The world leader in serving science

Advances in bioanalytical

LC-MS using the Orbitrap

mass analyzer

Dr. Michaela Scigelova

Bremen, Germany

2

Orbitrap by Application Areas

Perry RH, Cooks G, Noll RJ: Orbitrap mass spectrometry: instrumentation, ion

motion and applications. Mass Spec. Rev. 27, 661-699 (2008).

Proteomics

Metabolomics

Drug metabolism

Doping control

Lipids

Residue analysis

3

Application focus: Bioanalysis

Key figures of merit

Elemental composition determination

Structural characterisation

Quantitation

Application areas

• drug metabolism

• doping control

• food contaminants

BIOANALYSIS - Quantitative

measurement of a drug, drug

metabolite, or chemicals in

biological fluids

4

Orbitrap Analyzer – Key Figures of Merit

Mass accuracy

Resolution

Fidelity of isotope pattern abundancies

Dynamic range

Positive/negative switching

Multiple levels of fragmentation

5

MASS ACCURACY

Accurate mass measurement is

used to determine the elemental

composition of an analyte

• confirm the identification of

target compounds

• eliminate false positive

identification

• support the identification of

unknowns

• separation of possible

interferences

Example: mass 32

What can it be ??

S

O2

CH3OH

N2H4

6

Accurate Mass Is a Powerful Filter

Mass

measured

Tolerance

[Da]

Suggestions Calc Mass

32.0 +/- 0.2 O2

CH3OH

N2H4

S

31.9898

32.0261

32.0374

31.9721

32.02 +/- 0.02 CH3OH

N2H4

32.0261

32.0374

32.0257 +/- 0.002 CH3OH 32.0261

C = 12.0000H = 1.0078

N = 14.0031

O = 15.9949

S = 31.9721

7

Accurate Mass

Makes Life Easier

8

RESOLUTION

High resolution is necessary to separate peaks of one mass

from those of another and ensure that ions of only one kind

contribute to a particular measurement.

Example of pirimicarb (m/z 239)

Resolution Mass tolerance

(mmu)

Number of elemental

composition suggestions*

15,000 +/- 9 14

80,000 +/- 1.7 1

*Assuming CHNO elements

9

RESOLUTION

High resolution is necessary to separate peaks of one mass from

those of another and ensure that ions of only one kind contribute

to a particular measurement.

Experiments involving

complex mixtures

Accurate mass determination

R = 80,000

R = 15,000

0.32 ppm

6.50 ppm

10

RESOLUTION

High resolution is necessary to separate peaks of one mass from

those of another and ensure that ions of only one kind contribute

to a particular measurement.

Experiments involving

complex mixtures

Highly specific quantitation

Accurate mass determinationR = 80,000

11

Resolution

Enables Accurate Mass

and Accurate Quantitation

12

Stable Mass Accuracy

Stability of mass measurement

Positive/negative acquisition modes

13

Dynamic Range of Mass Accuracy

Azoxystrobin

Resolution 15,000

MH+ DM [ppm]

404.12390 -0.20

405.12723 5.27

406.12958 3.07

14

Azoxystrobin at resolution 80,000

The complete molecular ion cluster detected

correctly.

MH+ DM [ppm]

404.12390 -0.39

405.12723 -0.45

406.12958 -0.98

Dynamic Range of Mass Accuracy

15

On Isotopes, Their Abundancies, and How Can That Be Useful

16

Fidelity of Isotope Pattern Abundancies

Identification of an ‘unknown’

Accurately determine the mass of compound X and determine

sum formulae proposals

Exclude false positive hits by comparing the proposed sum

formulae to the theoretical isotope patterns

indicates >1 sulfur

indicates presence of sulfur

For details see Thermo Application note 30130

17

GOAL: Unique Elemental Composition

Kind T, Fiehn O: Metabolomic database annotations via query of elemental

compositions: Mass accuracy is insufficient even at less than 1 ppm.

