Identification and Structural Elucidation of Two Novel Glucosinolates in Aubrieta Deltoidea Using Uplc-Qtof-Ms with Ion Mobility

IDENTIFICATION AND STRUCTURAL ELUCIDATION OF TWO NOVEL GLUCOSINOLATES IN AUBRIETA DELTOIDEA USING UPLC-QTOF-MS WITH ION MOBILITY

A. Gledhill1 , Dominic Roberts1 & Dr. Don B. Clarke2 1 Waters Corporation, Atlas Park, Manchester, M22 5PP, UK. 2 The Food & Environment Research Agency, Sand Hutton, York, YO41 1LZ, UK.

The MSE data acquisition technique was used to acquire all When these exact masses were analysed by the elemental The precursor ion was selected in the quadrupole and the The predominant fragmentation ions were from desulfation and INTRODUCTION METHODS E data. In this mode the instrument alternates between low and composition calculator the formulas C17H31NO11S3 unique functionality of the SYNAPT G2 allowed time-aligned removal of the terminal methyl-sulfinyl groups. The MS high collision state on alternate scans (Figure 3). (methylsulfinyloxononyl-GLS) and C H NO S parallel (TAP) fragmentation experiments to be performed fragments m/z 182.9660, 228.0331, 312.0212 and 344.0103 When screening food samples for known and Samples 17 31 10 3 (methylthiooxononyl-GLS) were each top hits, using iFITTM, (Figure 6). suggest that the [absent] oxygen function is further along the unknown compounds the need to completely Myrinase hydrolysis was avoided by freeze drying Aubrieta with <1 ppm mass accuracy. alkyl chain, i.e. not on C1-C2. characterise each sample is difficult due to the deltoidea tissue prior to grinding and extraction with 70% The precursor ion was selected in the quadrupole and the OH o OH HS complexity of sample matrix. O O The high energy spectra, (which provides the fragmentation of unique functionality of the SYNAPT G2 allowed time aligned O aqueous methanol at 20 C. GLS were removed from extracts - HO 1 HO O HO S HO N C3H5NO4S2 HO S O - - OH O m/z 182.9660 - O C H NO S HO C H O 9 14 7 2 N 2 S ® 6 11 5 - m/z 312.0212 O - N HO the two compounds) are shown in figure 5. The fragmentation parallel (TAP) fragmentation experiments to be performed. m/z 163.0607 C5H9O O C9H14NO9S2 O O O O using SPE. Waters Oasis WAX Cartridges (6 cc, 150 mg, 30 m/z 85.0653- S m/z 344.0110 S OH O -O - O O O Chromatographic resolution is essential to O S HO - information can be used to provide further confirmation of the HO OH N C10H16NO3S OH OH μm) were loaded at a constant drip rate by increasing from HO -O m/z 230.0851 O- O - - Glucose fragments O O TAP, which is CID-IMS-CID, allows fragmentation to occur pre- C6H11O5 C4H5O3 HO HO resolve the compounds of interest from the m/z 163.0607 m/z 101.0239 S HO S suspected identification. OH gravity feed to full vacuum as required. - C9H10NO4S N OH N OH - O m/z 228.0331 O O O - IMS cell and post-IMS cell. The fragment ions produced in the O S C12H18NO10S2 endogenous background, as is high resolution O HO - m/z 400.0372 HO HO S O O HO S S OH 2- OH - C16H27NO11S3 - OSO3 N O Trap can be separated based on their size as they move O O m/z 505.0746 spectral data for similar reasons. After loading, cartridges were washed with ammonium acetate - Glc1 m/z 290.9844 C H O S S OH 6 11 9 2 OH O O OH O O 4 5 3 through the IMS cell. First generation ions can then be O HO 7 9 R (2 x 6 mL, 25 mM, pH 4.5), and methanol (1 x 6 mL) . The HO HO S HO SH OH 3 S Sulfate transfers 2 4 6 8 C H NO S- O N O 8 14 3 OH O C H NO S Exact Mass: 204.0694 1 For many analyses it has been shown that ion - O 17 30 11 3 1 3 5 fragmented further in the transfer T-wave. SO Exact Mass: 520.0981 3 S GLS were eluted with basic methanol (4 mL, 0.1% ammonia). - 7 3 4 Glc2 m/z 274.9895 C H O S - O + 2 6 11 8 2 O H 2 4 6 N - - N O C5H6NO5S o HS 1 O O O O m/z 191.9967 mobility coupled with these techniques provides OH S S The eluates were dried under a stream of nitrogen gas (30 C), - -O TM O HH O O HO O O This information can then be visualized in Driftscope , a - - .- Mclafferty HO OSO - C H SO 3 HO S O OSO 2 3 an additional degree of specificity and also OH m/z 74.9905 O rearrangement until dry and the residues taken up in water (2 mL, sonicated O O 7 9 E - Glc3 m/z 259.0124 C6H11O9S .- - 3 H 7 Figure 3. The MS acquisition technique SO4 m/z 95.9517HSO4 m/z 96.9596 6 8 O software package that works with 4D data and allows - N C H NO S 6 OH O 10 16 5 allows complex fragmentation experiments to be O O Exact Mass: 262.0749 for 10 min). O S 3 5 O -O 4 HO HS O N visualization of each sample in 2D and 3D plots. HO S O S - O - OH C H NO S S C8H12NO5S performed. N 10 16 2 - - m/z 234.0436 N O O m/z 214.0902 O O This allows collection of precursor and fragment ion C H NO S - - O OH 17 30 8 2 O m/z 440.1413 - O S O O O UPLC/HDMS Methodology HO 9 9 + O HO S SO .