IDENTIFICATION AND STRUCTURAL … and structural elucidation of two novel glucosinolates (GLS) in...
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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2012 Waters Corporation The MS E data acquisition technique was used to acquire all data. In this mode the instrument alternates between low and high collision state on alternate scans (Figure 3). Figure 3. The MS E acquisition technique This allows collection of precursor and fragment ion information for all species in an analysis without the sampling bias introduced with other common methods, such as DDA; where a specific m/z must be isolated before fragmentation. Further experiments were also run using the ion mobility separation (IMS) MS/MS functionality of the SYNAPT G2 to obtain more specific fragmentation information of the proposed new glucosinolate compounds. RESULTS AND DISCUSSION UPLC/MS E The BPI chromatogram of the extract is shown in figure 4. The extract was initially screened for known GLS compounds. The main peak at 2.77 minutes was identified as glucoaubrietin. During this process two unidentified peaks were detected in the chromatogram at 3.1 and 5.2 minutes. Figure 4. BPI chromatogram of SPE extract showing peaks of interest at 3.08 ad 5.18 minutes. Extraction of the low energy MS E spectra (provides the precursor ion information) showed major deprotonated ions at m/z 520.0981 and 504.1032. INTRODUCTION When screening food samples for known and unknown compounds the need to completely characterise each sample is difficult due to the complexity of sample matrix. Chromatographic resolution is essential to resolve the compounds of interest from the endogenous background, as is high resolution spectral data for similar reasons. For many analyses it has been shown that ion mobility coupled with these techniques provides an additional degree of specificity and also allows complex fragmentation experiments to be performed. The method presented here utilizes QuanTof detector technology and HDMS capabilities of the SYNAPT TM G2 mass spectrometer to overcome these challenges for the confident identification of new compounds. Here we present a strategy for the rapid identification and structural elucidation of two novel glucosinolates (GLS) in Aubrieta deltoidea. GLS are secondary plant metabolites found almost exclusively within the plant order Brassicales. They have been shown to be both health beneficial and toxic so methods for their accurate determination is essential. The nature of GLS with their almost endless range of side-chain modification makes identification and structural elucidation challenging. The total number of known individual GLS continues to increase and reached 204 in 2010. The discovery of novel GLS can be added to this database for future screening. While rock cress is not a food plant it was apparent that it contained new GLS and was considered suitable as a food model. IDENTIFICATION AND STRUCTURAL ELUCIDATION OF TWO NOVEL GLUCOSINOLATES IN AUBRIETA DELTOIDEA USING UPLC-QTOF-MS WITH ION MOBILITY A. Gledhill 1 , Dominic Roberts 1 & Dr. Don B. Clarke 2 1 Waters Corporation, Atlas Park, Manchester, M22 5PP, UK. 2 The Food & Environment Research Agency, Sand Hutton, York, YO41 1LZ, UK. Figure 1. Aubrieta deltoidea METHODS Samples Myrinase hydrolysis was avoided by freeze drying Aubrieta deltoidea tissue prior to grinding and extraction with 70% aqueous methanol at 20 o C. GLS were removed from extracts using SPE. Waters Oasis ® WAX Cartridges (6 cc, 150 mg, 30 μm) were loaded at a constant drip rate by increasing from gravity feed to full vacuum as required. After loading, cartridges were washed with ammonium acetate (2 x 6 mL, 25 mM, pH 4.5), and methanol (1 x 6 mL) . The GLS were eluted with basic methanol (4 mL, 0.1% ammonia). The eluates were dried under a stream of nitrogen gas (30 o C), until dry and the residues taken up in water (2 mL, sonicated for 10 min). UPLC/HDMS Methodology ACQUITY UPLC Separation prior to MS was performed using the ACQUITY UPLC ® system with an ACQUITY HSS T3 1.8 μM 2.1 x 100 mm column. The column oven was maintained at 45 o C and 5 μl of sample extract was