Multiple Stage Tandem Mass Spectrometryfor identification of plastic contaminations in blood plasma

using nano electrospray ionization

Tobias Kind and Kwang-Hyeon LiuFiehnLab UC Davis 2009

http://fiehnlab.ucdavis.edu/staff/kind/Metabolomics/LipidAnalysis

File under: Anecdotal mass spectrometryCC-by Free Reuse License granted; Tobias Kind FiehnLab 2009

Instrumentation – robotized chip based nano electrospray ionizationwith linear iontrap

Advion NanoMate robot not shown

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nanoESI chip LTQ Linear IonTrap

Plasma_Pos_LowMS_60MSMS_CE45_01 #35-73 RT: 0.07-0.15 AV: 39 NL: 2.53E4T: ITMS + p ESI Full ms [200.00-1200.00]

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650.55

703.64

678.64

654.18680.27

676.55 705.64690.55686.27664.55655.27648.55 701.64 706.55666.55660.45 707.55644.45

Full scan nanoESI positive mode with data dependent multiple stage tandem MS

We will fragment this ion with the awe-inspiring power ofMS/MS/MS/MS/MS/MS/MS/MS or MS8

Slowly repeat with me: MS1..MS2..MS3..MS4..MS5..MS6..MS7..MS8...

What could it be?

Plasma_Pos_LowMS_60MSMS_CE45_01 #184 RT: 1.15 AV: 1 NL: 2.29E3T: ITMS + c ESI d Full ms2 664.00@cid45.00 [170.00-675.00]

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607.32

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495.26

359.24 604.28 631.35540.52325.27 437.36212.44 283.20

MS^2precursor m/z 664.00 (ESI+)

-56.8

-112.76

-168.74

Plasma_Pos_LowMS_60MSMS_CE45_01 #190 RT: 1.27 AV: 1 NL: 1.16E2T: ITMS + c ESI d Full ms8 664.00@cid45.00 607.32@cid45.00 551.33@cid45.00 495.34@cid35.00 439.26 ...

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MS^8 (ESI+)precursor m/z 327.00

Plasma_Pos_LowMS_60MSMS_CE45_01 #184-190 RT: 1.15-1.27 AV: 7 NL: 2.99E3T: Composite spectrum MS8 664.00,607.32,551.33,495.34,439.26,383.17,327.06

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551.29

607.32495.31

327.08 439.26383.17

233.03359.24 631.35540.52153.09 251.07 480.52419.50

56 Da

56 Da

56 Da

56 Da

56 Da

MS^8 composite tandem mass spectrumof precursor m/z 664.00 (ESI+)

Sample: KH Liu (FiehnLab)

Plasma_Pos_LowMS_60MSMS_CE45_01.rawFriday, April 03, 2009

MS2 precursor 664.00|__MS3 precursor 607.32| |__MS4 precursor 551.33| | |__MS5 precursor 495.34| | | |__MS6 precursor 439.26| | | | |__MS7 precursor 383.17| | | | | |__MS8 precursor 327.06| | | | | | |__Composite spectrum MS8 664.00,607.32,551.33,495.34,439.26,383.17,327.06| | | | | | |__Single spectrum MS8 664.00,607.32,551.33,495.34,439.26,383.17,327.06 (190)| | | | | |__Composite spectrum MS7 664.00,607.32,551.33,495.34,439.26,383.17| | | | | |__Single spectrum MS7 664.00,607.32,551.33,495.34,439.26,383.17 (189)| | | | |__Composite spectrum MS6 664.00,607.32,551.33,495.34,439.26| | | | |__Single spectrum MS6 664.00,607.32,551.33,495.34,439.26 (188)| | | |__Composite spectrum MS5 664.00,607.32,551.33,495.34| | | |__Single spectrum MS5 664.00,607.32,551.33,495.34 (187)| | |__Composite spectrum MS4 664.00,607.32,551.33| | |__Single spectrum MS4 664.00,607.32,551.33 (186)| |__Composite spectrum MS3 664.00,607.32| |__Single spectrum MS3 664.00,607.32 (185)|__Single spectrum MS2 664.00 (184)

List of ions from precursor m/z 664.00 (ESI+)

List of product ions:m/z 664 , 607 ,551 ,495 ,439 ,383 ,327accurate masses see above

Solutions – well Plasma – well Lipids use LipidMaps tools!

