Understanding the Variability of Compound Quantification from Targeted Profiling Metabolomics of 1D-1H-NMR Spectra in Synthetic
Mixtures and Urine with Additional Insights on Choice of Pulse Sequences and Robotic Sampling
Stanislav Sokolenko, Ryan McKay, Eric JM Blondeel, Michael J Lewis, Ben George, David Chang, and Marc G Aucoin*
*Corresponding Author: Marc G Aucoin, Department of Chemical Engineering, University of Waterloo, maucoin@uwaterloo.ca
Supplementary information 1: Pulse sequence details
Presat 1D-1H A simple 1D-1H presaturation experiment was used for carrier frequency and
pulse width optimization, and is universally available on major manufacturer’s spectrometers.
The presaturation period (wide low rectangle denoting relatively low power) was comprised of
continuous wave irradiation at the carrier position tuned to the solvent peak via an empirical
array of the frequency and then observing the residual signal minima. The power setting was
adjusted to deliver ~80 Hz of gamma-B1 induced field strength for convenient periods of ~ 2.5
seconds. The optimized pulse width for high power (~33 kHz gamma-B1) excitation (shown as
tall narrow rectangles) was either determined by arraying the pulse period and observing a 360°
rotation, and then dividing by a factor of 4 for the 90° (Agilent Inova console), or by use of the
newer nutation single pulse calibration available on digital architectures (e.g. Agilent VNMRS
console). The pulse sequence typically performs little phase cycling except basic Cyclops (i.e.
rotation of the excitation and observed phase by 90° per increment). (b) 1D-NOESY_s1a4 The
1D-NOESY, or metnoesy, contained an initial delay (10ms), then a presaturation period of
990ms using the same power settings as the 1D-1H presaturation detailed above. Following the
presaturation period, two 90° high power pulses were followed by a “mixing” period (100ms)
also executed with saturation of the solvent peak (same power as presaturation period). Lastly a
90° high power pulse is executed before a 4 second acquisition period. The phase cycle and
subsequent magnetization behaviour can be deceptively complex, and has been detailed
elsewhere (McKay 2011). (c) grd-NOESY_s1a4 Lastly, for comparison, a modified 1D-NOESY
utilizing a simple composite inversion pulse (90°x180°y90°x (Bax 1985; Levitt 1986; Tate and
Inagaki 1992)) and pulsed field gradients during the mixing time was acquired for each sample.
The grd-NOESY has the same period and power presaturation as the standard 1D-NOESY but is
followed by the composite pulse that alternates between an effective 180° and 0° pulse on odd
and even transients, respectively via the phase cycle. This was followed by a “mix” delay
totalling 100ms in length, split into three solvent saturation of 59, 29, and 9ms each. These
sections were separated by pulse field gradients (Keeler et al. 1994) of 1ms duration and
amplitudes of 3.75, -18.75, and 5.625 gauss/cm (i.e. 2:-10:3) respectively. Lastly a 90° pulse
perturbed bulk magnetization into the transverse plane for the 4 second acquisition period. The
sweep width used on all experiments (600 MHz instrument) was 7225.4 Hz, with 4 steady states
before collection of 32 transients. Data were zero filled to twice the number of acquired points,
and an apodization window of 0.5 Hz line broadening was applied before fast fourier
transformation and analysis with Chenomx Suite software.
The authors would like to acknowledge that the grd-NOESY_s1a4 used in this study was first
presented as a poster by RM et al. (2009) at the Experimental Nuclear Magnetic Resonance
Conference in Pacific Grove, California and co-developed with Leo Spyracopoulos of the
Department of Biochemistry, University of Alberta.
References
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