Erfarenheter från att drogtesta med LC-HRMS. Olof Beck Klinisk farmakologi Karolinska Universitetssjukhuset Stockholm, Sweden

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1 Erfarenheter från att drogtesta med LC-HRMS Olof Beck Klinisk farmakologi Karolinska Universitetssjukhuset Stockholm, Sweden

2 Mass spectrometry For high throughput drug screening application 2

3 Analytisk teknikutveckling GC-MS/MS UPLC-MS/MS LC-MS/MS LC-MS EMIT, FPIA HPLC GC-MS LC-HRMS RIA TLC GC

4 Drogtestning Immunokemisk screening PROBLEM: För osäkert falska positiva resultat (1-15%) Begränsat sortiment Höga gränsvärden (tex. amfetamin) 4

5 24 Designer drugs - NPS 5

6 6 Enkel provberedning

7 Arbetet med NPS STRIDA-projektet LC-MS/MS 7

8 8 LC-TOFMS

9 9 Resultat (LC-TOFMS)

10 Selektivitet In conclusion, the potential of using LC-TOFMS as a complementary and alternative technology for screening in urine drug testing has been demonstrated. However, it should be pointed out that both sensitivity and mass resolution power still needs improvement. 1

11 Using LC-HRMS for urine drug screening Simple design in order to obtain a system to replace immunoassay screening Attraction of HRMS to collect universial data To apply HRSIM approach - XIC To obtain a more general screening method with improved sensitivity and accuracy 11

LC-HRMS Q Exactive Resolving power Max 14, @ m/z 2 (1.

Dynamic range > 5:1 Internal: < 1 ppm RMS External: < 5 ppm RMS Polarity switching Multiplexing

12 LC-HRMS Q Exactive Resolving power Max m/z 2 (1.5 scan/sec) Scan speed Max scan rate up to 12 Hz at resolution setting of m/z 2 Mass accuracy Dynamic range > 5:1 Internal: < 1 ppm RMS External: < 5 ppm RMS Polarity switching Multiplexing capability One full cycle in < 1 sec (one full scan positive mode and one full scan negative mode at resolution setting of 17,5) up to 1 precursors/scan

13 Resolution Resolution vs. scan speed Resolution (m/z 2) Max. scan speed (Hz) Scan speed (scan/s) Resolution Aquisition time ms ms ms ms

14 Spectral resolution (R) R = M/ΔM (5%) ΔM = Peak (FWHM Full width half maximum) Isotope A+3 för M3G: Internal standard M3G-d3: R = /.6 = ~ ΔM = A+3 of M3G Relative Abundance D3-M3G , R for m/z 2 NL: 1.75E6 Karolinska_Opiates14k_16#8-87 RT: AV: 8 SB: 2 4.1, 4.1 T: FTMS + p ESI Full lock ms [1.-5.] , R for m/z 2 NL: 4.8E6 karolinska_opiates_16# RT: AV: 1 SB: 2 4., 4. T: FTMS + p ESI Full lock ms [1.-5.] m/z , R for m/z 2 NL: 2.74E6 karolinska_opiates14k_16_ # RT: AV: 28 SB: 2 4., 4. T: FTMS + p ESI Full lock ms [1.-5.] m/z

15 Use of LC HRMS in full scan-xic mode for multi-component urine drug testing a step towards a black-box solution? 15

16 Method design Method: Postive ESI Full MS: Mass range 1-65 Da R=7 XIC (±1 ppm) Targeted MS/MS (1 analytes) R=175 Total analysis time: 6 min Lock mass Sample preparation: Dilution 1:5 with 9 internal standards Number of analytes: Plant alkaloids= 12 Therapeutic drugs= 16 NPS= 119 Total number: 147 analytes Reporting limit: Routine: 1 ng/ml Research: appr. 1 ng/ml

Detector response Exempel 1 RT 2.81 5 m/z 198.

17 Detector response Exempel 1 RT m/z Unknown Retention time (min)

18 Identification criteria 1. Mass accuracy within ± 2.5 ppm limit (using lock mass) 2. Absolute retentions time difference in the same batch compared to calibrators.3 min.

19 Selectivity at high mass resolution Relative Abundance d:\tracefinderdata\...\data\urin_r7 8/21/15 13::59 neg amf RT: SM: 9G Time (min) Relative Abundance NL: 8.34E9 TIC F: FTMS + p ESI Full ms [1.-65.] MS urin_amf_r7 RT: Time (min) NL: 6.59E9 TIC F: FTMS + p ESI Full ms [1.-65.] MS Genesis urin_r7 Relative Abundance Relative Abundance Relative Abundance RT: RT: Time (min) Time (min) RT: RT: 1.89 AA: Time (min) NL: 4.25E7 Base Peak m/z= F: FTMS + p ESI Full ms [1.-65.] MS urin_amf_r7 Relative Abundance NL: 8.15E7 Base Peak m/z= F: FTMS + p ESI Full ms [1.-65.] MS Genesis urin_r7 Relative Abundance NL: 1.71E6 Base Peak m/z= F: FTMS + p ESI Full ms [1.-65.] MS Genesis urin_r7 Relative Abundance RT: Time (min) urin_r7 #276 RT: 2.4 AV: 1 SB: , NL: 3.7E6 T: FTMS + p ESI Full lock ms [1.-65.] m/z urin_r7 #276 RT: 2.4 AV: 1 SB: , NL: 3.7E6 T: FTMS + p ESI Full lock ms [1.-65.] m/z NL: 2.67E5 Base Peak m/z= F: FTMS + p ESI Full ms [1.-65.] MS Genesis urin_r7

