96 well creatinine assay kit

Creatinine concentration in urine samples was analyzed using the 96-well Creatinine Assay Kit from Cayman Chemical (Ann Arbor, MI, USA). The analysis is based on a modified Jaffe colorimetric method that measures the difference in absorbance (495 nm) of the creatinine-picrate complex before and after acidification. The assay was carried out according to manufacturer’s instructions, and absorbance was measured using an ELx808 Absorbance Microplate Reader from BioTek Instruments, Inc. (Winooski, VT, USA).

Frozen urine samples were extracted and purified according to the previously developed protocol with FumoniTest immunoafinity column from VICAM (Watertown, MA, USA)12 16 (link). HPLC-fluorescence analysis was carried out on Agilent 1100 liquid chromatography system (Agilent Technologies, Wilmington, DE, USA) with the emission wavelength of 440 nm and excitation wavelength of 330 nm. An on-line automatic injector program performed the derivitization of FB₁. Chromatographic separations were performed on a Luna C18 column (5μm particle size, 250 × 4.6 mm) from Phenomenex (Torrance, CA, USA). The mobile phase consisted of a linear gradient starting from 0.1 M sodium phosphate monobasic (pH 3.4)-methanol (35/65, v/v) to 0.1 M sodium phosphate monobasic (pH 3.4)-methanol (20/80, v/v) over 13 min, with flow rate of 1.0 ml/min and injection volume of 100 μl. The limit of quantitation for this method was 20 pg per injection. The mean recovery rate was 83.4% ± 0.8%. The relative standard deviation (RSD) was 4.42%. FB₁ concentration was verified using LC/MS methods described in previous studies26 (link). Urinary creatinine levels were analysed using the 96-well Creatinine Assay Kit from Cayman Chemical (Ann Arbor, MI, USA).