accelerating the analysis of cyanotoxins sébastien sauvé environmental analytical chemistry –...
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Accelerating the Analysis of
CyanotoxinsSébastien Sauvé
Environmental Analytical Chemistry – Université de Montréal
Dipankar GhoshDirector for Environmental & Food safety
Thermo Fisher [email protected]
Audrey Roy-Lachapelle
Khadija Aboulfadl
Pascal Lemoine
Sherri Macleod
Liza Viglino
Arash Zamyadi
Michèle Prévost
Contributors
Context
• Microcystins are hepatotoxins produced by cyanobacteria (Blue-green Algae)
• These cyanotoxins are found in fresh waters and in drinking water reservoirs.
• A bloom can occur in warm, shallow, undisturbed surface water rich in nutrients.
Cyanobacterial bloom
Microcystis aeruginosa
K.Sivonen, G. Jones, in: 1. Chorus, J. Bartram (Eds.), Toxic Cyanobacteria in Water: A Guide to their Public Health Consequences, London, 1999.
http://www.aquarius-systems.com/Entries/View/349/bluegreen_algae.aspx http://www.plingfactory.de/index.html
•Multi-toxin online SPE-LC-MS/MS method
•Ultrafast laser diode thermal desorption methods (LDTD-APCI-MS/MS)
•Anatoxin-A
•Sum of microcystins
Objectives
Online SPE-Online SPE-LC-MS/MS LC-MS/MS
high high pressure pressure
multi-toxins multi-toxins methodmethod
Sébastien Sauvé, Département de chimie
SPE: EnrichmmentSPE: Enrichmment(solid phase extraction)(solid phase extraction)
The whole mass of analytes within the 1.0 ml sample will ne injected into the MS detector
Automated SPE Extraction Automated SPE Extraction (Online SPE)(Online SPE)
1.0 ml Chromatography MS/MSSPE
Waste
Detection:
Tandem mass spectrometry
(selected reaction monitoring - SRM)
LC-MS/MSLC-MS/MS
ThermoElectron TSQ Quantum Ultra EQuan MAX System
Tandem Mass Spectrometry (MS/MS)
Sulfamethoxazole+H+
m/z= 254.0
NH2 S
O
O
N
NO
HH
S
O
O
NH2
N
ONH2
m/z=156.0
m/z=108.0
m/z=92.0
Argon-induced Fragmentation
Cyanotoxins using LC-MS/MS
Challenge is to combine varied compounds into a single method for
the simultaneous determination of different cyanotoxins.
Target compounds
Compound pKa Molecular Weight (g mol-1)
Cylindrospermopsin 8.8 415
Anatoxin-a 9.4 165
Phenylalanine (interferent) 1.83 165
9.13
Nodularin 825
M-LR 3.5 995
1038
1045
981
1002
1025
986
M-LW
M-YR
M-LY
M-RR
Se
ve
n m
icro
cys
tin
M-LF
Dm-LR
3.5
3.5
Challenge
Speed!!
