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INTRODUCTION TO LC-MS/MS

Presented by: Huỳnh Khánh Duy – Application Manager

TP. HCM 09/2013HKDUY 2013

2

CHROMATOGRAPHY

Solvent

C ol u

mn

t

0

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3

Stationary phase

Flow of mobile phase

CHROMATOGRAPHY

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4

CHROMATOGRAPHY

Strong interaction

Weak interaction

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HPLC

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HPLC

A

B

C

D

E

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Sample

Chromatogram

0 5 10 15 20

Time (minutes)

A b u n d a n c e

A

B

C

D

E

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LC-MS

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LC-MS = LC (Liquid Chromatographer) coupled to MS (Mass Spectrometer).

LC: separation.

MS: detection.

Compounds have a unique mass number according to their structure. This mass

number is measured by a mass spectrometer.

8

MASS SPECTROMETER

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Molecules cannot be measured directly by MS → They have to be ionized.

From mass spectrometer:

Information about the molecules (molecular weight-related ions),

Information about forcibly dissociated fragment ions (Collision Induced

Dissociation; CID), and product ions generated from specific ions.

M

M

H+

M

H+

H+ bound H+

removed

CID

Fragment ions

Specific

compound

CID

Product ions generated from specific ions

Monitoring specific product ions = TQ MRM

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MASS SPECTROMETER

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CH3COCH3

Sample

Inlet

CH3+COCH3

Ionization

& Adsorptionof Excess Energy

Mass Analysis

CH3C+OCH3

+COCH3

+CH3

+

COH

Fragmentation

(Dissociation)

Detection

9

10

MASS SPECTROMETER

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194

67 109

5582

42

16513694

40 60 80 100 120 140 160 180 200

Mass (amu)

Mass Spectrum

N

C

C

NH

C

O

C

O

N

N

C H

C 3H

C3H

MassSpectrometer

Typical sample: isolatedcompound (~1 nanogram)

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LC-MS

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S i g n a l I n t e n s i t y

Time

mass-to-charge ratio (m/z) of ions

R e l a t i v e I n t e n s i t y

Chromatogram

Mass SpectrumMass spectra:

- Molecular weight

- Chemical structrure

Base peak: 100%intensity

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LC-MS

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M/Z

M/Z

M/Z

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LC-MS

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LC-PDA vs LC-MS

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10 ppb

(1)(2)(3)

(4)(5)

m/z

m/z

(5)

(4)

time

(3)m/z 193(2)m/z 582

(1)TIC

MS spectrainit.

init.

MS

20 ppm

time

(3) 210 nm(2) 580 nm

(2)

(3)

(4)

(5)

UV spectra

nm

nm

(5)

(4) AU

AU

PDA (photodiode array)

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LC vs LC-MS

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If an impurity

coincides with the

target component:

Mobile phase preparation errors

Fluctuations in peak retention times

Peak misidentification

A B

A B

An impurity coincides with the target:

Changes in area value

Incorrect quantitation

16

LC vs LC-MS

Merits

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The greatest merit in using an MS instrument as an LC detector:

In addition to retention times, mass information for each peak can be obtained simply at

the same time.

m/z 264 m/z 278m/z 267 m/z 281The peaks (including

those that cannot be

separated by time) can be

separated using mass

information.

This reduces the risk of

qualitative and

quantitative errors.

Mass information is a powerful tool for reducing the risks

associated with LC analysis, such as the following:

Peak identification (i.e., qualitative) errors

Quantitative errors due to the elution of

unpredicted impurities

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A:100

D:150B:100

C:150m/z=150

TIC

m/z=100

A

B

C D

LC vs LC-MS

18HKDUY 2013

LC vs LC-MS

- 1 8 -

MS

PDA

200 300 400 500 600 m/z

0e3

500e3

1000e3

1500e3

Int.

325.1

224.1156.0306.2 432. 5 564. 5608. 4476.0520.4388.4

Impurity Profiling of

medicine tablet

200 300 400 500 600 m/z

0e3

50e3

100e3

150e3

Int.

339.1

306.8

299.9180.2 564.0375.1 433.1

200 300 400 500 600 m/z

0e3

100e3

200e3

300e3

Int.

311.1

352.1262.1153.0 345.2 477.0391.7 592. 8 695. 7521.5

MW = 328 MW = 330

Impurity

Main product

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LC vs LC-MS

0.0 1.0 2.0 3.0 4.0 5.0 6.0

0.0

2.5

5.0

7.5

10.0

(x100,000)

VER:m/z 455.0 (2)PRO:m/z 331.9 (6) ALB:m/z 266.0 (60)PIR:m/z 260.0 (4) ANT:m/z 189.0 (6)

A L B

V E R

P R O

P I R

A N T

MS detector

min.

Int.

0.0 1.0 2.0 3.0 4.0 5.0

min.

-2.0

-1.0

0.0

1.0

2.0

3.0

4.0

mAU (x10)

254nm,4nm (1.00)

6.0

Int.

Chromatograms of plasma sample

A L B

V E R

P R O

P I R A

N

T

ANT :Antipyrine

PIR : Piroxicam

ALB : AlbedazolePRO : Propraolol

VER : Verapamil

How can these

compouinds can bequantified

compounds

PDA detector

20

LC vs LC-MS

A U

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

LansoprazoleUV: 254nm

Detection of 0.03% impurity in product

A U

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

Minutes

0 .5 0 1 .0 0 1 .5 0 2 .0 0 2 .5 0 3 .0 0 3 .5 0 4 .0 0 4 .5 0 5 .0 0 5 .5 0 6 .0 0 6 .5 0 7 .0 0 7 .5 0 8 .0 0 8 .5 0 9 .0 0 9 .5 0 1 0.0 0

SIR: 298.22 m/z

I n t e n s i t y

0.0

2.0x105

4.0x105

6.0x105

8.0x105

1.0x106

1.2x106

1.4x106

1.6x106

1.8x106

2.0x106

Minutes4. 80 5. 00 5.20 5.40 5.60 5.80 6.00

S/N = 870

Impurity

A U

-0.00180

-0.00175

-0.00170

-0.00165

-0.00160

-0.00155

-0.00150

-0.00145

-0.00140

-0.00135

-0.00130

-0.00125

Minutes

4.80 5.00 5.20 5.40 5.60 5.80 6.00

0.03%

S/N = 2

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LC-MS

Qualification

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Retention time of a compound is identical in same conditions (column,

tempearature, flow rate, etc.).

Sample Injection

Standard sample

(mixture of A and B)

Unknown sample

Compound A

t A

Compound B

tB

Mass spectrum:

Confirmation of existence.

Impurity and purity check.

Identification (library).

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LC-MS

Quantification

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Amount of a compound that passes through the detector is a function of Peak

area (or peak height)

Vμ L of 100 ppmstandard solution of A

Compound A

VμL of sample

Compound A t A

Area: 700

10

0

Conc.

(ppm)

100

0Peak

Area

700

70

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LC-MS

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HPLC Mass analyzer Interface

Aqueous/organicsolvent with buffers

Non-volatile analyte

molecules

A high vacuum

environmental

Analyze ions, m/z

Remove

solvent

Ionize analytes

Direct introduction of LC elute into MS and use of EI ion source (like in GC/MS) are

not the method of choice.

• Research on interfacing HPLC to MS began in the 1970s; API (atmospheric

pressure ionization) sources were commercialized after 1987.

• API interfaces: electrospray ionization (ESI), atmospheric pressure

chemical ionization (APCI) and AP photo ionization (APPI).

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LC-MS

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Mass Analyzer Interface

TMP TMP

Rotary Pump

Atmospheric

Pressure 10-310-4 Pa80150 Pa

Q-array Octopole Quadrupole Detector Ionization probe

HPLC

Mass spectrometer

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LC-MS

Configurations

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Mass Analyzer Interface

-Quadrupole (Q)

- Ion Trap (IT)

- TOF

- Tandem/Hybrid

- ESI

-APCI

-APPI

-Nanospray

HPLC Ion Transmission Ion Detector

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MASS SPECTROMETER

Ionization methods

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Electrospray Ionization (ESI)

Extremely soft ionization method. Suitable for theionization of high-polarity compounds.

Hard ionization method than ESI. Suitable for the

ionization of medium- to low-polarity compounds.

Atmospheric Pressure ChemicalIonization (APCI)

DUIS

Dual-ion source for both ESIand APCI.

