August 2011 found in many foods including corn and corn products. The contamination by the fungi can occur either
Received in revised form in the field or during storage. Corn and corn products represent a significant part of Hispanic diets.
11 January 2012
Hispanic females can become exposed to ZON during the consumption of these products. The estrogenic
Accepted 17 January 2012
property of ZON might promote precocious puberty in young girls and abnormal hormone balances in
women. Corn and corn products were procured from local area Hispanic markets. A single laboratory
Keywords:
validated method was used to determine ZON in these products. The limit of detection (LOD) for the
High-performance liquid chromatography
(HPLC)
method was 2.0 ng g
1. The recovery rate of ZON spiked tortillas was 95%, 76% and 114%, masa was 102%,
Fresh corn 98% and 107%, and whole corn was 92%, 89% and 94%, at concentrations of 5, 20, and 100 ng g
1
Corn tortillas respectively. A total of 35 corn products, collected from small family owned markets in the Spanish
Hispanic speaking areas most likely to sell products consumed by the local Hispanic population, were analyzed (8
Masa yellow and 7 white corn tortilla samples, 9 masa samples, 5 whole white corn and 5 whole yellow corn
Zearalenone samples and 1 nixtamalized corn sample). ZON was found in 10 of 15 tortilla samples with a mean of
1.60 ng g
1. Of the 9 masa samples, ZON was found in 7 samples with a mean of 4.52 ng g
1. ZON was
found in 4 fresh whole corn samples, averaging 0.71 ng g
1. The nixtamalized corn sample contained
a mean level of 10.31 ng g
1 ZON. ZON was more frequently found in the masa compared to the tortillas
and fresh whole corn.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction The International Agency for Research on Cancer (IARC) has

Zearalenone (ZON) is one of the major mycotoxins produced by
plant pathogenic Fusarium molds including Fusarium graminearum
and Fusarium culmorum (Miller, 1995) found in many cereal crops
worldwide including corn, wheat, barley, oats and rice (Hagler,
Bowman, Babadoost, Haney, & Swanson, 1987; Hagler, Towers,
Mirocha, Eppley, & Bryden, 2001; Jelinek, Pohland, & Wood, 1989;
Mirocha, Pathre, & Chrisensen, 1977). ZON in grains is a concern
to the health of humans and animals. Ear and kernel rot diseases of
maize caused by F. graminearum infection has caused major
mycotoxin problems including ZON in the U.S. (Leslie, Pearson,
Nelson, & Toussoun, 1990; Miller, 1994; Munkvold, 2003). More
than 90% of 114 isolates from corn of F. graminearum from the U.S.,
Australia, China, New Zealand, Norway, and Poland were found to
produce ZON (Mirocha, Abbas, Windels, & Xie, 1989).
* Corresponding author. 7301 Radcliffe Dr., College Park, MD 20740, USA.
Tel.: þ1 301 335 1056.
E-mail address: kdovidio@gmail.com (K.L. D’Ovidio).
categorized ZON as a class 2A carcinogen (IARC, 1993). ZON
produces estrogenic effects in humans and animals leading to
hyperestrogenism. Hyperestrogenism may be manifested as
enlargement of the reproductive organs. ZON can cause severe
reproductive and infertility problems in farm animals, particularly
in swine (CAST, 2003). Specifically, ZON can act as an estrogen
analog and cause alterations in the uterus of the reproductive tract
of swine and affect follicular and embryo development (Tiemann &
Danicke, 2007). The function of ZON as an estrogen analog is
important because swine are monogastric animals with responses
similar to humans in many respects (Dänicke, Swiech,
Buraczewska, & Ueberschär, 2005; Kuiper-Goodman et al., 1987).
At a molecular level, ZON and its metabolite zearalenol bind at
concentrations comparable to those of 17 b-estradiol (E2), natural
estrogen (Takemura et al., 2007). It is reliably known to affect many
animals at low exposure (Prelusky, Rotter, & Rotter, 1994).
