1. Academic Validation
  2. 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

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

  • J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Aug 15;998-999:40-4. doi: 10.1016/j.jchromb.2015.06.021.
Daniela Remane 1 Soeren Grunwald 2 Henrike Hoeke 3 Andrea Mueller 4 Stefan Roeder 5 Martin von Bergen 6 Dirk K Wissenbach 7
Affiliations

Affiliations

  • 1 Institute of Forensic Medicine, University Hospital Jena, Jena, Germany.
  • 2 Department of Metabolomics, Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany.
  • 3 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Leipzig, Leipzig, Germany.
  • 4 Department of Proteomics, Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany.
  • 5 Department of Environmental Immunology, Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany.
  • 6 Department of Metabolomics, Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany; Department of Proteomics, Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany; Department of Biotechnology, Chemistry and Environmental Engineering Aalborg University, Aalborg, Denmark.
  • 7 Department of Metabolomics, Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany. Electronic address: dirk.wissenbach@ufz.de.
Abstract

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.

Keywords

Creatinine; HPLC-DAD; Hippuric acid; Uric acid; Urine; Validation.

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