Gas capillary

Liquidsample

Nitrogen gas

Charged droplet formation

Ion evaporationHigh voltage (3 to 5 kV)+ creates positive ions- creates negative ions

Liquidsample

Nitrogen gas

Heater

Heater

Corona needle

Sample molecule

Solvent molecule

High voltage (3 to 5kV)+ creates positive ions- creates negative ions

Functionalgroups

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MASS SPECTROMETER

Ionization methods – ESI

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MASS SPECTROMETER

Ionization methods – APCI

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MASS SPECTROMETER

Ionization methods – ESI

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ESI & APCI normally do not cause fragmentation of

molecules - Soft ionization

Main ions generated:

Protonated molecule [M+H]+ at m/z(Mr+1).

Deprotonated molecule [M-H]- at m/z(Mr-1).

Other ions often observed:

Sodium adduct ions [M+Na]+.

Solavted ions [M+H+CH3CN]+

, [M+H+CH3OH]+.

Neutral lose of small molecules.

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MASS SPECTROMETER

Ionization methods

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50 100 150 200 250 300 350 400 m/z0e3

10e3

20e3

30e3

40e3

Int.

377

399

Riboflavin C17H20N4O6

Exact Mass: 376.14

m/z

Int.

(a) GCMS, EI spectrum of riboflavin (vitamine B2)

N

N

NH

N

O

O

OH

OH

HOOH

[M+H]+

[M+Na]+

(b) LCMS, ESI (API) spectrum of riboflavin

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MASS SPECTROMETER

Ionization methods

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NN

H

N

O

O

NH2(H3C)3C

Diketometribuzin (DK) C7H12N4O2

Exact Mass: 184.10Mol. Wt.: 184.20

50 100 150 200 250 300 350 400 450 0e3

50e3

100e3

150e3

200e3

250e3

300e3

350e3

185

197

207 227391105 295167 257142 268 317 425280 369128 155 243 404341 49766 82 46945357 482

50 100 150 200 250 300 350 400 450 0e3

100e3

200e3

300e3

400e3

500e3

183

243168153 265207113 228 28189 356297 368141 322193 461250 435 484416405 4997150

1

APCIPositive

mode

APCINegative mode

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MASS SPECTROMETER

Ionization methods

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150 200 250 300 350 400 450 500 550 m/z0e3

250e3

500e3

750e3

1000e3

Int.375

433 474

297315

357333 449255 393350237203141 457279163 411 490510 534 580553

150 200 250 300 350 400 450 500 550 m/z0

25000

50000

75000

100000

Int.409

349 445173 574537368273 335 395246225106 201 479426134 508

OH

H3C H

OHCH3

O

O

CH3

OH

CH3

CH3

CH2

O

H

O

CH3

C22H34O7

Forskolin

MW: 410

Positive

Negative

LC conditions: ODS column (75x4.6

mm), CH3CN/water = 80/20

Interface: ESI

[M-H]-

[MH-2H2O]+

[M+H]+

ESI-Q spectra by LCMS-2010EV

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MASS SPECTROMETER

Ionization methods

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MS mode Mass peak

(m/z)

Ion Type of Ion

Positive

474

433

411

[M+Na+ACN]+

[M+Na]+

[M+H]+

Solvated-Na-adduct ion

Na-adduct ion

Protonated ion

393

375

357

333

315

297

[M+H-H2O]+

[M+H-2H2O]+

[M+H-3H2O]+

[M+H-H2O-HAc]+

[M+H-2H2O-HAc]+

[M+H-3H2O-HAc]+

Protonated ion with neutral

loss of small molecules

Negative 409 [M-H]- Deprotonated ion

No simple Na-adduct ion can form in negative mode.

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MASS SPECTROMETER

Ionization methods

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Sodium adduct ions:

Na+ ions attach (non-covalent interaction) to the

target molecules M to form [M+Na]+

Solvent adduct ions:

Solvent molecules attach to target molecules to form

[M+Sol+H]+ (Sol: MeOH, CH3CN, THF etc); both ESIand APCI, more often for latter

Additive adduct ions:

Such as [M+NH3+H]+, [M+FA+H]+, [M+TFA+H]+

Dimmer ions [2M+H]+, [2M+Na]+

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MASS SPECTROMETER

Ionization methods

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In most case, adduct ions can be recognized easily: span

of adjacent mass peaks = adduct molecule:

e.g., [M+H]+ and [M+NH3+H]+: m/z = 17

[M+H]+ and [M+MeOH+H]+: m/z = 32

[M+H]+ and [M+Na]+: m/z = 22

[M+H]+ and [M+FA+H]+: m/z = 46

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MASS SPECTROMETER

Ionization methods

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• Natural pesticides – azadirachtin as the active component

• Neem extract mainly from seeds: very complex in composition

• Conventional method – reverse phase HPLC with UV detector (217

nm), for major components (Azadirachtin-A and -B, nimbin, salannin

etc)

• Difficulty in identification of minor components due to lack of

standards, such as other azadirachtins etc.

• LCMS: identification of components based on MW and neutral loss

spectrum

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MASS SPECTROMETER

Ionization methods

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5 10 15 20 25 30 35 40 45 50 55 min

-2.5e6

0e3

2500e3

5000e3

7500e3

10.0e6

12.5e6

15.0e6

17.5e6

20.0e6

22.5e6

25.0e6

27.5e6

Int.

597.00(2.00)541.00(2.00)555.00(2.00)499.00(2.00)645.00(2.00)703.00(2.00)TIC(1.00)

1 2

3

4 5 6

7

8 9

1 0

1 1

1 2

1 3

1 4

1 5

1 6

1 7

1 8

1 9

2 0 2 1

2 2

2 3

2 4

2 5

2 6

2 7

2 8

2 9

3 0

0 5 10 15 20 25 30 35 40 45 50 55 min

0

5

10

15

20

25

mAbs

UV, 217nm

MS-TICMS-MIC

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MASS SPECTROMETER

Ionization methods

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400 450 500 550 600 650 m/z

0e3

2500e3

5000e3

Int.

499

467562544449 485 578 677655403 623

Peak 22: 6-Desacetylnimbin

5 10 15 20 25 30 35 40 45 50 55 min-2.5e6

0e3

2500e3

5000e3

7500e3

10.0e6

12.5e6

15.0e6

17.5e6

20.0e6

22.5e6

25.0e6

27.5e6Int.

597.00(2.00)

541.00(2.00)555.00(2.00)499.00(2.00)645.00(2.00)703.00(2.00)TIC(1.00)

1 2

3

4 5 6

7

8 9

1 0

1 1

1 2

1 3

1 4

1 5

1 6

1 7

1 8

1 9

2 0 2 1

2 2

2 3

2 4

2 5

2 6

2 7

2 8

2 9

3 0

400 450 500 550 600 650 m/z

0.0e6

1.0e6

2.0e6

3.0e6

Int.555

618600572455437 634419 662537 679479497

Peak 23: 3-Desacetyl

salannin

450 500 550 600 650 700 m/z

0e3

500e3

1000e3

1500e3

Int.

625

688

670647525 607568549465 704585 721506

Peak 24: Ohichinolide-B

400 450 500 550 600 650 m/z

0e3

2500e3

5000e3

Int.541

509604

586481449 527 559421 621 645404 689665

Peak 27: Nimbin

400 450 500 550 600 650 m/z

0e3

2500e3

5000e3

Int.

597

660619419 642565497 519479437401 461 677541 694

Peak 28:Salannin

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MASS SPECTROMETER

Ionization methods

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500 550 600 650 700 750 m/z

0.0e6

1.0e6

2.0e6

3.0e6

Int.

703

685

585567 743

507 603 766667555 625 721 784525 642

500 550 600 650 700 750 m/z

0e3

250e3

500e3

750e3

Int.

