Overview of advanced instrumental techniques employed in food and

feed analysis Vit Kosek

2

Pesticide / veterinary drug residues

Environmental contaminants

Processing products

Natural components

Migrants from plastics

Additives

Nutrients

proteins lipids

saccharides minerals vitamins

Toxic metals Fiber

Primary sensorically active comp.

Antioxidants and other biologically

active components

Natural toxins

Antinutrition comps.

Contaminants

Biotechnology products

Food composition

3

Food and feed analysis Applications: Regulatory Food safety Quality control Research and development

4

We need the methods to be… Precise Reproducible Accurate Simple Cheap Fast

Sensitive Specific Safe Destructive/Non-destructive Online/Offline Official

Techniques must be always fit for purpose!

5

Elemental analysis Atomic absorption spectroscopy ICP-atomic emission spectroscopy ICP- mass spectrometry

AAS

ICP-MS ICP-AES

6

Molecular spectroscopy UV-VIS Infra-Red Nuclear Magnetic Resonance

UV-VIS

FTIR NMR

7

Gas chromatography Separation of sample constituents in gas phase On the basis of volatility and structure Analytes need to be sufficiently volatile and thermally stable Analytes usually up to 1000 Da

More on this topic: Michal Stupák

8

Liquid chromatography Separation of sample constituents in liquid state Wide range of analytes are separable No need for temperature stability Several mechanisms:

• Hydrophobic interactions (reverse phase) • Polar interactions and hydrogen bonds (normal phase, HILIC) • Charge interactions (ion Exchange)

More on this topic: Vojtěch Hrbek

9

Mobile phase - supercritical CO2 (Tcrit = 31 °C, Pkrit = 7390 kPa) Fluid with low viscosity and high diffusivity → high separation efficiency, shortened time of analysis

Polarity of supercritical CO2 ~ hexane

Amenable for analytes with wide range of polarities

More on this topic with: Beverly Bělková, Michaela Rektorisová

Supercritical fluid chromatography

10

Mass spectrometry Weighing molecules Molecules need to be ionised Ions can be manipulated with in electric or magnetic field Mass spectrum: m/z X intensity Destructive X very sensitive Specific 121210_VV10969AB_70_72_73AB_74_75AB_76_77AB_78AB_80_81_83_84_HT_poz_450 #52-68 RT: 0.38-0.50 AV: 17 SB: 1 0.06 NL: 1.37E7

T: FTMS + p NSI Full ms [55.00-1100.00]

100 150 200 250 300 350 400 450m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

348.2013

198.0971329.1744139.1116

180.0865114.0914364.1961268.1038 311.1638155.1065

376.3417222.148690.0916

296.1853 420.222460.0812

282.1181

202.1072

250.1798 392.2276

11

PARTS OF MASS SPECTROMETER Ion source

Mass analyser

Detector

Neutral molecules are transfered to charged particles → ionization

Ion separation in gaseous phase under a high vacuum conditions according to the mass-to-charge ratio

Ion detection (registration) after their previous separation based on m/z, determination of intensity of individual ions

(vacuum) vacuum

Electron Ionization Electrospray

Matrix Assisted Laser Desorption Ionization

Quadrupole Ion Trap

Time-of flight Orbitrap FT-ICR

12

Mass spectromectry and separation techniques

LC–MS ESI

LC–MS APCI

LC–MS APPI

Highly polar Non polar

Analyte polarity

Mol

ecul

ar w

eigh

100

1000

10000

100000

10

GC–MS EI, CI

13

TANDEM MS A method comprising at least two levels of mass analysis steps: either in

connection with a dissociation process or a chemical reaction that causes a change in the ion mass or ion charge MS/MS methods involve the activation of the selected ion (precursor) Activation of ions in space or in time

14

MODES OF TANDEM MS Product ion scan

Precursor ion scan

Neutral loss scan

Selected reaction monitoring

Multiple reaction monitoring

15

MODES OF TANDEM MS Scan MSn

Applicable for ion traps

16

Effect of tandem MS

source: R.G. Cooks, K.L. Busch, J. Chem. Educ. 59(11) (1982) 926–933

Scan MSn: selectivity × sensitivity

MS level

Inte

nsity

Signal

Noise

S/N

1 2 3 4

17

High resolution mass spectrometry Mass spectrometer has high resolving power Definiton according to the width of one peak Full Width at Half Maximum (FWHM)

