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Food Chemistry 182 (2015) 242–245

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Food Chemistry
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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
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
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
S5NH2 column. The developed method has been successfully applied to determine the concentration of
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 depends on total body skeletal muscle mass. Thus the urinary
creatinine concentration has been commonly used to normalize
Creatinine (Cr), uric acid (UA) and ascorbic acid (AA) are com- the concentrations of uric acid and other diagnostic markers in
mon components in human fluids. Their concentrations may affect urine in clinical and biomedical laboratories (Arndt, 2009; Burtis
human health and act as biomarkers for various diseases (Ascherio & Ashwood, 2001; Zuo, Yang, Zhu, He, & Aydin, 2011). The concen-
et al., 2009; Burtis & Ashwood, 2001; Choi, Mount, & Reginato, tration of creatinine in human fluids has been also frequently used
2005; Chonchol et al., 2007; Dehghan, van Hoek, Sijbrands, as a biomarker for renal function (Burtis & Ashwood, 2001; Kassirer,
Hofman, & Witteman, 2008; Gagliardi, Miname, & Santos, 2009; 1971; Mouton & Holder, 2006). As has been shown in numerous
Harper, 1977; Heinig & Johnson, 2006; Kassirer, 1971; Krishnan, studies in adults, human diet may also influences urinary creatinine
Kwoh, Schumarcher, & Kuller, 2007; Lapsia et al., 2012; Lin et al., and uric acid excretion (Burtis & Ashwood, 2001; Mayersohn,
2011; Mouton & Holder, 2006). The abnormal high concentrations Conrad, & Achari, 1983; Neubert & Remen, 1998; Zgaga,
of uric acid in human plasma and urine are associated with several Theodoratou, Kyle, et al., 2012; Zuo et al., 2011). Urinary creatinine
diseases, such as gouty arthritis, hyperuricemia, hypertension, concentration is affected by 3 dietary components in foods: crea-
pneumonia, type 2 diabetes, cardiovascular disease and kidney tinine, creatine, and protein. Ascorbic acid, also known as vitamin
damage (Ascherio et al., 2009; Burtis & Ashwood, 2001; Choi C is a natural antioxidant and plays an important role in various
et al., 2005; Chonchol et al., 2007; Dehghan et al., 2008; Gagliardi physiological processes. Although humans cannot produce AA
et al., 2009; Harper, 1977; Heinig & Johnson, 2006; Kassirer, themselves, they can and must take this nutrient from foods and
1971; Krishnan et al., 2007; Lapsia et al., 2012; Lin et al., 2011; beverages. A recent study has shown that consumption of antioxi-
Mouton & Holder, 2006). Creatinine, quantitatively excreted in dant-rich fruits can significantly enhance the excretion of uric acid,
the urine, is nonenzymatically formed from intracellular creatine a major antioxidant in humans (Zuo et al., 2011), and thus increase
and phosphocreatine in muscles. The creatinine excretion mainly 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
⇑ Corresponding author.
natural antioxidants, such as ascorbic acid, from food products. So
E-mail address: (Y. Zuo).
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
R. Zuo et al. / Food Chemistry 182 (2015) 242–245 243

