Creatinine standard

Blood urea nitrogen (BUN) and xanthine oxidase activity were determined using commercial kits according to the manufacturer indications (Sigma-Aldrich Co.). Serum creatinine was measured by HPLC method. Briefly 12.5 μl of sera were deproteinized by adding 1 ml of 100% acetonitrile. Samples were then vortexed and centrifuged twice for 20 minutes at 4°C, 16000 g. After vacuum evaporation of the supernatant fraction, dried samples were suspended in 12.5 μl of 5 mM NaH₂PO₄, pH 6.4 (mobile phase) and stored at -20°C. Samples were manually injected by a 10 μl loop on a Prominence HPLC system (Shimadzu, Kyoto, Japan). Analytes were separated on a SCX column (Zorbax SCX-300 4.6 x 250, Agilent Technologies, Santa Clara, CA, US) with an isocratic run and revealed at 234 nm and 240 nm. Data were collected and analyzed by LabSolutions software. The output peaks where identified and quantified by comparison with chromatographic runs of creatinine standards (Sigma-Aldrich).

For derivatization, a reagent prepared by BSA (N,O-bis(trimethylsilyl)acetamide), TMCS (trimethylchlorosilane), and TMSI (N-trimethylsilylimidazole) (3:2:3) was supplied by Supleco (Bellefonte, USA). A phosphate buffer was prepared with sodium dihydrogen phosphate and disodium hydrogen phosphate, both purchased from Panreac Quimica SLU (Barcelona, Spain). HPLC-grade solvents such as methanol, acetonitrile, ethyl acetate, and hexane, as well as sodium chloride (analytical grade), were acquired from Merck KGaA (Darmstadt, Germany). Sodium hydroxide was supplied by BDH Prolabo-VWR International (Barcelona, Spain). Creatinine standard and picric acid moistened with water (≥98%) were purchased from Sigma-Aldrich (St. Louis, USA).

Commercial Gentamicin injection (80 mg) was collected from a model pharmacy (Dhaka, Bangladesh). Laboratory standard Thiobarbituric acid, Trichloroacetic acid, Hydrochloric acid, 10% formalin, 0.9% saline, Phosphate buffer, Detergent, Uricase, Dichlorophenol sulphonate, Ascorbate oxidase, Peroxidase, Amino-antipyrine, Urease Enzyme reagent, EDTA, Sodium salicylate, Sodium nitroprusside, Alkaline hypochlorite, Sodium hypochlorite, Sodium hydroxide, Urea standard, Picric acid, Creatinine Standard, Nitrite standard solution, Griess reagent, and Alcohol were purchased from Sigma Aldrich (St. Louis, MO, USA) and used during the research.

Creatinine concentration in urine was measured using Jaffe's method (see Supplementary Materials). An eight-point calibration curve was prepared using a serial dilution of creatinine standard (Sigma-Aldrich, ON, Canada) in deionized water between 0.31 mg/dL and 20 mg/dL. Urine samples were diluted 40 times. A 50 μL volume of standards, urine samples, and method blanks were added to 100 μL of alkaline picric acid solution (Sigma-Aldrich, ON, Canada) in 96-well plates (VWR, ON, Canada), then incubated at room temperature for 30 min. Absorbance was measured at 490 nm using a microplate reader (Synergy H1, BioTek, VT, USA).