• Mass difference expressed as the peak width of a given mass peak measured (in mass units) at 50% of its height

m

∆ m

FWHM

RP = m∆ m�

18

Peak width at half maximum

High resolution mass spectrometry

19

High resolution mass spectrometry A mixture of xylene (m/z 92.0581) and toluene (m/z 92.0626) at

different settings of resolution

20

High resolution mass spectrometry Mass accuracy: The deviation between measured mass (accurate

mass) and calculated mass (exact mass) of an ion expressed as an error value (mDa, ppm) Important for structural interpretation (calculation

of elemental composition)

6

teor.

teor..exp 10)ppm( ⋅−

=∆m

mm

( ) 3teor..exp 10)mDa( ⋅−=∆ mm

FT-ICR • RP: up to 10,000 k

• MA: below 1 ppm

• COST: +++++

ORBITRAP • RP: up to 450 k

• MA: <1 – 3 ppm

• COST: ++++

TIME-OF-FLIGHT • RP: up to 50 k • MA: <1 – 5 ppm

• COST: +++(+)

21

[(arginine)1–5+H]+

m/z 523.3427

Molecular formulas based on a free selection among the elements C, H, N, O as a function of relative mass error vs. m/z.

The higher the mass error the larger number of candidates

Why do we need accuracy and precision?

22

23

Ambient mass spectrometry Sample ionization at atmospheric pressure Usually no separation Fast response MS imaging

Solvent

N2

Capillary (solvent)

Droplets

Surface

Capillary (gas)

V

Source of high voltage

Sample

Desorbed ions Entry to MS

Ion transfer

DESI

DART

24

REIMS Rapid Evaporative Ionization Mass Spectrometry First electrosurgical knife 1926 The hyphenation of electrosurgical knife and mass spec in 2010 Developed for cancer surgery

25

sample

REIMS system

iKnife 1st electrode

Current generator

PC

Source

Xevo G2-XS QTof

Metal mat 2nd electrode

Evaporation of the vicinity

of knife Electric circuit is closed

26

Time0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50

%

0

100

0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50

%

0

100170909_VK_POS_KURE1_K4565_2-5 TOF MS ES+

TIC9.35e6

2.06

0.75

0.270.25

0.51

0.97

1.841.20

1.43

1.381.64

1.60

2.29

170908_VK_NEG_VEPR5_PS80_2-5 TOF MS ES- TIC

1.93e80.40

0.190.22

0.73

0.56 1.71

0.751.55

0.95 1.37

1.16

0.98

1.351.18

1.88

1.90

REIMS data Chronograms

m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150

%

0

100170909_VK_POS_KURE1_K4565_2-5 265 (2.289) Cm (260:276) TOF MS ES+

1.46e6168.1380

154.1229

130.0665577.5158285.1563

232.1911220.1558 233.1086

369.3477339.2863 603.5303

m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150

%

0

100170908_VK_NEG_VEPR5_PS80_2-5 179 (1.552) Cm (176:185) TOF MS ES-

7.65e6281.2468

279.2346

255.2320

253.2175

699.5008

697.4875

283.2628

683.5025303.2340

554.2594419.2589391.2238 681.4857742.5395

744.5553

REIMS+ m/z 100-1200

REIMS- m/z 100-1200

REIMS+ TIC

REIMS- TIC

m/z500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960 980

%

0

100170908_VK_NEG_VEPR5_PS80_2-5 179 (1.552) Cm (176:185) TOF MS ES-

1.99e6699.5008

697.4875

683.5025

554.2594 681.4857

671.4833555.2626

657.4846642.4908

742.5395700.5085

725.5184

723.5010

709.5181

726.5337

727.5386

744.5553

750.5405885.5581

766.5464

776.5574 792.5602886.5578

m/z500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960 980

%

0

100170909_VK_POS_KURE1_K4565_2-5 265 (2.289) Cm (260:277) TOF MS ES+

1.32e5577.5158

575.4969

551.4978

548.5422 556.2741

603.5303

578.5242

579.5284604.5358

739.4647

737.4523897.7314

895.7097765.4698 923.7484898.7247

REIMS+ m/z 500-1000

REIMS- m/z 500-1000

TAG fragments

phospholipids TAG

phospholipids

27

REIMS method workflow iKnife

+ Mass spec

Model building

MVA

Tentative identification of important variables

Database creation spectra

28

Authentication by REIMS iKnife

+ Mass spec

Evaluation of the sample

Answer

? ?

