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Simultaneous determination of uric acid and creatinine in urine by an eco-friendly solvent-free high performance liquid chromatographic method
Talanta 58 (2002) 711 /717 www.elsevier.com/locate/talanta
Simultaneous determination of uric acid and creatinine in urine by an eco-friendly solvent-free high performance liquid chromatographic method Jen-Fon Jen *, Shih-Liang Hsiao, Kang-Hsiung Liu Department of Chemistry, National Chung-Hsing University, Taichung 40217, Taiwan, ROC Received 6 March 2002; received in revised form 18 June 2002; accepted 1 July 2002
Abstract A simple, rapid, and eco-friendly analytical method for simultaneous determination of uric acid and creatinine in urine applying high performance liquid chromatography (HPLC) is described. After dilution, de-protein, and filtration, the sample solution was injected to separate the species with C-18 column by an eluent containing 0.05 M ammonium phosphate buffer at pH 7.4. An UV detector was used to monitor the separation of species at 235 nm. Optimum conditions for separation and detection were investigated. Results indicated that under optimized condition measurements were achieved within 13 min. The detection limits were 0.127 and 0.156 mg ml 1 for uric acid and creatinine respectively. The recovery was 95% (0.57% RSD) for uric acid and 99.2% (0.98% RSD) for creatinine, from five measurements. Real urine specimens were tested. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Uric acid; Creatinine; Environmental-friendly; High performance liquid chromatography
1. Introduction Uric acid is the final product of catabolization of the purine nucleosides. Excess uric acid in urine is considered as a key factor in the development of renal calculus. Traditionally, uric acid is often quantified colorimetrically [1]. However, this approach is problematic due to the interference of other compounds present in biological fluids. Creatinine results from the irreversible, non-enzy-
* Corresponding author. Tel.: /886-4-2285-3148; fax: / 886-4-2286-2547 E-mail address: [email protected] (J.-F. Jen).
matic dehydration and loss of phosphate from phosphocreatine [2]. It is the most widely used clinical marker to assess renal function [3,4]. The determination method for creatinine widely accepted is Jaffe alkaline picrate procedure [5]. However, in addition to being time-consuming, this method has been reported to lead to overestimates due to interference by endogenous and exogenous pseudo-creatinine chromogens [4]. Besides, enzymatic assays have also been developed to enhance specificity for creatinine. But, these methods are still not free from interference. In the past decade, the use of chromatography and electrophoresis has been increasingly attractive for both uric acid and creatinine, with advantages
0039-9140/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 2 ) 0 0 3 7 7 - 6
712
J.-F. Jen et al. / Talanta 58 (2002) 711 /717
of simultaneous measurements and eliminating interfering species [6 /15]. In reverse-phase HPLC, organic solvents such as acetonitrile and methanol are generally used to achieve the separation of analytes. Since the organic solvents are considered as significant pollutants, some RP-HPLC techniques without the requirement of organic solvent in the mobile phase have thus eagerly been developed recently [16 /20]. Approaching this concept, Kock et al. [21] achieved the separation of uric acid and some bio-species within 20 min by using pH 4.60 phosphate buffer, with the requirement of replacing the guard column after every 30 injection. Through this process met the eco-friendly requirement, laboratory conditions seldom accept the frequent change of high-cost guard column. Chen et al. [10] determined uric acid and creatinine in urine by using C-18 column eluting with acetate buffer at pH 4.5. Although uric acid got eluted at 10 min, it took 45 min for one analytical run due to extensive washing and re-equilibration of the column. In the washing step, methanol was used. In HPLC, the presence of protein in the injected samples can cause modification of the column end in biased analytical results. However, the removal of protein from specimens is often the most time consuming step in the analysis. Because the extraction methods such as solvent, solid-phase, and Soxhlet extractions require large quantities of organic solvents, thus, the protein precipitation, centrifugation, filtration, and column switching or pre-column methods are often applied to remove interferences from protein. Despite, the time-expense is still the serious disadvantage. This paper describes a simple, rapid, precise, and eco-friendly analytical method for simultaneous determination of uric acid and creatinine in human urine specimens. The acid precipitation posterior to dilution and the filtration results in a satisfactory removal of protein from samples. The readily available C-18 stationary phase offers the retention and resolution of uric acid and creatinine only with a controlled pH aqueous buffer eluent.