645

685627

545

726701

663 743527563 585 609509 710 759 789

Peak 12

Peak 14

m/z Ion

703 [MH-H2O]+

685 [MH-2H2O]+

585 [MH-2H2O-TgOH]+

567 [MH-3H2O-TgOH]+

743 [MNa]+

m/z Ion

645 [MH-H2O]+

627 [MH-2H2O]+

545 [MH-H2O-TgOH]+527 [MH-2H2O-TgOH]

+

685 [MNa]+

Azadirachtin A, MW = 720

Azadirachtin B, MW = 662

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MASS SPECTROMETER

Ionization methods

HKDUY 2013

CH3

CH3

O

O

O

O

CH3

O

O

H3CO

OH

H

O OH

OH3CO

CH3

O

O

CH3

H

OHH

O

H

100

[MH]+ [MNa]+

[MH-2H2O]+

[MH-H2O]+[MH-H2O-TgOH]

+

[MH-2H2O-TgOH]+

m/z

23

18

18

18

H2O

H2O

TgOH

Mr 100

H2O

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MASS SPECTROMETER

Ionization methods

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Peak No [MH-H2O]+ Azadirchtin

(Base peak) Type Formula MW

7 601 I C32H42O12 618

8 645 G or H C33H42O14 662

10 641 D C34H44O14 676

12 703 A C35H44O16 720

14 645 B C33H42O14 662

15 687 L C35H44O15 704

16 647 F C32H40O14 664

42

MASS SPECTROMETER

Ionization methods

HKDUY 2013

Depend on structures and properties of compounds

High proton affinity: C-O, C-N double and triple bonds,

basic compound tend to form positive ions.

Low proton affinity: –COOH, -F, -Cl, -HSO3, phenols,

aniline and sugars tend to form negative ions due tostronger tendency to donate proton.

Many compounds form both positive & negative ions

due to multi function groups.

Acquire positive & negative mass spectra and compare

sensitivity and spectrum quality.

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MASS SPECTROMETER

Ionization methods

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protonation R--NH2

R--NH--R’

R--OHHR--SH

R--O--R’’R--C=O

[benzene ring]-]-OH

R--COOH

R--SO33HR--PO33H

Positive ion

Negative iondeprotonation

Functional group

44

MASS SPECTROMETER

Ionization methods

HKDUY 2013

ESI: CH3CN/H2O and MeOH/H2O

APCI: MeOH/H2O recommended; CH3CN/H2O may

cause coking on the corona needle in negative mode.

THF can be used for LCMS ionization.

The ratio is not so critical, but higher water content may

cause lower ionization efficiency.

Gradient elution with changing aqueous phase between

0 and 100% can be used.

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MASS SPECTROMETER

Ionization methods

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Non-volatile buffer like phosphate is not recommended

Using volatile buffer to replace phosphate buffer:

NH4 Ac/HAc, NH3/TFA, NH3/NH4 Ac etc

pH control:

• pH 1.8 ~ 2.5 : TFA, conc. < 0.1%

• pH 2.5 ~ 4 : FA, conc. ~ 0.1%

• pH 4 ~ 5 : HAc, conc. 0.1~5%

• pH 7 : NH4 Ac

• pH > 7 : NH3 aqueous solution

46

MASS SPECTROMETER

Other ionization methods

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• APPI : Atmospheric Pressure Photo ionisation [Non Polar]

• DART : Direct Analysis in Real Time [Solid samples]

• DESI : Desorption Electro Spray Ionisation [Min. sample prep

for bio fluids, Tablets, creams etc. [Thermo]

• ASAP : Atmospheric Solids Analysis Probe [Solid Sample

probe] Waters

• DUIS : Combined ESI + APCI [Shimadzu]

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MASS SPECTROMETER

Other ionization methods - DUIS

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MASS SPECTROMETER

Other ionization methods - DART

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IONIZATION METHODS

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Analysis target: Areas differ according to ionization method

High

polarity

Molecular weight GC/MS

(Also if derivatization is

required.)

Non-polar

Volatile

Thermally stable

LC/MS(MS)

(Simple, requiring almost

no pretreatment.)

High polarity

Non-volatile

Large mass numbers

Thermally unstable

Medium

polarity

Non-

polar

1,000

10,000

10,000

GC/MS

LC/MS

APCI

(Low/medium

polarity)

LC/MS

ESI

(High polarity)Contents:

polyvalent ions

50

MASS SPECTROMETER

Type of Mass filter

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Quadrupole

Time-of-flight (TOF)

Ion-trap

Magnetic-sector

Fourier-transform

Tandem MS (TQ, IT-TOF, …)

Qualification of unknown samples

Quadrupole < Ion trap < TOF

Sensitivity, quantitation

Quadrupole (SQ/SIM) < TOF (Scan) < Quadrupole (TQ/MRM)

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MASS SPECTROMETER

Type of Mass filter

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Quadrupole MS: Good for quantification. Ions selected by four poles.

Good for quantification of unknown substances.

Features: General-purpose, most widely used type. Low cost and easy

maintenance. Acquires SCAN and SIM data.

Advantages: (1) Compact and lightweight, (2) Easy to operate, (3) High sensitivity

excellent for quantification.

Disadvantages: Poor resolution. Little qualitative information (nominal mass only).

52

MASS SPECTROMETER

Type of Mass filter

HKDUY 2013


Advantages:

– Better detector than UV/PDA.

– More sensitive.

– Useful for screening and Quantitation.

Disadvantages:

– No Fragmentation.

– Not very sensitive.

– Not useful for Identification and confirmation of unknown

compounds.

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53

MASS SPECTROMETER

Type of Mass filter

HKDUY 2013


R&D Pharma / API

• Reaction Monitoring.

• Basic Impurity Identification.

Chemical Synthesis

• Reaction monitoring.

• High Thro put screening.

Food testing

• Analysis of permitted colours.• Mycotoxins.

Academia

54

MASS SPECTROMETER

Type of Mass filter

HKDUY 2013


min

(x100,000)

negative

1

2

3 4

5 6

7 8

109

1 2

3 4

5 6

7 8

9 10

Positive

m/z 307

m/z 291

m/z 459

m/z 443

m/z 473

m/z 305

m/z 289

m/z 457

m/z 441

m/z 471

Analysis of catechins in tea1: (-)-gallocatechin,

2: (-)-epigallocatechin,

3: (+)-catechin,

4: (-)-epicatechin,

5: (-)-epigallocatechin gallate,

6: (-)-gallocatechin gallate,

7: (-)-epicatechin gallate,

8: (-)-catechin gallate9, 10: methylated catechins

O

OH

HO

OH

OH

OH

(-)-epicatechin

Ultra-fast detection of catechins in Pos/Neg modes by LCMS-2020

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55

MASS SPECTROMETER

Type of Mass filter

HKDUY 2013

Triple Quadrupole MS

Full Scan MS

Q1

Selection of Ions in

selected m/z range

Q2 Collision cell

Only RF to move

ions

Q3

Only RF to move

ions to detector

56

MASS SPECTROMETER

Type of Mass filter

HKDUY 2013


Q1

Selection of

Precursor Ion

Q2 Collision cell

Fragmentation of

Precursor Ion

Q3

Scanning of all

Product Ions

Full Scan MSMS

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57

MASS SPECTROMETER

Type of Mass filter

HKDUY 2013


MRM

Q1

Selection of

Precursor Ion

Q2 Collision cell

Fragmentation of

Precursor Ion

Q3

Selection of

Product Ion

58

MASS SPECTROMETER

Type of Mass filter

HKDUY 2013

SIM

5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

(x100,000)

8:575.60>415.70 (1.00)7:563.60>403.70 (1.00)6:497.70>337.70 (1.00)5:485.70>325.70 (1.00)3:417.80>257.90 (1.00)2:405.80>245.90 (1.00)

14:971.40>811.30 (1.00)13:959.40>799.30 (1.00)12:733.40>573.60 (1.00)11:721.40>561.60 (1.00)10:655.60>495.70 (1.00)9:643.60>483.70 (1.00)

5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

0.25

0.50

0.75

1.00

1.25

(x10,000,000)

405.80 (10.00)TIC

575.60 (10.00)573.60 (10.00)563.60 (10.00)561.60 (10.00)497.70 (10.00)495.70 (10.00)485.70 (10.00)483.70 (10.00)417.80 (10.00)

971.40 (10.00)959.40 (10.00)811.50 (10.00)799.50 (10.00)TIC

MRM

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59

MASS SPECTROMETER

Type of Mass filter

HKDUY 2013

Tri-BDE (BDE-028)SIM MRM

Penta-BDE (BDE-100)SIM MRM

11.5 12.0 12.5 13.0

1.0

2.0

3.0

(x100,000)

245.90405.80

11.5 12.0 12.5 13.0

1.0

2.0

3.0

4.0

5.0

(x10,000)

405.80>247.90405.80>245.90

19.0 19.5 20.0 20.5

0.25

0.50

0.75

1.00

1.25(x100,000)