Chicken muscle

29

Why REIMS? Advantages: Quick alternative PCR, MS

a LC-MS methods Possibility of mobile instruments

Disadvantages: Low sensitivity Limited number of matrices

30

Ion mobility MS Additional separation dimension Standalone MS or hyphenated with LC Several types of ion mobility

31

tdrift

Detector

Analyte Ions

Gating Optics

Ion Mobility Cell

t0

VH VL

Electric Field

Stacked ring ion guide gives linear field

Basic operation of ion mobility

Source: Agilent Technologies

32

Adding Ion Mobility Spectrometry in LC/MS

Chromatography Ion Mobility Mass spectrometry

≈seconds ≈ 60 ms ≈ 100 μs

IMS fits between LC and TOF MS on the separation time scale! Source: Agilent Technologies

33

EIC: (827.2696-827.2917 m/z) - DONOligo_pivo IAC_konc v H2O_mravencan_HSS T3_inj20_-1700V.d

Counts vs. Acquisition Time (min)

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5 5.1 5.2 5.3 5.4 5.5

4x10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

IM separation of masked mycotoxins DON-3-triGlc [M+HCOO]-

LC-QTOF-MS 827.2827 +/- 20ppm

3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8Drift Time (ms) vs. Acquisition Time (min)

29

30

31

32

33

34

35

36

37

38

LC-IM-QTOF-MS 827.2827 +/- 20ppm

tr = 3.88 min td = 31.85 ms

tr = 4.08 min td = 32.65 ms

tr = 4.23 min td = 31.92 ms

tr = 4.30 min td = 33.35 ms

tr = 4.23 min td = 34.94 ms

Five isomers found in IM!

34

d

Ahigh = Vhigh · thigh

Alow = Vlow · tlow

v1 v2

d1=d2

d1 = d2

Differential Mobility Spectrometry SelexION

Easy hardware set-up: just two flat parallel electrodes

(SV=Separation Voltage)

asymmetric waveform d1 = v1 · thigh

d2 = v2 · tlow ⇔

⇒ v1 = K · E1

v2 = K · E2 if

DMS can be also understood as the miniaturization of IMS drift tube.

DMS takes advantage of the differences in the mobility of ions in high and low electric fields (Separation voltage)

The ion traveling from a starting point will return to exactly this same distance from the electrode after one cycle.

Separation Voltage

35

d

The ion traveling from a starting point will therefore not return to exactly this same distance from the electrode after one cycle and, thus, drift towards one of the electrodes.

However, if the waveform is applied by high SV, the mobility of the ion during application of the peak voltage deviate from its low-field value (dependance of K from E). In this instance, during the higher voltage portion of the waveform, the ion travels at a velocity different than it would absent; this change in mobility:

v = K(E)· E

d

CoV

Compensation voltage (CoV):

Restores the trajectory for a given ion to allow them to transmit through the DMS device and

enter the mass spectrometer

Separation Voltage

Separation Voltage

Differential Mobility Spectrometry

36

Heat Map Chromatograms PDO La Mancha (Spain) Sample PDO La Mancha (Spain) Reference Material

Labelled Spanish Saffron Labelled Spanish Saffron

PDO Krokos Kozani (Greece) Zafferano di Sardegna PDO (Italy)

m/z 100 to 1200 CoV -8 to 24

Run time 17 min

UHPLC-DMS-QTOF UHPLC-DMS-QTOF

UHPLC-DMS-QTOF UHPLC-DMS-QTOF

UHPLC-DMS-QTOF UHPLC-DMS-QTOF

37

Conclusions Wide array of techniques exist Domination of separation techniques

and mass spectrometry Manufacturesrs are routinely assisting

in development of methods More exciting instrumental

techniques to come!