2. Experimental section
2.1. Apparatus The HPLC employed in this work was a Shimadzu LC-9A system (Shimadzu, Kyoto, Japan), and a Waters 484 tunable absorbance detector (Waters, Milford, MA) with a 20-mlflow cell. The detection wavelength was set at 235 nm. A Supelcosil reverse-phase C-18 column (25 cm /4.6 mm id. 5 mm particle size) (Supelco, Bellefonte, PA) was used for separation. A RP-18 guard column was fitted up-stream of the analytical column. A Rheodyne 7125 sample injector (Cotati, CA) with a 20-ml external loop was used for sample introduction. A Chromatocorder 12 Integrator (SIC, Tokyo, Japan) was used to obtain the chromatogram, and perform data calculations. The U-3000 UV-VIS spectrophotometer (Hitachi, Tokyo, Japan) was used to obtain the spectra of uric acid and creatinine.
2.2. Chemicals and reagents Deionized water was produced using a Barnstead Nanopure Series 550 system (Barnstead, NY) for all aqueous solutions. All chemicals and solvents were of ACS reagent grade. Standard stock solutions (200 mg ml 1) of uric acid (Sigma, St. Louis, MO) and creatinine (Merck, Darmstadt, Germany) were prepared by dissolving 0.020 g in 50 ml water (for uric acid, pH 10.3) and then adding water to adjust the volume to 100 ml. The solutions were stored in brown glass bottles and kept at 4 8C. Fresh working solutions were prepared by appropriate dilution of the stock solutions before use. Phosphorous acid, sodium hydroxide, ammonium hydroxide, and ammonium hydrogen phosphate (Riedel-deHae¨n, Hanover, Germany) were used for the pH adjustments. The HPLC eluent was prepared as ammonium phosphate buffer solution (0.05 M) of pH 7.4 and it was filtered through a 0.45-mm poly (vinylidene difluoride) (PVDF) membrane filter and degassed ultrasonically.
J.-F. Jen et al. / Talanta 58 (2002) 711 /717
2.3. Sample preparation Urine sample was diluted by 200-fold and acidified to pH 2.24 with phosphorous acid to precipitate protein. A 2-ml aliquot of this mixture was passed through a 0.45-mm poly (vinylidene difluoride) (PVDF) disc filter and the filtrate was collected for HPLC analysis.
713
well as the reproducibility of qualitative and quantitative detection were studied thoroughly to optimize analytical conditions.
3. Results and discussions In order to verify the applicability of the ecofriendly analytical protocol, in addition to sample preparation, selections of separation column, detection wavelength, and the pH of mobile phase as
Fig. 1. The UV spectra of uric acid and creatinine in mobile phase solution. Spectrum: Solid-line for creatinine; dashed-line for uric acid; Concentration: 20 mg ml 1 each in pH 7.4 ammonium phosphate buffer solution; Scan speed: 600 nm min 1; Slit: 0.5 nm.
Fig. 2. Chromatogram for standard species of uric acid and creatinine. Mobile phase: 0.05 M ammonium phosphate buffer (pH 7.4) at 1.0 ml min 1 flow-rate. Concentration of uric acid and creatinine: 10 mg l 1 each for 20-ml injection; Peak 1: uric acid; Peak 2: creatinine.
J.-F. Jen et al. / Talanta 58 (2002) 711 /717
714
3.1. Pretreatment of urine specimens
3.2. Selection of column and detection wavelength
Having aimed at eco-friendly analytical protocol, the urine specimens are pretreated without organic solvents. After a series of tests, the acid precipitation of protein not only increases the resolution of peaks in chromatogram, it also increases the sensitivity of peak response. It depicts that the bound-uric acid and creatinine have been released in the acidic circumstance (pH 2.24). As the detection sensitivity is high enough (LODs are in sub-mg ml 1), the urine sample is diluted by 200-fold with pH 2.24 phosphorous acid solution. Accompanied by the filtration over 0.45-mm PVDF disc filter for further removal of particles in acidic diluted sample solution, the pressure of the HPLC system did not have any significant increase after several-hundred injections through the studies. It indicates that the eco-friendly pretreatment procedure is appropriate for urine specimens.
