Food Chemistry 182 (2015) 242–245

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Determination of creatinine, uric and ascorbic acid in bovine milk

and orange juice by hydrophilic interaction HPLC
Ruiting Zuo a,b, Si Zhou a, Yuegang Zuo a,⇑, Yiwei Deng c
a
Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA 02747, USA
b
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 Michigan–Dearborn, Dearborn, MI 48128, USA

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

Article history: Creatinine (Cr), uric (UA) and ascorbic acid (AA) are common constituents in human fluids. Their
Received 23 September 2014 abnormal concentrations in human fluids are associated with various diseases. Thus, apart from the
Received in revised form 28 February 2015 endogenous formation in human body, it is also important to examine their sources from food products.
Accepted 28 February 2015
In this study, a rapid and accurate HILIC method was developed for simultaneous determination of Cr, UA
Available online 6 March 2015
and AA in bovine milk and orange juice. Milk samples were pretreated by protein precipitation, cen-
trifugation and filtration, followed by HPLC separation and quantification using a Waters Spherisorb
Keywords:
S5NH2 column. The developed method has been successfully applied to determine the concentration of
Creatinine
Uric acid
UA, AA and Cr in milk and fruit juice samples. The milk samples tested were found to contain UA and
Ascorbic acid creatinine in the concentration range of 24.1–86.0 and 5.07–11.2 lg mL 1, respectively. The orange juices
Vitamin C contain AA over 212 lg mL 1.
Milk Ó 2015 Elsevier Ltd. All rights reserved.
Orange juice
Hydrophilic interaction liquid
chromatography (HILIC)

1. Introduction
Creatinine (Cr), uric acid (UA) and ascorbic acid (AA) are com-
mon components in human fluids. 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).
depends on total body skeletal muscle mass. Thus the urinary
creatinine concentration has been commonly used to normalize
the concentrations of uric acid and other diagnostic markers in
urine in clinical and biomedical laboratories (Arndt, 2009; Burtis
& Ashwood, 2001; Zuo, Yang, Zhu, He, & Aydin, 2011). The concen-
tration of creatinine in human fluids has been also frequently used
as a biomarker for renal function (Burtis & Ashwood, 2001; Kassirer,
1971; Mouton & Holder, 2006). As has been shown in numerous
studies in adults, human diet may also influences urinary creatinine
and uric acid excretion (Burtis & Ashwood, 2001; Mayersohn,
Conrad, & Achari, 1983; Neubert & Remen, 1998; Zgaga,
Theodoratou, Kyle, et al., 2012; Zuo et al., 2011). Urinary creatinine
concentration is affected by 3 dietary components in foods: crea-
tinine, creatine, and protein. Ascorbic acid, also known as vitamin
C is a natural antioxidant and plays an important role in various
physiological processes. Although humans cannot produce AA
themselves, they can and must take this nutrient from foods and
beverages. A recent study has shown that consumption of antioxi-
dant-rich fruits can significantly enhance the excretion of uric acid,
a major antioxidant in humans (Zuo et al., 2011), and thus increase
gouty arthritis and kidney stone risk. Therefore, besides the
endogenous production of uric acid and creatinine in human body,
it is also important to simultaneously examine their sources and
natural antioxidants, such as ascorbic acid, from food products. So