403.80563.60

18.5 19.0 19.5 20.0

0.5

1.0

1.5

2.0

2.5

(x100,000)

563.60>405.70563.60>403.70

Hexa-BDE (BDE-154)

22.0 22.5 23.0 23.5

2.5

5.0

7.5

(x1,000)643.60483.70

22.0 22.5 23.0 23.5

0.5

1.0

1.5

2.0

2.5

3.0

(x10,000)

643.60>485.70643.60>483.70

SIM MRM

Hexa-BDE (BDE-153)

Hepta-BDE (BDE-183)

Deca-BDE (BDE-209)

SIM MRM

23.0 23.5 24.0 24.5

1.0

2.0

3.0

4.0

(x10,000)

643.60>485.70643.60>483.70

23.0 23.5 24.0 24.5

0.5

1.0

1.5

2.0

(x10,000)

643.60483.70

26.0 26.5 27.0 27.5

1.0

2.0

3.0

4.0

5.0

6.0

(x1,000)

721.50561.60

SIM MRM

26.0 26.5 27.0 27.5

1.0

2.0

3.0

4.0

5.0

(x10,000)

721.40>563.60721.40>561.60

37.0 37.5 38.0 38.5

0.25

0.50

0.75

1.00

1.25

(x100,000)959.40799.50

37.0 37.5 38.0 38.5

2.5

5.0

7.5(x100,000)

959.40>801.30959.40>799.30

SIM MRM

60

MASS SPECTROMETER

Type of Mass filter

HKDUY 2013


Advantages:

– Very fast and highly sensitive.

– Useful for screening and Quantitation of trace level impurities.

– So far, the best tool for trace level quantitation.

Disadvantages:

– Low Resolution, Mass accuracy is less.

– Fragmentation is only up to MS2.

– Not useful for Identification and confirmation of unknown

compounds

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61

MASS SPECTROMETER

Type of Mass filter

HKDUY 2013


R & D Pharma / API

Quantitation of trace level impurities.

BA/BE studies.

PK/PD studies.

Impurity profiling [limited]

Chemical Synthesis

High Through put screening.

Environmental Food safety

Screening of wide range of impurities in water and food.

Analysis of Banned colours.

Mycotoxins.

Trace level impurities like pesticides.

62

MASS SPECTROMETER

Type of Mass filter

HKDUY 2013


Clinical

New born screening.

Immunosuppressant.

Vitamin D.

TDM.

Clinical trials phase I to III.

Forensic and Toxicology

Screening of banned substances in various matrices.

Quantitation and confirmation of banned substances.

Academia

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63HKDUY 2013

Ion-trap: Good for structure analysis. Ion-trap + MSn combination possible..

Good for structure analysis.

Features: Comparatively small and

cheap. Easy operation and maintenance.

Permits SCAN and SIM.

Advantages: (1) High sensitivity by

detecting all trapped ions.

(2) Permits MSn.

Disadvantages: Unsuited to quantitation.

Poor resolution. Inferior dynamic range to

quadrupole MS due to limit on trapped

quantity.

MASS SPECTROMETER

Type of Mass filter

64HKDUY 2013

Ion-trap: Good for structure analysis. Ion-trap + MSn combination possible..

MASS SPECTROMETER

Type of Mass filter

+V -V

0V

0V 0V

1. Selection of precursor ion

2. Fragmentation by

acceleration and collision with Argon

Ar Ar

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65HKDUY 2013

TOFMS: Good for qualification. Measures ion time of flight.

Good for qualification of unknown substances.

Features: Has become commonly used in recent years. Comparatively large

and expensive. Acquires SCAN data.

Advantages: (1) High resolution, (2) High mass measurement accuracy, (3) High

spectral sensitivity.

Disadvantages: Unsuited to quantitation. Requires high vacuum. Difficult to handle.

MASS SPECTROMETER

Type of Mass filter

66HKDUY 2013


MASS SPECTROMETER

Type of Mass filter

• Resolution (R ): R = m/ m

m = mass difference of two adjacent resolved peaks (typically m = mass

of first peak or average).

• Example: R = 500 (“low” resolution)

resolves m/z=50 and 50.1, and m/z=500 and 501

• Example: R = 150,000 (“high” resolution)

resolves m/z=50 and 50.0003, and m/z=500 and 500.0033

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67HKDUY 2013


MASS SPECTROMETER

Type of Mass filter

785.0 785.2 785.4 785.6 785.8 786.0 786.2 786.4 786.6 786.8 787.0 787.2 787.4 787.6 787.8 788.0 788.2

m/z

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

R e l a t i v e A b u n d a n c e

0

20

40

60

80

100

785.8419R=5901 786.3435

R=5900

786.8447R=5900

787.3463R=6000 787.8453

R=5800785.5934R=6200

785.8421R=23801

786.3434R=23900

786.8446R=24000

787.3457R=24100 787.8471

R=15600785.5992R=24300

785.8419R=48101 786.3435

R=47700

786.8446R=48200 787.3458

R=47500787.8477R=42000

785.5994R=47100

785.8413R=94801

786.3428R=95200

786.8442R=93600

787.3458R=98000

785.5989R=95800

787.8477R=89200

0.9 s

1.6 s

RP 75000.2 s

RP 300000.5 s

RP 60000

RP 100000

68


MASS SPECTROMETER

Type of Mass filter

Quadrupole& ion trap

Unregistered

461460459458457456455

100

90

80

70

60

50

40

30

20

10

Unregistered

461460459458457456455

90

80

70

60

50

40

30

20

10

0

R = 1,000

R = 3,000

R = 12,000

TOF

455.3

455.29

455.2910 Unregistered

461460459458457456455

90

80

70

60

50

40

30

20

10

0

HKDUY 2013

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69


MASS SPECTROMETER

Type of Mass filter

HKDUY 2013

Bovine Insulin spectrum [M+6H]6+

Inten.

1 Da

FWHM: 0.08 Da

Resolution: 12,000

< 5 ppm

453.0 454.0 455.0 456.0 457.0 458.0 459.0 460.0 461.0 462.0 463.0 464.0 m/z0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2.0

Inten.(x1,000,000)

455.2897

Mass accuracy = -2.9 ppm

R > 10,000

H3CO

H3CO

CH3H

3C

CN N

CH3

OCH3

OCH3

C27H38N2O4

Exact Mass: 454.2832, Mol. Wt.: 454.6017

Verapamil spectrum [M+H]+

High resolution means high mass accuracy - we can determine veryaccurately the mass of ion to 3rd or 4th decimal point.

70HKDUY 2013


MASS SPECTROMETER

Type of Mass filter

• Mass Accuracy :

• For exampleC9H12NO3 Theoretical mass = 182.0811

Observed mass = 182.0819

Mass Accuracy = 4.01 ppm

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71HKDUY 2013


MASS SPECTROMETER

Type of Mass filter

Identification and confirmation.

Non- Targeted Screening approach.

High level confidence in Research.

Analysis of compounds in Highly complex matrix.

72HKDUY 2013


MASS SPECTROMETER

Type of Mass filter

Advantages:

– High Resolution and Accurate mass.

– High speed of analysis.

Disadvantages:

– No Trapping of ions, No fragmentation.

– Just TOF is not enough for Identification and Confirmation.

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73

HKDUY 2013

MASS SPECTROMETER

Type of Mass filter - QTOF

74HKDUY 2013

MASS SPECTROMETER

Type of Mass filter - QTOF

Advantages:

High Resolution and Accurate mass.

High speed of analysis.

High Mass Range [20,000 or more].

MS2 fragmentation helps in better confirmation.

Works well for both Small Molecules and Big Molecules.

Disadvantages:

No MSn.

Quantitation is not so good.

