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Differential mobility spectrometry of paralytic shellfish toxins for enhanced selectivity of LC-MS analysis
Quantitation of PSTs by HILIC-DMS-MS/MS
• Limits of detection estimated from S/N ratios of low level matrix matched standards.
• Differences in LOD between runs with and without DMS were highly analyte dependent.
• Quantitation of PSTs in three different shellfish tissue reference
materials was compared by HILIC-MS/MS and HILIC-DMS-MS/MS. • Good agreement observed with reference values and especially with
HILIC-MS/MS results.
Source Fragmentation of Labile PSTs
• Sulphated PSTs exist as pairs of extremely labile 11-α and stable
11-β epimers.
• For each source parameter, a compromise setting is needed that will preserve fragile PSTs but sensitively detect stable ones.
• Similar trends are observed for energies, temperatures and gas flows elsewhere in the MS source, during CID and for DMS optimization.
Experimental
• SCIEX 5500 QTRAP operated in positive ionization mode • NRC HILIC Method – Agilent 1260 LC
o 5 μm TSKgel Amide-80 column (250 x 2 mm ID, Toso Hass)
o CH
3CN/H2O gradient, 50 mM HCOOH + 2 mM NH4OOCH
• Routine UHPLC HILIC Method – Agilent 1290 LC
o 1.7 μm BEH Amide (100 mm x 2.1 mm ID, Waters)
• A SelexION DMS was modified for external metering of carrier gas
modifier as described previously.1,2,3
Differential Mobility Spectrometry of Paralytic Shellfish Toxins for Enhanced
Selectivity of LC-MS Analysis
Fragmentation of Labile PSTs in DMS
• Fragmentation before or during DMS led to detection of
[M+H-SO3]+ product at a higher CV than the [M+H]+ precursor.
• Products formed after DMS are detected at the CV of the precursor.
• DMS parameters also cause fragmentation, but have an additional impact of changing the CV of precursor and product ions.
Conclusions and Future Work
• DMS showed excellent selectivity for PSTs and was able to separate all isomers from one another.
• Labile analytes are prone to dissociation during DMS separation. • DMS conditions with higher modifier concentrations and lower
temperatures help preserve labile analytes.
• Multidimensional HLIC-DMS-MS/MS method showed improved
selectivity compared to HILIC-MS/MS, but at the cost of sensitivity. • Planar DMS showed similar ability to separate PST epimers to
previous work using cylindrical FAIMS3, both of which showed
significantly higher resolution of epimers than linear traveling wave
ion mobility spectrometry4.
• Going forward, HILIC-DMS-MS/MS will be used for PST
quantitation in cases where matrix interference is suspected. • In the future, DMS could be used for direct screening by flow injection DMS-MS/MS, without the need for chromatography.
• The utility of DMS for other classes of algal biotoxins will continue to be investigated.
References
1. DG Beach, ES Kerrin, MA Quilliam. Anal. Bioanal. Chem. 2015, 407, 8397.
2. RW Purves et al. J. Am. Soc. Mass Spectrom. 2014, 1274.
3. DG Beach, JE Melanson, RW Purves. Anal. Bioanal. Chem. 2015, 407, 2473.
4. S Poyer et al. Int. J. Mass Spectrom. 2016, 402, 20.
Daniel G. Beach
Figure 4. Optimization of electrospray temperature for different classes of PSTs
Figure 7 Impact of ESI temperature and declustering potential on detection of labile (GTX2) and stable (GTX3) PST epimers by ESI-DMS-MS.
Figure 8. Impact of DMS parameters on detection of labile (GTX2) and stable (GTX3) PST epimers by ESI-DMS-MS.
Figure 2. Filtering of ions by DMS prior to MS detection
DMS electrode DMS electrode + + + selected ions to MS + all ions from ESI
DMS inlet on QTRAP cross-section of DMS separation
Introduction and Overview
• Paralytic Shellfish Toxins (PSTs) are a class of potent neurotoxin of algal origin that lead to intoxication and death annually as the result of consumption of contaminated shellfish.
• LC-MS is widely used for analysis of algal biotoxin, but challenges remain with small polar toxins like paralytic shellfish toxins (PSTs). • PSTs exist as a mixture of isomers and analogues (Fig. 1) that must
be separated prior to MS/MS analysis in complex biological and environmental samples prone to matrix interference.