Referred to the literature [21], a reversed-phase C-18 column has the potential to resolute uric acid and creatinine from other species very well. In order to obtain the highest detection sensitivity, the wavelength of detector is better set at or near the lmax. Fig. 1 demonstrates the UV spectra of uric acid and creatinine in the mobile phase solution. It can be seen, two characteristic absorption peaks appear at 290 and 235 nm for uric acid; and the creatinine has characteristic absorption at 232 nm. Hence, the wavelength of 235 nm was selected for the detection of both species. 3.3. Selection of mobile phase For the better conditions of an eco-friendly analytical protocol, the mobile phase should be either free from or in low level of organic solvents. Thus, an aqueous buffer solution was tested to be
Table 1 Reproducibility of the analytical method for uric acid and creatinine 0.25 mg ml 1
25 mg ml 1
Retention time (min)
Peak area (counts)
Retention time (min)
Peak area (counts)
Uric acid Average RSD%
3.56 0.65
16 365 1.28
3.57 0.42
1 442 768 0.79
Creatinine Average RSD%
4.82 1.15
17 668 1.39
4.82 0.32
1 810 279 1.06
Table 2 Calibration plots and standard addition plots for the proposed HPLC method Analyte
Regression equations
Linear coefficient
Concentration (mg ml 1)
Calibration plot Uric acid Creatinine
Y /58 258X/2627 Y /72 401X/1926
0.9999 0.9999
0.25 /25 0.25 /25
0.9999 0.9999
0 /40 (spiked) 0 /40 (spiked)
Standard addition Uric acid Y /59 903X/49 133 Creatinine Y /71 514X/176 255
Samples for the calibration plots of standard addition were spiked standard samples in sample No.2.
J.-F. Jen et al. / Talanta 58 (2002) 711 /717
the eluent. Because the C-18 stationary phase is hydrophobic, it retains the less-hydrophilic species, and the ionic analytes are hardly retained in the RP-column. Therefore, an eluent of modest elution strength with a pH to keeping the analytes in their molecular-forms is required. Being the pKa of uric acid and creatinine 5.40 and 3.55 respectively, the pH of eluent should be controlled as higher than the pH as possible to keep them in neutral forms. Considering the pH restriction of a silica-based column, the pH of eluent was selected at pH 7.40. From series of tests, the optimum separation and detection are achieved by eluting with 0.05 M ammonium phosphate buffer (pH 7.4) at 1.0 ml min 1 flow rate and monitoring at 235 nm by UV detector. The chromatograms for standard species of uric acid (peak 1) and creatinine (peak 2) are shown in Fig. 2. It can be seen, both species give sharp and symmetric peaks and well separated within 12 min. Here, it is worthy to mention that the separation was achieved only by using pH 7.4 phosphate buffer solution meeting the eco-friendly policy.
715
cients (g) are shown in Table 2. It can be seen, both calibration plots are with very good linear relationship (g /0.9999) in the concentration between 0.25 and 25 mg ml 1. The standard addition method with adding varied concentrations of analytes into dilute urine sample was used to determine chemical interference. The slopes found
3.4. Reproducibility Two different concentrations (0.25 and 25 mg ml 1) of uric acid and creatinine were injected seven times (200 ml sample solution each through the 20-ml sample loop) for testing the reproducibility of the proposed method. From the results listed in the Table 1, it is obvious that whatever be the qualitative detection (retention time) and the quantitative measurement (peak area) for uric acid and creatinine, the relative standard deviations are very low in both concentrations. It indicates that the reproducibility of the proposed ecofriendly analytical method is consistent and is acceptable. 3.5. Calibration plots of uric acid and creatinine Calibration plots of uric acid and creatinine measured by the proposed method were built-up in the range of 0.25 /25 mg ml 1. Linear-relationships were checked between the peak areas of the analytes and concentrations. The slopes and yintercepts of the regression, and correlation coeffi-
Fig. 3. Chromatogram for real samples. Mobile phase: 0.05 M ammonium phosphate buffer (pH 7.4) at 1.0 ml min 1 flowrate. Sample injection: 200 ml into the 20-ml sample loop; Peak 1: uric acid; Peak 2: creatinine.