http://dx.doi.org/10.1016/j.foodchem.2015.02.142
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
 R. Zuo et al. / Food Chemistry 182 (2015) 242–245
far little information is available in the simultaneous determination
of creatinine, uric acid and antioxidant ascorbic acid in food
products and their source strength.
Reversed-phase (RP) HPLC has been traditionally used for the
separation and determination of polar organic compounds in
human and other biological fluids (Chen, Pietrzyk, & Whitson,
1997; Hewavitharana & Bruce, 2003; Indyk & Woollard, 2004;
Jen, Hsiao, & Liu, 2002; Kochansky & Strein, 2000; Kwon, Kim, &
Suh, 2012; Moral, Diez, Resines, Bravo, & Arin, 2003; Sidorova &
Grigoriev, 2012; Yoshiura & Iriyama, 1986; Zhou et al., 2013;
Zuo, Wang, Zhou, Sachdeva, & Ruelos, 2008; Zuo et al., 2014).
However, the highly polar small molecules, like creatinine, uric
and ascorbic acid, have poor retention on a reversed-phase column.
To obtain some retention for these polar chemicals on RP-HPLC,
either an aqueous or ion-pairing agents containing mobile phase
are employed (Indyk & Woollard, 2004; Moral et al., 2003; Zuo
et al., 2008, 2014). Unfortunately, a high percentage of water in
the mobile phase results in dewetting and nonreproducibility
problems. A retention recovery process is usually required to be
integrated into the HPLC elution program (Zuo et al., 2008,
2014). Ion pairing reagents reduce the lifetime of the column and
the sensitivity and selectivity of mass spectrometric detectors.
Recent studies have demonstrated that hydrophilic interaction liq-
uid chromatography (HILIC) has advantages over RP-HPLC for the
analysis of the highly polar small organic compounds (Zuo et al.,
2014). In this study, a rapid and accurate hydrophilic interaction
HPLC method has been developed for simultaneous determination
of creatinine, UA and AA in bovine milk and orange juice.
2. Materials and methods
2.1. Chemicals and materials
Creatinine, uric acid and ascorbic acid standard chemicals were
purchased from Acros Organics (Geel, Belglum, NJ, USA). Sodium
dihydrogen phosphate obtained from Fisher Scientific (Fair Lawn,
NJ, USA) was used in preparing buffer solutions. Water and
acetonitrile, supplied by Pharmco Products (Brookfield, CT, USA),
and other solvents used in the experiments, were HPLC grade. All
standards were dissolved with distilled–deionized water. The
mobile phase solvents were vacuum-filtered and degassed before
use. Milk and orange fruit samples were purchased from local
supermarkets in Massachusetts and stored at 4 and 20 °C (room
temperature), respectively, until used in this study.
2.2. Standard solutions
Stock standard solutions (1.00 mg/mL) of creatinine and ascor-
bic acid were freshly prepared in deionized water and 5% phospho-
ric acid, respectively. Uric acid standard was dissolved in water
(1.00 mg/mL) by adding 0.1 M sodium hydroxide solution to adjust
to pH 10.35. Work solution contained 50.0 lg/mL of creatinine and
uric acid was prepared from the stock standard solution.
Calibration curves were prepared over the concentration of
0.0–20 lg/mL for all creatinine, ascorbic and uric acid.
2.3. Sample preparation
Milk sample (4.0 mL) was added into 2.5 mL of 3% (v/v) acetic
acid, mixed well and stood for 15 min, diluted to 10.0 mL with
water, mixed, and then centrifuged for 10 min at 1500 rpm. If clari-
fication was not achieved, extra drops of glacial acetic acid were
added. An aliquot of the supernatant was filtered through a
0.45 lm nylon filter membrane. Samples of orange juice were pre-
pared by freshly squeezing orange fruit in an all glass assembly
setup. The injection volume was 20 lL.
243
2.4. Chromatographic conditions
A Dionex high performance liquid chromatograph (Dionex
Corporation, Sunnyvale, CA, USA) equipped with a P680 pump, a
UVD-170U spectrophotometer detector and Chromeleon 6.60
software was used for analysis. The analytical column used in
the experiments was a Waters Spherisorb S5NH2 column
(250  4.6 mm, 5 l; Waters) fitted with a C18 guard column
(7.5  4.6 mm, 5 l). An isocratic elution was employed for
separation. The mobile phase consists of 50% acetonitrile and 50%
sodium phosphate buffer solution (10 mM) of pH 4.75 and the flow
rate was 1.2 mL/min. The detection of analytes was carried out by
UV absorbance at 205 nm.
2.5. Identification and quantification
The identification of creatinine and uric acid in sample was
achieved by comparing the retention times with those of authentic
standards, and by spiking the samples with the standard of each
analyte. In each sample, the quantification was carried out by
standard calibration curve method.
3. Results and discussion
After multiple preliminary tests with several hydrophilic sta-
tionary phases, a Waters Spherisorb S5NH2 column was selected
for the separation. Chromatographic conditions were optimized
with respect to mobile phase composition by varying the percent-
age of organic modifier from 20% to 90% and the pH value of aque-
ous buffer solution between 3.0 and 7.0. Optimal conditions for the
separation were observed with an isocratic elution containing 50%
acetonitrile and 50% phosphate buffer (10 mM) at pH 4.75. At this
pH, the analytes were eluted out in the order of creatinine (with
pKa values of 4.8 and 9.2 in water), uric acid (pKa value of 5.75
in water) and ascorbic acid (with a pKa value of 4.1 and 11.6 in
water) as presented in Fig. 1. This elution order indicates that
besides partition of the analytes between a water-enriched layer
on the surface of the polar stationary phase and the mobile phase,
hydrogen-bond formation and the ionic attraction/repulsion
between analytes and the amino functional group on the stationary
phase also plays a significant role in the HILIC separation (Alpert,
1990; Ares & Bernal, 2012; Zuo et al., 2011, 2014). The retention
times of creatinine, uric acid and ascorbic acid were: 3.09 ± 0.01,
4.67 ± 0.01 and 5.30 ± 0.01 min, respectively, which were deter-
mined from 21 consecutive injections during an analysis of a series
of standard solutions and milk samples. The relative standard
deviation (RSD) values of the retention times were smaller than
0.6%, and the precision in the peak area was better than 3.0% for
eight consecutive injections of the same milk sample, demonstrat-
ing that the analytical method developed was very stable and had
high reproducibility.
Fig. 1. HPLC chromatogram of a mixture of standards, creatinine, uric acid and
ascorbic acid.
244 R. Zuo et al. / Food Chemistry 182 (2015) 242–245

The quantitative measurements of the three analytes were Table 1

based on chromatographic peak area. Standard mixtures of The analytical recovery of analytes in the samples.

