ANALYTICAL SCIENCES DECEMBER 2008, VOL.

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2008 The Japan Society for Analytical Chemistry

1589

Simultaneous Determination of Creatinine and Uric Acid in

Human Urine by High-Performance Liquid Chromatography
Yuegang ZUO, Chengjun WANG, Jiping ZHOU, Amita SACHDEVA, and Vanessa C. RUELOS
Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth,
University of Massachusetts Graduate School of Marine Sciences and Technology,
285 Old Westport Road, North Dartmouth, MA 02747, USA

An environmentally friendly reversed-phase HPLC method for simultaneous determination of creatinine and uric acid in
human urine samples has been developed. Human urine samples were pretreated by dilution, protein precipitation,
centrifugation and filtration, followed by HPLC separations using a reversed-phase C18 column with an aqueous mobile
phase of phosphate buffer. The retention loss of a C18 column when using the highly aqueous mobile phases was avoided
by employing a gradient elution using a small volume (<0.23 mL) of acetonitrile and phosphate buffer at pH 4.75. This
developed method provides a simple, rapid separation and sensitive detection for the species of interest in 10 min with UV
detection at 205 nm. Quantitation was carried out by relating the peak areas of the identified compounds to that of
hypoxanthine as an internal standard. The detection limits for creatinine and uric acid were 0.045 and 0.062 mg mL1,
respectively. The recoveries of the standards added to urine samples were 87.3 102.2% for creatinine and 97.3 108.6%
for uric acid, and the relative standard deviation for both analytes was less than 1.0%. This method has been successfully
applied to estimating of creatinine and uric acid in human urine.
(Received March 24, 2008; Accepted June 23, 2008; Published December 10, 2008)

Introduction
Uric acid (Fig. 1) is the final oxidation product of purine
metabolism in humans and other higher primates. The
concentration levels of uric acid in serum and urine are
associated with various diseases, such as cardiovascular disease,
renal disease, diabetes, and hypertension,19 and are routinely
determined especially in clinical and biomedical laboratories.
Creatinine (Fig. 1) is a breakdown product of creatine phosphate
in muscle, and is usually produced at a fairly constant rate by
the body depending on muscle mass.
Therefore, the
concentration of uric acid and other diagnostic markers in urine
is commonly corrected by the urinary creatinine concentration,
and the creatinine ratio is often used as diagnostic markers.
The measurement of creatinine and uric acid in plasma and
urine is traditionally performed by colorimetrical methods, such
as those based on the Jaff alkaline picrate reaction for
creatinine10,11 and the reduction of phosphotungstate for uric
acid.12,13 However, these photometrical methods lack specificity
and suffer from interferences from various endogenous and
exogenous substances that react with alkaline picrate or reduce
phosphotungstate. A number of enzymatic methods have also
been adopted for routine clinical measurements of creatinine
and uric acid.11,14 Although the enzymatic methods generally
have less interference than colorimetrical methods, there have
been reports of various substances that still interfere.11,1315 To
avoid interferences in the sample matrices, many HPLC methods
for the isolation and quantitation of creatinine, uric acid, and
To whom correspondence should be addressed.
E-mail: yzuo@umassd.edu

other metabolites in biological fluids have been described,

including ion-exchange, normal-phase, reversed-phase, and
reversed-phase ion-pair chromatography.1621 Reversed-phase
HPLC methods with a highly aqueous mobile phase have been
increasingly attractive in light of the recent movement of green
analytical chemistry, which encourages the development of
analytical methods without or with reduced consumption of
organic solvent, energy and time.1719,2224 However, many
studies have reported that the use of a highly aqueous mobile
phase (>90% water) with C18 and C8 columns causes an anomalous
chromatographic behavior, and a retention loss of analytes.19,25
These anomalies have been attributed to (i) aggregation of the
bonded hydrocarbon chains in the presence of a highly aqueous
mobile phase, rendering them inaccessible to analytes, and (ii)
extrusion of the highly aqueous mobile phase from the pores of
the particles of the stationary phase.2527 Our preliminary
experiments in this study showed a rapid loss in the retention
time of creatinine and uric acid on the C18 column with highly
aqueous mobile phases reported in some recent publications.19,21
In this paper, a simple, fast, accurate and environmentally
friendly reversed-phase HPLC method is described for the

Fig. 1

Chemical structure of uric acid, creatinine and hypoxanthine.

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ANALYTICAL SCIENCES DECEMBER 2008, VOL. 24

Table 1 HPLC solvent gradient elution program

Time/min

Solvent A, %

Solvent B, %

Flow-rate/mL min1

0.00
3.50
4.00
5.00
5.50
10.00

100
100
70
70
100
100

0
0
30
30
0
0

0.45
0.45
0.5
0.5
0.5
0.5

simultaneous determination of creatinine and uric acid in human

urine. The method was optimized through gradient elution to
remove hydrophilic and hydrophobic residues, and to maintain
the C18 column resolution following separation of the analytes
using an aqueous mobile phase of phosphate buffer.

