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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA 02747, USA
College of Literature, Science, and the Arts, University of Michigan, 500 S. State St., Ann Arbor, MI 48109, USA
c
Natural Sciences Department, University of MichiganDearborn, Dearborn, MI 48128, USA
b
a r t i c l e
i n f o
Article history:
Received 23 September 2014
Received in revised form 28 February 2015
Accepted 28 February 2015
Available online 6 March 2015
Keywords:
Creatinine
Uric acid
Ascorbic acid
Vitamin C
Milk
Orange juice
Hydrophilic interaction liquid
chromatography (HILIC)
a b s t r a c t
Creatinine (Cr), uric (UA) and ascorbic acid (AA) are common constituents in human uids. Their
abnormal concentrations in human uids are associated with various diseases. Thus, apart from the
endogenous formation in human body, it is also important to examine their sources from food products.
In this study, a rapid and accurate HILIC method was developed for simultaneous determination of Cr, UA
and AA in bovine milk and orange juice. Milk samples were pretreated by protein precipitation, centrifugation and ltration, followed by HPLC separation and quantication using a Waters Spherisorb
S5NH2 column. The developed method has been successfully applied to determine the concentration of
UA, AA and Cr in milk and fruit juice samples. The milk samples tested were found to contain UA and
creatinine in the concentration range of 24.186.0 and 5.0711.2 lg mL 1, respectively. The orange juices
contain AA over 212 lg mL 1.
2015 Elsevier Ltd. All rights reserved.
1. Introduction
Creatinine (Cr), uric acid (UA) and ascorbic acid (AA) are common components in human uids. Their concentrations may affect
human health and act as biomarkers for various diseases (Ascherio
et al., 2009; Burtis & Ashwood, 2001; Choi, Mount, & Reginato,
2005; Chonchol et al., 2007; Dehghan, van Hoek, Sijbrands,
Hofman, & Witteman, 2008; Gagliardi, Miname, & Santos, 2009;
Harper, 1977; Heinig & Johnson, 2006; Kassirer, 1971; Krishnan,
Kwoh, Schumarcher, & Kuller, 2007; Lapsia et al., 2012; Lin et al.,
2011; Mouton & Holder, 2006). The abnormal high concentrations
of uric acid in human plasma and urine are associated with several
diseases, such as gouty arthritis, hyperuricemia, hypertension,
pneumonia, type 2 diabetes, cardiovascular disease and kidney
damage (Ascherio et al., 2009; Burtis & Ashwood, 2001; Choi
et al., 2005; Chonchol et al., 2007; Dehghan et al., 2008; Gagliardi
et al., 2009; Harper, 1977; Heinig & Johnson, 2006; Kassirer,
1971; Krishnan et al., 2007; Lapsia et al., 2012; Lin et al., 2011;
Mouton & Holder, 2006). Creatinine, quantitatively excreted in
the urine, is nonenzymatically formed from intracellular creatine
and phosphocreatine in muscles. The creatinine excretion mainly
Corresponding author.
E-mail address: yzuo@umassd.edu (Y. Zuo).
http://dx.doi.org/10.1016/j.foodchem.2015.02.142
0308-8146/ 2015 Elsevier Ltd. All rights reserved.
243
244
Table 1
The analytical recovery of analytes in the samples.
Compound
Creatinine
5
10
98.4 (5.0)
99.0 (3.4)
Uric acid
5
10
93.5 (0.7)
94.4 (0.6)
Ascorbic acid
5
10
93.7 (1.5)
96.2 (1.1)
a
Concentrations of standards are expressed as the equivalent concentrations
added in the nal injected solutions.
b
Calculated as % recovery = [(amount observed original amount)/amount
added] 100.
in Fig. 4. Individual analytes were identied by matching the retention times against those of authentic standards, and by spiking the
milk and orange juice samples with the standard of each analyte.
The concentrations of creatinine, uric and ascorbic in examined
milk and orange juice samples are presented in Table 2. The milk
samples examined were found to contain UA and creatinine in
the concentration range of 24.186.0 and 5.0711.15 lg mL 1,
respectively. The consumption of bovine milk may contribute to
the increased concentration of uric acid and creatinine in body
uids in adults slightly but could signicantly in children. No
Table 2
Analytical results of creatinine, uric and ascorbic acid in bovine milk and orange juice
samples.
Fig. 2. Calibration graphs for creatinine, uric acid and ascorbic acid.
Sample
Creatinine
Concentration/
lg mL 1
Uric acid
Concentration/
lg mL 1
Ascorbic acid
Concentration/
lg mL 1
Milk 1
Milk 2
Milk 3
Milk 4
Orange
8.26 0.08
11.2 0.10
6.98 0.17
5.07 0.08
ND
36.8 0.04
28.4 0.03
36.0 0.07
24.1 0.05
ND
ND*
ND
ND
ND
212 4.4
Not detected.
4. Conclusions
A selective, fast and accurate HILIC method has been developed
for the simultaneous determination of creatinine, uric and ascorbic
acid in bovine milk and orange juice samples. The HILIC method
used a Waters Spherisorb S5NH2 column and a mobile phase consisting of 50% acetonitrile and 50% of 10 mM phosphate buffer at
pH 4.75 at a ow rate of 1.2 mL min 1 and a detection wavelength
of 205 nm. This method has shown the advantages of the HILIC
over reversed-phase HPLC methods for the determination of highly
polar small organic compounds with good retention and without
ion pairing agent requirement. Good linearity and sensitivity were
obtained with this developed method. The detection limits for
creatinine, uric and ascorbic acid were 0.045, 0.10 and
0.18 lg mL 1, respectively. The described method was successfully
applied to the determination of creatinine, uric and ascorbic acid in
bovine milk and orange juice samples. The bovine milk contains
signicant levels of creatinine and uric acid, which may contribute
to the concentration of these metabolites in human uids. The
method has proved simple, robust and sensitive and can be also
employed for the routine analysis of other food products and
biouids.
Acknowledgements
The authors thank Dr. E. Ojadi for his contribution and support
to this work. This research project was partly supported by the
University of Massachusetts Dartmouth and University of
MichiganDearborn.
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