Quantification of Chlorinated Cysteine Metabolites

The ¹³C₃-¹⁵N labeled cysteine
(1 mM) was chlorinated for 48 h by applying 5 mM initial chlorine
in 200 mL of the phosphate buffer solution (10 mM, pH 7). The solution
was then enriched by freeze-drying following the procedure mentioned
in section 2.3 and
reconstituted in acetonitrile-d3 and water-d2 (9:1, v-v). For NMR
analyses, an aliquot (230 μL) of this extract was spiked with
100 μL of ¹³C-urea (4.23 mM) and 2 mg of Cr(acac)₃. The 100 MHz ¹³C NMR spectra were recorded using
a 400 MHz Bruker AVIII HD spectrometer with a 30° flip angle
and a repetition delay of 35 s (¹H decoupling only during
acquisition, pulse sequence: zgig30). The structures of the ¹³C₂-¹⁵N labeled mono- and dichloroacetonitrilesulfonic
acids (ClANSA and Cl₂ANSA) and the ¹³C₂ labeled dichloroacetaldehydesulfonic acid (Cl₂AcAlSA)
were further confirmed by their respective chemical shifts (¹H, ¹³C, and ¹⁵N NMR spectra) and 2D NMR correlation
spectra. Their concentrations were estimated based on their relative
abundances to ¹³C-urea (used as the internal quantification
standard) in ¹³C NMR spectra.
Another aliquot (10
μL) of this extract was used to make a serial dilution in acetonitrile
and water (9:1, v:v). Forty μL aliquots of these diluted solutions
were then spiked into the nondisinfected drinking water samples (40
mL each) from DWTPs 1 and 2. The processed calibration curves of the
above compounds were made on SFC-QTOF following the freeze-drying
enrichment of these spiked samples, which were then used to estimate
the concentration of novel sulfonated DBPs in water samples. The concentration
of brominated DBPs was estimated based on their chlorinated analogues.
The limit of quantification (LOQ) for ClANSA and Cl₂ANSA
was 20 ng/L, and it was 40 ng/L for Cl₂AcAlSA on SFC-QTOF.