Fig. 2 Chromatogram of a creatinine and uric acid standards (25.00

mg mL1) mixture with internal standard hypoxanthine (10.00 mg
mL1). Injection volume, 20 mL.

Experimental
Chemicals
Standards of creatinine, uric acid and hypoxanthine were
purchased from Acros Organics (Geel, Belglum, NJ). Sodium
phosphate and phosphorous acid were obtained from Fisher
Scientific (Fair Lawn, NJ). Sodium hydroxide was obtained
from CMS Inc. (Houston, TX). Acetonitrile was supplied by
Pharmco Products (Brookfield, CT). Except where noted, all
reagents were of analytical grade, and all solutions were
prepared using distilled-deionized water, and were filtered
through 0.45-mm membranes (Fisher Scientific) before HPLC
analysis. The mobile-phase solvents were degassed before use.
Standard solutions
Stock standard solutions (200 mg mL1) of creatinine and uric
acid (adjusted to pH 10.35) were freshly prepared in water.
Stock internal standard solutions (160 mg mL1 hypoxanthine)
were freshly prepared by dissolving 8.0 mg in 50 mL of water.
All solutions were stored in brown glass bottles and kept at 4C.
A series of 1.6 mL mixed working standard solutions containing
a 10.0 mg mL1 internal standard were prepared by adding a 100mL stock internal standard solution and appropriately diluting
the individual stock standard solutions to obtain concentrations
in the range of 0.0 25 mg mL1. Calibration curves were
constructed by linear regression of the peak area ratio of individual
standards to the internal standard versus the concentration.
Sample preparation
Urine samples were collected from four healthy volunteers in
plastic containers. Urine samples were diluted 100-fold with
distilled water and acidified to pH 2.35 with phosphorous acid
to precipitate protein before centrifugation at 5000g for 15 min.
The supernatants were filtered through 0.45 mm membrane
filters (Fisher Scientific brand) after adjusting the pH to 6.85
using 0.010 N sodium hydroxide and 10% phosphorous acid.
The 1.6 mL solutions containing pretreated samples and an
internal standard (10.0 mg mL1) were prepared in duplicate for
HPLC analysis. Twenty microliters of the pretreated urine samples
or standard solutions were directly injected into the HPLC system.
Precautions have always been taken to minimize sample
contamination. All sample containers, glassware and filtration
devices were thoroughly cleaned with a 0.10 M HCl solution,
and then finally with doubly distilled-deionized water.

HPLC analysis
A Dionex high-performance liquid chromatograph (Dionex
Corporation, Sunnyvale, CA) equipped with a P680 HPLC
pump, a UVD-170U spectrophotometer detector, a Gina 50
autosampler, and Chromeleon 6.60 software was used for all
experiments. The analytical column used was a symmetry C18
reversed-phase column (150 3.00 mm i.d., 5 mm; Waters) fitted
with a 10-mm C18 guard column. The detection of creatinine
and uric acid was carried out by direct UV absorbance at 205
nm. Solvent gradients were formed by a dual pumping system
by varying the proportion of solvent A (sodium phosphate
buffer; pH 4.75) to solvent B (acetonitrile; HPLC grade). The
solvent gradient elution program is presented in Table 1.
Identification and quantification
The identification of creatinine and uric acid in each urine
sample was achieved by comparing the HPLC chromatographic
retention times. In each sample, the quantification of both
creatinine and uric acid was carried out by relating the peak
areas of the identified compounds to that of the internal
standard, hypoxanthine, at a concentration of 10.0 mg mL1. A
trace amount of hypoxanthine may be present in urine, but its
concentration has always been found to be below the limit of
quantification, and thus does not significantly affect the
quantification of analytes (Fig. 3A). All of the calibration
standards and urine samples were measured in triplicate.

Results and Discussion

Chromatographic separation
It has been reported that the HPLC retention time gradually
decreases and even becomes almost no retention when reversedphase C18 columns are used for long-time continuous operation
with a highly aqueous mobile phase.2527 A similar retention loss
of uric acid and creatinine was also observed on a C18 column
with previously reported aqueous mobile phases after two runs,
or less than 20 min. The retention losses are attributed to (1) a
folding of the stationary phase alkyl chains in the presence of a
highly aqueous mobile phase, and (2) the highly aqueous mobile
phase being forced out of the pores when the flow is stopped
and the pressure released. Retention is lost because the mobile
phase is no longer in contact with the interior surface of the
particles, where most of the surface area is located.27 To
maintain good performance of the column, after multiple

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Table 2 Analysis results of creatinine and uric acid in human
urine samplesa
Urine
sample

Creatinine

Uric acid

Concentration/
mg mL1

RSD, %

Concentration/
mg mL1

RSD, %

2213
1487
1869
669

0.34
0.53
0.26
0.44

1036
647
1101
594

0.32
0.48
0.45
0.65

No. 1
No. 2
No. 3
No. 4

a. The urine samples were measured at 100-fold dilution.

