LC/QTOF Discovery of Previously Unreported Microcystins in

Alberta Lake Waters

Ralph HindleVogon Laboratory Services Ltd.

Cochrane, Alberta, Canada

Xu ZhangDavid W. Kinniburgh

Alberta Centre for Toxicology

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Vogon Laboratory Services Ltd.Calgary, Alberta, Canada

Vogon Laboratory Services Ltd.Calgary, Alberta, Canada

Vogon Laboratory Services Ltd.Cochrane, Alberta, Canada

Kananaskis Provincial Park

1. Identify and quantitate targeted microcystins (7 compounds) in lake water

2. Identify unknown peak in lake waters:a) Lake Water A (corresponding to nominal mass of YR)b) Lake Water B (corresponding to nominal mass of LY)

Confirmation Request

General Microcystin StructureOver 70 microcystins have been identified in the literature

MicrocystinProvided by ACFT

X Z Formula Neutral Mass

LR Leucine Arginine C49H74N10O12 994.5488

Desmethyl LR Leucine Arginine C48H72N10O12 980.5331

RR Arginine Arginine C49H75N13O12 1037.5658

YR Tyrosine Arginine C52H72N10O13 1044.5280

LW Leucine Phenylalanine C54H72N8O12 1024.5270

LF Leucine Tryptophan C52H71N7O12 985.5161

HtyR Homotyrosine Arginine C53H74N10O13 1058.5437

OCH3

CH3

NH

H3CO

HN

COHO

NNH

O CH2

OCH3

H3CHN

O

OHN

O

CH3

CH3

CH3HN

OCHO O

NH

NH2

HN

Microcystin-LR Structure(Most common microcystin reported)

C49H74N10O12Mass = 994.5488

Arginine (R)

Leucine (L)

MeAsp

Ala

MdhaGlu

Adda

QQQ MRM Parametersa

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a These patterns are useful, as they show that all supplied MCs fragment to the same 2 diagnostic ions

b RR readily shows a doubly‐charged precursors. Depending on source conditions, other MCs can show either singly‐ or doubly‐charged ions.

Microcystin Precursor b(m/z)

Product Ion(m/z)

Collision Energy(V)

LR 995.6 135.2213.2

8080

Desmethyl-LR 981.5 135.2213.2

8080

RR 520.0 135.2213.2

3040

YR 1045.5 135.2213.2

8070

LW 1025.5 135.2213.2

8060

LF 986.5 135.2213.2

7050

HtyR 1059.5 135.2213.2

8070

Common Microcystin Fragmentsm/z = 135.2 and 213.2

C9H11O+

m/z = 135.0804

Glu + Mdha = C9H13N2O4+

m/z = 213.0870

LC-QTOF Instrumentation

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HPLC Mass Spectrometer1290 Binary Pump 6540B QTOF1200 Degasser Jet Stream Electrospray Source1290 Autosampler1290 TCC

TOF MSQ‐TOF MS/MSQQQ MS/MSSingle Q MSTrap MSn

Acquire DataLC/MS

Transforming Data

Fully or Partially AutomatedFully or Partially Automated

Find CompoundsMFE

Find Compounds

Find CompoundsAuto MS/MS

Find Compounds LMFE (Proteins)

Find CompoundsTargeted MS/MS

Find CompoundsBy Formula

Compare Compounds

Compare compoundsbetween samples

• Metabolite ID• Mass Profiler

Compare compoundsbetween 2 sample sets

• Mass Profiler• Mass Profiler Professional

Compare compoundsbetween >2 sample sets

• Mass Profiler Professional

Identify Compounds

Molecular Formula Generation(MFG)

• Via accurate mass MS andMS/MS

MS/MS Structural Correlation (MSC) via accurate mass 

• Future Tools

Accurate Mass and RT(AMRT) Database Search• Endogenous Metabolites (METLIN)• Food, Forensics, Environmental

Protein DB Search• Spectrum Mill• Mascot• Others (via mzXML, mzData)

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16

Find By Formula Parameters

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Level 3 Calibrator (10 ng/mL)

Time of Flight MS Spectra

Database Score

Monoisotopic mass(varies in ppm)

Isotope spacing(varies in ppm)

Isotope distribution(varies in %) Score based on:

• Mass match – measured versus given.

• Abundance score – abundance pattern of measured isotope cluster compared withvalues predicted from the proposed formula.

