Validation of a multi-analyte HPLC-DAD method for determination of uric acid, creatinine, homovanillic acid, niacinamide, hippuric acid, indole-3-acetic acid and 2-methylhippuric acid in human urine

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 998-999; pp. 40 - 44
• Main Authors: Remane, Daniela, Grunwald, Soeren, Hoeke, Henrike, Mueller, Andrea, Roeder, Stefan, von Bergen, Martin, Wissenbach, Dirk K.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.08.2015
• Subjects: Chromatography, High Pressure Liquid - instrumentation; Chromatography, High Pressure Liquid - methods; Creatinine; Creatinine - urine; Hippurates - urine; Hippuric acid; Homovanillic Acid - urine; HPLC-DAD; Humans; Indoleacetic Acids - urine; Niacinamide - urine; Uric acid; Uric Acid - urine; Urine; Validation; Validation; Creatinine; Urine; HPLC-DAD; Hippuric acid; IAA; Uric acid; UA; HVA; QC; NA; CREA; HA; 2-MHA
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2015.06.021

•Extension of an HPLC-DAD for creatinine by additional six compounds.•Description of a fully validated multi-analyte procedure in human urine.•Comparison study (new vs existing method) showed low differences (∼3%) for creatinine.•Applicability proofed by analyzing authentic samples.•Additional information about lifestyle for epidemiological cohort studies obtained. During the last decades exposure sciences and epidemiological studies attracts more attention to unravel the mechanisms for the development of chronic diseases. According to this an existing HPLC-DAD method for determination of creatinine in urine samples was expended for seven analytes and validated. Creatinine, uric acid, homovanillic acid, niacinamide, hippuric acid, indole-3-acetic acid, and 2-methylhippuric acid were separated by gradient elution (formate buffer/methanol) using an Eclipse Plus C18 Rapid Resolution column (4.6mm×100mm). No interfering signals were detected in mobile phase. After injection of blank urine samples signals for the endogenous compounds but no interferences were detected. All analytes were linear in the selected calibration range and a non weighted calibration model was chosen. Bias, intra-day and inter-day precision for all analytes were below 20% for quality control (QC) low and below 10% for QC medium and high. The limits of quantification in mobile phase were in line with reported reference values but had to be adjusted in urine for homovanillic acid (45mg/L), niacinamide 58.5(mg/L), and indole-3-acetic acid (63mg/L). Comparison of creatinine data obtained by the existing method with those of the developed method showing differences from −120mg/L to +110mg/L with a mean of differences of 29.0mg/L for 50 authentic urine samples. Analyzing 50 authentic urine samples, uric acid, creatinine, hippuric acid, and 2-methylhippuric acid were detected in (nearly) all samples. However, homovanillic acid was detected in 40%, niacinamide in 4% and indole-3-acetic acid was never detected within the selected samples.