Current mass accuracy for unit mass resolution LTQ: 78 ppm (external calibration) or 0.046 Da @ 594.46 Da for reference purposesLipidMaps Tools used with M+H

CE(18:2(Ep)) 18 2 663.5711 0.4289 C45H75O3+CE(18:2(Ke)) 18 2 663.5711 0.4289 C45H75O3+CE(18:3(OH)) 18 3 663.5711 0.4289 C45H75O3+CE(18:1(Ep))-cyclo 18 1 663.5711 0.4289 C45H75O3+CE(18:1(Ke))-cyclo 18 1 663.5711 0.4289 C45H75O3+CE(18:2(OH))-cyclo 18 2 663.5711 0.4289 C45H75O3+CE(16:2(Ke,Ep2)) 16 2 663.4983 0.5017 C43H67O5+CE(16:2(Ke2,Ep)) 16 2 663.4983 0.5017 C43H67O5+CE(16:3(Ep,OO)) 16 3 663.4983 0.5017 C43H67O5+CE(16:3(Ke,OO)) 16 3 663.4983 0.5017 C43H67O5+CE(16:3(OH,Ep2)) 16 3 663.4983 0.5017 C43H67O5+CE(16:3(OH,Ke,Ep)) 16 3 663.4983 0.5017 C43H67O5+CE(16:3(OH,Ke2)) 16 3 663.4983 0.5017 C43H67O5+CE(16:4(OOH,Ep)) 16 4 663.4983 0.5017 C43H67O5+CE(16:4(OOH,Ke)) 16 4 663.4983 0.5017 C43H67O5+CE(16:4(OH,OO)) 16 4 663.4983 0.5017 C43H67O5+CE(16:4(OH2,Ep)) 16 4 663.4983 0.5017 C43H67O5+CE(16:4(OH2,Ke)) 16 4 663.4983 0.5017 C43H67O5+CE(16:5(OH,OOH)) 16 5 663.4983 0.5017 C43H67O5+CE(16:5(OH3)) 16 5 663.4983 0.5017 C43H67O5+CE(16:1(Ke,Ep2))-cyclo 16 1 663.4983 0.5017 C43H67O5+CE(16:1(Ke2,Ep))-cyclo 16 1 663.4983 0.5017 C43H67O5+CE(16:2(Ep,OO))-cyclo 16 2 663.4983 0.5017 C43H67O5+CE(16:2(Ke,OO))-cyclo 16 2 663.4983 0.5017 C43H67O5+CE(16:2(OH,Ep2))-cyclo 16 2 663.4983 0.5017 C43H67O5+CE(16:2(OH,Ke,Ep))-cyclo 16 2 663.4983 0.5017 C43H67O5+CE(16:2(OH,Ke2))-cyclo 16 2 663.4983 0.5017 C43H67O5+CE(16:3(OOH,Ep))-cyclo 16 3 663.4983 0.5017 C43H67O5+CE(16:3(OOH,Ke))-cyclo 16 3 663.4983 0.5017 C43H67O5+CE(16:3(OH,OO))-cyclo 16 3 663.4983 0.5017 C43H67O5+CE(16:3(OH2,Ep))-cyclo 16 3 663.4983 0.5017 C43H67O5+CE(16:3(OH2,Ke))-cyclo 16 3 663.4983 0.5017 C43H67O5+CE(16:4(OH,OOH))-cyclo 16 4 663.4983 0.5017 C43H67O5+CE(16:4(OH3))-cyclo 16 4 663.4983 0.5017 C43H67O5+

Solutions – well Plasma – well Lipids!