•Eliminate off line SPE
•Separate phenylanaline from anatoxin a (same SRM)
http://fav.me/dsk92w
Anatoxine-a and phenylalanin
•Separation of isobars using chromatography
•Quantification of specific fragment for anatoxin-a (166.10 > 43.3)
C:\Documents and Settings\...\anaphe3 2010-01-29 15:05:23
RT: 0.00 - 5.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0Time (min)
0
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1500000
0
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3000000
Inte
nsi
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NL: 5.02E5TIC F: + c ESI SRM ms2 [email protected] [42.800-43.800] MS anaphe3
NL: 3.22E6TIC F: + c ESI SRM ms2 [email protected] [119.500-120.500] MS anaphe3
NL: 1.82E6TIC F: + c ESI SRM ms2 [email protected] [130.600-131.600] MS anaphe3
NL: 1.16E6TIC F: + c ESI SRM ms2 [email protected] [148.600-149.600] MS anaphe3
anatoxine-a166.10 > 43.3quantificationphénylalanine166.10 > 120.0
anatoxine-a166.10 > 131.1
anatoxine-a166.10 > 149.1
Specific conditions for cyanotoxin determinations
Chromatograms obtained using SPE-UPLC/MSMS-ESI, in Milli-Q water spiked
@ 1 µg/l
TR: 3.63
TR: 3.42
TR: 3.98
TR: 5.31
TR: 3.95
TR: 3.63
TR: 5.10
TR: 3.63
TR: 1.81
TR: 1.67
TR: 3.63
TR: 3.42
TR: 3.98
TR: 5.31
TR: 3.95
TR: 5.10
TR: 5.29
TR: 3.83
Chromatograms obtained using SPE-UPLC/MSMS-ESI, in real sample (lab
culture)
Preliminary estimates of performance
Toxin Parent Fragment Recovery R2 Slope (x10-4)
MDL (ng/L)
cylindrospermopsin 416.10 194.10 98 0.9913 5.5 0.2 anatoxin-a 166.10 149.10 10 0.9949 8.6 10 MC-RR 519.76 135.00 56 0.9989 104.4 .01 MC-YR 1045.60 135.20 96 0.9997 2.5 17 nodularin 825.39 135.20 n/a - MC-LR 995.65 134.80 109 0.9936 5.7 1 dm-MC-LR 981.60 135.00 106 0.9933 8.8 3 MC-LY 1002.65 135.15 138 0.9984 2.0 - MC-LW 1025.67 891.40 140 0.9982 3.1 9 MC-LF 986.63 213.11 138 0.9911 2.6 1
•LDTD
Even faster?
Principles of the LDTD-APCI source: technique that combines thermal desorption (laser diode) and APCI sample is spotted (1-10 μL) into a 96-well plate and air-dried for 2 min uncharged analytes are thermally desorbed into the gas phase ionization takes place in the corona discharge region by APCI and the charged molecules will be transferred to the MS inlet
Source: www.chm.bris.ac.uk/ms/theory/apci-ionisation.html
→ e- + N2 → N2+. +
2e-
Primary ion formation
Secondary ion formation
Proton transfer
→ N2+. + H2O → N2 + H2O+.
→ H2O+. + H2O → H3O+ + HO.
→ H3O+ + M → (M+H)+ + H2O
Laser diode thermal desorption Laser diode thermal desorption (LDTD)(LDTD)
LDTD
Installation
Auto-sampler
960 samples
Corona needle position (APCI)
IR L
ase
r
(980 n
m,
20 W
)
Can ramp up to 3000oC/sec.Laser power is defined in %Normally ~100-150oC
Laser diode thermal desorption Laser diode thermal desorption (LDTD)(LDTD)
Laser diode thermal desorption Laser diode thermal desorption (LDTD)(LDTD)
LDTDLDTD ProcessProcess
(1) Infrared laser (980 nm, 20W) (2) LazWell Plate (96 wells): analyte desorption (1-10 µL spotted) (3) Transfer tube transporting the neutrally desorded analytes to the APCI region(4) Corona needle discharge region (APCI) (5) MS inlet
(1)
(2)
(3)
(4)
(5)
Parameters of the LDTD-APCI source are optimized for signal intensity :
solvent choice for analyte deposition in the well cavities laser power (%) carrier gas flow rate (L/min) mass deposition (deposition volume in µL) into plate well laser pattern
No need to optimize liquid chromatography - it has been completely eliminated!
A minimum of 2 SRM transitions were selected + their ion ratios
LDTD OptimizationLDTD Optimization
Optimization for MS (precursor) and MS/MS (SRM transitions) conditions in NI and PI mode.
Results / challenge
Only anatoxin-a can be vaprized and ionized by LDTD-APCI.
Interference from phénylalanine:
Different desorption patern (signal intensity vs laser power).
SRM Optimisation (main SRM identical).