J.-F. Jen et al. / Talanta 58 (2002) 711 /717
716
Table 3 Analysis results of real samples Specimens
No.1 (male) Uric acid 1
Content (mg ml ) RSD% Uric acid/creatinine
152 0.39 0.49
Creatinine 311 0.45
for the calibration and standard addition plots were similar for both analytes when the error range of 5% was taken into account. It indicates that no significant interference occurred in the measurements of both species. Detection limits (LODs) were evaluated at a signal-to-noise ratio of 3. The LODs of uric acid and creatinine in the standard solutions were 0.13 and 0.16 mg ml 1, respectively.
No.2 (male) Uric acid 858 0.49 0.44
Creatinine 1930 0.26
No.3 (female) Uric acid 223 0.29 0.52
Creatinine 432 0.33
4. Conclusion This paper investigated the potential of an ecofriendly analytical protocol to measure uric acid and creatinine by using HPLC-UV. With the traditional C-18 column, where no organic solvent was used to elute uric acid and creatinine, as well as in sample pretreatment. The results confirm that the proposed method can be a reference method to determine the uric acid and creatinine in urine with the advantages of easy operation, high recovery, less time-expense, and solvent-less.
3.6. Analysis of real samples and recovery In order to test the applicability of the proposed method in the determination of uric acid and creatinine in real samples, three urine specimens from students (two males and one female) were determined. It can be seen, the peaks of uric acid (peak 1) and creatinine (peak 2) were well separated from other peaks (Fig. 3), and no interference occurred. Results are listed in Table 3. The concentration of uric acid and creatinine in varied specimens were much different due to the varied amounts of water in-take. However, the ratio of uric acid to creatinine was similar in the range of 0.44 /0.52. The analytical recovery by the proposed method was evaluated by assaying urine sample spiked with standard solution. Analyses were done in five measurements for the specimen No.2 after being spiked uric acid 400 mg ml 1 and creatinine 800 mg ml 1. The observed value for the untreated sample was subtracted from that of the spiked sample. The recoveries for uric acid and creatinine were 95.0% with 0.57% RSD and 99.2% with 0.98% RSD, respectively.
Acknowledgements The authors thank the National Science Council of the R.O.C. (Taiwan) for financial support under the grant number NSC-90-2113-M-005-024.
References [1] M.A. Razzaque, J.H. Topps, J. Sci. Fd. Agric. 29 (1978) 935. [2] R.K. Murray, D.K. Granner, P.A. Mayes, V.W. Rodwell, Harper’s Biochemistry, 24th Edition, Appleton and Lange, Stanford, CT, 1996, p. 341. [3] A.S. Levey, R.D. Perrone, N.E. Madias, Annu. Rev. Med. 39 (1998) 465. [4] S. Narayanan, H.D. Appleton, Clin. Chem. 26 (1980) 1119. [5] M.H. Kroll, R. Chesler, C. Hagengruber, D.W. Blank, J. Kestner, M. Rawe, Clin. Chem. 32 (1986) 446. [6] G. Werner, V. Schneider, J. Emmert, J. Chromatogr. 525 (1990) 265. [7] Y. Yokoyama, M. Tsuchiya, H. Sato, H. Kakinuma, J. Chromatogr. 583 (1992) 1. [8] Y. Li, S. Wang, N. Zhong, Biomed. Chromatogr. 6 (1992) 191.