• Recent work on the polar neurotoxin β-N-methylamino-L-alanine
and its isomers showed the benefit to selectivity and limits of detection that could be achieved by using differential mobility
spectrometry (DMS) in combination with LC and MS/MS detection.1
• Here, this approach is extended to PSTs with the goal of investigating their behaviour during DMS separation and developing a quantitative LC-DMS-MS/MS method.
• Preliminary using cylindrical FAIMS showed the promise of
separating PSTs from one another using differential mobility.2
• Precise metering of low (< 1%) concentrations of solvent vapours as carrier gas modifiers has been critical to the optimization of DMS separations for small polar analytes.
• Insight is gained into the causes of in-source fragmentation of labile PSTs before, during and after DMS separation.
Figure 6. Impacts of varying acetonitrile concentration in carrier gas on CV of transmission and sensitivity of PSTs analyzed by ESI-DMS-MS.
(DV = 3250 V)
DMS Optimization
• The DMS carrier gas modifier delivery system was modified to allow for external metering of modifier solvent.
o allowed for a full range of modifier concentrations to be
investigated
o improved signal stability and DMS peak shape
• Improved selectivity was observed at high dispersion voltage at the cost of sensitivity, especially for labile PSTs.
• Improved selectivity was observed at low carrier gas concentration (~ 0.25 %), but higher concentrations (~ 1%) showed a protective effect on labile PSTs.
HLIC-DMS-MS/MS Method Development
• Two sets of DMS conditions investigated in HILIC-DMS-MS/MS: • Maximum selectivity at 0.35% MeCN modifier and DV = 3250 • Improved duty cycle and detection of labile PSTs at 2% MeCN
and DV = 3500
• Selected reaction monitoring (SRM) transitions become compound specific once a CV is assigned
• DMS CV switching time slow (msec) compared to SRM (μsec)
• LOD proportional to the number of SRM transitions monitored.1
Retention time scheduling used to maximize duty cycle in final method.
Figure 9. Improvements in selectivity of PST detection using two different HILIC-DMS-MS/MS methods.
Figure 1. Chemical structures of PST analogues N N H2N NH H N NH2 OH OH H R4 R1 R2 R 3 O O NH2 O O NHSO3 -OH R1 R2 R3 H H H STX H H OSO3- GTX2 H OSO3- H GTX3 OH H H NEO OH H OSO3- GTX1 OH OSO3- H GTX4 H H H GTX5 H H OSO3- C1 H OSO3- H C2 OH H H GTX6 OH H OSO3- C3 OH OSO3- H C4 H H H dcSTX H H OSO3- dcGTX2 H OSO3- H dcGTX3 OH H H dcNEO OH H OSO3- dcGTX1 OH OSO3- H dcGTX4 STXs = saxitoxins GTXs = gonyautoxins CTXs = N-sulfocarbamoyl gonyautoxins 1 3 7 9 10 12 6 -11 Toxin R4 carbamoyl decarbamoyl N-sulfocarbamoyl
gas from onboard flow meter
1/16” PEEK tubing
gas + modifier to DMS
¼"Brass Union Tee
½” to ¼” Brass reducer (female-female) ½” to ¼” Brass reducer (male-male) Glass wool DMS gas line
modifier flow from syringe or LC pump
Figure 3. Modified carrier gas modifier solvent delivery system
% MeCN in Carrier Gas
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0 20 40 60 80 100 GTX3 GTX4 STX dcSTX NEO dcNEO dcGTX3 C2 GTX5 GTX6 C4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 -30 -25 -20 -15 -10 -5 0 GTX3 GTX4 STX dcSTX NEO dcNEO dcGTX3 C2 GTX5 GTX5 C4
% MeCN in Carrier Gas