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75HKDUY 2013

MASS SPECTROMETER

Type of Mass filter – IT-TOF

Stage 2: The entry of the octopole “is closed”

Stage 1: The ions enter the octopole during a definite time

V

Lens 1

Skimmer (+8 V) ‘Open’ Lens 1

(+60 V)

Skimmer

Skimmer (-30 V) ‘Closed’

76HKDUY 2013

MASS SPECTROMETER


V

Lens 1Skimmer

Skimmer (-30 V) ‘Closed’Lens 1

(-80 V)

Stage 3: The ions are transferred to the trap

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77HKDUY 2013

MASS SPECTROMETER


- V 0 V0 V

Octopole

lens 1

Ion trapDuring entry of the ions, no RF isapplied to the ring electrode

78HKDUY 2013

MASS SPECTROMETER


0V 0V

Octopole

Ion trap

RF

When ions are in the trap, the RF is applied

and focuses them in the center

Ar Ar

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79HKDUY 2013

MASS SPECTROMETER


Ion trap

0V 0VRF

+V -V

0V

Simultaneous ejection (BIE: Ballistic Ion Extraction) of

all the ions into the TOF to obtain a TOF mass

spectrum

Detector

ReflectronTOF

80HKDUY 2013

MASS SPECTROMETER


ESIIon inlet CDL

DQ Array

Octopole

3D Ion trap Time of Flight

SIIon inlet CDL

Octopole

MCP detector

Ion Trap for ion

selection, CID

and MSn

TOF of high

resolution spectrum

of MS and MSn

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81HKDUY 2013

MASS SPECTROMETER


0 V 0 V

octopole

Lens 1

Ion trap

MS/MS duty cycle

• Ion trapping

• Ion cooling

• Ion selection

• Fragmentation by CID (Ar)

• Ion cooling

• Ejection to TOF

MS duty cycle

• Ion trapping

• Ion cooling

• Ejection to TOF

MS/MS/MS

MS/MS/MS/MS (MSn)

Multi stage CID

in the ion trap

82HKDUY 2013

MASS SPECTROMETER


Advantages:

High Resolution and Accurate mass.

High speed of analysis.

MS10 fragmentation helps in better confirmation.

Works well for small molecule applications [with less complex

matrix].

Disadvantages:

• Resolution is not enough if the matrix is complex.

• Specifications are lower compared to any HRAM instrument.

• Not suitable for proteomics applications.

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83HKDUY 2013

MASS SPECTROMETER


Reserpine Reserpine Degradation A

MS/MS

Analysis

MS/MS

Analysis

Side Reaction

Intact structure could be

observed as either common

product ion or neutral loss on

MS/MS spectra of reserpine and

its degradation products.

Degradation A

MS/MS

spectrum

Reserpine

MS/MS

spectrum

NH

N

O OO

O

OO

O OO

CH3

CH3

CH3

CH3

CH3

CH3

H

HH

NH

N

O OO

O

OO

O OO

CH3CH3

CH3

CH3

CH3

H

HH

m/z

Inten. (x1,000,000)

448.1967

195.0650

397.2104

236.1265 365.1851

577.2504336.1566

m/z

Inten. (x1,000,000)

381.1801

227.1168 349.1538

363.1688595.2684

NL212 NL212

NL212 NL212

84HKDUY 2013

MASS SPECTROMETER


Reserpine, Theoretical: 609.2807

Column : ODS column 2.0mmI.D.x50mmL

Mobile phase A : 5mmol/L ammonium formate - water

Mobile phase B : acetonitrile

Gradient program : 20%B (0min) →80%B (15min)

Flow rate : 0.2 mL/min

Injection volume : 1 uL

Column temp. : 40 deg. C

Ionization mode : ESI (+)

Nebulizing gas : 1.5 L/min

Drying gas press. : 100 kPa

Probe voltage : +4.5 kV

CDL temp. : 200 deg. C

BH temp. : 200 deg. C

Reserpine Degradation Products

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85HKDUY 2013

MASS SPECTROMETER


85

m/z

Inten. (x1,000,000)

448.1967

195.0650

397.2104

236.1265365.1851

577.2504336.1566

MS2

Precursor ion: m/z609.2802NL 161.0835

NL 212.0698

NL 244.0951NL 373.1537

NL 414.2152

NL 178.0853436.1949

NH

N

O OO

O

OO

O OO

CH3

CH3

CH3

CH3

CH3

CH3

H

HH

C10H11O4+

Theoretical:195.0652

C22H25N2O3+


C13H18NO3+


C23H29N2O4+


C22H30NO8+

Theoretical:436.1966C23H30NO8

+


Found Theoretical Error

PI 195.0650 195.0652 -0.0002

NL 414.2152 414.2155 -0.0003

PI 236.1265 236.1281 -0.0016

NL 373.1537 373.1526 0.0011

PI 365.1851 365.1860 -0.0009

NL 244.0951 244.0947 0.0004

PI 397.2104 397.2122 -0.0018

NL 212.0698 212.0685 0.0013

PI 436.1949 436.1966 -0.0017

NL 173.0853 173.0841 0.0012

PI 448.1967 448.1966 0.0001

NL 161.0835 161.0841 -0.0006

86HKDUY 2013

MASS SPECTROMETER


m/z

Inten. (x10,000,000)

415.2235

m/z

Inten. (x1,000,000)

254.1381

174.0905383.1990

Mass difference from Reserpine= -194.0567Da

…C10H10O4 (Theoritical: 194.0579)

Common neutral loss with Reserpine…161.08

The product ion which yields NLS 161 was m/z 448 in

reserpine

Peak #1 is supposed to have -194 Da difference

(C10H10O4) on the m/z 448 in the substructure of

reserpine.

NH

N

O OO

O

OO

O OO

CH3

CH3

CH3

CH3

CH3

CH3

H

HH

NL161

NH

N

OHO

OO

O

CH3CH3

CH3

H

HH

C23H30N2O5M+H]+: 415.2227

Putative Structure of Peak #1

MS1

MS2

m/z 448

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87HKDUY 2013

MASS SPECTROMETER


m/z

Inten. (x10,000,000)

593.2496

436.1966

m/z

Inten. (x10,000,000)

381.1801

227.1168 349.1538

MS1

MS2

NL212

Difference from reserpine = -16.0304Da

…CH4 (Theoritical: 16.0313)

Common NLS with reserpine:…212.07

The product ion which yields NLS 212 was

m/z 397 in reserpine

Peak #3 is supposed to have -16 Da

difference (CH4) on the m/z 397 in the

substructure of reserpine.

N

H

N

O OO

O

OO

O OO

CH3

CH3

CH3

CH3

CH3

CH3

H

HH

m/z397 of

reserpine

88HKDUY 2013

MASS SPECTROMETER


m/z

Inten. (x1,000,000)

236.1266 365.1845174.0911

192.1008265.1341159.0668 350.1629

396.1950

Reserpine

m/z609

397

MS3 measurement for the PI which yields NLS 212

Peak

m/z593

381

305.1609

305.1628

Common product ion onreserpine and Peak #3 was

m/z305.16.

It is supposed that peak #3

has -16 Da (CH4) difference

from reserpine except the

structure of m/z305.

= demethylation

NH

N

O OO

O

OO

O OO

CH3CH3

CH3

CH3

CH3

H

HHC32H36N2O9M+H+:593.2494

Putative structure of Peak #3

NH

N

O OO

O

OO

O OO

CH3

CH3

CH3

CH3

CH3

CH3

H

HH

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89HKDUY 2013

MASS SPECTROMETER

Ion Detection

90HKDUY 2013

MASS SPECTROMETER

Ion Detection

TOF detector

–6.5KV

∆650 V

∆6KV

–700V

Optical lens

Scintillator

Microchannel

Plate (MCP)

e –e –

e –

e –

hν

Overall gain ~ 2x106

Ground

Photomultiplier

tube (PMT)

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93HKDUY 2013

MASS SPECTROMETERS

Triple quadrupoles

94

LC-MS/MS

Configuration

HKDUY 2013

Ionization probe

Solvent delivery module(gradient)

Note) The LC detector can be removed, if not required.

Mobile phase

Mobile phase

Mixer

Autosampler

Controller

LC detector

Autosampler rinsing solutionColumn oven

Column

Degasser

Reservoir tray

Rotarypump

Nitrogen gasgenerator

LCMS-8040

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95

LC-MS/MS

HKDUY 2013

Positive ions(protonatedmolecules)

Protonation Removeprotons

Negative ions(deprotonated

molecules)

Protons (hydrogen ions)

Separation in column

Inject mixed sample

Components enter MS sequentiallyfrom components with weak retention

Mobile phase

Atmosphericpressure region

Mass separation unit(Q1)

DetectorIonization probe

Vacuum region

Mass separation unit(Q3)

Collision cell(q2)

Ionization probe

Ionization probe

Ionization probe q2

Q1

Q3

Detector

96

LC-MS/MS

SIM Mode

HKDUY 2013

Ions pass through MS1 and collision cell, but only specified ions pass through

MS2.

As the detection time for the specified ions exceeds the scan time, sensitivity

increases 10 to 100 times.

Analysis method for quantitation of target compounds.