In Eastern Europe where chronic exposure to ZON takes place
there are reliable case reports of early puberty in girls (Szu } ts,
Mesterházy, Falkay, & Bartók, 1997) as well as a reliable report on
the effect of ZON on girls in Northern Italy Hungary and Belgium

0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2012.01.035
 T.C. Hewitt et al. / Food Control 26 (2012) 300e304
also known for ZON exposure (Massart, Meucci, Saggese, & Soldani,
2008). These reports in humans are quite important because they
have reliable indications and reliable measures of exposure. Case
reports in Puerto Rico from 1978 to 1984 documented an epidemic
of premature thelarche and precocious puberty (Comas, 1982; Ingle
& Martin, 1986; Larriuz-Serrano, Perez-Cardona, Ramos-Valencia, &
Bourdony, 2001; Sáenz de Rodríguez, Bongiovanni, & Conde de
Borrego, 1985; Sáenz de Rodríguez, 1984). There were implications
that dairy and meat products could be contaminated with anabolic
estrogens to increase muscle mass in cattle and poultry (Sáenz de
Rodríguez, 1984; Sáenz de Rodríguez, Bongiovanni, & Conde de
Borrego, 1985). Contamination of grain products by Fusarium
toxins was proposed as the causative agent (Schoental, 1983). Early
puberty is an established risk factor for breast cancer, and puberty
may be an important window of susceptibility to the cancer
(National Cancer Institute (NCI), 2006).
There have been increasing numbers of reports of finding ZON in
corn and cereal grains worldwide (Zinedine, Soriano, Molto, &
Manes, 2007). Alternating low and moderate temperatures in
storage promote production of this toxin and optimum production
occurs at 81  F (Ohio Agricultural Research and Development
Center (OARDC), 2009; Vincelli & Parker, 2002). F. graminearum is
the main Fusarium species found in warmer regions of the U.S. such
as the corn-belt and California (Chelkowski, 1989, p. 63). ZON has
been reported found in cereals and corn worldwide including The
Netherlands, Indonesia, Ghana and Argentina (Gonzalez, Resnik, &
Pacin, 2003; Kpodo, Thrane, & Hald, 2000; Nuryono, Noviandi,
Bohm, & Razzazi-Fazeli, 2005; Tanaka et al., 1988, 1990). ZON has
been found in U.S. processed food products including breakfast
cereal, snack foods, popcorn, and corn meal (Warner & Pestka,
1987).
Dietary patterns of Mexican American populations in the U.S.
indicate that these individuals consume corn and corn products
as a significant portion of their diet (Block & Norris, 1995;
Plasencia, 2004). Mexico is ranked third in the world for the
highest total production of corn of approximately 24 million tons
per year. They also have one of the highest yearly per capita
consumptions of corn. For the year 2005, the corn consumption
was 122.9 kg/capita/yr based on Food and Agriculture Organi-
zation data (FAOSTAT, 2010). From 60% to 70% of the total corn
production in Mexico is for human consumption, depending on
socioeconomic status. Tortillas (the most common corn-based
form of alkaline-cooked products) can have an average
consumption of as high as 325 g/d per person (Plasencia, 2004).
Dietary studies of Mexican American women have demonstrated
that their consumption of corn tortillas has remained relatively
stable over time. Ninety-eight out of 100 (98%) of the immigrants
studied incorporated corn tortillas in their main diet after
immigration to the United States, following the same consump-
tion pattern for corn tortillas as while living in Mexico (Romero-
Gwynn & Gwynn, 1997).
The aim was to: 1. Evaluate and validate a method for ZON in
corn and corn products, 2. apply this method to conduct a small
survey of these products collected in southern California.
2. Materials and methods
2.1. Reagents and materials
2.1.1. Materials
Corn and corn based products were purchased from local
Hispanic markets in and around San Diego that were all located in
cities with large Hispanic populations including Chula Vista and
Escondido. Corn samples included whole ears of white and yellow
corn and a bag of nixtamalized white corn (5 lb/bag). Corn products
301
included ground corn flour (masa) (5 lb/bag) yellow corn tortillas
and white corn tortillas (4.59 lb/bag).
2.1.2. Chemicals and supplies
All solvents were suitable for LC analysis and were purchased
from Fisher Scientific Company LLC (Hanover Park, IL). Phosphate
buffered saline (PBS) 10 mM and Tween 20 were purchased from
SigmaeAldrich Chemical CO. (St. Louis, MO). Water was purified on
a Millipore Milli-Q-P Plus system.