0.0 0.2 0.4 0.6 0.8 1.0 1.2 % R el at iv e Int ens it y 0 20 40 60 80 100 GTX2 GTX1 dcGTX2 C1 C3 0.0 0.2 0.4 0.6 0.8 1.0 1.2 C om pens at ion V ol tag e (V ) -30 -25 -20 -15 -10 -5 0 GTX2 GTX1 dcGTX2 C1 C3 Labile PSTs Stable PSTs Se le c ti v it y Se n s it iv it y Dispersion Voltage(V) 2500 2750 3000 3250 3500 3750 4000 0 20 40 60 80 100 GTX3 GTX4 STX dcSTX NEO dcNEO dcGTX3 C2 GTX5 GTX6 C4 2500 2750 3000 3250 3500 3750 4000 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 STX GTX4 GTX3 C4 GTX6 GTX5 C2 dcGTX3 dcNEO NEO dcSTX Dispersion Voltage (V) 2500 2750 3000 3250 3500 3750 4000 % R el at iv e Int ens it y 0 20 40 60 80 100 GTX2 GTX1 dcGTX2 C1 C3 2500 2750 3000 3250 3500 3750 4000 C om pens at ion V ol tag e (V ) -30 -25 -20 -15 -10 -5 0 GTX2 GTX1 dcGTX2 C1 C3 Labile PSTs Stable PSTs Se le c ti v it y Se n s it iv it y
Figure 5. Impacts of varying dispersion voltage on CV of transmission and sensitivity of PSTs analyzed by ESI-DMS-MS. (0.4 % MeCN modifier)
Dispersion Voltage DMS Temperature
-30 -20 -10 2900 V 3300 V 3500 V 3700 V [GTX3+H]+ [GTX2+H-SO3]+ [GTX2+H]+ Compensation Voltage (V) -30 -20 -10 0.03% 0.2% 0.5% 1% [GTX3+H]+ [GTX2+H-SO3]+ [GTX2+H]+ Compensation Voltage (V) -30 -25 -20 -15 -10 -5 0 150 ○C 225 ○C 300 ○C Compensation Voltage (V) % MeCN Modifier [GTX3+H]+ [GTX2+H-SO3]+ [GTX2+H]+ 22 23 24 25 26 27 28 0 2000 4000 6000 8000 10000 Time (min) 22 23 24 25 26 27 28 0 1000 2000 3000 4000 5000 dcGTX2 dcGTX3 dcGTX3 dcGTX2 Time (min) 5.0 5.5 6.0 6.5 7.0 7.5 8.0 0 2000 4000 6000 8000 5.0 5.5 6.0 6.5 7.0 7.5 8.0 0 20000 40000 60000 80000 0 0 NEO NEO m/z > m/z, CV 316 > 296, -19.2 V H IL IC -DMS -MS /MS H IL IC -MS /MS Int ens it y ( c ps ) Int ens it y ( c ps )
routine UHPLC-HILIC NRC 5 μm HILIC
variab le r .t . ma trix in te rf e re n ce variab le r .t . ma trix in te rf e re n ce m/z > m/z, CV 353 > 273, -16.4 V 353 > 255, -13.4 V
Figure 10 Comparison of quantitative analysis by MS/MS and HILIC-DMS-MS/MS to reference values for three shellfish tissue reference materials. PSTs were extracted using a dispersive single step extraction and quantitated using matrix matched calibration.
1 2 3 4 5 6 7 8 9 μ m ol /k g t is s ue blue mussel RM HILIC-DMS-MS/MS LC-pCox-FLD HILIC-MS/MS 0 1 2 3 4 5 6 μ m ol /k g t is s ue NRC CRM-PSP-Mus HILIC-DMS-MS/MS Certified Value HILIC-MS/MS 0.0 0.2 0.4 0.6 0.8 1.0 μ m o/ k g t is s ue
CEFAS pacific oyster CRM
HILIC-DMS-MS/MS Certified Value HILIC-MS/MS LOD STX dcSTX NEO GTX1 GTX4 GTX2 GTX3 dcGTX2 dcGTX3 C2 C1 GTX5 HILIC-MS/MS (nM) 132 95 73 23 7.8 8.0 8.4 5.2 6.6 0.60 5.4 3.8 HILIC-DMS-MS/MS (nM) 23 48 22 2.8 36 5.0 3.0 1.5 1.6 1.5 0.62 5.4 HILIC-MS/MS (nmol/kg tissue) 661 476 364 116 39 40 42 26 33 3.0 27 19 HILIC-DMS- MS/MS (nmol/kg tissue) 114 240 111 14 181 25 15 7.4 8.0 7.6 3.1 27
Measurement Science and Standards, National Research Council Canada, Halifax, NS
glass wool
diffuser gas + modifier to DMS
gas from onboard flow meter
modifier solvent from LC or syringe pump STXs 11α-GTXs 11β-GTXs Compensation Voltage (V) -30 -25 -20 -15 -10 -5 0 600 ○C 400 ○C 200 ○C Compensation Voltage (V) -30 -25 -20 -15 -10 -5 0 20 V 150 V 300 V
ESI Temperature Declustering Potential
[GTX3+H]+ [GTX2+H-SO3]+ [GTX2+H]+ [GTX3+H]+ [GTX2+H-SO3]+ [GTX2+H]+
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