Determination of 3-hydroxypropylmercapturic acid in urine by three column-switching high-performance liquid chromatography with electrochemical detection using a diamond electrode

- Three column-switching HPLC-ECD allowed the sensitive detection of 3-HPMA.
- SAX, C8 and ODS columns were required for the on-line separation of 3-HPMA.
- The LOD of 3-HPMA by column-switching HPLC-ECD detection was 0.1 pmol.
- The% recovery of 3-HPMA (0.5 nmol) by on-line separation was 94%.
- The% recovery of 3-HPMA (0.1 nmol) in SPE-column-switching HPLC-ECD was 55%.

A three column-switching high-performance liquid chromatography (HPLC) using an electrochemical detector (ECD) equipped with a diamond electrode was established to determine 3-hydroxypropylmercapturic acid (3-HPMA) in urine. An extracted urine sample was consecutively fractionated using a strong anion-exchange column (first column) and a C8 column (second column) via a switching valve before application on an Octa Decyl Silyl (ODS) column (third column), followed by ECD analysis. The% recovery of 3-HPMA standard throughout the three-column process and limit of detection (LOD) were 94 ± 1% and 0.1 pmol, respectively. A solid phase extraction step is required for the sensitive analysis of 3-HPMA in urine by column-switching HPLC-ECD despite a decreased% recovery (55%) of urine sample spiked with 100 pmol of 3-HPMA. To test the utility of our column-switching HPLC-ECD method, 3-HPMA levels of 27 urine samples were determined, and the correlation between HPLC-ECD and LC-Electrospray ionization (ESI)-MS/MS method was examined. As a result, the median values of μmol 3-HPMA/g Creatinine (Cre) in urine obtained by column-switching HPLC-ECD and LC-MS/MS were 2.19 ± 2.12 μmol/g Cre and 2.13 ± 3.38 μmol/g Cre, respectively, and the calibration curve (y = 1.5171x − 1.007) exhibited good linearity within a defined range (r2 = 0.907). These results indicate that the combination of column-switching HPLC and ECD is a powerful tool for the specific, reliable detection of 3-HPMA in urine.