2.7. Urine Collection and Analysis for 3,5,6-Trichloro-2 Pyridinol (TCPy)

Spot urine samples were collected from the study participants at each timepoint before starting their work shift. The collected samples were transferred on wet ice to the Menoufia University laboratories in Shebin El-Kom, Egypt. The samples were then stored at −20 °C until they were shipped on dry ice for analysis at the University at Buffalo (Buffalo, NY, USA). The urine samples were analyzed for the specific metabolite of CPF, 3,5,6-trichloro-2 pyridinol (TCPy), as explained in detail previously [30]. The analysis method included hydrolysis, extraction, derivatization, and analysis by negative-ion chemical ionization gas chromatography-mass spectrometry (GC/MS) utilizing ¹³C-¹⁵N-3,5,6-TCPy as an internal standard. Creatinine was colorimetrically analyzed using the Jaffe reaction [36], and urine TCPy concentrations are expressed as micrograms TCPy per gram creatinine. The precision of the analysis method was high, with a 0.997 intraclass correlation coefficient and 0.5 ng/mL detection level. TCPy levels from 7 timepoints across the 3 years of study are available for the purpose of this work (times 1, 3, 6, 9, 11, 13, 14).