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Serum Creatinine Determination by High Performance Liquid Chromatography and Five Automated Chemistry Analyzers

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Serum Creatinine Determination by High Performance Liquid Chromatography and Five Automated Chemistry Analyzers ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 13, No. 6 Copyright © 1983, Institute for Clinical Science, Inc. Serum Creatinine Determination by High Performance Liquid Chromatography and Five Automated Chemistry Analyzers EARLE W. HOLMES, Ph.D.*, TRUDE H. OESER*, STEPHEN E. KAHN, Ph.D.*, LEONAS BEKERIS, M.D.t, and EDWARD W. BERMES, JR., Ph.D.* *Departments of Pathology, Biochemistry and Biophysics, Loyola University Medical Center, Maywood, IL 60525 and tDepartment of Pathology, Mac Neal Memorial Hospital Berwyn, IL 60402 ABSTRACT A sensitive and specific procedure is described for the determination of serum creatinine by reverse phase high performance liquid chromatog­ raphy. The method involves sample pretreatment with a cation exchange resin followed by the isocratic separation of creatinine and an external standard which are detected by their absorbances at 254 nm. The analyt­ ical recovery of creatinine from serum was 99.8 ±4.9 percent using this method. The between-day coefficients of variation for creatinine concen­ trations of 0.83 and 8.63 mg per dl were 3.07 percent and 3.2 percent, respectively. Results obtained with this reference method were compared to results obtained with five automated chemistry analyzers. The results showed that while the comparison methods correlated well with the ref­ erence method (r > 0.98 in all cases), those comparison methods based on the Jaffe reaction showed negative biases. In some cases, these biases were highly statistically significant. The difference between paired results obtained with some of the automated Jaffe-based assays exceeded 0.20 mg per dl in a substantial number of specimens with creatinine concentration in the range of 0.5 to 2.3 mg per dl. The measurement of acetoacetate concentrations of specimens sub­ mitted to our laboratory for serum creatinine assays showed that interfer­ ence by this substance with certain Jaffe-based assays could result in clin­ ically significant false elevations in serum creatinine concentrations. Introduction Jaffe in 1886. Since that time, this color reaction has been used with numerous The reaction of creatinine with alkaline modifications for the determination of sodium picrate was first described by serum creatinine. As might be expected 503 0091-7370/83/1100-0503 $01.20 © Institute for Clinical Science, Inc. 504 HOLMES, OESER, KAHN, BEKERIS, AND BERMES in the case of a well-studied, widely-used Lim et al.9 The first part of the procedure assay, a number of substances that inter­ is a sample pretreatment designed to iso­ fere with this reaction have been iden­ late creatinine from other serum constit­ tified. 2’3,14,16 uents. The basis of this pretreatment The nonspecificity of the Jaffe reaction step has already been described.1 for serum creatinine is dealt with in Mini-columns containing 100 mg of several ways. The first of these involves Dowex AG 50W X-12 were poured in pretreatment of serum specimens with 2.5M NaOH and washed with 2.0 ml of adsorbents, organic solvents, protein H20 and 4.0 ml of adsorption buffer (0.04 précipitants or dialysis.10 However, these M citric acid, 0.02 M dibasic sodium techniques have met with variable suc­ phosphate, pH 3.0). Exactly 0.5 ml of cess. With the exception of dialysis, none sample was mixed with 5.0 ml of adsorp­ is suitable for use with automated instru­ tion buffer, and 5.0 ml of the mixture was mentation. A second and more popular applied to the minicolumn. Each column approach to increasing specificity in was washed with 4.0 ml H20, and the serum creatinine assays is to use kinetic adsorbed creatinine was then eluted with versions of the Jaffe reaction. These ki­ 3.0 ml of 0.5 M sodium acetate, pH 8.6. netic assays aim to differentiate chro­ One hundred jjlI of a solution of 0.58 mM mogen formation by creatinine from that allopurinol in 0.5 M sodium acetate was due to interfering substances by differ­ added to each eluent as an external stan­ ences in their reaction rates.7 A third ap­ dard. 12 proach to a more specific assay for cre­ Twenty (Jil of each eluent was injected atinine is to abandon the Jaffe reaction onto a 4 mm x 30 cm column of 10 (jiM entirely in favor of creatinine methods silica particles to which a unimolecular based on the use of enzymes as layer of octadecyltrichlorosilane was reagents10 or methods based on high bonded.