| sampling |
overnight fasting urine
collection |
ensures
more stable homeostatic
concentrations of metabolites |
| mid stream
urine collection |
avoids unwanted contamination
from urinary tract |
| collecting urine sample
in labeled tube containing sodium azide (NaN3) |
to stop bacterial growth
in samples; final concentration of 0.05% wt/vol |
| store
immediately in to
−40 to –80 °C until
NMR experiments are performed |
helps arrest metabolic
activities
and sample degradation |
| sample
processing |
centrifugation/filtration |
centrifuge
at 1000 rpm to remove the turbidity
from unwanted particulates,
or filter using 0.22 μ filter
to remove any macromolecular content in the sample |
| phosphate buffer |
phosphate buffer helps in
avoid chemical shift drift that occurs due to pH variations |
| internal reference
standard;
e.g., TSP or DSS |
in
protein/lipid free urine
sample, TSP and DSS are a good choices as internal standards for quantification
and normalization |
| use of deuterated EDTA |
only recommended when variation
of ionic concentration urine is very large and drift in the chemical
shifts is causing quantitative errors. |
| acquisition
parameters |
one-dimensional
gradient
NOESY with water presaturation experiment. |
|
| time domain points (TD): 64K |
Increased
resolution |
| line broadening (lb): 0.1–0.5 Hz |
| relaxation delay >5.0 s |
relaxation delay depends
on longitudinal relaxation time (T1) of
metabolite resonances; it should be five times T1 for absolute quantitation or matched to the T1 of the reference spectra used for deconvolution. |
| acquisition time: 2.5 s |
increased resolution |
| spectral width (sw): 12 ppm |
|
| number of scan (ns): 64 |
for desired S/N, more
are
required for diluted samples |
| dummy scan (ds): 8 |
to achieve steady state
prior to acquisition |
| excitation pulse: 90 deg |
shorter pulse widths can
be used for single pulse NMR analysis |
| receiver gain (rg): optimal |
either a constant
RG for
all or auto optimized for every sample |
| mixing time (tm): |
pulse sequence
requirements for NOESY; minor loss in signal intensity due to transverse
relaxation |
| 100 ms for standard experiment |
| 10 ms for gradient experiment |
| sample temperature: 300 K |
kept constant throughout
the study |
| shimming, tune, and match: for every sample |
increased
accuracy, precision,
and reproducibility |
| processing
parameters |
windowing:
exponential window
function with line broadening of 0.3–1.0 Hz |
|
| zero filling: a factor of
2 of TD |
increased
resolution |
| phase correction: manual
phasing is preferred |
optimal for accurate integration
of peaks area |
| baseline correction: automatic/manual |
increased accuracy of peak
integration |
| chemical shift referencing |
both TSP and DSS can be
used for chemical shift referencing (δ 0.0), although DSS is
the IUPAC standard |