J.-F. Jen et al. / Talanta 58 (2002) 711 /717 [9] T. Seki, Y. Orita, K. Yamaji, S. Moriguchi, A. Shinoda, J. Chromatogr. A 730 (1996) 139. [10] Y.-M. Chen, R.A. Pietrzyk, P.A. Whitson, J. Chromatogr. A 763 (1997) 187. [11] K.J. Shingfield, N.W. Offer, J. Chromatogr. B 723 (1999) 81. [12] T. Fujii, S. Kawabe, T. Horike, T. Taguchi, M. Ogata, J. Chromatogr. B 730 (1999) 41. [13] C.J. Kochansky, T.G. Strein, J. Chromatogr. B 747 (2000) 217. [14] A. Schmutz, W. Thormann, Electrophoresis 15 (1994) 51.
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[15] M.K. Shirao, S. Suzuki, J. Kobayashi, H. Nakazawa, E. Mochizuki, J. Chromatogr. B 693 (1997) 463. [16] M.D. Foster, R.E. Synovec, Anal. Chem. 68 (1996) 2838. [17] R.M. Smith, R.J. Burgess, J. Chromatogr. A 785 (1997) 49. [18] D.J. Miller, S.B. Hawthorne, Anal. Chem. 69 (1997) 623. [19] W. Hu, P.R. Haddad, Anal. Commun. 35 (1998) 49. [20] T. Umemura, K. Tsunoda, A. Koide, T. Oshima, N. Watanabe, K. Chiba, H. Haraguchi, Anal. Chim. Acta 419 (2000) 87. [21] R. Kock, B. Delvoux, H. Greiling, Eur. J. Clin. Chem. Clin. Biochem. 31 (1993) 303.
Simultaneous determination of uric acid and creatinine in urine by an eco-friendly solvent-free high performance liquid chromatographic method Jen-Fon Jen *, Shih-Liang Hsiao, Kang-Hsiung Liu Department of Chemistry, National Chung-Hsing University, Taichung 40217, Taiwan, ROC Received 6 March 2002; received in revised form 18 June 2002; accepted 1 July 2002
Abstract A simple, rapid, and eco-friendly analytical method for simultaneous determination of uric acid and creatinine in urine applying high performance liquid chromatography (HPLC) is described. After dilution, de-protein, and filtration, the sample solution was injected to separate the species with C-18 column by an eluent containing 0.05 M ammonium phosphate buffer at pH 7.4. An UV detector was used to monitor the separation of species at 235 nm. Optimum conditions for separation and detection were investigated. Results indicated that under optimized condition measurements were achieved within 13 min. The detection limits were 0.127 and 0.156 mg ml 1 for uric acid and creatinine respectively. The recovery was 95% (0.57% RSD) for uric acid and 99.2% (0.98% RSD) for creatinine, from five measurements. Real urine specimens were tested. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Uric acid; Creatinine; Environmental-friendly; High performance liquid chromatography
1. Introduction Uric acid is the final product of catabolization of the purine nucleosides. Excess uric acid in urine is considered as a key factor in the development of renal calculus. Traditionally, uric acid is often quantified colorimetrically [1]. However, this approach is problematic due to the interference of other compounds present in biological fluids. Creatinine results from the irreversible, non-enzy-
* Corresponding author. Tel.: /886-4-2285-3148; fax: / 886-4-2286-2547 E-mail address: [email protected] (J.-F. Jen).
