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PubMed · 2077096

Analyses with the KODAK-Ektachem. Accuracy control using reference method values and the influence of protein concentration. Part II. Substrates.

Abstract

The reliability of the determination of the most common substrates with the Ektachem 700 was evaluated. Accuracy control was performed in various ways including the comparison of results with reference method values. The influence of protein concentration was investigated systematically using sera containing varying amounts of protein obtained by ultracentrifugation. Bilirubin. Deviation from the reference method values was within limits of the guidelines (Dt. Arztebl. 85 (1988) B517-B532). The influence of protein concentration was negligible. Cholesterol. The results agreed well with the reference method values. The method was not influenced by different protein concentrations. Creatinine. The results were in good agreement with reference method values, when the method was calibrated with the primary assigned values of the calibrators. At high protein concentrations, the results were higher than those of the comparative method. Glucose. The mean deviation from the reference method values was 1.0%. There was a clear positive bias at high protein concentrations. Protein. The results from the Ektachem deviated from the reference method values by -6.8%. Total glycerol (triacylglycerols). In the analysis of control sera, the Ektachem values differed greatly (+32.1%) from the reference method values. This difference was not found in a comparative study with native sera. At high protein concentrations the Ektachem results were higher than those of the comparative method. Urea. The results were lower than the method-dependent assigned values (-16.6%). This deviation was not observed in a comparative study with native sera. Interference by protein was not observed. Uric acid. In accuracy control with reference method values, a small bias was observed (+3.3%), which increased at high protein concentrations.

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BibTeXRIS

W R Külpmann, P Maibaum, O Sonntag, G Schumann, L Siekmann. 1990. Analyses with the KODAK-Ektachem. Accuracy control using reference method values and the influence of protein concentration. Part II. Substrates.. https://pubmed.ncbi.nlm.nih.gov/2077096/

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Bilirubin↗

Solvent partition of 14C-unconjugated bilirubin to remove labeled polar contaminants.

Polar derivatives contaminating unconjugated bilirubin (UCB) are customarily extracted with weak alkali. As UCB degrades rapidly at alkaline pH, regeneration of polar derivatives may limit purification. This problem is especially important when trying to eliminate radiolabeled contaminants from 14C-UCB. As polar derivatives of UCB should have a much greater aqueous to CHCl3 partition ratio (PR) than UCB even at neutral pH, where degradation of UCB is minimal, 14C-UCB in CHCl3 was serially extracted with an aqueous buffer at pH 7.0 to determine whether labeled derivatives could be preferentially removed. A single extraction of customarily purified 14C-UCB removed 0.18+/-0.06% of the radioactivity as labeled derivatives. Subsequent serial extractions yielded a stable, 67% lower 14C-PR with only 0.03% of radioactivity as labeled derivatives. Reverse-phase high-performance liquid chromatography (HPLC) of phases from later extractions revealed, however, that up to 1.1% of the disintegration per minute (dpm) in CHCl3 phases and up to 50% in aqueous phases were polar impurities. HPLC of partition phases spiked with purified 14C-UCB revealed that these impurities derived from incomplete extraction of the least polar impurities and their regeneration during HPLC. In the dark under argon, 14C-UCB in CHCl3or dimethyl sulfoxide (DMSO) solution degraded very slowly to polar derivatives. Extraction of impurities from a solution of 14C-UCB in CHCl3 is best done using pH 7.0 buffer, with removal of over 80% of the labeled contaminants remaining after customary purification by alkaline extraction.

Bilirubin↗