Aliquots of amniotic fluid were thawed at room temperature for 1 hour prior to the analysis. The amniotic fluid analysis was proceeded with 200 μL of amniotic fluid reconstituted into 400 μL of phosphate buffer (0.045 M NaH2PO4/K2HPO4, pH 7.4, 100% D2O). The mixture was then centrifuged at 16,000× g at 4 °C for 10 minutes, and 550 μL of the supernatant was transferred into 5 mm NMR tubes (Norell, Landisville, NJ, USA).
NMR spectra were recorded using an Agilent DD2 600 MHz NMR spectrometer (Agilent Technologies, Inc., CA, USA) operating at 599.93 MHz 1H at 298 K. A standard water-suppressed one-dimensional NMR spectrum was obtained for amniotic fluid using a standard NOESY pulse sequence (recycle delay −G1−90°−t1−90°−tm−G2−90°−acquisition) with a 2 s recycle delay and a 100 ms tm. The 90° pulse length was adjusted to about 10 μs for each sample and 64 transients were collected into 32,000 data points over a 20 ppm spectral width. Metabolite assignments were usually obtained by considering chemical shifts, coupling constants and relative intensities.
NMR spectra were recorded using an Agilent DD2 600 MHz NMR spectrometer (Agilent Technologies, Inc., CA, USA) operating at 599.93 MHz 1H at 298 K. A standard water-suppressed one-dimensional NMR spectrum was obtained for amniotic fluid using a standard NOESY pulse sequence (recycle delay −G1−90°−t1−90°−tm−G2−90°−acquisition) with a 2 s recycle delay and a 100 ms tm. The 90° pulse length was adjusted to about 10 μs for each sample and 64 transients were collected into 32,000 data points over a 20 ppm spectral width. Metabolite assignments were usually obtained by considering chemical shifts, coupling constants and relative intensities.