Bioinformatics 7, 234-244 (2006)*

Assuming better than 1 ppm mass deviation, generally, a

unique elemental composition can be obtained for

compounds < 300 Da*

But with an additional information from Isotopic

Abundance Ratios, unique elemental composition can be

obtained for compounds up to 2200 Da

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Elemental

Composition

Structural Elucidation

19

LTQ Orbitrap Velos Technology

High accuracy/resolution

detection

Fragmentation of ions

Higher energy

Parent isolation

Fragmentation

ETD module

Peptides with PTM

Fast ion trap

detection

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Fragmentation ‘Menu’

CID

In an ion trap

‘Resonance’ fragmentation

Detection in ion trap (fast)

Detection in Orbitrap (high

resolution/accurate mass)

MSn

x

HCD

In multipole collision cell

Multiple collisions possible

x

Detection in Orbitrap (high

resolution/accurate mass)

x

No low mass cut-off

Each fragmentation technique has its pros and cons

Select what best suits your application

You have a choice

21

Roxithromycin C41H76N2O15 m/z 837.53185

100 200 300 400 500 600 700 800

m/z

0

10

20

30

40

50

60

70

80

90

1000

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

679.43610

558.36337

522.33837 716.45652603.38458

679.43717

158.11769

522.33884398.25443

603.38541

233.15379

342.22706

O

O

OH

NO O O

OHOH

O

O

OH

O

O

O

NOH

O

NOH

0.9 ppm

0.8 ppm0.5 ppm

1.8 ppm 4.2 ppm

3.8 ppm0.6 ppm

0.5 ppm

0.9 ppm

0.6 ppm

1.8 ppm

2.0 ppm

CID

HCD

22

Quercetin – Higher energy fragmentation HCD

80 100 120 140 160 180 200 220 240 260 280 300m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

303.04996C15H11O70.09 ppm

153.01808C7 H5 O4-1.00 ppm

229.04961C13H9 O40.31 ppm

137.02316C7 H5O3-1.15 ppm 257.04446

C14H9O50.05 ppm

201.05466C12H9O30.18 ppm

165.01808C8 H5O4-0.93 ppm 285.03955

C15H9 O60.65 ppm

183.02908C8H7 O51.55 ppm

121.02819C7 H5O2

-1.78 ppm95.04887C6 H7O

-2.81 ppm

O

OOH

OH

OH

OH

O

O

OHOH

OH

247.06023C13 H1

1

O50.51ppm

OHOH

OH

OH

OH

OH

OH

OH OH

OH

OH

OH

OH

O

OH

OH

O

OHOH

OH

O

OH

OH

O

OH

OH

111.00747C5H3O3-1.81 ppm

OH

OH

O

Mass Frontier - Fragmentation mechanisms and spectrum interpretation

O

OH

OH

OH

O

OH

OH

H

23

Traditional library

search only useful for

compounds represented

in the library

Unknown compounds

not in the library can not

be identified

Use MSn to obtain

structural arrangements

of unknowns

Sheldon et al.: Determination of ion structures in structurally related compounds using

precursor ion fingerprinting. J. Am. Soc. Mass Spectrom. 20, 370-376, (2009).

Multiple Levels of Fragmentation - MSn

“Spectral Tree”

Level = MS stage

Node = product ion spectrum

Branch = connects precursor

and its product ion spectrum

24

Group of compounds with structural similarity

Different structures

Different masses

Different MS/MS spectra

BUT

Share some structural features

Consistent CID behaviour

Important structural information

Sheldon et al.: Determination of ion structures in structurally related compounds using

precursor ion fingerprinting. J. Am. Soc. Mass Spectrom. 20, 370-376, (2009).

25

Identical MSn spectra of analogues

Sheldon et al.: Determination of ion structures in structurally related compounds using

precursor ion fingerprinting. J. Am. Soc. Mass Spectrom. 20, 370-376, (2009).

MS4

MS3

MS3

MS4

26

Getting to Know the ‘Unknown’

Apomorphine not in the database

MSn n = 1 - 4

Data indicated two precursor ions m/z 239 and 193 common to

heroin, codeine and morphine

Substructures identified

by comparison to existing

‘spectra trees’ in the library

Sheldon et al.: Determination of ion structures in structurally related compounds using

precursor ion fingerprinting. J. Am. Soc. Mass Spectrom. 20, 370-376, (2009).