The extraction solution was prepared using methanolewater
(75 þ 25 v/v), mixed, and equilibrated to room temperature. The
standards solution was prepared using methanolewater (1 þ 1 v/v).
The washing solution was prepared by dissolving 1 pouch PBS in
1.0 L water and adding 5 mL Tween 20.
The following supplies used during the course of this research
were obtained from various suppliers, as follows: Centrifuge tubes
e 50 mL, polypropylene (Fisher Scientific Company LLC, Hanover
Park, IL), Glass microfiber filter paper, 11 cm (Whatman 934AH,
Whatman, Inc., Clifton, NJ), IACeZearalaTest WB columns (G 1026,
Vicam Corp. Watertown, MA). Column manifold (Vicam G1104) 12-
position stand and column reservoir with a 10 mL syringe with luer
tip or equivalent (Vicam Corp. Watertown, MA). The ZON IAC
contains monoclonal antibodies that are cross reactive with ZON:
the columns should have capacity of level as high as 1500 ng total
ZON, and should give a recovery of not less than 80% for ZON (ZON)
when 1000 ng of ZON is applied in 10 mL of PBS. The columns
should have a shelf life of 18 months at 4  C or 12 months at room
temperature. ZON 10 mg powdered form (Z2125-10MG,
SigmaeAldrich Chemical CO. St. Louis, MO).
ZON intermediate stock standard solution (approximate
20 mg mL
1) was prepared and exact concentration of intermediate
stock standard solution calculated by determining the absorbance
using the similar AOAC Official Methods of Analysis 971.22 for
aflatoxins using the following formula:
ZON mg=mL ¼ fðA  MW  1000Þ=εg
where A ¼ the absorbance of the 10 mg mL
1 solution;
MW ¼ molecular weight of ZON (318.4); ε ¼ molar absorptivity
(12,623 in acetonitrile). The solution was stored at
18  C. The
solution was equilibrated to room temperature for at least 30 min
before use (Trucksess, 2005, Ch. 49).
ZON working standard solution: A 1.0 mg mL
1 ZON working
standard solution was prepared by measuring 1 mL of the
5 mg mL
1 standard stock solution, which was prepared from the
20 mg mL
1 ZON intermediate stock standard solution and placing it
into a 5 mL volumetric flask and diluted to volume using the
standards solution. The contents of the flask were mixed on
a vortex and stored at
18  C. The solution was equilibrated to
room temperature for at least 30 min before use.
Test samples of the three types of corn materials were spiked
with ZON at concentrations of 5, 20, and 100 ng g
1. A spiking
solution containing 10 ug ZON/mL was used. All spiked test
samples were kept at room temperature for at least 1 h before
analysis.
2.2. Apparatus
ZON was detected and quantified by reverse phase liquid
chromatography (RPLC) analysis using a Waters Model 2695 Alli-
ance LC system consisting of the pump and injector, Waters 2475
fluorescence detector, and Empower 2 control and data system
(Waters Corp., Milford, MA). Separation was performed on a YMC
ODS-AQ LC reverse-phase column (Waters AQ12S031546WT,
4.6  150 mm, 3 um, Waters Corp., Milford, MA), or equivalent.
302 T.C. Hewitt et al. / Food Control 26 (2012) 300e304

2.3. Experimental design 2.4.4. Calibration curves

Calibration curves were prepared for ZON using the working
Each bag of masa was mixed separately in a KitchenAid Tilt- standard solution. These solutions covered the range of
Head Stand Mixer using the paddle and then sampled. Each bag 3.125e200 ng mL
1 ZON. The following working standard solutions
of tortillas was dried separately in a THELCO Model 17 Serial were prepared by serial dilution of the 1000 ng mL
1 standard
Number: 21AG-6, GCA/Precision Scientific (Chicago, IL) Convection (200 ng mL
1, 100 ng mL
1, 50 ng
1 mL, 25 ng mL
1, 12.50 ng mL
1,
Oven for 24 h at 60  C or until completely dried. Tortillas were 6.25 ng mL
1, 3.125 ng mL
1) and were prepare daily in separate
ground separately in a Thomas Wiley Laboratory Mill Model 4, 2 mL HPLC vials to make serial dilutions to prepare the 5 standards.