Cannot analyze unknown compounds as the mass spectra are not saved.

Effective for simultaneous full scan and SIM.

Optimal parameters must be confirmed in advance.

Pass through SIM

Q 1 Collision cell Q 3 Detector

Pass

through

Same as LC/MS (SQ) SCAN

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97

LC-MS/MS

MRM Mode

HKDUY 2013

Specific ions selected by MS1 undergo collision-induced dissociation (CID) in the

collision cell and specific ions can also be selected by MS2.

Precursor ion-derived product ions are selected.

Despite reduced ion quantity, significantly reduced chemical noise enhances S/N.

High selectivity is ideal for highly sensitive quantitative analysis.

Analysis method for target compounds.

Q 1 Q 3 Detector

SIMCollision cell

Fragmentation

SIM

98

LC-MS/MS

MRM Mode

HKDUY 2013

Ionization probe

Eliminates background fortrace-level quantitation withhigh S/N

MRM10 ppb

1.0 1.5 2.0 2.5 min0

2500

5000

7500

High sensitivity buthigh background

10 ppb

1.0 1.5 2.0 2.5 min0

10000

20000

30000

40000

50000 SIM

Quadrupole Q1

Scan/SIM

Detector Collision cell

FragmentQuadrupole Q3

Scan/SIM

Ion source Detector

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99

LC-MS/MS

Progesteron

HKDUY 2013

300 305 310 315 320 325 330m/z0

100

%

315.1

316.1

Mass Spectrum from

MS1

100 125 150 175 200 225 250 275 300 325m/z0

100

%

109.097.0

Product ion spectrum from MS2Product ions

OCH

2

CH2

CH3

O

CH3

CH3

O

O

CH3

CH3

CH3

Precursor ion

100

LC-MS/MS

HKDUY 2013

UV

MS-TIC

Tuning condition

MS-TIC

CID condition

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101

LC-MS/MS

HKDUY 2013

Peak 2, RT = 7.31

100 200 300 400 500 600 m/z0.0e6

5.0e6

10.0e6

Int. 232

171 200 246270119 332 384354 482416 611536 667

100 200 300 400 500 600 m/z0e3

50e3

Int. 232

144169

187201

246215101 129 398269 343 479442

N

H3C

H3CCH2 C N C

H2

CS

CH

S

H2

C CH2

NH2

m/z155

m/z187

m/z215

m/z144

m/z187

NH3C

H3C

CH2 C NC

H2C

SCH

CH2

SCH2

NH2

m/z169

m/z201

m/z215

Normal

CID

102

LC-MS/MS

HKDUY 2013

Peak 13, RT = 24.81 (Main-3)

100 200 300 400 500 600 m/z0.0e6

5.0e6

10.0e6

Int. 343

365 517407 482298255 460155 214106 553326132 623 671 700597

100 200 300 400 500 600 m/z0e3

50e3

100e3

Int. 343

298255

365155 286187 391112 482367223 328 434

N

H3C

H3C

CH2 C NC

H2C

SCH

S

H2C

CN C

S

CH2

CH

N

CH3

CH3

m/z187

m/z155

m/z255

m/z298

Normal

CID

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103HKDUY 2013

MS vs MS/MS

Tetra-Dioxins Tetra-Furans

RT: 16.93 - 20.35 SM: 3G

1 7. 0 1 7. 2 1 7.4 1 7. 6 1 7.8 1 8. 0 1 8.2 1 8. 4 1 8. 6 1 8. 8 1 9. 0 1 9. 2 1 9.4 1 9.6 1 9. 8 2 0.0 2 0. 2

Time (min)

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100


NL:

2.25E5

m/z=

321.50-

322.50

MS

pcb1248-

20psi-05

NL:

4.70E4

m/z=

333.50-

334.50

MS

pcb1248-

20psi-05

RT: 16.93 - 20.35 SM: 5G

1 7. 0 1 7. 2 1 7. 4 1 7.6 1 7. 8 1 8. 0 1 8. 2 1 8. 4 1 8.6 1 8. 8 1 9. 0 1 9. 2 1 9.4 1 9.6 1 9.8 2 0. 0 2 0. 2

Time (min)

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100


NL:

2.66E5

m/z=

305.50-

306.50

MS

pcb1248-

20psi-05

NL:

5.20E4

m/z=

317.50-

318.50

MS

pcb1248-

20psi-05

2,3,7,8

TCDD-IS2,3,7,8

TCDF-IS

2,3,7,8

TCDD 2,3,7,8

TCDF

104HKDUY 2013

MS vs MS/MS

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106

LCMS-8040

HKDUY 20138030 UF Lens UF Sweeper II

UF Lens

UF Sweeper II

107

LC-MS/MS

Cross Talk

HKDUY 2013

Compound A

m/z 402 Q1)>167Q3)

Compound B

m/z 382

Q1)>167Q3)

Ghost peaks may appear or performance in

quantitation may be deteriorated.

Compound C

m/z 215

Q1)>167Q3)

Detector

Ionizationprobe

Collision cellq2

Q1 Q3 Cross Talk: The phenomenon where ions

loses momentum by collisions with the

collision gas remain in the collision cell and

are detected by the subsequent analysis.

MRM

Transition 1

MRM

Transition 2

MRM

Transition 3

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112

Environment

Economy

Economy

• Production Capacity

• Cost

Environment

• Solvent.

• Energy.

Time.

Solvent.

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LC-MS/MS

Ultra-fast Analysis

113

Mobile phase

• Mobile phase changing → re-built of analysis method.

• Flow → Pressure.

Stationary phase

• Stationary phase changing → Column, Analysis methods.

• Column length → Separation.

Efficiency ?

Amount of used solvent ?

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LC-MS/MS

Ultra-fast Analysis

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Column

Packing particles < 3 μm

System

Pressure ≥ 1000 bar

Detector

High response (> 50 Hz)

Flow-cell 2.5 μL, 5 mm (traditional detector)

Higher resolution.

Shorter analysis time.

Higher sensitivity.

Lower solvent consumption.

Use as HPLC

Easy to migrate from HPLC → UHPLC

HKDUY 2013

LC-MS/MS

Ultra-fast LC

HKDUY 2013

LC-MS/MS

Ultra-fast LC

Column size example:Reducing column sizeto 1/5 reduces analysistime to approx. 1/5.

Mobile phaseexample:Doubling pumpingrate reducesanalysis time toapprox. 1/2.

50 km

Both measures reduceanalysis time to 1/10.

Maintaining the resolution

Passing through 100 m tunnel at 100 km/h

Time to pass through tunnel

reduced to 1/2


Time to pass through tunnel

reduced to 1/10

Particle size:Reducing particle sizeimproves resolution(at constant pumpingrate).

Pumping rate:Reducing particlesize maintains highresolution, even ifpumping rate isincreased.

R e s o l u t i o n

Poor

Good

Small Large

A n a l y s i s t i m e

( m i n u t e s )

Column length (mm)250

150100

75

5030

Note) Relative time, taking the time

of analysis with 4.6 x 150

mm column as 100 minutes

(at constant pumping rate).150

100

50

I.D.6.0 mm4.6 mm3.0 mm2.0 mm

Particle size5.0 m3.0 m2.2 m

Speed 50 km/h

Speed 100 km/h


Speed 50 km/h

Speed 50 km/h

Select a column with

small particle size.

But the resolution becomes poorer.

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HKDUY 2013

LC-MS/MS

Ultra-fast LC

Flowrate

H = A dp + B / + C dp2 H : Height equivalent to a theoretical

plate dp : Particle size of packing : Linear flowrate A dp : Vortex diffusion B / : Molecular diffusion C dp 2 : Resistance to mass transfer

A

B

C

(Fast )(Slow )

N

u m b e r o f

t h

e o r e t i c a l

p l a t e s

( H i g h )

( L o w

)

H

Vortex diffusionDiffusion occurring between packing particles

Resistance to mass transfer Diffusion occurring within packing particles

Molecular diffusionNatural diffusion along mobile phase flow.

Particle size(large)

1. Size of gaps between particles

2. Variability in particle size (particle size distribution)

Particle size(small)

Particle size

distribution(large)

Particle sizedistribution

(small)

Particle size(large)

Particle size(small)

Injection Analysis Injection Analysis Injection Analysis

Start

0 min 0 sec

Injection Analysis Injection Analysis Injection Analysis

Other UHPLC

30sec for injection

Simadzu UFLC

10sec for injection

Injection Analysis

Injection Analysis

Injection Analysis

Finish

3 min 0 sec

Finish

2 min 0 sec

Finish

1 min 40 sec

33% Faster

44% FasterNexera with

overlapping

injection

The fastest Autosampler: 10 sec/injection.

