FUTURE CONFIRMATORY CRITERIA

Transcription

1 FUTURE CONFIRMATORY CRITERIA Revision CD 2002/657 Marco Blokland, Bjorn Berendsen, Hans Mol, Leen van Ginkel, Saskia Sterk

2 REVISION 2

3 Revision 2002/657 EU Commission has explicitly asked for a revision of 2002/657 Status of new document: not yet decided on EURLs in the area are tasked with making a proposal State of the art criteria for confirmatory analysis Should stand for a long time 3

4 EXAMPLE: Ambient Ionization-MS Ambient ionization methods permit direct ionization of samples outside the mass spectrometer in the open atmosphere There are >70 different types of AIMS described Minimal sample preparation No chromatography 4

5 What would this mean for new criteria? Since no chromatography is involved it will break the retention time criteria Without the use of retention time the number of false positives will increase?? Solution 1: The most selective MS measurement should be used in confirmatory analysis using AIMS Solution 2: Inclusion of another selective orthogonal technique 5

6 Ion Ratio 6

7 LC-MS/MS relative ion intensity In 2002/657 MS/MS criteria are based on expert opinions, at that time no scientific data available Experimental data is now available Relative intensities Allowed Dev. [rel] >50 % ± 20 % >20 % to 50 % ± 25 % >10 % to 20 % ± 30 % <10 % ± 50% Relative Intensity Berendsen et al: Drug Testing and Analysis, 2016, 8,

8 LC-MS/MS relative ion intensity The data shows deviations depend on the intensity of the less abundant ion From: Berendsen et al: Drug Testing and Analysis 2016, 8,

9 LC-MS/MS relative ion intensity The S/N of the lowest product ion is very important in obtaining stable ion ratio s Maximum allowed deviation a compromise between the false positive and false negative rate New guideline proposed ± 40 % (relative) for all relative ion intensity In case the precursor selection has a mass selection window of more than one Dalton (e.g. in case of Data Independent Acquisition) the technique is considered as full-scan confirmatory analysis. 9

10 Retention Time 10

11 Revision: Retention times Almost all current guidelines have a relative requirement (e.g. 2.5%) for the maximum deviation of the Rt between sample and standard Using gradient elution, empirical studies (Mol et al. Berendsen et al.) show deviation is absolute over the whole retention time range 11

12 Revision: Retention times From: Mol et al: Analytica Chimica Acta, 2015, 873, 1-13 From: Berendsen et al: Drug Testing and Analysis 2016, 8,

13 Fast chromatography: one-minute gradiënt 1 minute Oven: 60 C Typical run times seconds on 2-5 mm ID column Flow ml/min 13

14 Revision: Retention times All mass spectrometric analyses are combined with a sufficiently orthogonal separation (e.g. chromatography) or any separation technique that shows enough separation power for the specific application Possible to use of an absolute or relative criterion ± 0.1 minute for LC and GC In case retention time is below 1 minute then 5%, allowing the use of fast chromatography At least twice the retention time corresponding to the void volume of the column If an internal standard is used the relative retention time of the analyte, shall correspond to that of the calibration standard at a tolerance of ± 0.5 % for GC and ± 1 % for LC 14

15 Separation in the first dimension 1 n c 15

16 Coupling of second separation dimension 1 n c 16

17 Fully coupling of two separation techniques When truly orthogonal n c,2d = 1 n c x 2 n c 1 n c 17

18 Example added value of LC as orthogonal technique Kaabia, Z., Laparre, J., Cesbron, N., Le Bizec, B., & Dervilly-Pinel, G. (2018). Comprehensive steroid profiling by liquid chromatography coupled to high resolution mass spectrometry. The Journal of Steroid Biochemistry and Molecular Biology, 183,

19 Ion Mobility 19

20 Ion Mobility Mass spectrometry Ions separated by mass-to-charge ratio Ion mobility Ions separated by size, shape, and charge Size and charge: ions driven through gas filled chamber using electric field Shape: compact ions travel faster, less hindered by drift gas 20

21 Ion Mobility Drift times related to collisional cross sections (CCS) 21 Drift time

22 Collisional Cross Sections (CCS) CCS = Effective area of interaction between individual ion and neutral gas molecule Related to chemical structure and dimensions Can be obtained from experimental ion mobility data, and by molecular modelling Not all types of ion mobility yield CCS data Different protonation sites can yield different CCS values Boschmans et al., Analyst, 2016, 141,

23 Drift tube ion mobility (DT-IMS) Types of ion mobility High field asymmetric IMS (FAIMS) Differential Mobility Spectrometry (DIMS) Traveling wave ion mobility (TWIM) Trapped ion mobility (TIMS) López et al., New J. Chem., 2013, 37, Silveira et al., Analytical Chemistry, 2014, 86,

24 Drift tube ion mobility (DT-IMS) Types of ion mobility Agilent 6560 High field asymmetric IMS (FAIMS) Differential Mobility Spectrometry (DIMS) AB Sciex SelexIon Traveling wave ion mobility (TWIM) Waters Synapt Trapped ion mobility (TIMS) Bruker timstof López et al., New J. Chem., 2013, 37, Silveira et al., Analytical Chemistry, 2014, 86,

25 Separation of isomers: Diethylstilbestrol cis-des DTIMS trans-des Observed CCS difference ~ 5% TWIMS 25

26 Experimental determination of CCS value CCS can be calculated from drift time using Mason-Schamp equation: Ω / z = charge state of the analyteion e= charge of an electron N= number density of the drift gas µ= reduced mass of the ion-neutral pair k B = Boltzmann constant T = gas temperature K 0 = reduced mobility; mobility measured at standard temperature and pressure Only valid for drift tube instruments TWIMS and TIMS instruments need to be calibrated CCS calculation is not possible from DIMS data 26

