UPLC-HRMS: A tool for multi-residue veterinary drug methods

Transcription

1 AOAC, Paris, November 23-24, 2009 UPLC-HRMS: A tool for multi-residue veterinary drug methods Anton Kaufmann Official Food Control Authority of the Canton of Zurich (Kantonales Labor Zürich)

2 The challenge Multi-residue and Multi-class 100 veterinary drugs in different matrices Japan positive list: 600 pesticides Non-target Pesticides in infant formula (mycotoxins, preservatives)

3 The answer was LC-MS/MS LC-MS/MS became the standard tool for trace analysis in complex matrices because of its high selectivity and sensitivity However, Each analyte has to be carefully tuned. Transitions can not be calculated. The instrument is completely blind for untuned compounds (even if they are present in very high concentrations).

4 Swiss alpine scenery as seen by an MS/MS (MRM) P. del Corvo P. Uomo Schepadüi Piz Roduc Pécianett P. del Sole Val Priora

5 Discovering the unexpected:? (full scan HRMS data)

6 Limitation of LC-MS/MS Monitoring dozens of MRMs reduces sensitivity. Managing these MRMs can become a difficult task..

7 Single MRM (one analyte) Analyte: A Retention time

8 Multiple MRM (several analytes) Analyte: A B C D E F Dwell time versus number of data points across a peak Retention time

9 MRM windows (about hundred analytes) Analyte: A B C D E F Retention time

10 Scheduled MRM (one thousend analytes) Analyte: A B C D E F G H I J Retention time

11 Can we or our software manage all these transitions? An analyte might produce more than one peak Some analytes might produce the same MRM A ph sensitive analyte might move out of a window A labile analyte might have degraded in the standard solution.

12 How did this device evolve? Full Scan Data from HRMS Single MRM Still more individual disks? multiple MRM Rather unlimited download from the web

13 Residue analysis by LC-MS Ayers Rock type: Chocolate hill type: thewith many matrix related peaks are possibly a clear analyte peak a lot of baseline covering the analyte peak

14 Is HRMS an alternative? Providing similar selectivity and sensitivity? Rugged and user friendly? Affordable to a wide range of laboratories?

15 HR-Techniques Fourier Transformation Ion Cyclotron Time of Flight Orbitrap

16 Schematic of the Orbitrap

17 Characteristics of the Orbitrap (Exactive) Resolution FWHM with 10 Hz FWHM with 1 Hz High mass accuracy and stability Good sensitivity Ease of use

18 Importance of Resolution High resolution permits narrow mass windows Narrow mass windows produce high selectivity

19 RT: Medium Resolution FWHM Liver extract spiked with marbofloxacin (TIC) NL: 3.88E8 TIC MS 09_10_08_ 79 Relative Abundance Time (min) Liver extract spiked with marbofloxacin (m/z = 363 window: 500 md ) RT: Relative Abundance Time (min) NL: 1.51E6 m/z= MS 09_10_08_ 79

20 Liver extract spiked with marbofloxacin (m/z = 363 window: 500 md) RT: Relative Abundance Time (min) RT: Relative Abundance Medium Resolution FWHM Time (min) NL: 1.51E6 m/z= MS 09_10_08_ 79 Liver extract spiked with marbofloxacin (m/z = window: 50 md) NL: 1.51E6 m/z= MS 09_10_08_ 79

21 Medium Resolution FWHM Liver extract spiked with marbofloxacin (m/z = window: 50 md) RT: Relative Abundance Time (min) NL: 1.51E6 m/z= MS 09_10_08_ 79 Liver extract spiked with marbofloxacin (m/z = window: 2 md) RT: Relative Abundance NL: 8.21E Time (min) m/z= MS 09_10_08_ 79

22 Medium Resolution FWHM Liver extrakt spiked with norfloxacin (m/z = window: 2 md ) RT: Relative Abundance RT: RT: Time (min) Time (min) Relative Abundance Relative Abundance NL: 9.95E4 m/z= MS 09_10_08_ 79 NL: 9.95E4 m/z= MS 09_10_08_ 79 NL: 5.30E4 m/z= MS 09_10_08_ Time (min)

23 Medium Resolution FWHM Norfloxacin spectrum (m/z = ) 09_10_08_79 #4124 #4125 #4126 #4127 #4128 #4129 #4130 #4131 RT: AV: 1 NL: 4.64E4 4.84E4 3.66E4 4.27E4 4.90E4 3.58E4 3.41E4 4.43E4 T: FTMS {0,0} + p ESI Full ms [ ] > 2 md Relative Abundance m/z.

