Candidate reference method for determining creatinine in serum: method development and interlaboratory validation

Abstract

We describe a "high-performance" liquid chromatographic (HPLC) method for accurately determining creatinine in serum. After prechromatographic precipitation of protein, we performed isocratic ion-exchange chromatography with ultraviolet detection (234 nm). Analytical results showed linearity up to 1770 mumol/L, a detection limit of 22 mumol/L, an average analytical recovery of 101%, and a CV ranging from 3% to 11%. We used certified human serum (National Institute of Standards and Technology), and additional lyophilized serum pools also assayed by definitive isotope-dilution mass spectrometry, to validate the accuracy of the HPLC method. In addition, the isocratic HPLC results showed close agreement with those obtained with a step-gradient HPLC method. We also compared the isocratic HPLC method with alkaline picrate and enzymatic methods. Our findings with samples from nonuremic, uremic, and diabetic ketoacidotic patients confirmed the positive bias previously reported with the alkaline picrate method. Interlaboratory transferability of the method was demonstrated with various commercial instruments and analytical columns. We evaluated column stability and possible interference from endogenous or exogenous compounds. On the basis of our analytical findings, we recommend the isocratic HPLC method as a candidate Reference Method for determining creatinine in serum.

The influence of ketoacids on plasma creatinine assays in diabetic ketoacidosis

“… Analysis of agreement according to Bland and Altman [21]. In this analysis, the difference between two assays is plotted against their mean for each plasma sample.…”

“…Automated enzymatic creatinine assays were introduced in clinical medicine more than 10 years ago, but are still not generally used, for financial reasons [17–20]. The ion‐exchange high‐performance liquid chromatography (HPLC) procedure with ultraviolet detection at 234 nm has been proposed as a candidate reference method, because results obtained with it are similar to the definitive isotope‐dilution mass spectrometry [21, 22].…”

The influence of ketoacids on plasma creatinine assays in diabetic ketoacidosis

“…For instance, this led to a plasma creatinine concentration of 152 mmol L 21 (Jaffe Â) instead of 107 mmol L 21 (HPLC) or 102 mmol L 21 (PAP+) in a severely dehydrated female patient, who had an HPLC creatinine of 51 mmol L 21 after correction of the ketoacidosis and hydration status. In a normovolaemic female patient, the positive error was even more pronounced: 75 (Jaffe Â) vs. 40 [21]. In this analysis, the difference between two assays is plotted against their mean for each plasma sample.…”

“…Prior to measurement, these samples were deproteinized within 10 min after venepuncture with a 1:1 mixture of fluoride plasma and 0.1 N perchloracetic acid and stored at ±208C for 3±10 days. In the arterial blood sample, pH and PCO 2 were measured on a blood-gas analyser ( 21 . Plasma concentration units are given in mmol L 21 , except for creatinine (mmol L 21 ).…”

“…Automated enzymatic creatinine assays were introduced in clinical medicine more than 10 years ago, but are still not generally used, for financial reasons [17±20]. The ion-exchange high-performance liquid chromatography (HPLC) procedure with ultraviolet detection at 234 nm has been proposed as a candidate reference method, because results obtained with it are similar to the definitive isotope-dilution mass spectrometry [21,22].…”