Trisaminohexyl isocyanurate, a urinary biomarker of HDI isocyanurate exposure

Abstract

Biological monitoring of occupational exposure to 1,6-hexamethylene diisocyanate (HDI)-containing spray-paints is limited to analysis of metabolites of HDI monomer although polymeric HDI isocyanurate constitutes the predominant inhalation and skin exposure for workers in the automotive paint industry. A novel method using nanoflow ultra-performance liquid chromatography coupled to nano-electrospray ionization tandem mass spectrometry (nano-UPLC-ESI-MS/MS) was developed to quantify trisaminohexyl isocyanurate (TAHI), a hydrolysis product of HDI isocyanurate, in the urine of spray-painters. Analytical and internal standards were synthesized in-house and weighted linear regression calibration curves were generated using spiked control urine from non-exposed persons (0.06-7.98 μg/L; N = 13; w = x^-2; r = 0.998). Urine samples collected from 15 exposed workers (N = 111) were subjected to acid hydrolysis and extracted with dichloromethane, then derivatized with acetic anhydride. The derivatized product, trisacetamidohexyl isocyanurate (TAAHI), was analyzed using nano-UPLC-ESI-MS/MS. The protocol was sensitive and specific for analysis of TAHI in the urine of exposed workers with a method detection limit at 0.03 μg/L. TAHI was detected in 33 of 111 urine samples and in 11 of 15 workers. This biomarker for HDI isocyanurate is critical to determine the relative potency and dose-relationships between the monomer and oligomer exposure on the development of diisocyanate induced health effects in future studies.

Keywords: 1,6-Hexamethylene diisocyanate; Biomarker; Exposure; Isocyanate; Isocyanurate; Liquid chromatography–mass spectrometry.

Fig. 1.

Molecular structures of 1,6-hexamethylene diisocyanate monomer and its oligomers uretdione, biuret, and isocyanurate.

Fig. 2.

Chemical structures of [A] trisaminohexyl isocyanurate (TAHI), [B] trisacetamidohexyl isocyanurate (TAAHI), [C] trisaminoheptyl isocyanurate (TAHpI), and [D] trisacetamidoheptyl isocyanurate (TAAHpI).

Fig. 3.

A: Fragmentation spectrum of precursor ion [M + H]⁺ for TAAHI (m/z 553.3) obtained by direct injection ESI-MS/MS operated in positive ion-mode with electrospray ionization (scan range, m/z 100–600; collision energy, 25 eV). B: Fragmentation spectrum of precursor ion [M + H]⁺ for TAAHI (m/z 553.3) obtained by direct injection ESI-MS/MS operated in positive ion-mode with electrospray ionization (scan range, m/z 100–600; collision energy, 50 eV).

Fig. 4.

A: Fragmentation spectrum of precursor ion [M + H]⁺ for TAAHpI (m/z 595.3) obtained by nano-UPLC-ESI-MS/MS operated in positive ion-mode with nano-electrospray ionization (scan range, m/z 100–600; collision energy, 25 eV). B: Fragmentation spectrum of precursor ion [M + H]⁺ for TAAHpI (m/z 595.3) obtained by nano-UPLC-ESI-MS/MS operated in positive ion-mode with nano-electrospray ionization (scan range, m/z 100–600; collision energy, 50 eV).

Fig. 5.

[A] TAAHI mass spectral fragments and [B] TAAHpI mass spectral fragments.

Fig. 6.

[A] Extracted ion chromatograms acquired by selected reaction monitoring for TAAHI: m/z 553.3 → 130.0, m/z 553.3 → 212.1, and m/z 553.3 → 494.4, and total ion chromatogram for TAAHpI (combined m/z 595.3 → 130.0, 226.1, and 536.4); obtained for control urine spiked with TAHpI (2.0 μg/L). [B] Extracted ion chromatograms acquired by selected reaction monitoring for TAAHI: m/z 553.3 → 130.0, m/z 553.3 → 212.1, and m/z 553.3 → 494.4, and total ion chromatogram for TAAHpI (combined m/z 595.3 → 130.0, 226.1, and 536.4); obtained for urine sample 8 from worker #7 spiked with TAHpI (2.0 μg/L) and a calculated concentration of 0.36 μg/L for TAHI.

