Rapid determination of creatinine in serum and urine by ion-pair high-performance liquid chromatography

  • R. Marsilio
  • R. Dall’Amico
  • D. Giordano
  • L. Murer
  • G. Montini
  • M. Ros
  • N. Dussini
  • G. Zacchello
  • L. Bacelle
  • M. Plebani


We report a simple and reliable high performance liquid chromatography method for measuring creatinine in serum and urine. The chromatographic run is performed on a C18 column after protein precipitation with acetone and addition of cimetidine as an internal standard. The separation is carried out in 20 min at a flow rate of 0.8 ml/min, with a mobile phase consisting of 100 mmol/l sodium dihydrogen phosphate solution, containing 30 mmol/l sodium lauryl sulfate pH 3.0 and acetonitrile (60:36, v/v). The absorbance is monitored at 200 nm. The relationship between creatinine concentration and the creatinine/internal standard peak area is linear up to 1,088 µmol/l. Within-run precision measured at three different creatinine concentrations ranges from 0.89% to 2.34% in serum and from 0.34% to 1.10% in urine. Between-run precision varies from 1.68% to 3.17% in serum and from 1.58% to 1.85% in urine over a wide range of concentrations. Analytical recovery is between 98.71% and 101.25% in serum and between 98.96% and 100.27% in urine. The detection limit is 3.24 µmol/l for a signal-to-noise ratio of 3. The method shows a good linearity with the reference isotope dilution gas chromatography-mass spectrometry procedure (r=0.999), without interferences, even in the presence of high bilirubin concentrations.

Key words

Creatinine High-performance liquid chromatography Gas chromatography-mass spectrometry 