injected. Mobile phase A consisted of water + 0.1% formic acid and mobile phase B methanol + 0.1% formic acid. A 10-minute gradient, with a flow rate of 0.5 mL/min was run from 2% to 98% B holding for 1 minute before returning to start conditions. SYNAPT G2 HDMS The mass spectrometer, a SYNAPT G2 HDMS, was operated in negative ion electrospray mode with activated ion mobility. Capillary voltage was set at 0.7 kV, cone voltage 30 V, desolvation temperature 450 o C, desolvation gas flow 800 L/ Hr. Figure 2. Instrument schematic of the SYNAPT G2 HDMS The precursor ion was selected in the quadrupole and the unique functionality of the SYNAPT G2 allowed time-aligned parallel (TAP) fragmentation experiments to be performed (Figure 6). The precursor ion was selected in the quadrupole and the unique functionality of the SYNAPT G2 allowed time aligned parallel (TAP) fragmentation experiments to be performed. TAP, which is CID-IMS-CID, allows fragmentation to occur pre- IMS cell and post-IMS cell. The fragment ions produced in the Trap can be separated based on their size as they move through the IMS cell. First generation ions can then be fragmented further in the transfer T-wave. This information can then be visualized in Driftscope TM , a software package that works with 4D data and allows visualization of each sample in 2D and 3D plots. Figure 7. Driftscope visualization of m/z 520 and fragments after fragmentation in the Trap region and IMS separation. Inset showing the 3D visualization. Figure 8. TAP fragmentation of m/z 520 fragments. m/z 262 was selected to isolate the oxygen function on the alkyl chain. Fragments were assigned using MassFragment. When these exact masses were analysed by the elemental composition calculator the formulas C 17 H 31 NO 11 S 3 (methylsulfinyloxononyl-GLS) and C 17 H 31 NO 10 S 3 (methylthiooxononyl-GLS) were each top hits, using iFIT TM , with <1 ppm mass accuracy. The high energy spectra, (which provides the fragmentation of the two compounds) are shown in figure 5. The fragmentation information can be used to provide further confirmation of the suspected identification. Figure 5. High energy spectra of peaks at 3.08 (lower) and 5.18 minutes (upper). Accurate mass fragments are assigned using MassFragment TM to provide evidence for the proposed identification. Fragment analysis of the high energy data was performed using MassFragment. MassFragment employs a systematic bond disconnection approach to assign fragment ions to proposed structures giving a score to the most probable. A total of 26 accurate mass fragments were assigned to methylsulfinyloxononyl-GLS and 18 to methylthiooxononyl- GLS, providing significant structural information to support the proposed new GLS. UPLC/IMS/MS/MS UPLC/IMS/MS/MS was also performed to provide more detailed fragmentation information of the two proposed new glucosinolates. Figure 6. Time-aligned parallel (TAP) fragmentation technique. The predominant fragmentation ions were from desulfation and removal of the terminal methyl-sulfinyl groups. The MS E fragments m/z 182.9660, 228.0331, 312.0212 and 344.0103 suggest that the [absent] oxygen function is further along the alkyl chain, i.e. not on C 1 -C 2 . Figure 9. Structural assignment and fragmentation pathway for methylsulfinyloxononyl-GLS C 17 H 31 NO 11 S 3 . Experiments taking fragments such as m/z 262.0749 provided an unambiguous diagnostic ion at m/z 191.9967 where the complete removal of the C 5 -C 9 chain positions the oxo-function on C 3 . CONCLUSIONS SYNAPT G2 HDMS technology has enabled the true complexity of Aubrieta deltoidea to be observed using UPLC-IMS-MS e . Two new glucosinolates were identified and confirmed from a single UPLC/MS E acquisition. The use of ion mobility helps clean up the mass spectra of the identified and unidentified compounds—and this allows the for much easier assignment and structural elucidation. The MassFragment software also provides a fast and accurate approach to solving complex structural elucidation problems. The use of MS/MS and time-aligned parallel (TAP) fragmentation provides further structural information. Intact GLS after WAX SPE Time 2.00 4.00 6.00 8.00 10.00 % 0 100 RC_006 