matic dehydration and loss of phosphate from phosphocreatine [2]. It is the most widely used clinical marker to assess renal function [3,4]. The determination method for creatinine widely accepted is Jaffe alkaline picrate procedure [5]. However, in addition to being time-consuming, this method has been reported to lead to overestimates due to interference by endogenous and exogenous pseudo-creatinine chromogens [4]. Besides, enzymatic assays have also been developed to enhance specificity for creatinine. But, these methods are still not free from interference. In the past decade, the use of chromatography and electrophoresis has been increasingly attractive for both uric acid and creatinine, with advantages
0039-9140/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 2 ) 0 0 3 7 7 - 6
712
J.-F. Jen et al. / Talanta 58 (2002) 711 /717
of simultaneous measurements and eliminating interfering species [6 /15]. In reverse-phase HPLC, organic solvents such as acetonitrile and methanol are generally used to achieve the separation of analytes. Since the organic solvents are considered as significant pollutants, some RP-HPLC techniques without the requirement of organic solvent in the mobile phase have thus eagerly been developed recently [16 /20]. Approaching this concept, Kock et al. [21] achieved the separation of uric acid and some bio-species within 20 min by using pH 4.60 phosphate buffer, with the requirement of replacing the guard column after every 30 injection. Through this process met the eco-friendly requirement, laboratory conditions seldom accept the frequent change of high-cost guard column. Chen et al. [10] determined uric acid and creatinine in urine by using C-18 column eluting with acetate buffer at pH 4.5. Although uric acid got eluted at 10 min, it took 45 min for one analytical run due to extensive washing and re-equilibration of the column. In the washing step, methanol was used. In HPLC, the presence of protein in the injected samples can cause modification of the column end in biased analytical results. However, the removal of protein from specimens is often the most time consuming step in the analysis. Because the extraction methods such as solvent, solid-phase, and Soxhlet extractions require large quantities of organic solvents, thus, the protein precipitation, centrifugation, filtration, and column switching or pre-column methods are often applied to remove interferences from protein. Despite, the time-expense is still the serious disadvantage. This paper describes a simple, rapid, precise, and eco-friendly analytical method for simultaneous determination of uric acid and creatinine in human urine specimens. The acid precipitation posterior to dilution and the filtration results in a satisfactory removal of protein from samples. The readily available C-18 stationary phase offers the retention and resolution of uric acid and creatinine only with a controlled pH aqueous buffer eluent.
2. Experimental section
2.1. Apparatus The HPLC employed in this work was a Shimadzu LC-9A system (Shimadzu, Kyoto, Japan), and a Waters 484 tunable absorbance detector (Waters, Milford, MA) with a 20-mlflow cell. The detection wavelength was set at 235 nm. A Supelcosil reverse-phase C-18 column (25 cm /4.6 mm id. 5 mm particle size) (Supelco, Bellefonte, PA) was used for separation. A RP-18 guard column was fitted up-stream of the analytical column. A Rheodyne 7125 sample injector (Cotati, CA) with a 20-ml external loop was used for sample introduction. A Chromatocorder 12 Integrator (SIC, Tokyo, Japan) was used to obtain the chromatogram, and perform data calculations. The U-3000 UV-VIS spectrophotometer (Hitachi, Tokyo, Japan) was used to obtain the spectra of uric acid and creatinine.
2.2. Chemicals and reagents Deionized water was produced using a Barnstead Nanopure Series 550 system (Barnstead, NY) for all aqueous solutions. All chemicals and solvents were of ACS reagent grade. Standard stock solutions (200 mg ml 1) of uric acid (Sigma, St. Louis, MO) and creatinine (Merck, Darmstadt, Germany) were prepared by dissolving 0.020 g in 50 ml water (for uric acid, pH 10.3) and then adding water to adjust the volume to 100 ml. The solutions were stored in brown glass bottles and kept at 4 8C. Fresh working solutions were prepared by appropriate dilution of the stock solutions before use. Phosphorous acid, sodium hydroxide, ammonium hydroxide, and ammonium hydrogen phosphate (Riedel-deHae¨n, Hanover, Germany) were used for the pH adjustments. The HPLC eluent was prepared as ammonium phosphate buffer solution (0.05 M) of pH 7.4 and it was filtered through a 0.45-mm poly (vinylidene difluoride) (PVDF) membrane filter and degassed ultrasonically.
J.-F. Jen et al. / Talanta 58 (2002) 711 /717
2.3. Sample preparation Urine sample was diluted by 200-fold and acidified to pH 2.24 with phosphorous acid to precipitate protein. A 2-ml aliquot of this mixture was passed through a 0.45-mm poly (vinylidene difluoride) (PVDF) disc filter and the filtrate was collected for HPLC analysis.
713
well as the reproducibility of qualitative and quantitative detection were studied thoroughly to optimize analytical conditions.