Fig. 7.

A: Fragmentation spectrum of precursor ion [M + H]⁺ for TAAHI (m/z 553.3) in urine sample 2 from worker #13 (TAHI 3.98 μg/L). Spectrum was obtained by nano-UPLC-ESI-MS/MS operated in positive ion-mode with nano-electrospray ionization (scan range, m/z 100–600; collision energy, 25 eV). B: Fragmentation spectrum of precursor ion [M + H]⁺ for TAAHI (m/z 553.3) in urine sample 2 from worker #13 (TAHI 3.98 μg/L). Spectrum was obtained by nano-UPLC-ESI-MS/MS operated in positive ion-mode with nano-electrospray ionization (scan range, m/z 100–600; collision energy, 50 eV). C: Fragmentation spectrum of precursor ion [M + H]⁺ for TAAHI (m/z 553.3) in urine sample 3 from worker #14 (TAHI 9.89 μg/L). Spectrum was obtained by nano-UPLC-ESI-MS/MS operated in positive ion-mode with nano-electrospray ionization (scan range, m/z 100–600; collision energy, 25 eV). D: Fragmentation spectrum of precursor ion [M + H]⁺ for TAAHI (m/z 553.3) in urine sample 3 from worker #14 (TAHI 9.89 μg/L). Spectrum was obtained by nano-UPLC-ESI-MS/MS operated in positive ion-mode with nano-electrospray ionization (scan range, m/z 100–600; collision energy, 50 eV).

Fig. 7.

A: Fragmentation spectrum of precursor ion [M + H]⁺ for TAAHI (m/z 553.3) in urine sample 2 from worker #13 (TAHI 3.98 μg/L). Spectrum was obtained by nano-UPLC-ESI-MS/MS operated in positive ion-mode with nano-electrospray ionization (scan range, m/z 100–600; collision energy, 25 eV). B: Fragmentation spectrum of precursor ion [M + H]⁺ for TAAHI (m/z 553.3) in urine sample 2 from worker #13 (TAHI 3.98 μg/L). Spectrum was obtained by nano-UPLC-ESI-MS/MS operated in positive ion-mode with nano-electrospray ionization (scan range, m/z 100–600; collision energy, 50 eV). C: Fragmentation spectrum of precursor ion [M + H]⁺ for TAAHI (m/z 553.3) in urine sample 3 from worker #14 (TAHI 9.89 μg/L). Spectrum was obtained by nano-UPLC-ESI-MS/MS operated in positive ion-mode with nano-electrospray ionization (scan range, m/z 100–600; collision energy, 25 eV). D: Fragmentation spectrum of precursor ion [M + H]⁺ for TAAHI (m/z 553.3) in urine sample 3 from worker #14 (TAHI 9.89 μg/L). Spectrum was obtained by nano-UPLC-ESI-MS/MS operated in positive ion-mode with nano-electrospray ionization (scan range, m/z 100–600; collision energy, 50 eV).

Fig. 8.

[A] Total ion chromatograms acquired by selected reaction monitoring for TAAHI (combined m/z 553.3 → 130.0 and 494.4) and TAAHpI (combined m/z 595.3 → 130.0, 226.1, and 536.4); obtained for control urine spiked with TAHI (0.25 μg/L) and TAHpI (2.0 μg/L). [B] Total ion chromatograms acquired by selected reaction monitoring for TAAHI (combined m/z 553.3 → 130.0 and 494.4), and TAAHpI (combined m/z 595.3 → 130.0, 226.1, and 536.4); obtained for urine sample 8 from worker #7 spiked with TAHpI (2.0 μg/L) and a calculated concentration of 0.36 μg/L for TAHI.