Sm (Mn, 1x2) 1: TOF MS ES- BPI 1.32e6 2.77 438.0544 5.18 504.1037 3.08 520.0989 Drift time Product ions - separated by IMS m/z Precursor ion fragmented Drift time m/z Precursor & fragments share same drift time Q1 mass selection O HO HO OH OSO3 - OH O HO HO O SH OH SO3 - O HO HO OH S OH H + O - HS H H S O O HO S O O O - O S O O O - O HO HO OH S OH N O S - O O O O O HO HO OH S OH N - O O HS N - O O O HO HO HO S OH N O S - O O O O HO HO OH S OH OSO3 - N O S - O O O O HO HO OH S OH S O O - N O S O O O N O S - O O O O N O S - O O N HO O SO3 - + HS O N O S - O O O S OH O - S O HO HO O - OH O N O S - O O O O HO HO OH S OH S O O N - O OH N O S - O O O N O S HO O O HS Glc3 m/z 259.0124 Glc2 m/z 274.9895 Sulfate transfers HSO4 - m/z 96.9596 SO4 .- m/z 95.9517 C6H11O8S2 - C6H11O9S - C16H26NO7S - m/z 376.1430 C10H16NO2S - m/z 214.0902 C2H3SO .- m/z 74.9905 Glc1 m/z 290.9844 C6H11O9S2 - C16H26NO10S2 - Exact Mass: 456.0998 C16H27NO11S3 2- m/z 505.0746 C10H16NO5S - Exact Mass: 262.0749 C17H30NO11S3 Exact Mass: 520.0981 C10H17NO2 Exact Mass: 183.1259 C8H14NO3S - Exact Mass: 204.0694 1 5 7 9 8 6 4 2 3 1 2 1 2 3 4 5 6 7 3 9 9 9 9 3 3 C10H16NO5S2 - m/z 294.0470 C11H17O2S2 - m/z 245.0670 C6H11O5 - m/z 163.0607 C8H12NO5S - m/z 234.0436 3 4 6 7 5 C5H6NO5S - m/z 191.9967 C3H5NO4S2 m/z 182.9660 7 8 6 C17H30NO8S2 - m/z 440.1413 S O O O - SO3 .- m/z 79.9568 + O HO HO OH S OH O N O S - O O O C12H18NO10S2 - m/z 400.0372 O OH S N O S - O O O C9H14NO7S2 - m/z 312.0212 HO S O OH N - O C10H16NO3S - m/z 230.0851 HO O - C4H5O3 - m/z 101.0239 - O C5H9O - m/z 85.0653 O HO HO O - OH C6H11O5 - m/z 163.0607 O HO S OH N - O C9H10NO4S - m/z 228.0331 Glucose fragments 3 4 2 1 C9H14NO9S2 - m/z 344.0110 OH 3 4 R H Mclafferty rearrangement Size Shape Charge
Transcript of IDENTIFICATION AND STRUCTURAL … and structural elucidation of two novel glucosinolates (GLS) in...
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The MSE data acquisition technique was used to acquire all data. In this mode the instrument alternates between low and high collision state on alternate scans (Figure 3).
Figure 3. The MSE acquisition technique
This allows collection of precursor and fragment ion information for all species in an analysis without the sampling bias introduced with other common methods, such as DDA; where a specific m/z must be isolated before fragmentation.
Further experiments were also run using the ion mobility separation (IMS) MS/MS functionality of the SYNAPT G2 to obtain more specific fragmentation information of the proposed new glucosinolate compounds.
RESULTS AND DISCUSSION
UPLC/MSE
The BPI chromatogram of the extract is shown in figure 4. The extract was initially screened for known GLS compounds. The main peak at 2.77 minutes was identified as glucoaubrietin. During this process two unidentified peaks were detected in the chromatogram at 3.1 and 5.2 minutes.
Figure 4. BPI chromatogram of SPE extract showing peaks of interest at 3.08 ad 5.18 minutes.
Extraction of the low energy MSE spectra (provides the precursor ion information) showed major deprotonated ions at m/z 520.0981 and 504.1032.
INTRODUCTION When screening food samples for known and unknown compounds the need to completely characterise each sample is difficult due to the complexity of sample matrix.
Chromatographic resolution is essential to resolve the compounds of interest from the endogenous background, as is high resolution spectral data for similar reasons.
For many analyses it has been shown that ion mobility coupled with these techniques provides an additional degree of specificity and also allows complex fragmentation experiments to be performed.
The method presented here utilizes QuanTof detector technology and HDMS capabilities of the SYNAPTTM G2 mass spectrometer to overcome these challenges for the confident identification of new compounds.
Here we present a strategy for the rapid identification and structural elucidation of two novel glucosinolates (GLS) in Aubrieta deltoidea.
GLS are secondary plant metabolites found almost exclusively within the plant order Brassicales. They have been shown to be both health beneficial and toxic so methods for their accurate determination is essential.