creatinine, uric and ascorbic acid in the concentration range of Compound Standard added (lg/mL)a Mean recoveryb (%) (±SD)
0.00–20.0 lg mL 1 were prepared for calibration graphs. Three Creatinine 5 98.4 (±5.0)
replicate injections of standards at each concentration were 10 99.0 (±3.4)
performed. A good linearity was observed for each of the Uric acid 5 93.5 (±0.7)
analytes examined as illustrated in Fig. 2. A typical calibration 10 94.4 (±0.6)
graph for creatinine followed the equation: y = 1.009x + 0.047, Ascorbic acid 5 93.7 (±1.5)
with the coefficients of determination, R2 = 1.000; for uric acid, 10 96.2 (±1.1)
y = 0.383x + 0.0173 with R2 = 1.000; and for ascorbic acid, a
Concentrations of standards are expressed as the equivalent concentrations
y = 0.189x + 0.0444 with R2 = 0.999. The detection limits examined added in the final injected solutions.
at three times of the background noise were 0.045, 0.10 and b
Calculated as % recovery = [(amount observed original amount)/amount
0.18 lg mL 1 for creatinine, uric and ascorbic acid, respectively. added]  100.
The corresponding limits of quantification were 0.15 lg mL 1 for
creatinine, 0.33 lg mL 1 for uric acid and 0.60 lg mL 1 ascorbic
acid. The analytical recovery of the described method was tested
by adding known amounts of analyte standards into the samples,
and the percentage recoveries were found to be 93.4–103.4% for
creatinine, 92.8–95.0% for uric acid and 92.2–97.3% for ascorbic
acid as presented in Table 1. Day-to-day precision was evaluated
by performing six injections of standard solutions and milk sam-
ples each day on five different days within a 2-week period.
Inter-day precision (RSD) on the basis of retention time and peak
area was better than 0.9% and 4.0%, respectively. Repeatability of
the method was performed by three analysts (five determinations
by each analysts) using the described method and the same instru-
Fig. 3. A typical HPLC chromatogram of a bovine milk sample. Injection volume,
ment. The results shown no significant differences (RSD% < 3.5). 20 lL.
The method described above has been applied to determine
creatinine, uric and ascorbic acid in bovine milk and orange juice
samples. As milk is a highly complex biological fluid, some pre-
treatment is mandatory to remove protein, simplify the matrix,
protect the analytical column and facilitate unambiguous signal
interpretation. Post-secretory enzymatic activity in milk, which
can cause alteration in the concentration of target analytes, can
be generally prevented by deproteinization with any of several
recommended organic solvents and acids, such as methanol,
acetonitrile, perchloric, trichloroacetic, or acetic acid, during
sample preparation (Fritz & Zuo, 2007; Gill & Indyk, 2007;
Hewavitharana & Bruce, 2003; Indyk & Woollard, 2004). In the pre-
sent study, an acid precipitation of protein with acetic acid was
employed, which could improve the specificity of the HILIC Fig. 4. A typical HPLC chromatogram of an orange juice sample. Injection volume,
20 lL.
determination by removing protein-bound interferents, when
compared with other organic solvents tested such as methanol
and acetonitrile in the preliminary experiments in this study. in Fig. 4. Individual analytes were identified by matching the reten-
Excellent selectivity and sensitivities were achieved for creatinine, tion times against those of authentic standards, and by spiking the
uric and ascorbic acid for milk samples when this sample prepara- milk and orange juice samples with the standard of each analyte.
tion technique was combined with the described HILIC procedures The concentrations of creatinine, uric and ascorbic in examined
(Fig. 3). A typical chromatogram for an orange sample is illustrated 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 24.1–86.0 and 5.07–11.15 lg mL 1,
respectively. The consumption of bovine milk may contribute to
the increased concentration of uric acid and creatinine in body
fluids in adults slightly but could significantly in children. No

Table 2
Analytical results of creatinine, uric and ascorbic acid in bovine milk and orange juice
samples.

Sample Creatinine Uric acid Ascorbic acid

Concentration/ Concentration/ Concentration/
lg mL 1 lg mL 1 lg mL 1
Milk 1 8.26 ± 0.08 36.8 ± 0.04 ND*
Milk 2 11.2 ± 0.10 28.4 ± 0.03 ND
Milk 3 6.98 ± 0.17 36.0 ± 0.07 ND
Milk 4 5.07 ± 0.08 24.1 ± 0.05 ND
Orange ND ND 212 ± 4.4
*
Fig. 2. Calibration graphs for creatinine, uric acid and ascorbic acid. Not detected.
 R. Zuo et al. / Food Chemistry 182 (2015) 242–245
detectable amount of ascorbic acid was found in any of the tested
milk samples. The orange juices contain AA over 212 lg mL 1. Our
previous study has shown that consumption of antioxidant rich
fruits can enhance the excretion of uric acid in urine (Zuo et al.,
2011).
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 con-
sisting of 50% acetonitrile and 50% of 10 mM phosphate buffer at
pH 4.75 at a flow 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
significant levels of creatinine and uric acid, which may contribute
to the concentration of these metabolites in human fluids. The
method has proved simple, robust and sensitive and can be also
employed for the routine analysis of other food products and
biofluids.
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
Michigan–Dearborn.
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