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An Ultrasensitive LCMS NMR Platform Utilizing SegmentedFlow Microcoil NMR, Nanospray ESI, and 4 mm L

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Title: An Ultrasensitive LCMS NMR Platform Utilizing SegmentedFlow Microcoil NMR, Nanospray ESI, and 4 mm L


1
An Ultrasensitive LC-MSNMR Platform Utilizing
Segmented-Flow Microcoil NMR, Nanospray ESI, and
4 mm LC ColumnsYiqing Lin, Paul Vouros, Jimmy
Orjala1, Roger Kautz and NIH R01 GM075856-01
The Barnett Institute of Chemical and Biological
Analysis
Boston, Massachusetts1Dept. Pharmacognosy, U. IL
Chicago
An offline approach to LC-NMR accommodates the
disparate timescales and sample mass requirements
of LC-MS and NMR. The 10-fold better mass
sensitivity of microcoil NMR probes is accessible
by concentrating collected LC fractions a robust
automated system for loading samples from 96-well
plates is commercially available. This poster
describes further optimizations to this approach
using two recently-developed technologies from
the Barnett Institute.
The combination of MS and NMR data are the gold
standard for identifying unknown compounds.
LC-MS-NMR is thus desirable for profiling trace
constituents of complex mixtures however, where
MS analysis takes under 1 second with 1 ng of
analyte, NMR at the microgram level requires
hours to days, depending on the information
required (1D, 2D, Heteronuclear).
NanoSplitter LS-MS
High Throughput Microcoil NMR Using Segmented
Flow Sample Loading
The nanosplitter was developed to provide the
advantages of true nano-electrospray MS with LC
separations on normal bore (2 mm and larger)
columns. The nanosplitter consumes only 0.1 of
the LC eluent, allowing 99 to be collected for
NMR. If the column capacity is 100 µg of the
largest peak, then a 0.2 constituent will
produce an interpretable spectrum in LC-NMR
(below).
This microplug automated loading system can
pick up 2.0 µL of 2.5 µL in a 96-well plate, and
load it into a microcoil NMR probe without
additional dilution.
  • An offline NMR approach has advantages
  • LC-MS is performed routinely, on familiar (or
    validated) equipment.
  • NMR data can be requested retrospectively, after
    review of LC-MS data.
  • All available sample mass from the entire LC
    peak width is pooled for NMR.
  • The most mass-sensitive (smallest) NMR probes
    can be used.
  • NMR time can be allocated according to the
    information needed Time-based collection
    resembles on-line LC-NMR data, or Peak-based
    collection of components of specific interest.

Microanalyical Methods In Natural Product
Discovery (right). The traditional method of
natural product drug discovery would be to use
bioassay-guided fractionation to purify an active
compound, then to scale up growth and
fractionation to obtain enough of the compound
for analysis and identification. With automated
microanalytical methods, MS and NMR data can be
obtained non-destructively during the separation
before bioassay. Data may be examined
retrospectively for fractions that are positive
in subsequent bioassay, and known compounds can
be recognized from MS and NMR data. Novelty and
potency can thus be established prior to the
laborious scale-up preparation.
The flow system is filled with a fluorocarbon
fluid, immiscible with common NMR solvents.
Sample plugs are formed by drawing alternately
from a 96-well plate of samples and a vial of
the immiscible fluid (Fluorinert FC43). 'Wash'
plugs of clean DMSO are inserted between samples
to eliminate 50 nL of carryover that occurs in
non-ideal components. The wash plugs also
provide a reproducible signal for positioning the
following sample plug. Commercially available
components were used to acquire all data below
a Protasis/MRM microcoil NMR probe and a Gilson
215 sample handler. (The figure above was made
with a home built probe, from Kautz et al. 2005).
It is planned to implement the microplug method
with Protasis 1-minute NMR platform.
LC-MS-NMR of entire separation
Comprehensive LC- NMR is illustrated in the
following analysis of standards, a mixture of
taxol, indapamide, and digitoxin. Time based
fraction collection was used and NMR was acquired
of all fractions, with 2 hr per fraction in an
overnight automated NMR run. This comprehensive
LC-NMR approach would be used to detect compounds
with poor electrospray ionization and UV
absorbance, such as glycans and lipids.
NMR of fractions selected after LC-MS
Evaluation
A cyanobacterial extract found to be active in a
proteasome inhibition assay was analyzed by LC-MS
(30 µg loaded to LC). Fractions were collected
of LC peaks and analyzed by microplug NMR (2
hr/peak). A number of known metabolites could
be identified from the combined MS and NMR
spectra one was established as novel.
Correlation of MS, UV, and NMR data. Retention
times of standards MS and UV chromatogram
correlated within 0.6 sec, in 6 replicates of 4
standards examined. Fraction breakpoints for NMR
are recorded on the UV chromatogram. Recovery
Comparing the amount of indapamide loaded onto
the column with the amount resuspended for NMR
analysis, the recovery was 92 with an RSD of
1.1 over six repetitions.
Limit of Detection. Injecting 250 ng of
indapamide on-column produced an NMR spectrum
with a S/N of 3 for its lowest-intensity peak.
The limit of detection was confirmed by spiking
the cyanobacterial extract (at right) with 300 ng
of taxol (350 nmol). A 2 hr NMR acquisition of
the fraction recovered after LC is shown below.
Note the NMR sensitivity may be doubled or
quadrupled by pooling 2 or 4 LC runs (30 min
each).
Linearity. From 0.25 to 25 µg indapamide were
loaded onto LC. The NMR integral of recovered
indapamide is plotted below against the amount
loaded. The plot is linear with an R2 of 0.9999.
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