The nature of GLS with their almost endless range of side-chain modification makes identification and structural elucidation challenging. The total number of known individual GLS continues to increase and reached 204 in 2010.
The discovery of novel GLS can be added to this database for future screening. While rock cress is not a food plant it was apparent that it contained new GLS and was considered suitable as a food model.
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.
Figure 1. Aubrieta deltoidea
METHODS Samples
Myrinase hydrolysis was avoided by freeze drying Aubrieta deltoidea tissue prior to grinding and extraction with 70% aqueous methanol at 20 oC. GLS were removed from extracts using SPE. Waters Oasis® WAX Cartridges (6 cc, 150 mg, 30 μm) were loaded at a constant drip rate by increasing from gravity feed to full vacuum as required.
After loading, cartridges were washed with ammonium acetate (2 x 6 mL, 25 mM, pH 4.5), and methanol (1 x 6 mL) . The GLS were eluted with basic methanol (4 mL, 0.1% ammonia). The eluates were dried under a stream of nitrogen gas (30 oC), until dry and the residues taken up in water (2 mL, sonicated for 10 min).
UPLC/HDMS Methodology
ACQUITY UPLC
Separation prior to MS was performed using the ACQUITY UPLC® system with an ACQUITY HSS T3 1.8 µM 2.1 x 100 mm column.
The column oven was maintained at 45 oC and 5 µl of sample extract was injected.
Mobile phase A consisted of water + 0.1% formic acid and mobile phase B methanol + 0.1% formic acid. A 10-minute gradient, with a flow rate of 0.5 mL/min was run from 2% to 98% B holding for 1 minute before returning to start conditions.
SYNAPT G2 HDMS
The mass spectrometer, a SYNAPT G2 HDMS, was operated in negative ion electrospray mode with activated ion mobility. Capillary voltage was set at 0.7 kV, cone voltage 30 V, desolvation temperature 450 oC, desolvation gas flow 800 L/Hr.
Figure 2. Instrument schematic of the SYNAPT G2 HDMS
The precursor ion was selected in the quadrupole and the unique functionality of the SYNAPT G2 allowed time-aligned parallel (TAP) fragmentation experiments to be performed (Figure 6).
The precursor ion was selected in the quadrupole and the unique functionality of the SYNAPT G2 allowed time aligned parallel (TAP) fragmentation experiments to be performed.
TAP, which is CID-IMS-CID, allows fragmentation to occur pre-IMS cell and post-IMS cell. The fragment ions produced in the Trap can be separated based on their size as they move through the IMS cell. First generation ions can then be fragmented further in the transfer T-wave.
This information can then be visualized in DriftscopeTM, a software package that works with 4D data and allows visualization of each sample in 2D and 3D plots.
Figure 7. Driftscope visualization of m/z 520 and fragments after fragmentation in the Trap region and IMS separation. Inset showing the 3D visualization.
Figure 8. TAP fragmentation of m/z 520 fragments. m/z 262 was selected to isolate the oxygen function on the alkyl chain. Fragments were assigned using MassFragment.
When these exact masses were analysed by the elemental composition calculator the formulas C17H31NO11S3 (methylsu l f iny loxononyl-GLS) and C1 7H3 1NO1 0S3 (methylthiooxononyl-GLS) were each top hits, using iFITTM, with <1 ppm mass accuracy.
The high energy spectra, (which provides the fragmentation of the two compounds) are shown in figure 5. The fragmentation information can be used to provide further confirmation of the suspected identification.
Figure 5. High energy spectra of peaks at 3.08 (lower) and 5.18 minutes (upper). Accurate mass fragments are assigned using MassFragmentTM to provide evidence for the proposed identification.
Fragment analysis of the high energy data was performed using MassFragment. MassFragment employs a systematic bond disconnection approach to assign fragment ions to proposed structures giving a score to the most probable.
A total of 26 accurate mass fragments were assigned to methylsulfinyloxononyl-GLS and 18 to methylthiooxononyl-GLS, providing significant structural information to support the proposed new GLS.
UPLC/IMS/MS/MS
UPLC/IMS/MS/MS was also performed to provide more detailed fragmentation information of the two proposed new glucosinolates.
Figure 6. Time-aligned parallel (TAP) fragmentation technique.