166.03>149.06
166.06>149.02
Separation using a gradient of LDTD laser power
1.0E+04
1.1E+05
2.1E+05
3.1E+05
0.0E+00
4.0E+06
8.0E+06
1.2E+07
0 10 20 30 40 50 60
PHE Peak A
rea
AN
A-a
Pea
k A
rea
Laser Power (%)
ANA-a
PHE
Performances (anatoxin-a)
Calibration
type R2
Linearity range
(µg/L)
MDL
(µg/L)
MLQ
(µg/L)
Standards
Avg. RSD
(%)
External 0.999 3 – 250 1 3 8
Internal 0.998 5 – 250 1 4 5
1
Microcystins
• The are over 80 known microcystins.
• A unique structural feature: Adda (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) which plays an important role in its toxicity.
O
NH
HN
N
NH
HN
HN
HN
O
COOH
O CH2
O
O
OCOOHO
NH
HN
NH2
O
Microcystin-LR
Adda
Arginine (R)
Leucine (L)
MMPB
X. Wu, C. Wang, B. Xiao, Y. Wang, N. Zheng, J. Liu. Analytical Chimica Acta, 709 (2012) 66-72.
Context
• The presence of microcystins can pose an health risk for humans and animals:
–Skin irritation, vomiting, diarrhea, asthma, headache, fever, and muscle weakness.
–Inhibiting protein phosphatases in tissues, causing serious damage to the liver from bioaccumulation.
The World Health Organisation (WHO) recomends a guideline for MC-LR of 1 g lL-1 in drinking water.
K.Sivonen, G. Jones, in: 1. Chorus, J. Bartram (Eds.), Toxic Cyanobacteria in Water: A Guide to their Public Health Consequences, London, 1999.
Alternatives
Analytical Methods Advantages Disadvantages
HPLC-UVHPLC-MS
• Specific analysis• Time consuming• Stantards limitation• Expensive
GC-MS • Total MC analysis• More steps (need
of derivatization)• Time consuming
ELISA• Fast and easy• Unexpensive• Total MC analysis
• Binding constants of the MC with the anti-body may vary
• Cross-selectivity
Need of robust detection methods to evaluate and control the risks due to the presence of microcystins in water.
D.O. Mountfort, P. Holland, J. Sprosen. Toxicon 45 (2005) 199-206K. Kaya, T. Sano. Analytica Chimica Acta 386 (1999) 107-112.
Objective
• Objective: Analysis of total microcystins using LDTD-APCI-MS/MS technology.
• The method provides:
–Instant information about risks of contamination
–Information about the whole spectrum of
cyanobacterial peptide toxins congeners
Oxydation
Experimental workflow:
–Lemieux oxidation of microcystins into MMPB
–Liquid-liquid extraction (Ethyl acetate)
–Desorption by LDTD
–Negative ionisation by APCI
–Detection with a TSQ Quantum Ultra AM triple quadrupole mass
spectrometerHOOC
OCH3
CH3
erythro-2-Methyl-3-methoxy-4-phenylbutyric AcidMMPB
X. Wu, C. Wang, B. Xiao, Y. Wang, N. Zheng, J. Liu. Analytical Chimica Acta, 709 (2012) 66-72.M-R. Neffling, E. Lance, J. Meriluoto. Environmental Pollution, 158 (2012) 948-952
Lemieux oxidation
Mdha
D-Ala
X
Masp Z
D-Glu
O
HN
CH3OCH3
CH3CH3
KMnO4 + NaIO4HOOC
OCH3
CH3
MMPBMicrocystin
Adda
• 0,05 M Potassium permanganate (KMnO4) and 0,05 M Sodium periodate (NaIO4)