• Spacing match – how the m/z spacingbetween the lowest m/z ionand the A+1 and A+2 ions compared withthe values predicted from the proposed formula.

Score Evaluation for Find by Formula

RT  Name Mass  m/z  Score Diff from Tgt (ppm)  Ions  Flags (Tgt)

Flag Severity (Tgt) 

4.60 HtyR 1058.5429 1059.5505 98.4 ‐0.7 14 Pass

4.62  YR 1044.5275 1045.5347  99.3 ‐0.5 18 Pass

4.68 LR 994.5487  995.5560  99.2 ‐0.1 15 Pass

4.80 RR 1037.5654  519.7900  99.2 ‐0.4  16 Pass

4.87 dm‐LR 980.5332  981.5404  99.7 ‐0.1 13 Pass

5.27 LW 1024.5265  1025.5340  99.3 ‐0.5 8 Pass

5.40 LF 985.5159  986.5232  99.8 ‐0.2 12 Pass

• Scores range from 98.4 – 99.8• Mass error all < 1 ppm; up to 18 ions found to help confirm molecular formula• MassHunter table includes Flags and Severity ratings to help interpret results (no flags 

identified in this data)

Compounds-at-a-Glance (20 uL)

HtyR

YR

LR

RR

0.1 ng/mL 1 ng/mL 10 ng/mL

Calibration Curves

Calibration Accuracies

Creating a Quant method is easy:• Review the data in Qual• Export a Compound Exchange Format (CEF) file• Select “New Method from File” in Quant• Add calibration levels• That’s it!

Water Sample Results – Lake A

Cal 1

Cal 2

Cal 3

Lake A

Lake B

HtyR YR LR RR

Lake A – YR Investigation in Qual

• The raw spectrum shows that the unknown contains a distinct A ion at 1045.5342• This is ‐0.5 ppm mass error and score of 99.4 for YR formula: C52H72N10O13• Because RT is wrong, suspect structural isomer• Run Targeted MS/MS on each sample (next slide)

YR vs Unknown Compound: MS/MS

• Notable ions missing from Lake A compound:• 127.0862• 155.0803• 157.1094• 213.0864

• Notable ions present in Lake A compound:• 150.0904• 361.1730

• Conclusion: Unknown peak is not YR

Zoom in on common MC fragments - 135

135.0804 = C9H11O+

• De‐methylation is common in microcystins• Two 135 ions are present in both YR and in 

unknown compound in Lake A, from 2 different fragments

• Indicates that Adda group is intact and not de‐methylated

OCH3

CH3

NH

H3CO

HN

COHO

NNH

O CH2

OCH3

H3C

X

O

Z

O

CH3HN

CHO O135.1168 = C10H15

+

Zoom in on common MC fragments - 213

• De‐methylation can also occur on Mdha group

• 213.0870 ion is very low in Lake A unknown

• Indicates a de‐methylated microcystin

Glu + Mdha = C9H13N2O4+

m/z = 213.0870

Zoom in on common MC fragments - 199• Predict: C8H11N2O4

+ fragment ion from [Mdha – CH2] de‐methylation• Result: Found in Unknown, not in YR• Des‐methyl HtyR has same formula as YR, and so is a strong 

candidate• If 1045.5 ‐> 213.1 is monitored as qualifier ion by QQQ, ratio will be 

low compared to YR standard

Additional unknowns detected – Lake A

• MS‐only• PCD of 52 microcystins (in‐house)• Mass Tolerance: 5 ppm• Min Score: 70• H+ adduct required

Additional unknowns detected – Lake B

• MS‐only• PCDL of 52 microcystins (in‐house)• Mass Tolerance: 5 ppm• Min Score: 70• H+ adduct required

1. All calibrators were detected at 0.1 ng/mL on 6540 QTOF2. Calibration curves were linear (R2 > 0.998)3. Microcystins confirmed:

1. Lake A: dm-LR = 15.8 ng/mL2. Lake B: LR = 0.4 ng/mL

4. Identify unknown peak in lake waters:I. Lake Water A

i. HtyR tentatively identified, corresponding to nominal mass of YRii. 7 additional MCs were tentatively identified

II. Lake Water Bi. suspected LY confirmed to be different compoundii. 7 additional MCs were tentatively identified

5. In-house PCDL of 52 compounds was created from WHO list and used to tentatively identify previously unreported microcystins

Summary

Acknowledgements

• ACFT sample prep staff