The predominant fragmentation ions were from desulfation and removal of the terminal methyl-sulfinyl groups. The MSE fragments m/z 182.9660, 228.0331, 312.0212 and 344.0103 suggest that the [absent] oxygen function is further along the alkyl chain, i.e. not on C1-C2.
Figure 9. Structural assignment and fragmentation pathway for methylsulfinyloxononyl-GLS C17H31NO11S3.
Experiments taking fragments such as m/z 262.0749 provided an unambiguous diagnostic ion at m/z 191.9967 where the complete removal of the C5-C9 chain positions the oxo-function on C3.
CONCLUSIONS SYNAPT G2 HDMS technology has enabled the true complexity of Aubrieta deltoidea to be observed using UPLC-IMS-MSe. Two new glucosinolates were identified and confirmed from a single UPLC/MSE acquisition. The use of ion mobility helps clean up the mass spectra of the identified and unidentified compounds—and this allows the for much easier assignment and structural elucidation. The MassFragment software also provides a fast and accurate approach to solving complex structural elucidation problems. The use of MS/MS and time-aligned parallel (TAP) fragmentation provides further structural information.
Intact GLS after WAX SPE
Time2.00 4.00 6.00 8.00 10.00
%
0
100
RC_006 Sm (Mn, 1x2) 1: TOF MS ES- BPI
1.32e62.77
438.0544
5.18504.1037
3.08520.0989
Drift timeProduct ions -
separated by IMS
m/z
Precursor ionfragmented
Drift time
m/z
Precursor & fragmentsshare same drift time
Q1 mass selection
OHO
HOOH
OSO3-
OH
OHO
HOO
SH
OH
SO3-
OHO
HOOH
S
OH
H+
O-HS
H H
S
O
O
HO S
O
O
O-O S
O
O
O-
OHOHO
OHS
OH
NO
S-O
O
O
O
OHO
HOOH
S
OH
N-O
O
HS
N-O
O
OHO
HO
HO
S
OH
NO
S-O
O
O
OHO
HOOH
S
OH
OSO3-
NO
S-O O
O
OHO
HOOH
S
OH
S
O
O-NO
SO O
O
NO
S-O
O
O
O
NO
S-O
O
NHO
O
SO3- +
HS
O
NO
S-O O
O
S
OH
O
-S
OHO
HOO-
OH
O
NO
S-O O
O
OHO
HOOH
S
OH
S
O
ON-O
OH
NO
S-O O
O
NO
SHO O
O
HS
Glc3 m/z 259.0124
Glc2 m/z 274.9895
Sulfate transfers
HSO4- m/z 96.9596SO4
.- m/z 95.9517
C6H11O8S2-
C6H11O9S-
C16H26NO7S-
m/z 376.1430
C10H16NO2S-
m/z 214.0902
C2H3SO.- m/z 74.9905
Glc1 m/z 290.9844 C6H11O9S2-
C16H26NO10S2-
Exact Mass: 456.0998
C16H27NO11S32-
m/z 505.0746
C10H16NO5S-
Exact Mass: 262.0749
C17H30NO11S3Exact Mass: 520.0981
C10H17NO2Exact Mass: 183.1259
C8H14NO3S-
Exact Mass: 204.0694
1
5 7 9
86423
1
2
1
2
3
4
5
6
7
39
9
9
9
3
3
C10H16NO5S2-
m/z 294.0470
C11H17O2S2-
m/z 245.0670
C6H11O5-
m/z 163.0607
C8H12NO5S-
m/z 234.0436
3 4
6
7
5
C5H6NO5S-
m/z 191.9967
C3H5NO4S2m/z 182.9660
7
86
C17H30NO8S2-
m/z 440.1413 S
O
O
O-
SO3.- m/z 79.9568
+
OHO
HOOH
S
OH
O
NO
S-O O
OC12H18NO10S2
-
m/z 400.0372
O
OHS
NO
S-O
O
O
C9H14NO7S2-
m/z 312.0212
HO
S
O
OHN-O
C10H16NO3S-
m/z 230.0851HO
O-
C4H5O3-
m/z 101.0239
-O
C5H9O-
m/z 85.0653
OHO
HOO-
OH
C6H11O5-
m/z 163.0607
OHO
S
OH
N-OC9H10NO4S-
m/z 228.0331
Glucose fragments
3 42
1
C9H14NO9S2-
m/z 344.0110
OH
3 4R
H
Mclafferty rearrangement
Size
Shape
Charge