far little information is available in the simultaneous determination 2.4. Chromatographic conditions
of creatinine, uric acid and antioxidant ascorbic acid in food
products and their source strength. A Dionex high performance liquid chromatograph (Dionex
Reversed-phase (RP) HPLC has been traditionally used for the Corporation, Sunnyvale, CA, USA) equipped with a P680 pump, a
separation and determination of polar organic compounds in UVD-170U spectrophotometer detector and Chromeleon 6.60
human and other biological fluids (Chen, Pietrzyk, & Whitson, software was used for analysis. The analytical column used in
1997; Hewavitharana & Bruce, 2003; Indyk & Woollard, 2004; the experiments was a Waters Spherisorb S5NH2 column
Jen, Hsiao, & Liu, 2002; Kochansky & Strein, 2000; Kwon, Kim, & (250  4.6 mm, 5 l; Waters) fitted with a C18 guard column
Suh, 2012; Moral, Diez, Resines, Bravo, & Arin, 2003; Sidorova & (7.5  4.6 mm, 5 l). An isocratic elution was employed for
Grigoriev, 2012; Yoshiura & Iriyama, 1986; Zhou et al., 2013; separation. The mobile phase consists of 50% acetonitrile and 50%
Zuo, Wang, Zhou, Sachdeva, & Ruelos, 2008; Zuo et al., 2014). sodium phosphate buffer solution (10 mM) of pH 4.75 and the flow
However, the highly polar small molecules, like creatinine, uric rate was 1.2 mL/min. The detection of analytes was carried out by
and ascorbic acid, have poor retention on a reversed-phase column. UV absorbance at 205 nm.
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 2.5. Identification and quantification
et al., 2008, 2014). Unfortunately, a high percentage of water in
the mobile phase results in dewetting and nonreproducibility The identification of creatinine and uric acid in sample was
problems. A retention recovery process is usually required to be achieved by comparing the retention times with those of authentic
integrated into the HPLC elution program (Zuo et al., 2008, standards, and by spiking the samples with the standard of each
2014). Ion pairing reagents reduce the lifetime of the column and analyte. In each sample, the quantification was carried out by
the sensitivity and selectivity of mass spectrometric detectors. standard calibration curve method.
Recent studies have demonstrated that hydrophilic interaction liq-
uid chromatography (HILIC) has advantages over RP-HPLC for the
3. Results and discussion
analysis of the highly polar small organic compounds (Zuo et al.,
2014). In this study, a rapid and accurate hydrophilic interaction
After multiple preliminary tests with several hydrophilic sta-
HPLC method has been developed for simultaneous determination
tionary phases, a Waters Spherisorb S5NH2 column was selected
of creatinine, UA and AA in bovine milk and orange juice.
for the separation. Chromatographic conditions were optimized
with respect to mobile phase composition by varying the percent-
2. Materials and methods age of organic modifier from 20% to 90% and the pH value of aque-
2.1. Chemicals and materials ous buffer solution between 3.0 and 7.0. Optimal conditions for the
separation were observed with an isocratic elution containing 50%
Creatinine, uric acid and ascorbic acid standard chemicals were acetonitrile and 50% phosphate buffer (10 mM) at pH 4.75. At this
purchased from Acros Organics (Geel, Belglum, NJ, USA). Sodium pH, the analytes were eluted out in the order of creatinine (with
dihydrogen phosphate obtained from Fisher Scientific (Fair Lawn, pKa values of 4.8 and 9.2 in water), uric acid (pKa value of 5.75
NJ, USA) was used in preparing buffer solutions. Water and in water) and ascorbic acid (with a pKa value of 4.1 and 11.6 in
acetonitrile, supplied by Pharmco Products (Brookfield, CT, USA), water) as presented in Fig. 1. This elution order indicates that
and other solvents used in the experiments, were HPLC grade. All besides partition of the analytes between a water-enriched layer
standards were dissolved with distilled–deionized water. The on the surface of the polar stationary phase and the mobile phase,
mobile phase solvents were vacuum-filtered and degassed before hydrogen-bond formation and the ionic attraction/repulsion
use. Milk and orange fruit samples were purchased from local between analytes and the amino functional group on the stationary
supermarkets in Massachusetts and stored at 4 and 20 °C (room phase also plays a significant role in the HILIC separation (Alpert,
temperature), respectively, until used in this study. 1990; Ares & Bernal, 2012; Zuo et al., 2011, 2014). The retention
times of creatinine, uric acid and ascorbic acid were: 3.09 ± 0.01,
2.2. Standard solutions 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
Stock standard solutions (1.00 mg/mL) of creatinine and ascor- of standard solutions and milk samples. The relative standard
bic acid were freshly prepared in deionized water and 5% phospho- deviation (RSD) values of the retention times were smaller than
ric acid, respectively. Uric acid standard was dissolved in water 0.6%, and the precision in the peak area was better than 3.0% for
(1.00 mg/mL) by adding 0.1 M sodium hydroxide solution to adjust eight consecutive injections of the same milk sample, demonstrat-
to pH 10.35. Work solution contained 50.0 lg/mL of creatinine and ing that the analytical method developed was very stable and had
uric acid was prepared from the stock standard solution. high reproducibility.
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 Fig. 1. HPLC chromatogram of a mixture of standards, creatinine, uric acid and
setup. The injection volume was 20 lL. 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

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) 242–245 245

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