Overlapping injection.

Minimized carryover.

HKDUY 2013

LC-MS/MS

Ultra-fast LC

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HKDUY 2013

LC-MS/MS

Ultra-fast LC

Low Carryover

Low carryover, even with LC/MS/MS

Automatic sample pretreatment functions: Auto injection of internal standard(measurement by LC/MS)

Supports simple addition of an internal standard for correction of

suppression(1) Take in internal standard

(2) Rinse

(3) Take in sample (4) Start analysis

To columnSample

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

25

50

75

100

125

mAU

C18

(4.6mm ID, 150mm,

5μm)

1.0 mL/min

Water/acetonitrile = 45/55

40245nm

5.4 MPa

TPN at peak 5 = 11352

Rs (4,5 = 10.849)

C18

(2.1mm ID, 50mm, 1.8 μm)

1.8 mL/min

Water/acetonitrilen = 45/55

40245nm

1. Acetophenone

2. Propiophenone

3. Butyrophenone4. Valerophenone

5. Hexanophenone

25 times faster

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 min

1050

1075

1100

1125

1150

1175

mAU

45.0

46.0

47.0

48.0

49.0

50.0

51.0

52.0

MPa

105 MPa


Rs (4,5 = 8.40)

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LC-MS/MS

Ultra-fast LC

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0.0 1.0 2.0 3.0 4.0 5.0 min

0

25

50

75

100

125

mAU

C18

(4.6mm ID, 150mm, 5 μm)

1.0 mL/minWater/acetonitrile = 30/70

40245nm

115 MPa


0.0 2.5 5.0 7.5 10.0 12.5 min

1

2 3

4

5

67

4.7 MPa


C18(2.1mm ID, 300mm, 1.8 μm)

0.5 mL/min


40245nm

1. Acetophenone

2. Propiophenone

3. Butyrophenone

4. Valerophenone

5. Hexanophenone

6. Heptanophenone

7. Octanophenone

5 times higher separation efficiency

HKDUY 2013

LC-MS/MS

Ultra-fast LC

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

25

50

75

100

125

mAU

C18

(4.6mm ID, 150mm, 5μm)

1.0 mL/min


40245nm

5.4 MPa


Rs (4,5 = 10.849)

C18

(2.1mm ID, 100mm, 1.8 μm)

1.4mL/min


60245nm

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min0

25

50

75

100

125

150

175

mAU

100 MPa


Rs (4,5 = 12.21)

1. Acetophenone

2. Propiophenone

3. Butyrophenone

4. Valerophenone

5. Hexanophenone

14 times faster1.5 times higher separation effciency

HKDUY 2013

LC-MS/MS

Ultra-fast LC

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Peak capacity : 244 in 8 minutes

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 min

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

mAU

Bovin serum albumin triyptic digest

Column : C182.1mm, 100mm, 1.8 μm

Mobile phase : A : 0.03 % TFA in water

B : 0.03 % TFA in acetonitrile

Gradient : B 5% → 40 % (8 min)

Mixer : 180 μL


Column temp. : 40

Detection : 214 nm

Sample : Trypsin digested BSA

(total of 1 pmol / uL)

HKDUY 2013

LC-MS/MS

Ultra-fast LC

Water as mobile phase → Green LC

LC

Green LC 1 : Theophylline2 : Caffeine

1

2

1

2

150

Water only

4030% methanol

Green LC

Column : Shodex ET-RP1 (3.0mm, 150mm)

Mobile phase : water


Column temp. : 150 oC

LC

Column : C18 (4.6mm, 150mm, 5 μm)

Mobile phase : methanol / water = 3/7


Column temp. : 40 oC

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LC-MS/MS

Ultra-fast LC

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0 min 60 min

0 min 30 min

(15 min)

Column: 150 mm L x 4.6 mm I.D., 5 μm

Flow rate: 1 mL/min

Injection volume: 10 μL

Analytical time: 60 min

Consumption volume: 60 mL/sample

Column: 75 mm L x 4.6 mm I.D., 2.2 μm

Flow rate: 1 mL/min (or 2 mL/min)


Analytical time: 30 min (or 15 min)


HKDUY 2013

LC-MS/MS

Ultra-fast LC

0 min 60 min

0 min 60 min

Column: 150 mmL x 4.6 mmI.ID., 5 μm

Flow rate: 1 mL/min




Column: 150 mmL x 2.0 mmI.ID., 5 μm

Flow rate: 0.2 mL/min




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LC-MS/MS

Ultra-fast LC

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Acetonitirile consumption reduced up to > 80% while remaining the

separation efficiency

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 min0

50

100

150

200

250mAU

mAU

0.0 5.0 10.0 15.0 20.0 25.0 min0

50

100

150

200

250

Mobile phase : A: 0.1% formic acid-water, B: acetonitrile, Gradient elution

Temperature : 40

Flow rate : 1.0mL/min

Peaks : 1:cefadroxil, 2:cephaprin, 3:cefaclor, 4:cefalexin, 5:cephradine, 6:cefotaxime. 7:cefazolin, 8 :cefuroxime, 9

:cefoperazone, 10 :cefloxitin, 11 :cefamandole A, 12 :cephalothin, 13: cefamandole B

HPLCShim-pack VP-ODS (4.6 mm 250 mm , 5μm)

UHPLCShim-pack XR-ODS (3.0mm 100 mm , 2.2μm)

Acetonitrile: 12.8mL

Acetonitrile: 1.7mL

HKDUY 2013

LC-MS/MS

Ultra-fast LC

Maximized Throughput

25 times faster analysis cycle, 10 sec injection speed , 2300 samples/day.

Solvent consumption reduced to 1/40.

Cost saving and environmentally-friendly UHPLC

Maximized Performance

5 times higher separation efficiency.

Carryover reduced to one/third.

Maximized Expandability

Expanded configurations by more than 100 optional units.

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LC-MS/MS

Ultra-fast LC

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Solvent Saving

Flow Rate : 3/5 reduction x Analysis time : 1/25 reduction

= Mobile phase consumption/analysis is reduced to 1/40 Cost for power

Analysis Time : 1/25 reduction

= Power consumption is reduced to 1/25

Cost for performance

Initial cost + 5 years maintenance cost up to x 1.3 only

Sample / System up to x 5

= Cost / performance is reduced to 1/15

Cost saving for method migration

Method migration conventional LC ↔ UHPLC) tool available

HKDUY 2013

LC-MS/MS

Ultra-fast LC

HKDUY 2013

LC-MS/MS

Ultra-fast LC

S4-Plunger surface(x50)

S4 –Plunger Super Smooth Surface Sapphire

Minimized frictional heat on plunger surface

Optimized material for UHPLC

Plunger, Plunger seal, Needle seal, High-pressure valve, etc.

New high pressure valve design

High--strength

seal materialS4-Plunger

High-pressure valve

The World’s highest pressure 130MPa

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HKDUY 2013

LC-MS/MS

MS responding to performance of UFLC

What makes an MS instrument suitable for UFLC?

The ability to acquire data at high speed without sacrificing data quality is

required!

Three things that enable ultrafast analysis:

Ability to perform scan measurement at high speed

UFscanning

Ability to switch between positive and negative ion measurement at

high speed

UFswitching

High sensitivity in high-speed measurement

UFsensitivity

HKDUY 2013

LC-MS/MS


20 points 10 points

4 to 5 points

Influence of Data Sampling Pointson Peak Form

1.0min.

UFLC Data

If the number of data points decreases,

the sensitivity also decreases.

This adversely affects the reproducibility.

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HKDUY 2013

LC-MS/MS


• Increase the scan speed.

With conventional instruments,the sensitivity decreases.

• Decrease the scan speed.

It is difficult to handle high-speedanalysis.

Scan: Data is acquired in the desired m/z range.m/z

100

1,100

Scan speed

(scan cycle)

t(s)

Scan speed

(scan cycle)m/z

100

1,100

t(s)

Since LCMS-2020, it has become possible to

maintain sensitivity when the scan speed is

increased.