- m/z 79.9568 3 information for all species in an analysis without the sampling 3 OH - C16H26NO10S2 N C10H17NO2 Exact Mass: 456.0998 SO - + HO Exact Mass: 183.1259 N 3 The method presented here utilizes QuanTof OH O O O - S bias introduced with other common methods, such as DDA; S HS -O ACQUITY UPLC S O C H O S - detector technology and HDMS capabilities of the 11 17 2 2 N - OH m/z 245.0670 O O O C10H16NO5S2 m/z 294.0470 S O TM where a specific m/z must be isolated before fragmentation. -O O O HO 9 HO S SYNAPT G2 mass spectrometer to overcome 3 OH C H NO S- Separation prior to MS was performed using the ACQUITY - N 16 26 7 O m/z 376.1430 these challenges for the confident identification UPLC® system with an ACQUITY HSS T3 1.8 µM 2.1 x 100 mm Further experiments were also run using the ion mobility of new compounds. column. separation (IMS) MS/MS functionality of the SYNAPT G2 to Figure 9. Structural assignment and fragmentation pathway obtain more specific fragmentation information of the o for methylsulfinyloxononyl-GLS C17H31NO11S3. Here we present a strategy for the rapid The column oven was maintained at 45 C and 5 µl of sample proposed new glucosinolate compounds. identification and structural elucidation of two extract was injected. Experiments taking fragments such as m/z 262.0749 provided novel glucosinolates (GLS) in Aubrieta deltoidea. Mobile phase A consisted of water + 0.1% formic acid and RESULTS AND DISCUSSION an unambiguous diagnostic ion at m/z 191.9967 where the GLS are secondary plant metabolites found mobile phase B methanol + 0.1% formic acid. A 10-minute Figure 5. High energy spectra of peaks at 3.08 (lower) and complete removal of the C5-C9 chain positions the oxo-function gradient, with a flow rate of 0.5 mL/min was run from 2% to E 5.18 minutes (upper). Accurate mass fragments are assigned on C3. almost exclusively within the plant order UPLC/MS TM 98% B holding for 1 minute before returning to start using MassFragment to provide evidence for the proposed Brassicales. They have been shown to be both conditions. identification. health beneficial and toxic so methods for their The BPI chromatogram of the extract is shown in figure 4. The extract was initially screened for known GLS compounds. The accurate determination is essential. SYNAPT G2 HDMS Fragment analysis of the high energy data was performed CONCLUSIONS main peak at 2.77 minutes was identified as glucoaubrietin. using MassFragment. MassFragment employs a systematic Figure 7. Driftscope visualization of m/z 520 and fragments During this process two unidentified peaks were detected in The nature of GLS with their almost endless The mass spectrometer, a SYNAPT G2 HDMS, was operated in bond disconnection approach to assign fragment ions to after fragmentation in the Trap region and IMS separation. SYNAPT G2 HDMS technology has enabled the Inset showing the 3D visualization. range of side-chain modification makes negative ion electrospray mode with activated ion mobility. the chromatogram at 3.1 and 5.2 minutes. proposed structures giving a score to the most probable. true complexity of Aubrieta deltoidea to be e identification and structural elucidation Capillary voltage was set at 0.7 kV, cone voltage 30 V, observed using UPLC-IMS-MS . desolvation temperature 450 oC, desolvation gas flow 800 L/ challenging. The total number of known A total of 26 accurate mass fragments were assigned to Hr. Intact GLS after WAX SPE RC_006 Sm (Mn, 1x2) 1: TOF MS ES- methylsulfinyloxononyl-GLS and 18 to methylthiooxononyl- individual GLS continues to increase and reached 2.77 100 BPI 204 in 2010. 438.0544 1.32e6 GLS, providing significant structural information to support the Two new glucosinolates were identified and proposed new GLS. confirmed from a single UPLC/MSE acquisition. The discovery of novel GLS can be added to this UPLC/IMS/MS/MS database for future screening. While rock cress is not a food plant it was apparent that it UPLC/IMS/MS/MS was also performed to provide more detailed The use of ion mobility helps clean up the mass

contained new GLS and was considered suitable % fragmentation information of the two proposed new spectra of the identified and unidentified as a food model. glucosinolates. compounds—and this allows the for much easier 5.18 assignment and structural elucidation. 504.1037 3.08 520.0989

0 Time The MassFragment software also provides a fast 2.00 4.00 6.00 8.00 10.00 Q1 mass Size selection and accurate approach to solving complex Shape structural elucidation problems. Figure 4. BPI chromatogram of SPE extract showing peaks of Charge interest at 3.08 ad 5.18 minutes. m/z m/z Extraction of the low energy MSE spectra (provides the The use of MS/MS and time-aligned parallel (TAP) precursor ion information) showed major deprotonated ions at fragmentation provides further structural Figure 2. Instrument schematic of the SYNAPT G2 HDMS m/z 520.0981 and 504.1032. Drift time Drift time information. Precursor ion Product ions - Precursor & fragments Figure 8. TAP fragmentation of m/z 520 fragments. m/z 262 Figure 1. Aubrieta deltoidea fragmented separated by IMS share same drift time was selected to isolate the oxygen function on the alkyl chain. Fragments were assigned using MassFragment. Figure 6. Time-aligned parallel (TAP) fragmentation technique. TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2012 Waters Corporation