accelerating the analysis of cyanotoxins sébastien sauvé environmental analytical chemistry –...

Accelerating the Analysis of

CyanotoxinsSébastien Sauvé

Environmental Analytical Chemistry – Université de Montréal

[email protected]

Dipankar GhoshDirector for Environmental & Food safety

Thermo Fisher [email protected]

mailto:[email protected]

mailto:[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

500000

1000000

0

500000

1000000

1500000

0

1000000

2000000

3000000

Inte

nsi

ty

0

200000

400000

NL: 5.02E5TIC F: + c ESI SRM ms2 [email protected] [42.800-43.800] 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


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


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


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:

Questions?Questions?

[email protected]

[email protected]

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


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


Sample residue

Plate well

Deposition solvent

Carrier gas flow rate

Gaz flow at 3,0 L/min

Ethyl Acetate is the best deposition solvent

accelerating the analysis of cyanotoxins sébastien sauvé environmental analytical chemistry –...

Documents

thermal desorption laser

apci sample

analyte desorption

ldtdapci source

mass of analytes

laser power

lcmsms challenge

drinking water reservoirs