Arthur H. Thomas Co. (Philadelphia, PA), mixed using the Kitch- The blank was prepared by placing 800 mL of the standards solution
enAid mixer and then sampled. Each ear of fresh corn and the bag into a 2 mL HPLC vial. The standard curve was constructed and the
of nixtamalized corn were dried separately in the convection oven plot checked for linearity by examining the correlation coefficient
for 48e72 h at 60  C or until completely dried. Kernels on fresh (R2 > 0.99) of concentration and responses.
cobs were removed by hand from the ears and then all corn
samples were ground separately in the Wiley Laboratory Mill, 2.4.5. Quantitation and calculation
mixed in the KitchenAid mixer and then sampled. All samples Quantitation of ZON was performed by measuring peak area at
were stored in the refrigerator in 50 mL polypropylene centrifuge ZON retention time and comparing them with the relevant stan-
tubes. dard curve. Peak area (response, Y-axis) of ZON standard was
plotted against concentration (ng/mL, X-axis) slope (S) and Y-
intercept (a) were determined. The sample level of ZON was
2.4. Method of analysis calculated with the following formula, where R is the test solution
peak area, and V is the final volume (3 mL) of the injected test
2.4.1. Sample extraction solution. F is the dilution factor. If no further dilution then F is 1. W
Finely ground sample (20 g) was weighed and placed in a glass is 1.6 g for corn and corn products, passed through the IAC.
blender jar. After addition of 100 mL extraction solution the sample
was blended at high speed for 3 min. The extract was filtered ZON ug=kg ¼ ð½ðR
aÞ=S  V=WÞ  F
through an 11 mm folded rapid filter paper (Fisher Brand P8) into
a clean 125 mL Erlenmeyer flask. The filtrate (20 mL) was trans- 3. Results and discussion
ferred into a 250 mL Erlenmeyer flask, then 80 mL of 10 mM PBS
containing 0.5% Tween 20 was added, mixed, and filtered through 3.1. Validation
glass microfiber paper. Diluted filtrate (40 mL) (equivalent to 1.6 g
test sample) was collected in a 50 mL graduate cylinder and saved A method developed by Trucksess (2011) was evaluated and
for IAC separation and isolation. validated to apply to the determination of ZON in corn and corn
products (Table 1). Mean recoveries for ZON added to tortillas, masa
2.4.2. Immunoaffinity column isolation and corn at levels of 5, 10 and 20 ng g
1 were >70%. The method
After removing from storage at 4  C, the IAC must be equili- demonstrated satisfactory precision with the corn and corn product
brated to room temperature for at least 15 min before use. IAC samples during extraction and recovery of spiked levels of ZON. The
contains monoclonal antibodies that are cross reactive with ZON mean RSDr was <10%. This demonstrated that the method
at optimum temperature ranging from 20 to 30  C. The top cap exhibited good accuracy and reproducibility for corn and corn
was removed from column and attached to the syringe end cap products. Recovery data were similar to that of a single laboratory
(the fit must be tight) that is placed in the manifold. The bottom method for ZON in grains and grain products (Trucksess, 2011).
cap of column was removed and liquid in column was allowed to Fig. 1 shows LC chromatogram of ZON standard (25.00 ng mL
1)
pass through until liquid was about 2e3 mm above column bed. and ZON naturally contaminated masa (19.46 ng g
1). The chro-
Diluted filtrate sample (40 mL) was added to the IAC and was matograms of the other test samples were similar. There was no
allowed to pass through the column by gravity. After the column other interference peak near the ZON peak area. The retention time
was allowed to run dry the column was washed with 10 mL of ZON was approximately 11.3 min.
