Methods for Quantitative Creatinine Determination

John F. Moore1, J. Daniel Sharer1

1 Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Appendix 3O
DOI:  10.1002/cphg.38
Online Posting Date:  April, 2017
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Reliable measurement of creatinine is necessary to assess kidney function, and also to quantitate drug levels and diagnostic compounds in urine samples. The most commonly used methods are based on the Jaffe principal of alkaline creatinine‐picric acid complex color formation. However, other compounds commonly found in serum and urine may interfere with Jaffe creatinine measurements. Therefore, many laboratories have made modifications to the basic method to remove or account for these interfering substances. This appendix will summarize the basic Jaffe method, as well as a modified, automated version. Also described is a high performance liquid chromatography (HPLC) method that separates creatinine from contaminants prior to direct quantification by UV absorption. Lastly, a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method is described that uses stable isotope dilution to reliably quantify creatinine in any sample. This last approach has been recommended by experts in the field as a means to standardize all quantitative creatinine methods against an accepted reference. © 2017 by John Wiley & Sons, Inc.

Keywords: creatinine; glomerular filtration rate; HPLC; LC‐MS/MS; picric acid

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Table of Contents

  • Introduction
  • Basic Protocol 1: Manual Picric Acid Assay
  • Basic Protocol 2: Automated Picric Acid Assay
  • Basic Protocol 3: Creatinine Determination by HPLC with UV Detection
  • Basic Protocol 4: Creatinine Determination: LC‐MS/MS Method
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Manual Picric Acid Assay

  • Urine samples
  • 1.3% picric acid, saturated solution in water (Sigma‐Aldrich)
  • 0.75 N sodium hydroxide (NaOH) solution (6 g in 200 ml water)
  • Urine chemistry control (human; Sigma‐Aldrich)
  • Creatinine standard solution, 100 mg/ml in 0.1 N HCl

Basic Protocol 2: Automated Picric Acid Assay

  • Creatinine Reagent kit (Peripheral Visions)
  • Creatinine standards at 3 and 10 mg/dl
  • Quality control (QC; 50 pooled remnant samples of known creatinine concentration)
  • Pipet tips (Peripheral Visions)
  • Creatinine Analyzer 2 (Beckman‐Coulter)

Basic Protocol 3: Creatinine Determination by HPLC with UV Detection

  • Urine samples
  • Serum or plasma samples
  • 1‐Octanesulfonic acid, sodium salt, 10 mmol/liter (2.2 g/liter) in 95:5 water/acetonitrile
  • Orthophosphoric acid
  • Creatinine standards, 0 to 200 µmol/liter
  • Pharmacia LKB Model 2248 pump
  • Kratos Analytical Model Spectroflow 783 detector (other HPLC/detector systems can be used, although results may vary)
  • Nucleosil 120 × 3 mm C 18 analytical column (Macherey‐Nagel)

Basic Protocol 4: Creatinine Determination: LC‐MS/MS Method

  • d 3‐Creatinine (ISTD; CDN Isotopes)
  • Creatinine
  • Acetonitrile
  • Ammonium acetate
  • Quality control (QC; pooled remnant samples)
  • HPLC, model Alliance 2795 (Waters)
  • Tandem mass (MS/MS) spectrometer, model Quattro Micro API (Waters; other HPLC/MS/MS systems than those referenced here can be used, although results may vary)
  • HPLC column, TSK gel Amide‐80 (Tosoh Biosciences)
  • Costar Spin‐X HPLC microcentrifuge filter (Corning)
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Literature Cited

Literature Cited
  Bartels, E., & Cikes, M. (1969). Über Chromogene der Kreatininbstimmung nach Jaffe [Chromogens in the creatinine determination by Jaffe]. Clinica Chimica Acta, 26, 1–10. doi: 10.1016/0009‐8981(69)90278‐2.
  Diskin, C. (2006). Creatinine and glomerular filtration rate: An imperfect marriage of convenience. Nephrology Dialysis Transplantation, 21, 3338–3339. doi: 10.1093/ndt/gfl374.
  Fabiny, D., & Ertingshausen, G. (1971). Automated reaction‐rate method for determination of serum creatinine with the centrifichem. Clinical Chemistry, 17, 696–700.
  Folin, O., & Morris, J. (1914). On the determination of creatinine and creatine in urine. Journal of Biological Chemistry, 17, 469–473.
  Heinegard, D., & Tiderstrom G. (1973). Determination of serum creatinine by a direct colorimetric method. Clinica Chimica Acta, 43, 305–310. doi: 10.1016/0009‐8981(73)90466‐X.
  Jaffe, M. (1886). Uber den Niederschlag, welchen Pikrisaure in normalen Harn erzeugt und uber eine neue Reaction des Kreatinins [A new reaction of creatinine in normal generated urine with picric acid and its precipitation products]. Zeitschrift fur Physiologische Chemie, 10, 391–400. doi: 10.1515/bchm1.1886.10.5.391.
  Lehninger, A. (1975). Biochemistry, the molecular basis of cell structure and function 2nd edition (p. 716, 767, 840). New York: Worth Publishers.
  Peake, M., & Whiting, M. (2006). Measurement of serum creatinine ‐ current status and future goals. The Clinical Biochemist, 27, 173–184.
  Rahn, K., Heidenreich, S., & Bruckner, D. (1999). How to assess glomerular function and damage in humans. American Journal of Hypertension, 17, 309–317. doi: 10.1097/00004872‐199917030‐00002.
  Stevens, L., Coresh, J., Greene, T., & Levey, A. (2006). Assessing kidney function—measured and estimated glomerular filtration rate. The New England Journal of Medicine, 354, 2473–2483. doi: 10.1056/NEJMra054415.
  Takahashi, N., Boysen, G., Li, F., Li, Y., & Swenburg, J. (2007). Tandem mass spectrometry measurements of creatinine in mouse plasma and urine for determining glomerular filtration rate. Kidney International, 71, 266–271. doi: 10.1038/
  Tsikas, D., Wolf, A., & Frolich, J. (2004). Simplified HPLC method for urinary and circulating creatinine. Clinical Chemistry, 50, 201–203. doi: 10.1373/clinchem.2003.024141.
  Wyss, M., & Schulze, A. (2002). Health implications of creatine: Can oral creatine supplementation protect against neurological and atherosclerotic disease? Neuroscience, 112, 243–260. doi: 10.1016/S0306‐4522(02)00088‐X.
  Young, S., Struys, E., & Wood, T. (2007). Quantification of creatine and guanidinoacetate using GC‐MS and LC‐MS/MS for the detection of cerebral creatine deficiency syndromes. Current Protocols in Human Genetics, 54, 17.3.1‐17.3.18. doi: 10.1002/0471142905.hg1703s54.
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