In this paper we have investigated and optimized a non-enzymatic fluorometric creatinine assay. The method was originally described by Blass in the 90s, but we found that besides the reagents mentioned in the paper, an addition of hydrogen peroxide is required to obtain a fluorescent compound. The excitation and emission maxima of the fluorophore are 405 nm and 475 nm, respectively. The optimal conditions for creatinine quantification are as follows: 25 mmol L-1 3,5-dinitrobenzoic acid dissolved in 1,4-butanediol with 58 mmol L-1 H2O2 and aqueous solution of 2 mol L-1 NaOH mixed in 1:1 ratio. A linear calibration curve (y = 18,7x + 446 for 300 s of incubation) was obtained in the range from 2.6 to 750 μmol L-1 of creatinine with LOD and LOQ equal to 0.7 and 2.6 μmol L-1, respectively. The method was found to be selective towards the analyte in the presence of compounds such as urea, uric acid, bilirubin, albumin and glucose. The developed protocol was applied for creatinine determination in 13 real human serum samples. A correlation (y = (0.94 ± 0.03) x + (3.66 ± 3.22) with Pearson's r 0.996) and statistical agreement (two-tailed Student's t-test at 95% confidence interval with 12 degrees of freedom) was reached between the obtained results and the reference enzymatic method.

中文翻译：

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来自旧罐底的荧光法测定人血清中肌酐

在本文中，我们研究并优化了非酶促荧光肌酐测定。该方法最初由 Blass 在 90 年代描述，但我们发现除了论文中提到的试剂外，还需要加入过氧化氢才能获得荧光化合物。荧光团的激发和发射最大值分别为 405 nm 和 475 nm。肌酐定量的最佳条件如下： 25 mmol L-1 3,5-二硝基苯甲酸溶于 1,4-丁二醇，58 mmol L-1 H2O2 和 2 mol L-1 NaOH 水溶液按 1:1 混合比率。在 2.6 至 750 μmol L-1 的肌酐范围内获得线性校准曲线（y = 18,7x + 446，孵育 300 秒），LOD 和 LOQ 分别等于 0.7 和 2.6 μmol L-1。发现该方法在尿素、尿酸、胆红素、白蛋白和葡萄糖等化合物存在下对分析物具有选择性。开发的方案用于测定 13 份真实人血清样品中的肌酐。获得的结果之间达到了相关性 (y = (0.94 ± 0.03) x + (3.66 ± 3.22) 与 Pearson's r 0.996) 和统计一致性（双尾学生 t 检验，置信区间为 95%，自由度为 12）和参考酶法。