Table 3 The recoveries of creatinine and uric acid in human

urine samples
Standard added in
No. 1 samplea/
mg mL1
1.00
5.00
10.0

Recovery, %
Creatinine
87.3 88.2 87.4
99.2 100.1 102.2
95.4 96.1 96.7

Uric acid
97.4 97.8 97.3
104.9 107.7 108.6
105.3 105.1 104.2

a. Concentrations of standards are expressed as the equivalent

concentrations added in the final injected solutions.

Fig. 3 HPLC chromatogram of a human urine sample. (A) Without

the addition of an internal standard; (B) with the internal standard
hypoxanthine. Injection volume, 20 mL; hypoxanthine (internal
standard) concentration, 10.0 mg mL1.

preliminary assays, gradient elution with a small volume of

acetonitrile (less than 0.23 mL) was employed, following an
isocratic elution of analytes using an aqueous phosphate buffer
mobile phase in this study.
Figure 2 shows a chromatogram of a standard mixture of
creatinine and uric acid. A base-line separation can be achieved
in a short elution time of less than 10 min. Figures 3A and 3B
present a typical HPLC separation of a freshly colleted urine
sample without and with the addition of internal standard,
respectively. Chromatogram B was obtained through about 20
chromatographic runs after chromatogram A. No detectable
change in the retention time of the analytes has been observed.
The mean values of the retention time for creatinine, uric acid
and internal standard hypoxanthine were: creatinine, 2.970
0.031 min; uric acid, 3.920 0.038 min; and hypoxanthine,
5.540 0.054 min, respectively. The relative standard deviation
(RSD) values of the retention times and the peak areas were
generally smaller than 1%, indicating that the developed
separation method was very stable and had high reproducibility.
The chosen wavelength of 205 nm provides a higher sensitivity
with a clean chromatogram than the wavelength of 220 or 235
nm employed in previous studies.1921 The peak purity was
confirmed by constant signal ratios (relative absorbances at
different wavelength) across the peak profile of creatinine, uric
acid and the internal standard.
Quantitative analysis
The calibration curves for both creatinine and uric acid were
linear over the concentration ranges tested: 0.00 25.0 mg mL1.

A linear relationship between the ratio of the peak area of the

standards to that of the internal standard and the concentration
of the standards in aqueous solutions was obtained. For
creatinine, a typical calibration curve followed the equation
y = 0.083x + 0.0223, with the square of the correlation
coefficient being R2 = 0.9994, while for uric acid, y = 0.065x +
0.0147 with R2 = 0.9997 (y is the ratio of peak area of standards
to that of internal standard; x is the concentration). The
detection limits measured as three-times the background noise
were 0.045 mg mL1 creatinine and 0.062 mg mL1 uric acid.
Determination of creatinine and uric acid in human urine samples
Although several authors have described direct column
injection without the deproteinization step, an acid precipitation
of protein with phosphorous acid at pH 2.35 was employed in
this study, which could improve the specificity of the HPLC
measurement by removing protein-bound interferents. Excellent
selectivity and sensitivities were achieved for creatinine, uric
acid and hypoxanthine for human urine samples when this
sample pretreatment was coupled with the described HPLC
procedures (Figs. 3A and 3B). Creatinine and uric acid were
identified by matching the retention times against those of
authentic standards, and by spiking the urine samples with the
standard of each analyte. The concentrations of creatinine and
uric acid in tested human urine samples are given in Table 2.
The analytical recovery of the described method was tested by
adding three levels of known amounts of creatinine and uric acid
standards into the No. 1 urine sample, and the percentage
recoveries were found to be 87.3 102.2% for creatinine and
97.3 108.6% for uric acid, as shown in Table 3.

Conclusions
The described direct photometric HPLC method has proved to
be a rapid, sensitive, accurate and environmentally friendly
technique for the simultaneous determination of creatinine and

1592
uric acid in human urine. This method has successfully
prevented a retention loss of reversed-phase C18 column
encountered in previous studies using a highly aqueous mobile
phase, by introducing a brief gradient elution with a small
volume of acetonitrile (<0.23 mL), following analyte separation.
The method developed in this study could also be used in the
separation and quantitative measurement of creatinine and uric
acid in other biological fluids.

Acknowledgements
This work was partly supported by the Massachusetts Agriculture
Research Fund and the University of Massachusetts Dartmouth.

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