  1. 1.
    Jaffé M. Ueber den Niederschlag, welchen Pikrinsäure in normalen Harn erzeugt und über eine neue Reaction des Kreatininis. Z Phisiol Chem 1886;10:391.Google Scholar
  2. 2.
    Owen JA, Iggo B, Scondrett FS, Steward CP. The determination of creatinine in plasma or serum and urine: a critical examination. Biochem J 1954;58:426.PubMedGoogle Scholar
  3. 3.
    Tager-Nilsson AC. Serum creatinine determination using an ion exchange resin. Scand J Clin Lab Invest 1961;13:326.CrossRefGoogle Scholar
  4. 4.
    Chromson A, Grady HJ, Stonley MA. Determination of creatinine by means of automatic chemical analysis. Am J Clin Pathol 1961;35:83.Google Scholar
  5. 5.
    Tanganelli E, Prencipe L, Bassi D, Cambiaghi S, Murador E. Enzymatic assay of creatinine in serum and urine with creatinine iminohydrolase and glutamate dehydrogenase. Clin Chem 1982;28:1461.PubMedGoogle Scholar
  6. 6.
    Toffaletti J, Blosser N, Hall T, Smith S, Tompkins D. An automated dry-slide enzymatic method evaluated for measurement of creatinine in serum. Clin Chem 1983;29:684.PubMedGoogle Scholar
  7. 7.
    Fossati P, Prencipe L, Berti G. Enzymatic creatinine assay: a new colorimetric method based an hydrogen peroxide measurement. Clin Chem 1983;29:1494.PubMedGoogle Scholar
  8. 8.
    Guder WG, Hoffmann GE, Hubbuch A, Poppe WA, Siedel J, Price CP. Multicentre evaluation of an enzymatic method for creatinine determination using a sensitive colour reagent. J Clin Chem Clin Biochem 1986;24:889.PubMedGoogle Scholar
  9. 9.
    Patel CP, George RC. Liquid chromatographic determination of creatinine in serum and urine. Anal Chem 1981;53:734.PubMedCrossRefGoogle Scholar
  10. 10.
    Ziemniak JA, Chiaromonte DA, Scentag JJ. Liquid chromatographic determination of cimetidine, its known metabolites, and creatinine in serum and urine. Clin Chem 1981;27:272.PubMedGoogle Scholar
  11. 11.
    Thienpont LM, Van Landuyt KG, Stöckl D, De Leenheer AP. Candidate reference method for determining serum creatinine by isocratic HPLC: validation with isotope dilution gas chromatography-mass spectrometry and application for accuracy assessment of routine test kits. Clin Chem 1995;41:995.PubMedGoogle Scholar
  12. 12.
    Lim CK, Richmond W, Robinson DP, Brown SS. Towards a definitive assay of creatinine in serum and urine: separation by a high performance liquid chromatography. J Chromatogr 1978;145:41.PubMedCrossRefGoogle Scholar
  13. 13.
    Lam RH, Tarding F. Specific high-performance liquid chromatographic method for the determination of creatinine in rat plasma. J Chromatogr 1988;426:358.PubMedCrossRefGoogle Scholar
  14. 14.
    Achari R, Mayerson M, Conrad KA. HPLC analysis of human plasma and urine. J Chromatogr Sci 1983;21:278.PubMedGoogle Scholar
  15. 15.
    Spierto FW, MacNeil ML, Culbreth P, Duncan I, Burtis CA. Development and validation of a liquid chromatographic procedure for serum creatinine. Clin Chem 1980;26:286.PubMedGoogle Scholar
  16. 16.
    Krstulovic AM, Bertani-Dziedic L, Caporusso JM. Rapid assessment of endogenous creatinine in physiological samples analyzed by reversed-phase liquid chromatography with spectroscopic detection. Clin Chim Acta 1979;99:189.PubMedCrossRefGoogle Scholar
  17. 17.
    Werner ER, Fuchs D, Hausen A, Reibenegger G, Wachter H. Simultaneous determination of neopterine and creatinine in serum with solid-phase extraction and on line elution liquid chromatography. Clin Chem 1987;33:2028.PubMedGoogle Scholar
  18. 18.
    Buchanan DN, Tait SK, Domino EF. High performance liquid chromatographic assay of creatinine in human urine. J Chromatogr 1979;163:212.PubMedCrossRefGoogle Scholar
  19. 19.
    Okuda T, Oie T, Nishida M. Liquid chromatographic measurement in plasma and urine. Clin Chem 1983;29:851.PubMedGoogle Scholar
  20. 20.
    A Zhiri, Houot O, Wellman-Bednawska M, Siest G. Simultaneous determination of uric acid and creatinine in plasma by reversed-phase liquid chromatography. Clin Chem 1985;31:109.PubMedGoogle Scholar
  21. 21.
    Bjerve KS, Egense J, Lampinen LM, Masson P. Evaluation of several creatinine methods in search of a suitable secondary reference method: report from the subcommittee on reference method for creatinine, Nordic Society for Clinical Chemistry. Scand J Clin Lab Invest 1988;48:365.PubMedCrossRefGoogle Scholar
  22. 22.
    Linnet K, Bruunshuus I. HPLC with enzymatic detection as a candidate reference for serum creatinine. Clin Chem 1991;37:1665.Google Scholar
  23. 23.
    Rosano TG, Ambrose RT, Wu AHB, Swift TA, Yadegari P. Candidate reference method for determining creatinine in serum: method development and interlaboratory validation. Clin Chem 1990;36:1951.PubMedGoogle Scholar
  24. 24.
    Ambrose RT, Ketchum DF, Smith JW. Creatinine determined by “high-performance” liquid chromatography. Clin Chem 1983;29:256.PubMedGoogle Scholar
  25. 25.
    Guy JM, Legg EF. An improved cation exchange HPLC method for the measurement of serum creatinine. Ann Clin Biochem 1990;27:223.PubMedGoogle Scholar
  26. 26.
    Scott PH. High-performance liquid chromatography measurement of plasma creatinine in newborns. Clin Chem 1992;38:101.PubMedGoogle Scholar
  27. 27.
    Harmoinen A, Sillanaukee P, Jokela H. Determination of creatinine in serum and urine by cation-exchange high-pressure liquid chromatography. Clin Chem 1991;37:563.PubMedGoogle Scholar
  28. 28.
    Soldin SS, Hill JG. Micromethod for determination of creatinine in biological fluids by high-performance liquid chromatography. Clin Chem 1978;24:747.PubMedGoogle Scholar
  29. 29.
    Murakita H. Simultaneous determination of creatine and creatinine in serum by high-performance liquid chromatography. J Chromatogr 1988;431:471.PubMedCrossRefGoogle Scholar
  30. 30.
    Paroni R, Arcelloni C, Fermo I, Bonini PA. Determination of creatinine in serum and urine by a rapid liquid-chromatographic method. Clin Chem 1990;36:830.PubMedGoogle Scholar
  31. 31.
    Ekelund S, Påby P. High-performance liquid chromatographic determination of creatinine. Scand J Clin Lab Invest 1991;51:67.PubMedCrossRefGoogle Scholar
  32. 32.
    Kock R, Seitz S, Delvoux B, Greiling H. A method for the simultaneous determination of creatinine and uric acid in serum by high-performance-liquid-chromatography evaluated versus reference methods. Eur J Clin Chem Clin Biochem 1995;33:23.PubMedGoogle Scholar
  33. 33.
    Björkhem I, Blomstrand R, Öhman G. Mass fragmentography of creatinine proposed as a reference method. Clin Chem 1977;23:2114.PubMedGoogle Scholar
  34. 34.
    Siekmann L. Determination of creatinine in human serum by isotope dilution-mass spectrometry. Definitive methods in clinical chemistry. IV. J Clin Chem Clin Biochem 1985;23:137.PubMedGoogle Scholar
  35. 35.
    Stöckl D, Reinauer H. Candidate reference methods for the determination of target values for cholesterol, creatinine, uric acid, and glucose in external quality assessment and internal accuracy control. I. Method setup. Clin Chem 1993;39:993.PubMedGoogle Scholar
  36. 36.
    Welch MJ, Cohen A, Hertz HS, Ng KJ, Shaffer R, VanDerLijn P, White EV. Determination of serum creatinine by isotope dilution mass spectrometry as a candidate definitive method. Anal Chem 1986;58:1681.PubMedCrossRefGoogle Scholar
  37. 37.
    Thienpont LM, Van Nieuwenhove B, Stockl D, Reinauer H, De Leenheer AP. Determination of reference method values by isotope dilution-gas chromatography/mass spectrometry: a five years’ experience of two European Reference Laboratories. Eur J Clin Chem Clin Biochem 1996;34:853.PubMedGoogle Scholar
  38. 38.
    Molnar I, Horvath C. Separation of amino acids and peptides on non polar stationary phases by high-performance liquid chromatography. J Chromatogr 1977;142:623.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1999

Authors and Affiliations

  • R. Marsilio
    • 1
  • R. Dall’Amico
    • 1
  • D. Giordano
    • 1
  • L. Murer
    • 1
  • G. Montini
    • 1
  • M. Ros
    • 1
  • N. Dussini
    • 1
  • G. Zacchello
    • 1
  • L. Bacelle
    • 2
  • M. Plebani
    • 2
  1. 1.Department of PediatricsUniversity Hospital of PaduaPaduaItaly
  2. 2.Department of Laboratory MedicineUniversity of PaduaPaduaItaly

Personalised recommendations