MeOH-PBS (15 þ 85 v/v) and then was washed with 5 mL water. The limit of detection (LOD) for the method was 2.0 ng g
1 based
After forcing 3 mL air through the column with a syringe the ZON on signal (standard solution) to noise (reagent blank) ratio of
was eluted with 0.75 mL methanol and allowed to drip freely and greater than 3 to 1 for a concentration of ZON at 2.0 ng mL
1. There
collected into a 3 mL volume flask. The column was then allowed
to stand for 1 min and then eluted with additional 0.75 mL
Table 1
methanol and collected into the same flask. After elution 10 mL air
Recovery of zearalenone added to corn and corn products.
was forced through column with a syringe. The elute was diluted
to 3 mL volume with HPLC water, mixed and was saved for LC Commodity Level addeda Mean Recoveryb SDc RSDrd %c
(ng/g) (ng/mL) (%)
analysis.
Tortilla 5 94.8 1.5 1.6
20 76.4 5.6 7.3
2.4.3. LC analysis 100 114.4 2.3 2.0
The LC mobile phase was a mixture of acetonitrilee Masa 5 102.4 5.2 5.1
methanolewater in a ratio of (45 þ 10 þ 45 v/v/v). The flow rate 20 97.6 7.2 7.3
was set at 1.0 mL/min. The fluorescence detector was set at exci- 100 106.7 9.0 8.4
Whole corn 5 91.5 6.7 7.3
tation wavelength 274 nm, emission wavelength 440 nm. The ZON
20 89.5 6.3 7.1
working standards, reagent blank, and test solution were injected 100 93.6 7.4 7.9
into LC column in 50 mL amounts. The ZON peak in test solution was a
Spiking levels of 5, 20, and 100 ng g
1.
identified by comparing retention time with those of standards. The b
Average recovery of the 4 replicates at the spiking level.
retention time was approximately 11e12 min. The standard curve c
Standard deviation, n ¼ 4.
d
of ZON was constructed. Relative standard deviation.
 T.C. Hewitt et al. / Food Control 26 (2012) 300e304 303

2.50 3.3. Occurrence and exposure

2.00
Past studies have suggested that ZON consumed in muscle
1.50 A
meats, dairy products and grains may be responsible for precocious
1.00
puberty in young girls in Puerto Rico and Eastern Europe (Sáenz de
EU

0.50 B Rodríguez, Bongiovanni, & Conde de Borrego, 1985; Sáenz de

0.00 Rodríguez, 1984; Szu } ts et al., 1997). ZON contamination of these
commodities is a sporadic environmental problem therefore addi-
-0.50
tional years of analyzing these products are warranted (Ciegier,
-1.00
1976). Although ZON was detected only in low levels in the corn
-1.50 and corn products analyzed in this study ZON might form
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
byproducts after processing which have not yet been isolated. The
Minutes
effects of the byproducts are largely unknown.
Fig. 1. Chromatogram of (A) ZON standard (31.25 ng mL
1) and (B) naturally There are currently no regulatory guidelines in the U.S. for
contaminated masa (19.46 ng g
1). levels of ZON in food or feed. Since ZON has been determined to
have high biological activity and is frequently found in grains,
mainly in wheat and maize, guidelines or maximum tolerable
was no noise response from the reagent blank. The limit of quan- levels for ZON (predominantly in grains), ranging from 0 to
titation (LOQ) was 6.0 ng mL
1 (approximately 3.0 times of the 1000 mg kg
1 have been adopted by nine countries (Krska, 1999). A
LOD) (Keith et al., 1983). provisional maximum tolerable daily intake (PMTDI) for ZON and
its metabolites (including a-zearalanol) of 0.5 mg/kg of body
3.2. Survey weight per day has been instituted by JEFCA (Joint FAO/WHO
Expert Committee on Food Additives) (Codex Alimentarius
A limited survey of 35 corn products were collected from Commission, 2000). Since the highest possible level of corn
small family owned markets in the Spanish speaking areas most tortilla consumption is 325 g/d/person, the highest level of ZON
likely to sell products produced and consumed by the local found in a tortilla in this study was 3.44 ng g
1, and the Mexican
Hispanic population. The results of the survey are given in American diet may include 4 meals consisting of two or more 20 g
Table 2. The amount of ZON present in the samples of masa was tortillas each meal, the total ZON exposure could be 550 ng ZON/d/
higher than in tortillas and corn, except for the nixtamalized person which is significantly lower than 0.5 mg/kg (60 kg average)
corn. If the masa and tortillas were prepared from the same batch per day. However, this study did not look at the total ZON found in
of corn then the level of ZON in corn could be higher than that in all foods consumed in the typical MexicaneAmerican diet, which
masa or tortillas. includes beans, rice, cheese, eggs, milk, meat and vegetables.
