An Ultrasensitive LC-MS+NMR Platform Utilizing Segmented-Flow
Microcoil NMR, Nanospray ESI, and 4 mm LC Columns
Yiqing Lin, Paul Vouros, Jimmy Orjala1, Roger Kautz* and NIH R01 GM075856-01
The Barnett Institute
of Chemical and Biological Analysis
Boston, Massachusetts
1Dept.
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:
The nanosplitter was developed to provide the advantages of true nanoelectrospray 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).
High Throughput Microcoil NMR Using
Segmented Flow Sample Loading
This “microplug” automated loading system can pick up 2.0 µL of 2.5 µL in a 96well 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.
40-fold Better S/N.
1000-fold better mass sensitivity.
Reduced Ion Suppression
Sampling Flat Region of Parabolic Flow
Preserves Chromatographic Resolution.
LC–MS + offline microcoil NMR
Metabolite Identification Platform
Segmented Flow Loading
Culture
Resuspend in 2-4 uL NMR solvent
Bioactive
Fraction
Microcoil
NMR
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 Separation
UV-DAD
Sample Recovery
NMR Lab
(Store in Freezer)
Nanosplitter ESI-MS
LC-MS
Evaporate LC Solvent
Bioactivity
Fraction Collection
LC-MS-NMR of entire separation
200 µL Fractions
LC-MS Lab
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.
Total Ion Chromatogram
4.42
12.89
10.26
Relative Abundance
9.49
4.93
3
7.18
5.25
4.09
7.50
6.16
4
5
6
10.65
7
8
Time (min)
9
10
11
12.24
12
13.30
13
14.13
14
14.55
15
NMR
5.091
1.618
1500
7.332
1000
centrifugation
11.878
750
500
250
X
0
2
4
6
8
DAD1 A, Sig=254,4 Ref=360,100 (YL_090707_1903_FRC2_2.D)
mAU
80
9.657
1250
Cyanobacterial Culture
13.049
UV
4.611
1750
10.437
mAU
2000
Example: Fraction 2 (30 μg injection)
Sample Preparation:
16
DAD1 C, Sig=210,8 Ref=360,100 (YIQING \YIQING_092706_5.D)
23.246
2.52
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.
8.59
20.941
RT: 0.06- 16.05
SM: 7B
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
1.18
5
2.33
0.84
1
2
Biochem Lab
10
12
14
ambiguine E
ambiguine I
70
freeze-dry
.2 µg
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.
1.5x y-scale
min
60
organic solvent
extraction
Hapalindole H
18.274
50
40
1 µg
new compound?
30
1x y-scale
20 µg
(1/15 y-scale)
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.
24.268
32.439
17.261
16.200
12.705
5.858
10
Silica gel column
prefractionation
14.069
14.567
taxol
20
1 hr /fraction
25.980
SPE cleanup
0
5
10
6 Fractions
15
20
25
30
35
min
UV chromatogram
cycloheximide
yl_082707_mix3_MS2 #1019-1092 RT: 21.63-22.12 AV: 14 NL: 6.81E6
F: + c d Full ms2 305.12@35.00 [ 70.00-625.00]
288.16
100
Me
95
90
85
80
Me
Me
75
70
65
Relative Abundance
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).
60
55
50
HRMS: m/z 304.1945
Calculated for C21H24N2, error 0.6ppm
H
NC
H
45
40
262.13
35
30
25
305.22
20
15
N
H
10
5
100
246.21
182.19 196.12
94.10 122.20 158.11
0
306.22
323.16
150
200
250
300
364.91
350
m/z
405.35
520.16
400
450
500
550
600
Hapalindole H
278.14:
m/z 305.22
loss of HCN
taxol (300 ng onto LC)
yl_082707_mix3_MS2 #1178-1267 RT: 24.74-25.19 AV: 13 NL: 5.81E6
F: + c d Full ms2 407.03@35.00 [ 100.00-825.00]
339.17
100
95
90
new compound?
85
80
75
70
380.14
Relative Abundance
65
60
55
371.22
50
45
40
35
30
25
301.19
20
15
10
237.24
5
taxol (reference)
0
100
158.13 181.36
150
337.13
407.20
289.22
221.19
200
408.25
250
300
350
m/z 407.20
loss of HCN
400
458.16
450
m/z
595.06
500
550
600
380.14:
650
700
750
800
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.