• Oxidation, at room temperature and pH 9 for 1 hour
• Reaction quenched with saturated sodium bisulfite
• Use of sulfuric acid 10% to reach pH 2
T. Sano, K. Nohara, F. Shiraishi, K. Kaya. J. Environ. Anal. Chem., 49 (1992) 163-170.
Microcystins Oxidation Optimisation
0,0E+00
5,0E+03
1,0E+04
1,5E+04
2,0E+04
2,5E+04
0,01 M 0,02 M 0,05 M 0,1 M
Reagents Concentration
MM
PB
Pea
k A
rea
(arb
itra
ry u
nit
)
Reagents concentrations
KMnO4 and NaIO4 optimised at 0,05 M
Microcystins Oxidation Optimisation
0,0E+00
5,0E+03
1,0E+04
1,5E+04
2,0E+04
2,5E+04
3,0E+04
3,5E+04
4,0E+04
4,5E+04
0 1 2 3 4 5 6
Oxidation time (h)
MM
PB
Pea
k A
rea
(arb
itra
ry u
nit
)
Oxidation time
Optimal oxidation time at 1h
LDTD parameter optimisation
0,0E+00
5,0E+04
1,0E+05
1,5E+05
2,0E+05
2,5E+05
3,0E+05
3,5E+05
0 10 20 30 40 50 60 70
Laser Power (%)
MM
PB
Pea
k A
rea
(arb
itra
ry u
nit
)Laser power
Best laser power at 35%
pH during oxidation
Microcystins Oxidation Optimisation
0,0E+00
5,0E+03
1,0E+04
1,5E+04
2,0E+04
2,5E+04
3,0E+04
3,5E+04
1 3 5 7 9 11
pH
MM
PB
Pea
k A
rea
(arb
itra
ry u
nit
)
Optimised conditions at pH 9
Microcystin detection and quantification
Quantification of MMPB by internal calibration with 4-phenylbutyric acid
MMPB 4-phenylbutyric acid (4-PB)(Internal standard)
Optimal Selected Reaction Monitoring (SRM) parameters for the analysis of MMPB and 4-PB by MS/MS
APCI (-)Scan time: 0,005 sQ1 width: 0,70 amuQ3 width: 0,70 amu
Analysis of MMPB with LDTD-APCI-MS/MS
Internal Calibration (MMPB / 4-PB ratio)
Calibration curve showing the linearity of the LDTD experiment
Oxidation reaction yield of Microcystins : 111% MMPB recovery yield : 48%
Method Validation
n=6R2 : 0,9995Linearity range: 1 – 500 g/LLOD: 1 g/LLOQ: 3 g/LStandards Avg. RSD < 9%
WHO Guideline: 1g/L
• An 8-min automated online SPE-LC-MS/MS method for many toxins (but excluding saxitoxins)
•Ultrafast laser diode thermal desorption methods (LDTD-APCI-MS/MS) (15 sec per sample but with simple oxydation for MC)
•Anatoxin-A
•Sum of microcystins
Conclusions
AcknowledgementsAcknowledgements
Parterns and funding agencies:
Analysis with LDTD-APCI-MS/MS
LDTD Source
http://ldtd.phytronix.com/
(980 nm, 20 W)
(0,5-3 L/min)
Analysis with LDTD-APCI-MS/MS
–Minimal sample preparation
–Small volume of sample needed (1-5 L)
–15 sec / sample (no chromatographic
separation)
–No carryover
–Combined with atmospheric ionisation
(APCI)
–High-thoughput
10 plates in the loader = 960 samples
LDTD Source 10-plate sample loader
LazWell sample plate
http://ldtd.phytronix.com/
LDTD a sample introduction method using thermal desorption
LDTD parameter optimisation
Laser pattern
0
10
20
30
40
50
0 1 2 3 4 5 6 7
Time (s)
La
se
r P
ow
er
(%)
Laser Power: 35%Gas Flow: 3 L/minDeposition volume: 2LLaser pattern duration: 6 s
LDTD peak shape
Laser desorption parameters
GazFlow2-3 - TIC - RT: 0,00 - 0,15 NL: 4,55E5F: - c APCI SRM ms2 207,100@cid12,00 [ 130,600-131,600]
0,02 0,04 0,06 0,08 0,10 0,12 0,14Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e In
ten
sity
RT: 0,07
0,14 0,140,050,040,030,02
LDTD parameter optimisation
Sample residue
Plate well
Deposition solvent
Carrier gas flow rate
Gaz flow at 3,0 L/min
Ethyl Acetate is the best deposition solvent