24 Isobaric interferences Dom (Analyte) Täschhorn (Matrix Compound)? m/z

25 High Resolution FWHM = Liver with norfloxacine m/z = mda RT: Relative Abundance NL: 3.02E4 m/z= MS 09_10_08_ Time (min) RT: Relative Abundance NL: 3.02E4 m/z= MS 09_10_08_ Time (min)

26 Comparison of Spectra 09_10_08_28 # RT: AV: 4 NL: 1.75E4 T: FTMS {0,0} + p ESI Full ms [ ] Norfloxacin Relative Abundance FWHM = Matrix _10_08_ # RT: 6.39 AV: NL: 4.43E T: FTMS {0,0} + p ESI Full ms [ ] m/z Quad peak width Relative Abundance FWHM = m/z

27 How to compare the selectivity between an MS/MS and HRMS? MS/MS Two dimensional HRMS One dimensional

28 (MS/MS): Two dimensions How empty is it out there?

29 (HRMS): One dimension How wide are these gaps?

30 The approach Producing extracts from blank matrices Separating the extracts by UPLC Monitoring dummy transitions (MS/MS) dummy exact masses (HRMS) Make observed peak areas comparable

31 Testing new fishing grounds Another matrix related compound!

32 Evaluating the catch Evaluating Undesired peak areas (matrix) versus Desired peak areas (analyte)

33 Dummy transitions (MS/MS) 550 >=18 Dalton m/z (product ion) Reihe m/z (precursor ion) Some of these transitions two dimensions are probably impossible (not existing elemental composition for neutral loss)

34 Dummy exact masses (HRMS) mass defect Reihe m/z nominal mass one dimension Only a relatively narrow mass defect range was tested

35 Comparing apples and oranges Select 6 drugs (tetracyclins, chinolons and sulfonamides) Determine their response peak area / concentration Calculate average response for these 6 drugs Divide each dummy peak area by the average response Do this for MS/MS and HRMS dummy peaks

36 Effect of resolution elution region of analytes conc FWHM = 10' FWHM = 25' FWHM = 50'000 rt Honey matrix FWHM = 100'000

37 Effect of resolution conc FWHM = 10' FWHM = 25' FWHM = 50'000 rt Honey matrix FWHM = 100'000

38 Effect of matrix 100 Complexity of matrix Orbitrap: FWHM = 10' conc fish honey kidney rt liver

39 HRMS versus MS/MS conc FWHM = 10' FWHM = 50'000 rt FWHM = 25' FWHM = 100'000 conc Fish (easy matrix) rt

40 HRMS versus MS/MS conc FWHM = 10' FWHM = 25'000 conc FWHM = 50'000 rt FWHM = 100' rt Liver (difficult matrix)

41 Selectivity crossover point HRMS One dimensional MS/MS Two dimensional Crossover point: HR-MS reaches MS/MS selectivity at a resolution of FWHM (corresponds to 2 data-points per second)

42 MS/MS 5 µg/kg dimetridazole in Honey? Standard 142 > > / / 1.19 Sample Delta Retention time = 0.08 min (UPLC) MRM ratio match

43 HRMS No dimetridazole in honey Standard 5 µg/kg? Sample Sample + spike 5 µg/kg

44 This is not yet the end of the flagpole!

45 Information at your fingertips A virtual unlimited number of compound can be monitored without sacrificing sensitivity Full scan data can be accesses again and again. There is no need for an apriori hypothesis We find a sulfonamide. Is there also trimethoprim? There is chloramphenicol. We can prove that this is not a contamination of the sample by showing the presence of the metabolite

46 No reference compound available Some utilized drugs or pesticides are not commercially available as reference substances Designer drugs are never commercially available We might not use the 600 analyte pesticide standard for screening purposes but only one analyte per compound family

47 Further confirmation potential Exact mass: The accuracy of the exact measurement is well below the mass extraction window. Confirmation and determination of elemental composition

48 Further confirmation potential calculated isotopic pattern of chloramphenicol glucuronide measured isotopic pattern of chloramphenicol glucuronide

49 The generic detector Penicillines Amoxicillin H 2 N H N H S CH 3 (variable) O O N H CH 3 OH O (generic) H S CH 3 C 6 H 10 NO 2 S [M+H] + = H H N + O CH 3 OH

50 Quantification Ronidazol Y = *X R^2 = W: 1/X Ronidazol 0 to 10 µg/l Area

51 Unification of screening, quantification and confirmation Weak fragmentation conditions produce product ions Monitoring the precursor and product ion at a resolution of FWHM produces 4 identification points according to Commission Decision 2002/657/EC

52 What are the limitations? Limitations of software! Capacity of Trap Discrimination free interface and transfer optic Discrimination free extraction clean-up procedures required Residue control plans which do not demand multi-residue methods Reluctance of analysts to leave established trails

53 Conclusion HRMS is within the reach for residue control laboratories HRMS can compete with MS/MS (Selectivity and Sensitivity) HRMS breaks exisiting MS/MS limitations.

54 MS/MS (single analyte: MRM) He will observe the fish under his hole

55 MS/MS (multi residue: MRM s) They will observe the fishes beneath many holes

56 HRMS (multi residue: Full scan) She observes the whole picture at a glance