3. Results and discussions In order to verify the applicability of the ecofriendly analytical protocol, in addition to sample preparation, selections of separation column, detection wavelength, and the pH of mobile phase as
Fig. 1. The UV spectra of uric acid and creatinine in mobile phase solution. Spectrum: Solid-line for creatinine; dashed-line for uric acid; Concentration: 20 mg ml 1 each in pH 7.4 ammonium phosphate buffer solution; Scan speed: 600 nm min 1; Slit: 0.5 nm.
Fig. 2. Chromatogram for standard species of uric acid and creatinine. Mobile phase: 0.05 M ammonium phosphate buffer (pH 7.4) at 1.0 ml min 1 flow-rate. Concentration of uric acid and creatinine: 10 mg l 1 each for 20-ml injection; Peak 1: uric acid; Peak 2: creatinine.
J.-F. Jen et al. / Talanta 58 (2002) 711 /717
714
3.1. Pretreatment of urine specimens
3.2. Selection of column and detection wavelength
Having aimed at eco-friendly analytical protocol, the urine specimens are pretreated without organic solvents. After a series of tests, the acid precipitation of protein not only increases the resolution of peaks in chromatogram, it also increases the sensitivity of peak response. It depicts that the bound-uric acid and creatinine have been released in the acidic circumstance (pH 2.24). As the detection sensitivity is high enough (LODs are in sub-mg ml 1), the urine sample is diluted by 200-fold with pH 2.24 phosphorous acid solution. Accompanied by the filtration over 0.45-mm PVDF disc filter for further removal of particles in acidic diluted sample solution, the pressure of the HPLC system did not have any significant increase after several-hundred injections through the studies. It indicates that the eco-friendly pretreatment procedure is appropriate for urine specimens.
Referred to the literature [21], a reversed-phase C-18 column has the potential to resolute uric acid and creatinine from other species very well. In order to obtain the highest detection sensitivity, the wavelength of detector is better set at or near the lmax. Fig. 1 demonstrates the UV spectra of uric acid and creatinine in the mobile phase solution. It can be seen, two characteristic absorption peaks appear at 290 and 235 nm for uric acid; and the creatinine has characteristic absorption at 232 nm. Hence, the wavelength of 235 nm was selected for the detection of both species. 3.3. Selection of mobile phase For the better conditions of an eco-friendly analytical protocol, the mobile phase should be either free from or in low level of organic solvents. Thus, an aqueous buffer solution was tested to be
Table 1 Reproducibility of the analytical method for uric acid and creatinine 0.25 mg ml 1
25 mg ml 1
Retention time (min)
Peak area (counts)
Retention time (min)
Peak area (counts)
Uric acid Average RSD%
3.56 0.65
16 365 1.28
3.57 0.42
1 442 768 0.79
Creatinine Average RSD%
4.82 1.15
17 668 1.39
4.82 0.32
1 810 279 1.06
Table 2 Calibration plots and standard addition plots for the proposed HPLC method Analyte
Regression equations
Linear coefficient
Concentration (mg ml 1)
Calibration plot Uric acid Creatinine
Y /58 258X/2627 Y /72 401X/1926
0.9999 0.9999
0.25 /25 0.25 /25
0.9999 0.9999
0 /40 (spiked) 0 /40 (spiked)
Standard addition Uric acid Y /59 903X/49 133 Creatinine Y /71 514X/176 255
Samples for the calibration plots of standard addition were spiked standard samples in sample No.2.
J.-F. Jen et al. / Talanta 58 (2002) 711 /717
the eluent. Because the C-18 stationary phase is hydrophobic, it retains the less-hydrophilic species, and the ionic analytes are hardly retained in the RP-column. Therefore, an eluent of modest elution strength with a pH to keeping the analytes in their molecular-forms is required. Being the pKa of uric acid and creatinine 5.40 and 3.55 respectively, the pH of eluent should be controlled as higher than the pH as possible to keep them in neutral forms. Considering the pH restriction of a silica-based column, the pH of eluent was selected at pH 7.40. From series of tests, the optimum separation and detection are achieved by eluting with 0.05 M ammonium phosphate buffer (pH 7.4) at 1.0 ml min 1 flow rate and monitoring at 235 nm by UV detector. The chromatograms for standard species of uric acid (peak 1) and creatinine (peak 2) are shown in Fig. 2. It can be seen, both species give sharp and symmetric peaks and well separated within 12 min. Here, it is worthy to mention that the separation was achieved only by using pH 7.4 phosphate buffer solution meeting the eco-friendly policy.