27

Concept of ‘Spectral Trees’

Identification of ion structures and sub-structures

Subsequent reconstruction of the molecular structure of small

organic compounds

Characterisation of structurally similar compounds

Identification of designer drugs

Novel chemical analogues

Mass Frontier sw for creating spectral tree libraries

28

SUMMARY

Accurate mass is useful

For precursor and fragment ions

Useful only if reliable

• Calibration stability (days)

• Dynamic range (> 3 orders of magnitude)

• Stable in alternating positive/negative mode

Reliable only if measured at adequate resolution

29

Drug

metabolism, 17

Doping control,

16

Food

contaminants/

residue analysis,

5

Orbitrap in Bioanalysis

Published research articles

(2006- 2008)

30

Doping control

Thevis M, Kamber M, Schanzer W: Screening for metabolically stable aryl-propionamide-derived selective androgen receptor modulators for doping control purposes. Rapid Comm. Mass Spectrom. 20, 870-876 (2006).

Thevis M, Kohler M, Maurer J, Schlorer N, Kamber M, Schanzer W: Screening for 2-quinolinone-derived selective androgen receptor agonists in doping control analysis. Rapid Comm. Mass Spectrom. 21, 3477-3486 (2007).

Thevis M, Kohler M, Thomas A et al.: Determination of benzimidazole- and bicyclic hydantoin-derived selective androgen receptor antagonists and agonists in human urine using LC-MS/MS. Anal. Bioanal. Chem. 391, 251-261 (2008).

Thevis M, Kohler M, Schlorer N et al.: Mass spectrometry of hydantoin-derived selective androgen receptor modulators. J. Mass Spectrom. 43, 639-650 (2008).

Thevis M, Schanzer W: Mass spectrometry of selective androgen receptor modulators. J. Mass Spectrom. 43, 865-876 (2008).

Thevis M, Kohler M, Thomas A, Schlorer N, Schanzer W: Doping control analysis of tricyclic tetrahydroquinoline-derived selective androgen receptor modulators using liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 22, 2471-2478 (2008).

Thevis M, Wilkens F, Geyer H, Schanzer W: Determination of therapeutics with growth-hormone secretagogue activity in human urine for doping control purposes. Rapid Comm. Mass Spectrom. 20, 3393-3409 (2006).

Thevis M, Sigmund G, Schiffer AK, Schanzer W: Determination of N-desmethyl- and N-bisdesmethyl metabolite of Sibutramine in doping control analysis using liquid

chromatography-tandem mass spectrometry. Eur. J. Mass Spectrom. 12, 129-136 (2006).

31

Doping control

Thevis M, Krug O, Schanzer W: Mass spectrometric characterization of efaproxiral (RSR13) and its implementation into doping controls using liquid chromatography atmospheric pressure ionization-tandem mass spectrometry. J. Mass Spectrom. 41, 332-338 (2006).

Thevis M, Makarov AA, Horning S, Schanzer W: Mass spectrometry of stanozolol and its analogues using electrospray ionization and collision-induced dissociation with quadrupole-linear ion trap and linear ion trap-Orbitrap hybrid mass analyzers. Rapid Comm. Mass Spectrom. 19, 3369-3378 (2005).

Virus ED, Sobolevsky TG, Rodchenkov GM: Introduction of HPLC/Orbitrap mass spectrometry as screening method for doping control. J. Mass Spectrom. 43, 949-957 (2008).

Thomas A, Geyer H, Kamber M, Schanzer W, Thevis M: Mass spectrometric determination of gonadotrophin-releasing hormone (GnRH) in human urine for doping control purposes by means of LC ESI-MS/MS. J. Mass Spectrom. 43, 908-915 (2008).

Thevis M, Thomas A, Schanzer W: Mass spectrometric determination of insulins and their degradation products in sports drug testing. Mass Spectrom. Rev. 27, 35-50 (2008).

Bredehoft M, Schanzer W, Thevis M: Quantification of human insulin-like growth factor-1 and qualitative detection of its analogues in plasma using liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Comm. Mass Spectrom. 22, 477-485 (2008).

