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.