715
cients (g) are shown in Table 2. It can be seen, both calibration plots are with very good linear relationship (g /0.9999) in the concentration between 0.25 and 25 mg ml 1. The standard addition method with adding varied concentrations of analytes into dilute urine sample was used to determine chemical interference. The slopes found
3.4. Reproducibility Two different concentrations (0.25 and 25 mg ml 1) of uric acid and creatinine were injected seven times (200 ml sample solution each through the 20-ml sample loop) for testing the reproducibility of the proposed method. From the results listed in the Table 1, it is obvious that whatever be the qualitative detection (retention time) and the quantitative measurement (peak area) for uric acid and creatinine, the relative standard deviations are very low in both concentrations. It indicates that the reproducibility of the proposed ecofriendly analytical method is consistent and is acceptable. 3.5. Calibration plots of uric acid and creatinine Calibration plots of uric acid and creatinine measured by the proposed method were built-up in the range of 0.25 /25 mg ml 1. Linear-relationships were checked between the peak areas of the analytes and concentrations. The slopes and yintercepts of the regression, and correlation coeffi-
Fig. 3. Chromatogram for real samples. Mobile phase: 0.05 M ammonium phosphate buffer (pH 7.4) at 1.0 ml min 1 flowrate. Sample injection: 200 ml into the 20-ml sample loop; Peak 1: uric acid; Peak 2: creatinine.
J.-F. Jen et al. / Talanta 58 (2002) 711 /717
716
Table 3 Analysis results of real samples Specimens
No.1 (male) Uric acid 1
Content (mg ml ) RSD% Uric acid/creatinine
152 0.39 0.49
Creatinine 311 0.45
for the calibration and standard addition plots were similar for both analytes when the error range of 5% was taken into account. It indicates that no significant interference occurred in the measurements of both species. Detection limits (LODs) were evaluated at a signal-to-noise ratio of 3. The LODs of uric acid and creatinine in the standard solutions were 0.13 and 0.16 mg ml 1, respectively.
No.2 (male) Uric acid 858 0.49 0.44
Creatinine 1930 0.26
No.3 (female) Uric acid 223 0.29 0.52
Creatinine 432 0.33
4. Conclusion This paper investigated the potential of an ecofriendly analytical protocol to measure uric acid and creatinine by using HPLC-UV. With the traditional C-18 column, where no organic solvent was used to elute uric acid and creatinine, as well as in sample pretreatment. The results confirm that the proposed method can be a reference method to determine the uric acid and creatinine in urine with the advantages of easy operation, high recovery, less time-expense, and solvent-less.
3.6. Analysis of real samples and recovery In order to test the applicability of the proposed method in the determination of uric acid and creatinine in real samples, three urine specimens from students (two males and one female) were determined. It can be seen, the peaks of uric acid (peak 1) and creatinine (peak 2) were well separated from other peaks (Fig. 3), and no interference occurred. Results are listed in Table 3. The concentration of uric acid and creatinine in varied specimens were much different due to the varied amounts of water in-take. However, the ratio of uric acid to creatinine was similar in the range of 0.44 /0.52. The analytical recovery by the proposed method was evaluated by assaying urine sample spiked with standard solution. Analyses were done in five measurements for the specimen No.2 after being spiked uric acid 400 mg ml 1 and creatinine 800 mg ml 1. The observed value for the untreated sample was subtracted from that of the spiked sample. The recoveries for uric acid and creatinine were 95.0% with 0.57% RSD and 99.2% with 0.98% RSD, respectively.
Acknowledgements The authors thank the National Science Council of the R.O.C. (Taiwan) for financial support under the grant number NSC-90-2113-M-005-024.
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