LC/QTOF Discovery of Previously Unreported Microcystins in Alberta Lake Waters

Transcription

1 LC/QTOF Discovery of Previously Unreported Microcystins in Alberta Lake Waters Ralph Hindle Vogon Laboratory Services Ltd. Cochrane, Alberta, Canada Xu Zhang David W. Kinniburgh Alberta Centre for Toxicology 1

2 Vogon Laboratory Services Ltd. Calgary, Alberta, Canada

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4 Vogon Laboratory Services Ltd. Calgary, Alberta, Canada

5 Vogon Laboratory Services Ltd. Cochrane, Alberta, Canada

6 Kananaskis Provincial Park

7 Confirmation Request 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)

8 General Microcystin Structure Over 70 microcystins have been identified in the literature Microcystin Provided by ACFT X Z Formula Neutral Mass LR Leucine Arginine C 49 H 74 N 10 O Desmethyl LR Leucine Arginine C 48 H 72 N 10 O RR Arginine Arginine C 49 H 75 N 13 O YR Tyrosine Arginine C 52 H 72 N 10 O LW Leucine Phenylalanine C 54 H 72 N 8 O LF Leucine Tryptophan C 52 H 71 N 7 O HtyR Homotyrosine Arginine C 53 H 74 N 10 O

9 Microcystin-LR Structure (Most common microcystin reported) C 49 H 74 N 10 O 12 Mass = Glu Mdha Adda O CH 3 CH 3 H 3 C O HO HN NH C O O H N O CH 3 N O NH O CH 2 H 3 C CH 3 HN H N C HO O O O CH 3 CH 3 Ala Leucine (L) HN NH MeAsp Arginine (R) NH 2

10 QQQ MRM Parameters a Microcystin Precursor b (m/z) Product Ion (m/z) LR Desmethyl-LR RR YR LW LF HtyR Collision Energy (V) 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. 10

11 Common Microcystin Fragments m/z = and Glu + Mdha = C 9 H 13 N 2 O 4 + m/z = C 9 H 11 O + m/z =

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13 LC-QTOF Instrumentation HPLC Mass Spectrometer 1290 Binary Pump 6540B QTOF 1200 Degasser Jet Stream Electrospray Source 1290 Autosampler 1290 TCC 13

14 Transforming Data Acquire Data LC/MS Find Compounds Compare Compounds Identify Compounds TOF MS Q TOF MS/MS QQQ MS/MS Single Q MS Trap MS n Find Compounds MFE Find Compounds LMFE (Proteins) Find Compounds Auto MS/MS Compare compounds between samples Metabolite ID Mass Profiler Compare compounds between 2 sample sets Mass Profiler Mass Profiler Professional Protein DB Search Spectrum Mill Mascot Others (via mzxml, mzdata) Accurate Mass and RT (AMRT) Database Search Endogenous Metabolites (METLIN) Food, Forensics, Environmental Find Compounds Targeted MS/MS Find Compounds By Formula Compare compounds between >2 sample sets Mass Profiler Professional Molecular Formula Generation (MFG) Via accurate mass MS and MS/MS MS/MS Structural Correlation (MSC) via accurate mass Future Tools Fully or Partially Automated 14

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16 Find By Formula Parameters 16

17 Level 3 Calibrator (10 ng/ml) 17

18 Time of Flight MS Spectra

19 Database Score Monoisotopic mass (varies in ppm) Isotope distribution (varies in %) Isotope spacing (varies in ppm) Score based on: Mass match measured versus given. Abundance score abundance pattern of measured isotope cluster compared with values predicted from the proposed formula. Spacing match how the m/z spacing between the lowest m/z ion and the A+1 and A+2 ions compared with the values predicted from the proposed formula.

20 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 Pass 4.62 YR Pass 4.68 LR Pass 4.80 RR Pass 4.87 dm LR Pass 5.27 LW Pass 5.40 LF Pass Scores range from 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)

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22 Compounds-at-a-Glance (20 ul) 0.1 ng/ml 1 ng/ml 10 ng/ml HtyR YR LR RR

23 Calibration Curves

24 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!

25 Water Sample Results Lake A HtyR YR LR RR Cal 1 Cal 2 Cal 3 Lake A Lake B

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27 Lake A YR Investigation in Qual The raw spectrum shows that the unknown contains a distinct A ion at This is 0.5 ppm mass error and score of 99.4 for YR formula: C 52 H 72 N 10 O 13 Because RT is wrong, suspect structural isomer Run Targeted MS/MS on each sample (next slide)

28 YR vs Unknown Compound: MS/MS Notable ions missing from Lake A compound: Notable ions present in Lake A compound: Conclusion: Unknown peak is not YR

29 Zoom in on common MC fragments = C 9 H 11 O + 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 O CH 3 H 3 C CH = C 10 H 15 + HO C O HN O NH Z O CH 3 O N O CH 2 H 3 C CH 3 HN C HO O NH X O

30 Zoom in on common MC fragments Glu + Mdha = C 9 H 13 N 2 O 4 + m/z = De methylation can also occur on Mdha group ion is very low in Lake A unknown Indicates a de methylated microcystin

31 Zoom in on common MC fragments Predict: C 8 H 11 N 2 O 4+ fragment ion from [Mdha CH 2 ] de methylation Result: Found in Unknown, not in YR Des methyl HtyR has same formula as YR, and so is a strong candidate If > is monitored as qualifier ion by QQQ, ratio will be low compared to YR standard

32 Additional unknowns detected Lake A MS only PCD of 52 microcystins (in house) Mass Tolerance: 5 ppm Min Score: 70 H + adduct required

33 Additional unknowns detected Lake B MS only PCDL of 52 microcystins (in house) Mass Tolerance: 5 ppm Min Score: 70 H + adduct required

34 1. All calibrators were detected at 0.1 ng/ml on 6540 QTOF 2. Calibration curves were linear (R 2 > 0.998) 3. Microcystins confirmed: 1. Lake A: dm-lr = 15.8 ng/ml 2. Lake B: LR = 0.4 ng/ml 4. Identify unknown peak in lake waters: I. Lake Water A II. Summary i. HtyR tentatively identified, corresponding to nominal mass of YR ii. 7 additional MCs were tentatively identified Lake Water B i. suspected LY confirmed to be different compound ii. 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

35 Acknowledgements ACFT sample prep staff