Elix 5 system

The reagents urea, 1,4,5,8-naphthalene tetracarboxylic dianhydride (NTCDA), horseradish peroxidase (HRP), N,N-diethyl-1,4-phenylenediamine sulfate (DPD), nitro blue tetrazolium (NBT), NaH₂PO₄·H₂O, Na₂HPO₄·7H₂O, hydrogen peroxide (30% aqueous solution), glycerol, toluene, diethyl ether, hexanes and phosphate buffered saline (PBS, 10× concentration) were purchased from Sigma Corporation or Fisher Scientific and used as received without additional purification. Pure strain samples of E. coli O157:H7 (ATCC 43894), S. aureus (ATCC BAA-44) and Ps. aeruginosa PAO1 (ATCC BAA-47) were purchased from ATCC (Manassas, VA). Deionized water (DI H₂O, 18 MΩ) was purified using a Millipore Elix 5 system. Pre-purified nitrogen gas (≥99.99%) was obtained from Norco Corporation (Boise, Idaho). Samples of CN were prepared via thermal polymerization of anhydrous urea at 575 °C, as previously described.^{21 (link)}

HA, MA, and PGA were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). o‐MHA, m‐MHA, p‐MHA, and DMP were purchased from Tokyo Kasei Kogyo (Tokyo, Japan). N‐Benzoyl‐DL‐alanine was used as an internal standard (IS) and purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Hydrochloric acid (HCl) and pyridine were analytical grade. Water was purified with an Elix 5 system (Millipore, Bedford, MA, USA). A ClinChek^®—Control (Urine Control lyophilized) for occupational medicine was purchased from RECIPE Chemicals +Instruments GmbH (Munich, Germany). A mixed standard stock solution of MA, PGA, HA, and MHAs (each 10 g/L) and an IS solution (5 g/L) were prepared in methanol and stored at 4°C. Blank urine samples were collected from healthy adult volunteers. These volunteers were not occupationally exposed to toluene, xylene, styrene, or ethylbenzene. Informed consent was obtained from the volunteers before collection of urine. This study was approved by the Ethics Committee of the Japan Industrial Safety and Health Association.