HKDUY 2013

APPLICATIONS

Pesticides

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HKDUY 2013

APPLICATIONS

Pesticides

HPLC : Nexera UHPLC system

Column

Mobile phase

Flow rate

Gradient program

Column temperature

: Shim-pack XR-ODSII (75 mm x 2 mmI.D., 2.2 um)

: 0.2 mL / min.

: 5% B (0-2.5 min.)→55% B (2.51-6 min.)→80% B (6.01-12 min.)

→100% (12-15 min.)→5% (15.01-20 min.)

: 40 C

MS : LCMS-8040 Triple quadrupole mass spectrometer

Ionization : ESI (Positive / Negative)

Ion spray voltage : +4.5 kV / -3.5 kV

MRM : 276 MRM transitions (2 MRMs / compound)

Dwell time 5 msec. / Pause time 1 msec.

: A ; 2 mM ammonium formate containing 0.1 % formic acid – water

B ; Methanol

DL temperature : 250 C

HB temperature : 400 CNebulizing Gas : 3 L / min.

Drying Gas : 15 L / min.

HKDUY 2013

APPLICATIONS

Pesticides

Number of compounds: 138

LOQs of 138 pesticides in the EURL method by LCMS-8040

Technique LOQs < 10 ppb LOQs > 10 ppb Not Ionization

by LC/MS/MS 72 (100 %) 0 (0 %) 0 (0 %)

by GC/MS/MS 47 (71 %) 6 (9 %) 13 (20 %)

80 % of compounds which was refered to GC-QqQ

were possible to be measured byLC-QqQ

0.089.9 ppb

72: LC/MS/MS

66: GC/MS/MS

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HKDUY 2013

APPLICATIONS

Pesticides

HKDUY 2013

APPLICATIONS

Pesticides

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HKDUY 2013

APPLICATIONS

Veterinary Drugs

1 Salbutamol 25 Sulfamethoxazole

2 5-Propylsulfonyl-1H-benzimidazole-2-amine 26 Tiamulin

3 Lincomycin 27 Florfenicol

4 Trimethoprim 28 Chloramphenicol

5 Thiabendazole 29 Clorsulon6 Sulfacetamide 30 Ethopabate

7 Ormetoprim 31 Sulfaquinoxaline

8 Ractopamine 32 Sulfadimethoxine

9 Sulfadiazine 33 Prednisolone

10 Xylazine 34 Hydrocortisone

11 Clenbuterol 35 Dexamethasone

12 Sulfathiazole 36 Penicillin-G

13 Sulfapyridine 37 Sulfanitran

14 Sulfamerazine 38 Emamectin B1a

15 Carbadox 39 beta-Trenbolone

16 Pyrimethamine 40 alpha-Trenbolone

17 Thiamphenicol 41 Zeranol

18 Sulfadimidine 42 Oxacillin

19 Sulfamonomethoxine 43 Famphur 20 Trichlorfon (DEP) 44 Fenobucarb (BPMC)

21 Sulfamethoxypyridiazine 45 Phenylbutazone

22 Sulfachlorpyridazine 46 Melengestrol Acetate

23 Erythromycin 47 Temephos (Abate)

24 Sulfadoxine 48 Allethrin

HKDUY 2013

APPLICATIONS

Veterinary Drugs

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HKDUY 2013

APPLICATIONS

Veterinary Drugs

HKDUY 2013

APPLICATIONS

Veterinary Drugs

Compounds

Calibration curve

Range (ng / g) r

2 LOQ (ng / g)

Compounds

Calibration curve

Range (ng / g) r

2 LOQ (ng / g)

Salbutamol 5-500 0.999 1 .1 Sulfamethoxazole 5-500 0.997 4 .5

5-Propylsulfonyl-1H-benzimidazole-2-amine 5-500 0.997 2.5 Tiamulin 5-500 0.999 5.0

Lincomycin 10-1000 0.999 1.6 Florfenicol 50-500 0.990 16.3

Trimethoprim 5-500 0.996 3.2 Chloramphenicol 25-500 0.999 12.4

Thiabendazole 5-500 0.998 2.5 Clorsulon 50-500 0.997 50.0

Sulfacetamide 25-500 0.999 21.3 Ethopabate 5-500 0.997 5 .3

Ormetoprim 5-500 0.998 1 .9 Sulfaquinoxaline 5-500 0.998 1 .3

Ractopamine 5-500 0.999 2.0 Sulfadimethoxine 5-500 0.998 1.0

Sulfadiazine 10-500 0.998 5.8 Prednisolone 10-500 0.999 6.2

Xylazine 5-500 0.999 1.6 Hydrocortisone 10-500 0.996 5.2

Clenbuterol 5-500 0.997 0.8 Dexamethasone 20-1000 0.999 15.3

Sulfathiazole 5-500 0.996 1.5 Penicillin-G 5-500 0.999 1.4

Sulfapyridine 5-500 0.998 2.5 Sulfanitran 5-500 0.989 2.0

Sulfamerazine 10-1000 0.998 1.9 Emamectin B1a 5-500 0.999 5.0

Carbadox 10-500 0.999 4.3 beta-Trenbolone 10-500 0.997 5.8

Pyrimethamine 5-500 0.997 1.1 alpha-Trenbolone 10-500 0.998 5.4

Thiamphenicol 50-500 0.989 16.7 Zeranol 25-500 0.995 18.9

Sulfadimidine 10-1000 0.995 1.2 Oxacillin 5-500 0.999 0.3

Sulfamonomethoxine 5-500 0.995 0.8 Famphur 10-500 0.999 7.9

Trichlorfon (DEP) 25-500 0.999 10.3 Fenobucarb (BPMC) 5-500 0.999 1.1

Sulfamethoxypyridiazine 5-500 0.997 2.7 Phenylbutazone 5-500 0.996 6.4

Sulfachlorpyridazine 25-500 0.996 21.5 Melengestrol Acetate 5-500 0.997 3.1

Erythromycin 5-500 0.999 0.1 Temephos (Abate) 10-500 0.999 3.4

Sulfadoxine 5-500 0.993 1.1 Allethrin 5-500 0.999 3.9

Calibration point (conc.)

5, 10, 25, 50, 100, 250, 500 ng / g (=1, 2.5, 5, 10, 25, 50, 100 ng / mL)

Veterinary drug standards were spiked into the pork matrix.

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HKDUY 2013

APPLICATIONS

Veterinary Drugs

HKDUY 2013

APPLICATIONS

Veterinary Drugs

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HKDUY 2013

APPLICATIONS

Veterinary Drugs

APPLICATIONS

Veterinary Drugs

Compounds Spike level

(ng / g P ork Chicken Salmon Compounds

Spike level

(ng / g P ork Chicken Salmon

Salbutamol 5 75 74 89 Sulfamethoxazole 5 76 100 99

5-Propylsulfonyl-1H-benzimidazole-2-amine 5 108 99 101 Tiamulin 5 104 118 121

Lincomycin 5 120 129 124 Florfenicol 50 122 134 113

Trimethoprim 5 92 114 116 Chloramphenicol 50 121 156 119

Thiabendazole 5 7 1 77 103 Clorsulon 50 108 108 93

Sulfacetamide 50 104 91 157 Ethopabate 5 8 5 116 112

Ormetoprim 5 97 144 150 Sulfaquinoxaline 5 79 91 108

Ractopamine 5 61 133 107 Sulfadimethoxine 5 81 93 91

Sulfadiazine 50 52 55 71 Prednisolone 50 114 122 155

Xylazine 5 90 91 82 Hydrocortisone 50 139 114 127

Clenbuterol 5 90 76 76 Dexamethasone 50 105 126 149

Sulfathiazole 5 84 62 74 Penicillin-G 50 116 116 116

Sulfapyridine 50 88 76 85 Sulfanitran 5 58 107 76

Sulfamerazine 50 86 74 83 Emamectin B1a 5 101 129 130

Carbadox 50 76 64 91 beta-Trenbolone 50 110 109 103

Pyrimethamine 5 101 86 97 alpha-Trenbolone 50 108 109 110

Thiamphenicol 50 95 90 92 Zeranol 50 116 111 122

Sulfadimidine 5 104 84 83 Oxacillin 5 123 133 116

Sulfamonomethoxine 50 90 78 82 Famphur 50 107 112 128

Trichlorfon (DEP) 50 99 123 151 Fenobucarb (BPMC) 5 9 8 109 116

Recovery (%) Recovery (%)

LC-MSMS (EN)

Documents

Transcript of LC-MSMS (EN)