The processing effects of boiling and nixtamalization of corn ZON has been found to occur naturally in almost every
to produce masa reduces ZON levels significantly (Abbas, agricultural product, including cereals, beans, rice, as well as in
Mirocha, Rosiles, & Carvajal, 1988). In the making of tortillas, milk, and meats, however the most frequently contaminated
the masa is then formed into dough, pressed and baked or fried commodity was found to be corn (JECFA, 2000; Kuiper-
which slightly reduces ZON levels. It was observed that very low Goodman, Scott, & Watanabe, 1987).
to no levels of ZON were found in whole fresh corn except for the
nixtamalized corn sample. A possible explanation for higher 4. Conclusion
levels of ZON in the nixtamalized corn sample could be because it
was highly contaminated. It has been determined that the nix- The risk to Hispanic women due to exposure to ZON in corn and
tamalization process produces tortillas with less pericarp and corn products was important to ascertain as it has been proposed to
aleurone thus lower fiber levels (Gomez Adalpa, Martinez Bustos, cause precocious puberty in young girls and abnormal hormone
Figueroa Cardenas, Ordorica Falomir, & Gonzalez Hernandez, balances in women which could lead to breast cancer. Since corn
1996). ZON has been shown to be found in the outer portion of has been demonstrated to be one of the most frequently consumed
the grain, which is removed during the nixtamalization process, foods in the Mexican/Hispanic diet it is important to survey the
with less present in the endosperm layer (Ryu, Jackson, & corn and corn products that they consume to determine exposure
Bullerman, 2002). to ZON (Garcia & Heredia, 2006). There were very few Hispanic
Markets that produced their own tortillas or that purchased their
corn products from very small operations in Mexico or in the San
Table 2 Diego metropolitan area during the time of the sample collection.
ZON found in white and yellow corn tortillas, masa, whole white and yellow corn There were very few markets that carried fresh corn from Mexico or
and nixtamalized corn.
Southern California as well. The important aspect of this study was
Commodity Number Zearalenone levels (ng g
1)b to collect corn and corn products produced in the local area as it
analyzeda was the key to determining exposures to ZON by the local Hispanic
>2 Min Max Averaged
White tortillas 7 2 0.05 3.1 1.2
population as this population tends to prefer corn and corn prod-
Yellow tortillas 8 4 0.78 6.8 2.0 ucts that are from local Hispanic Markets as their native language
Brand A masa 7 5 0.91 19.5 5.8 and products are represented there. Low levels of ZON were found
Brand B masa 2 0 0.4 0.8 0.2 in corn and corn products in this study. These specific results
White corn 5 1 1.04 4.3 1.4
indicate a low risk to the greater Hispanic population. As a result of
Yellow corn 5 0 0.31 0.3 0.1
Nixtamalized cornc 1 1 n/a 10.3 10.3 this study, it may be useful to significantly expand the number of
a
samples of products, to include year round collections of
Two subsamples were analyzed per sample.
b
Limit of Detection, 2 ng g
1 zearalenone.
commodities, expand the study to multiple years, expand collec-
c
only 1 sample analyzed. tions to a larger geographical area consisting of locally owned
d
Average calculated by using 0 ng g
1 for samples < 2 ng g
1. Hispanic Markets and add a greater variety of corn based
304 T.C. Hewitt et al. / Food Control 26 (2012) 300e304
commodities such as tostadas and tamales as well as adding dairy
and meat products commonly consumed by Hispanic women that
could be subject to ZON contamination.
Acknowledgments
This work was supported by the San Diego State University
Grants Program, San Diego, CA, USA. The authors would like to
thank Dr. Mary W. Trucksess, U.S. Food and Drug Administration,
Center for Food Safety and Applied Nutrition, College Park, MD for
providing advice on this project; to San Diego State University,
School of Exercise and Nutritional Sciences students: Sarah Cole,
Melissa Collins and Khristina Teope for their assistance in sample
preparation.
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