27 CCS repeatability using TWIMS CCS of eight compounds determined on 7 different days using TWIMS instrument Compound Detected ion m/z Average Experimental CCS (Å 2, n=7) Cimaterol [M+H] % Clenbuterol [M+H] % Isoxsuprine [M+H] % THC [M+H] % RR-p-chloramphenicol [M-H] % THC-COOH [M-H] % Salmeterol [M+H] % Okadaicacid [M-H] % Repeatability is < 1% for all compounds, with the exception of RR-p-CAP (1.1%) RSD No significant effect of matrix observed Literature: <2%, inter-laboratory <5% Article in preparation 27

28 CCS as extra identification point? + Highly reproducible, no effect of matrix + Possibility to compare with literature or database + Reduction of matrix - Large deviation between DTIMS and TWIMS data found for some compounds -High correlation between CCS and m/z, which reduces uniqueness of CCS value Drift time (bins) β-agonists R² = m/z 28

29 Orthogonality of ion mobility Hernández-Mesa, M., Le Bizec, B., Monteau, F., García- Campaña, A. M., & Dervilly- Pinel, G. (2018). Collision Cross Section (CCS) database: An additional measure to characterize steroids. Analytical chemistry, 90(7), % error bars Only [M+H] + 29

30 Evaluation future Ion Mobility 1 cycle DTX-2 Okadaic acid 2 cycle 3 cycle 4 cycle 5 cycles 30

31 Serpentine UltralongPath with Extended Routing (SUPER) High Resolution Traveling Wave Ion Mobility-MS using Structures for Lossless Ion Manipulations (SLIM) 1 pass 13.5 m R= ms peak capacity > pass R=340 31

32 Examples of SLIM 32

33 Ion-mobility MS Cross-section additional confidence in confirmatory analysis Future alternative for chromatography? Criteria proposed: Cross-section is within 2 percent of a reference standard 0.5 identification point Not (yet) in compliance with orthogonality criteria 33

34 High Resolution Mass Spectrometry 34

35 Definition full scan hrms Current Definition of hrms: 657/2002 Resolution > 10,000 10% valley FDA FVM guidance Resolution > 10,000 FWHM SANTE/11945 not defined 35

36 full scan hrms Define resolving power, mass accuracy or both? Resolving power: Sante/11945: use enough resolving power to achieve a mass accuracy <5 ppm Mass accuracy: 657/2002: no mass accuracy requirement SANTE/11945: 5 ppm (< m/z mda) FDA FVM: MS: 5 ppm MS/MS: 10 ppm 36

37 Proposal: full scan hrms 2002/657 revision In high-resolution mass spectrometry (HRMS), the resolution shall typically be greater than 10,000 for the entire mass range at 10 % valley or 20,000 at FHWM. Only diagnostic ions with a relative intensity of more than 10 % in the reference spectrum of the calibration standard or MMS are suitable For confirmation of identity the mass deviation of all diagnostic ions should be below 5 ppm (or in case M/z <200 below 1mDa). Adducts and isotopes of selected diagnostic ions are excluded. In full scan mode, the isotopic pattern should match the molecular formula. 37

38 Suggestions for identification points Technique IPs earned Chromatography (GC or LC) 1 Ion mobility 0.5 LR-MS ion 1 Precursor ion selection at <±0.5 Da mass range 1 (indirect) LR-MS n product ion 1.5 HR-MS ion 1.5 HR-MS n product ion 2.5 Non MS techniques 1 38

39 Examples Technique(s) Separation Number of ions IP GC-MS (EI or CI) GC n GC-MS (EI and CI) GC 2 (EI) + 2 (CI) GC-MS (EI or CI) 2 derivates GC 2 (Derivate A) + 2 (Derivate B) LC-MS LC n (MS) GC-or LC-MS/MS GC or LC 1 precursor + 2 products GC-or LC-MS/MS GC or LC 2 precursor + 2 products GC-or LC-MS 3 GC or LC 1 precursor + 1 MS 2 product + 1 MS 3 product GC-or LC-HRMS GC or LC n IM-HRMS IM 3 GC-or LC-HRMS/MS GC or LC 1 precursor (<±0.5 Da mass range) + 2 products GC-or LC-HRMS and HRMS/MS GC or LC 1 full scan ion + 1 HRMS product ion a GC-and LC-MS GC and LC 2 ions (GCMS) + 1 ion (LCMS) 39

40 Examples Technique(s) Separation Number of ions IP GC-MS (EI or CI) GC n 1+n GC-MS (EI and CI) GC 2 (EI) + 2 (CI) 1+4 GC-MS (EI or CI) 2 derivates GC 2 (Derivate A) + 2 (Derivate B) 1+4 LC-MS LC n (MS) 1+n GC-or LC-MS/MS GC or LC 1 precursor + 2 products *1.5 = 5 GC-or LC-MS/MS GC or LC 2 precursor + 2 products *1.5 = 6 GC-or LC-MS 3 GC or LC 1 precursor + 1 MS 2 product + 1 MS 3 product = 5 GC-or LC-HRMS GC or LC n 1 + n*1.5 IM-HRMS IM *1.5 = 5 GC-or LC-HRMS/MS GC or LC 1 precursor (<±0.5 Da mass range) + 2 products *2.5 = 7 GC-or LC-HRMS and HRMS/MS GC or LC 1 full scan ion + 1 HRMS product ion a = 5 GC-and LC-MS GC and LC 2 ions (GCMS) + 1 ion (LCMS) = 5 40

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