CE(18:2(Ep)) 18 2 663.5711 0.4289 C45H75O3+CE(18:2(Ke)) 18 2 663.5711 0.4289 C45H75O3+CE(18:3(OH)) 18 3 663.5711 0.4289 C45H75O3+CE(18:1(Ep))-cyclo 18 1 663.5711 0.4289 C45H75O3+CE(18:1(Ke))-cyclo 18 1 663.5711 0.4289 C45H75O3+CE(18:2(OH))-cyclo 18 2 663.5711 0.4289 C45H75O3+

Sphingosine CerP 18:1 644.5013 0.5013 C36H71NO6P+Sphingosine HexCer 12:0 644.5096 0.5096 C36H70NO8+Sphinganine Cer 24:4 644.5976 0.5976 C42H78NO3+

Problem: Our mass accuracy is 0.046 Da but the solutions are 0.5 Da difference

Without Google Scholar we would be lost to search single m/z valuesor numbers. Still its a horrible way to do mass spectral library search.Has anybody a Google API example?

Solutions – well Plasma – well Google It!

On the lipid composition of human meibum and tears: Comparative analysis of nonpolar lipidsIgor A. Butovich http://www.iovs.org/cgi/content/abstract/iovs.08-1889v1

Researcher working with tears = The Dacriologist (Book by Djerassi)Dakryology, the neologism for the science of tears10.1007/BF00137944also CRYING: The Natural and Cultural History of Tears By Tom Lutz Norton

"However, rushing to this conclusion would have been a mistake because subsequent fragmentation of ion m/z 663 in sequential MSn experiments gave us the following chain of product ions differing by 56 amu: 663 (MS1) -> 607 (MS2) -> 551 (MS3) -> 495 (MS4) -> 439 (MS5) -> 383 (MS6) -> 327 (MS7) (not shown). Note that the first four ions (663, 607, 551, 495) were also easily detectable in a simple MS1 experiment (Fig. 11A) . These transformations were indicative of a sequential loss of several up to six) t-butyl groups and were formerly described for oxidized form of Irgafos 168 (C42H63O4P, Fig. 11A , inset) a polymer additive present in polyethylene and polypropylene and its derivatives."

Solutions – well Plasma – well Plasticizers!

Analysis of Additives in Polyethylene with DesorptionChemical Ionization/Tandem Mass Spectrometry

S. W. CHEN and G. R. HER*Appl. Spectrosc. 47, 844-851 (1993) http://www.opticsinfobase.org/abstract.cfm?URI=as-47-6-844

Also Oxidized Naugard 524 - http://dx.doi.org/10.1016/0003-2670(95)00183-Z

Picture Source: S. W. CHEN and G. R. HER Appl. Spectrosc. 47, 844-851 (1993)

Solution – probably Irganox / Irgafos contamination

Picture Source: Analysis of Additives in Polyethylene with DesorptionChemical Ionization/Tandem Mass SpectrometryS. W. CHEN and G. R. HER*Appl. Spectrosc. 47, 844-851 (1993) http://www.opticsinfobase.org/abstract.cfm?URI=as-47-6-844

Plasma_Pos_LowMS_60MSMS_CE45_01 #184-190 RT: 1.15-1.27 AV: 7 NL: 2.99E3T: Composite spectrum MS8 664.00,607.32,551.33,495.34,439.26,383.17,327.06

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Our MS^8"Lipid"

Problem solved, "lipid" is oxidized plasticizer.

Is the problem really solved?

CID 14572930

InChIKey: AZSKHRTUXHLAHS-UHFFFAOYSA-N

No, because for the formula = C42H63O4Pthe exact mass = 662.446397016.

The [M+H]+ would be 663.4542 Da and not 664.0

In this case an ultrazoom investigation orinfusion into FT-ICR-MS with resolving power50,000 to 100,000 would probably help.

MS/MS on isotopic peak, ion suppression of isobars, overlap with multiple ceramides or cholesterol and oxycholesterol species possible

Final question – How can we avoid rediscovery (reinventing the wheel)?

• sharing experiments (free)• sharing mass spectra (free or sell)• putting publications into OA for easier searching

How can we prevent plasticizer contaminations?

• running solvent blanks with each experiment• running methods blanks with each experiment• using only (baked) glassware

Other solutions?

• library search (but compound is not in NIST)• remember the awesome power of MS/MS/MS/MS/MS/MS/MS/MS• modern iontraps are currently not used to the full extend Data Dependent • exclusion libraries for unwanted "contaminations"

See: XLS supplement from Keller, B.O.; Sui, J.; Young, A.B.; Whittal, R.M. Interferences and contaminants encountered in modern mass spectrometry.