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, Massachusetts1Dept. Pharmacognosy, U. IL Chicago

High Throughput Microcoil NMR Using Segmented Flow Sample Loading

NanoSplitter LS-MS:

Sampling Flat Region of Parabolic FlowPreserves Chromatographic Resolution.

40-fold Better S/N.1000-fold better mass sensitivity. Reduced Ion Suppression

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.

Total Ion Chromatogram

min2 4 6 8 10 12 14

mAU

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DAD1 C, Sig =210,8 Ref=360,100 (YIQING \ YIQING_092706_5.D)

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RT: 0.06 -16.05 SM: 7B

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5101520253035404550556065707580859095

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ativ

e A

bund

ance

4.42

12.8910.26

9.49

4.93 8.597.185.251.18 7.504.09 6.162.33 2.52 10.65 12.24 13.300.84 14.13 14.55

UV

NMR

cycloheximide

20 µg

1 µg

.2 µg

(1/15 y-scale)

1.5x y-scale

1x y-scale

X

1 hr /fraction

Sample Preparation:

Cyanobacterial Culture

centrifugation

freeze-dry

SPE cleanup

organic solventextraction

Silica gel columnprefractionation

6 Fractions

Example: Fraction 2 (30 μg injection)

UV chromatogram

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DAD1 A, Sig=254,4 Ref=360,100 (YL_090707_1903_FRC2_2.D)

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20.9

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32.4

39

taxol

Hapalindole H

ambiguine I ambiguine E

new compound?

NH

Me

HMe

Me

NCH

yl_082707_mix3_MS2 #1019-1092 RT: 21.63-22.12 AV: 14 NL: 6.81E6F: + c d Full ms2 305.12@35.00 [ 70.00-625.00]

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288.16

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305.22

246.21 306.22196.12182.19158.11122.2094.10 364.91323.16 520.16405.35

yl_082707_mix3_MS2 #1178-1267 RT: 24.74-25.19 AV: 13 NL: 5.81E6F: + c d Full ms2 407.03@35.00 [ 100.00-825.00]

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339.17

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237.24337.13 407.20

289.22221.19 408.25181.36158.13 458.16 595.06

HRMS: m/z 304.1945Calculated for C21H24N2, error 0.6ppm

Hapalindole Hm/z 305.22 278.14:

loss of HCN

m/z 407.20 380.14:

loss of HCN

new compound?

taxol (300 ng onto LC)

taxol (reference)

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.

LC-MS-NMR of entire separation

NMR of fractions selected after LC-MSEvaluation

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).

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.

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.

UV-DAD

LC-MS

Nanosplitter ESI-MS

Culture

Bioactivity

Segm

ented Flow

Loading

Sample Recovery

LC–MS + offline microcoil NMRMetabolite Identification Platform

Bioactive Fraction

LC Separation

Fraction Collection200 µL Fractions

NMR

Microcoil

Evaporate LC Solvent

Resuspend in 2-4 uL NMR solvent

LC-MS Lab

NMR Lab

Biochem Lab

(Store in Freezer)

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.

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).

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.

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.

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.