* High pressure liquid chroma­ performance liquid chromatography tography was carried out using 0.01 M (HPLC).1,4,9,11,12 ammonium acetate as the mobile phase The existence of a variety of assays for at a flow rate of 1.0 ml per min. Creati­ serum creatinine as well as the variability nine and allopurinol were detected by among the automated kinetic Jaffe assays their absorbances at 254 nm. The creat­ with respect to their precise assay inine concentration of a sample was conditions3 suggests the possibility of calculated by comparing its peak height intra-method and inter-instrument bias ration (creatinine peak height per allo­ in creatinine results. The purpose of this purinol peak height) to those of a series study was: (1) to develop a sensitive, spe­ of creatinine standards! that were car­ cific, and accurate HPLC assay for serum ried through the entire procedure. creatinine and (2) to use this assay as a The analytical recovery of creatinine reference method for the evaluation of by this method was assessed by com­ the creatinine test on five automated paring the peak height ratios of extracted chemistry analyzers. and unextracted aqueous creatinine stan­ dards and by adding pure creatinine in Materials and Methods increments of 0.3 to 18 mg per dl to pooled human serum. The average re­ S er u m C r e a t in in e by HPLC coveries are based on results from tests The following HPLC assay for serum * u-Bondapak C18, Waters Associates, Milford, MA. creatinine was developed by the present t New England Reagent Laboratory, East Provi­ authors based on the previous work of dence, RI. CREATININE DETERMINATION BY HPLC 505 performed in duplicate on two separate was separated into aliquots for each of occasions. The precision of the method the comparison methods and frozen. was established by assaying commercial Since it was not possible to assay each control sera on 10 occasions over a one specimen by all of the methods, compar­ month period. ison results for the MCA, ASTRAN and EKTA methods are based on subsets of C o m pa r iso n M e t h o d s the group of 72. Acetoacetate was measured on specimens sent to the lab­ Creatinine results determined by oratory for creatinine determination. HPLC were compared with the following Specimens from known diabetics were automated methods using a split-sample excluded from this group. protocol: Dupont ACA III (ACA),$ Technicon SMAC (SMAC),§ Beckman I nterference S t u d ie s ASTRA (ASTRA),» Multistat (MCA),! and EKTACHEM 400 (EKTA).** Unless The responses of the reference and specified otherwise, each automated in­ test methods to several known inter­ strument was operated according to the fering substances were tested using manufacturer’s instructions using the cal­ pooled serum spiked with the substances ibrators and reagents that they supplied. of interest in the concentrations indi­ The ASTRA instrument was also oper­ cated in the Results section. Acetoace­ ated using reagents manufactured by tate concentrations in clinical specimens Nobel Scientific (ASTRAN).ft were determined by an enzymatic A series of aqueous standards were as­ method.8 sayed as samples on each instrument (ex­ cept EKTA) to check for systematic bias S ta tistic a l E v a lu atio n that might occur as a result of differences between calibrators. Method comparison data were ana­ lyzed by linear regression techniques.5 C l in ic a l S p e c im e n s Since the differences between paired re­ sults for individual specimens assayed by The 72 serum specimens used in the various combinations of reference and method comparison study were obtained comparison method were not normally from blood drawn in red-topped vacutain- distributed according to the Kolmo- ers and submitted to the laboratory for gorov-Smirnov Test,15 the significance of biochemical profiling. Specimens with these differences was tested using the glucose values greater than 140 mg per Sign Test. Differences that were signifi­ dl were excluded from this group. The cant at the 0.05 level by the Sign Test creatinine concentrations of the speci­ were further evaluated using Wilcoxin’s mens as determined by HPLC ranged Test.15 from 0.4 to 23.2 mg per dl (mean, 4.5 mg per dl, median, 1.0 mg per dl). Serum Results HPLC A ssay f o r S er u m C r e a t in in e Dupont Company, Wilmington, DE. § Technicon Instruments Corp., Tarrytown, NY. I Beckman Instruments, Inc., Brea, CA. In figure 1 is shown the elution profile II Instrumentation Laboratory Inc., Lexing­ obtained when a serum specimen with a ton, MA. creatinine concentration of 3 mg per dl ** Eastman Kodak Company, Rochester, NY. ft Nobel Scientific Industries, Inc., Alexan­ was extracted and chromatographed as dria, VA. described in the Methods section. Cre­ 506 HOLMES, OESER, KAHN, BEKERIS, AND BERMES aqueous creatinine standards were ana­ lyzed. The mean recovery of creatinine from the cation exchange mini-columns was 100.3 percent (sd, 6.0 percent; range 94 to 106 percent) for a series of aqueous E standards with creatinine concentrations c of 1.0 to 10.0 mg per dl.
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