Thevis M, Bredehoft M, Geyer H, Kamber M, Delahaut P, Schanzer W: Determination of Synacthen in human plasma using immunoaffinity purification and liquid chromatography/ tandem mass spectrometry. Rapid Comm. Mass Spectrom. 20, 3551-3556 (2006).

Thevis M, Maurer I, Kohler M, Geyer H, Schanzer W: Proteases in doping control analysis. Intl. J. Sports Med. 28, 545-549 (2007).

32

Drug metabolism

Dernovics M, Lobinski R: Speciation analysis of selenium metabolites in yeast-based

food supplements by ICPMS - Assisted hydrophilic interaction HPLC - Hybrid linear ion

trap/Orbitrap MSn. Anal. Chem. 80, 3975-3984 (2008).

Erve JCL, DeMaio W, Talaat RE: Rapid metabolite identification with sub parts-per-

million mass accuracy from biological matrices by direct infusion nanoelectrospray

ionization after clean-up on a ZipTip and LTQ/Orbitrap mass spectrometry. Rapid

Commun. Mass Spectrom. 22, 3015-3026 (2008).

Zhang H, Zhang D, Ray K: A software filter to remove interference ions from drug

metabolites in accurate mass liquid chromatography/mass spectrometric analyses. J.

Mass Spectrom. 38, 1110-1112 (2003).

Zhu MS, Ma L, Zhang HY, Humphreys WG: Detection and structural characterization of

glutathione-trapped reactive metabolites using liquid chromatography-high-resolution

mass spectrometry and mass defect filtering. Anal. Chem. 79, 8333-8341 (2007).

33

Drug metabolism

Nielen MWF, van Engelen MC, Zuiderent R, Ramaker R: Screening and confirmation

criteria for hormone residue analysis using liquid chromatography accurate mass time-

of-flight, Fourier transform ion cyclotron resonance and Orbitrap mass spectrometry

techniques. Anal. Chim. Acta 586, 122-129 (2007).

Bateman KP, Kellmann M, Muenster H, Papp R, Taylor L: Quantitative-qualitative data

acquisition using a bench-top Orbitrap mass spectrometer. J. Am. Soc. Mass Spectrom.

(2009)doi:10.1016/j.jasms.2009.03.002.

Dunn WB, Broadhurst D, Brown M et al.: Metabolic profiling of serum using ultra

performance liquid chromatography and the LTQ-Orbitrap mass spectrometry system. J

Chrom. B 871, 288-298 (2008)

(interfacing sub-2 μm liquid chromatography to the LTQ Orbitrap; detection of metabolites

in a complex mammalian biofluid, serum)

Li AC, Shou WZ, Mai TT, Jiang XY: Complete profiling and characterization of in vitro

nefazodone metabolites using two different tandem mass spectrometric platforms.

Rapid Comm. Mass Spectrom. 21, 4001-4008 (2007)

Chen GD, Khusid A, Daaro I, Irish P, Pramanik BN: Structural identification of trace

level enol tautomer impurity by on-line hydrogen/deuterium exchange HR-LC/MS in a

LTQ Orbitrap hybrid mass spectrometer. J. Mass Spectrom. 42, 967-970 (2007).

34

Drug metabolism

Cuyckens F, Balcaen LIL, De Wolf K et al.: Use of the bromine isotope ratio in HPLC-

ICP-MS and HPLC-ESIMS analysis of a new drug in development. Anal. Bioanal. Chem.

390, 1717-1729 (2008).

Lim HK, Chen J, Cook K, Sensenhauser C, Silva J, Evans DC: A generic method to

detect electrophilic intermediates using isotopic pattern triggered data-dependent

high-resolution accurate mass spectrometry. Rapid Comm. Mass Spectrom. 22, 1295-

1311 (2008). Cuyckens F, Balcaen LIL, De Wolf K et al.: Use of the bromine isotope

ratio in HPLC-ICP-MS and HPLC-ESIMS analysis of a new drug in development. Anal.

Bioanal. Chem. 390, 1717-1729 (2008).

