Quantification of N-methylmalonamic acid in urine as metabolite of the biocides methylisothiazolinone and chloromethylisothiazolinone using gas chromatography-tandem mass spectrometry

J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Feb 15;1044-1045:185-193. doi: 10.1016/j.jchromb.2017.01.019. Epub 2017 Jan 18.


Methylisothiazolinone and the mixture of chloromethylisothiazolinone/methylisothiazolinone (MCI/MI, 3:1) are widespread biocides used in cosmetic and household products. Due to their skin permeability, they might be taken up by the general population via use of products containing these biocides. As both compounds are known skin sensitizers, the use of these products is under discussion by regulatory agencies. In order to evaluate the possible uptake of MI and/or MCI/MI by human biomonitoring, we have developed and validated a highly sensitive and specific GC/MS/MS-method for the quantification of N-methylmalonamic acid (NMMA), a known metabolite of MI and MCI in urine of rats. After freeze-drying of urine, the analyte is derivatised with pentafluorobenzyl bromide in anhydrous solution and the PFB-derivative is extracted into n-hexane. After concentration, the derivative is finally quantified by GC/MS/MS in EI-mode using 13C3-NMMA as internal standard. The limit of quantification for NMMA was 0.5ngmL-1 urine. Precision within and between-series was determined to range between 3.7-10.9% using native and spiked quality control samples. Accuracy ranged between 89 and 114%. In a pilot study we applied this method to spot urine samples of 63 persons not knowingly exposed to MI and/or MCI/MI. NMMA was quantifiable in every urine sample analysed, with no significant difference in urinary levels between male and female participants. The median (95th percentile) levels for urinary NMMA were 3.6 (7.4) ngmg-1 creatinine and 2.9 (9.1) ngmg-1 creatinine for males (n=32) and females (n=31), respectively. In a volunteer experiment, a relation of exposure to MI and/or MCI/MI and subsequent NMMA-excretion was shown. Our method is the first to report human urinary background levels of NMMA. However, the possibility of formation and urinary excretion of NMMA within physiological processes cannot be ruled out.

Keywords: Biocides; Human biomonitoring; Metabolism; Pentafluorobenzyl bromide; Urine.

MeSH terms

  • Adult
  • Animals
  • Disinfectants / urine*
  • Female
  • Gas Chromatography-Mass Spectrometry / methods*
  • Humans
  • Linear Models
  • Male
  • Malonates / urine*
  • Middle Aged
  • Rats
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Tandem Mass Spectrometry / methods*
  • Thiazoles / urine*
  • Young Adult


  • Disinfectants
  • Malonates
  • Thiazoles
  • 2-methyl-4-isothiazolin-3-one
  • N-methylmalonamic acid