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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
18
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
20
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