Peterman SM, Duczak N, Kalgutkar AS, Lame ME, Soglia JR: Application of a linear ion trap/Orbitrap mass spectrometer in metabolite characterization studies: Examination of the human liver microsomal metabolism of the non-tricyclic anti-depressant nefazodone using data-dependent accurate mass measurements. J. Am. Soc. Mass Spectrom. 17, 363-375 (2006).

Lim HK, Chen J, Sensenhauser C, Cook K, Subramanyam V: Metabolite identificationby data-dependent accurate mass spectrometric analysis at resolving power of 60,000 in external calibration mode using an LTQ/Orbitrap. Rapid Comm. Mass Spectrom. 21,1821-1832 (2007).

Wang YY, Chen XY, Li Q, Zhong DF: Characterization of metabolites of a novel histamine H-2-receptor antagonist, lafutidine, in human liver microsomes by liquid chromatography coupled with ion trap mass spectrometry. Rapid Commun. Mass Spectrom. 22, 1843-1852 (2008).

35

Drug metabolism

Zhang NR, Yu S, Tiller P, Yeh S, Mahan E, Emary WB: Quantitation of small molecules using high-resolution accurate mass spectrometers – a different approach for analysis of biological samples. Rapid Commun. Mass Spectrom. 23, 1085-1094 (2009).

(direct comparison of quantitative performance between API 4000 triple quadrupole and the LTQ Orbitrap)

Bluemlein K, Raab A, Meharg AA, Charnock JM, Fledmann J: Can we trust mass spectrometry for determination of arsenic peptides in plants: comparison of LC-ICP-MS and LC-ES-MS/ICP-MS with XANES/EXAFS in analysis of Thunbergia alata. Anal. Bioanal. Chem. 390, 1739-1751 (2007).

Ruan Q, Peterman S, Szewc MA et al.: An integrated method for metabolite detection and identification using a linear ion trap/Orbitrap mass spectrometer and multiple data processing techniques: Application to indinavir metabolite detection. J. Mass Spectrom. 43, 251-261 (2008).

36

Food contaminants/residue analysis

Nielen MWF, van Engelen MC, Zuiderent R, Ramaker R: Screening and confirmation criteria for hormone residue analysis using liquid chromatography accurate mass time-of-flight, Fourier transform ion cyclotron resonance and Orbitrap mass spectrometry techniques. Anal. Chim. Acta 586, 122-129 (2007).

Pico Y, Barcelo D: The expanding role of LC-MS in analyzing metabolites and degradation products of food contaminants. Trac-Trends in Anal. Chem. 27, 821-835 (2008).

van der Heeft E, Bolck YJC, Beumer B, Nijrolder AWJM, Stolker AAM, Nielen MWF: Full-scan accurate mass selectivity of ultra-performance liquid chromatography with time-of-flight and Orbitrap mass spectrometry in hormone and veterinary drug residue analysis. J. Am. Soc. Mass Spectrom. 20, 451-463 (2009).

Le Breton MH, Rochereau-Roulet S, Pinel G et al.: Direct determination of recombinant bovine somatotropin in plasma from a treated goat by liquid chromatography/high-resolution mass spectrometry. Rapid Commun. Mass Spectrom. 22, 3130-3136 (2008).

Hogenboom AC, van Leerdam JA, de Voogt P: Accurate mass screening and identification of emerging contaminants in environmental samples by liquid chromatography–hybrid linear ion trap Orbitrap mass spectrometry. J Chrom. A 1216, 510–519 (2009).

Entry of the Exactive instrument in late 2008

37

Executive Summary

Mass spectrometric detection will be employed in most bioana-lytical assays.

The Orbitrap mass spectrometer has been used for a wide range of applications: in the discovery phase and during the preclinical and clinical stages of drug development, for detection of food contaminants, and in doping control.

The desirable attributes of the Orbitrap-based analyzers most quoted in published literature are:

• reliable high mass accuracy and its dynamic range

• very high resolving power

• MS/MS or MSn fragmentation capabilities with accurate mass

Development continues in the areas of Orbitrap design, mass analyzer hybridization, coupling to novel ionization techniques, and advancing data processing tools