20 Escaline. XIC and PRM (time ) Z:\TraceFinderData\...\Data\Linj_25 2/11/15 :18:8 1 5 RT: 2.83 AA: NL: 1.67E7 Base Peak m/z= F: FTMS + p ESI Full ms [1.-65.] MS Genesis Linj_ Time (min) m/z R 32 to resolve m/z 1 5 RT: 2.83 AA: NL: 1.13E6 Base Peak m/z= F: FTMS + p ESI Full ms @hcd5. [5.-25.] MS Genesis Linj_ Time (min) m/z

21 Pitfalls EIC, Mass accuracy (R=7) Orbitrap_ULMVXT_ _1 8/11/15 15:54:24 K_1 RT: Harmaline: m/z= Extracted: ± 2 mda m/z: RT: 3.13 AA: NL: 3.2E8 Base Peak m/z= F: FTMS + p ESI Full ms [1.-65.] MS Genesis Orbitrap_ULMVXT_ _ Time (min) RT: Harmaline: m/z= Extracted: ±.1 mda m/z: RT: 3.14 MA: NL: 2.54E8 Base Peak m/z= F: FTMS + p ESI Full ms [1.-65.] MS Orbitrap_ULMVXT_ _ Time (min)

22 Pitfalls False negative results: Mass accuracy fail within a batch (Hardware problems) orbitrap_ulmvxt_ _1 1/22/15 14:31:31 1 RT: Mass accuracy for dextromethorphane is outside ±1 ppm False neg. sample NL: 7.8E4 Base Peak m/z= F: FTMS + p ESI Full ms [1.-65.] MS OM_Orbitrap_ULMVXT_ _ The same sample reinjected: Mass accuracy is within ±1 ppm RT: 3.74 AA: NL: 5.51E8 Base Peak m/z= F: FTMS + p ESI Full ms [1.-65.] MS ICIS orbitrap_ulmvxt_ _ Time (min)

23 Pitfalls False negative results: Integration fail (Software buggs) Psilocyn is not integrated in the sample view false negative But integrated in the method parameter settings

24 Pitfalls False positive/negative results within a low concentration levels integration difficulties False positve findings (integration difficulties) Spike

25 Selectivity and need for reporting limit Low concentrations? False positive? Reporting limit adjustment?? Sample is positive for desmethyltramadol. Background peaks with respons below limit for other analytes

26 QC-prover i drift Nominal concentration ng/ml Mean measured concentration CV% N Nominal concentration ng/ml Mean measured concentration CV% N DMT , , NBOMe , ,9 187 alfa-pvp , ,5 187 Atropine , ,4 397 Hydrocodone , ,8 397 Hydromorphone , ,6 397 Mitraphylline , ,2 397 Zolpidem-COOH , ,8 397

27 Method comparison LC-HRMS Neg Pos LC-MS/MS Neg 57 2* Pos 153 *O-desmethyltramadol, interference in LC-MS/MS qualifier 27

28 Erfarenhet Metoden används i rutin >18. prover analyserade Turbopumpen går ibland sönder 28

29 Felresultat 3371Prover = kromatogram Missade toppar vid integrering i 3 fall 3 Fall av 813 möjliga positiva fall =.3 % falskt negativa Numera korrigerat! I 48 fall hittade systemet en topp som var fel 48/18766 =.2% Men 48/3371 = 1.4% och 48/(81+48) = 5.6% Falska positiva sorterades bort med manuell granskning! 29

30 Nuvarande möjliga strategi LC-HRMS Negativt 3

31 Resultat Substance N Substance N Pregabalin 169 Metylon 6 Ritalinsyra 444 Metedron 5 Zopiklon 339 Hydrokodon 4 Gabapentin 38 3-MMC/4-MMC/6-APDB/2-MMC 3 Metylfenidat 271 Akrylfentanyl 3 Zolpidemsyra 17 Alfa-PVT 3 Dextrometorfan 67 Etylon 3 O-desmetyltramadol 39 MDPV 3 Fentanyl 36 Metiopropamin 3 Alfa-PVP 17 Mitragynin 3 Psilocin 15 4-CMC 2 Hydromorfon 12 4-MEC 2 Atropin 11 5-APDB/6-APDB 2 Efedrin 1 5-EAPB 2 MDPHP 1 5-MeO-NiPT 2 Etylfenidat 8 Alfa-PBP 2 Pyrovaleron 8 Bufedron/5-APDB/2-MMC/6-2 APDB/3-MMC/4-MMC Ketamin 6 Dihydrocodeine 2 Single findings (22): 1-(4-fluorofenyl)-2-(1-metyletylamino)pentan-1-on/Desoxy- D2PM 2-fluoroamfetamin/4-fluoroamfetamin /3-MeO-PCP/4-MeO-PCP /4-fluoro-alfa- PVP /4-Metylamfetamin /Alfa-PPP /Allyleskalin /Butylon /Meprobamat /Methoxetamine /Metkatinon /Metoxetamin /Metoxipiperamid /Metoxyfenidin /MPHP /Nafyron /N- Etylbufedron /para-fluoro-isobutyrylfentanyl /Pentylon /Tetrahydrofuran-fentanyl

32 Maintenance Clean: S-lens and ion source once/1-1.5 months (after 15 25samples) Mass calibration Every third day with use of internal mass calibration (lock mass) Clean: sweep cone and ion transfer tube once/1-2 week

33 Ekonomi 18 h per dygn tillgänglig analystid 6 h för underhåll 18 injektioner 15 okända prover 22 dgr per månad x prover per år Kostnad ~ 2 SEK/prov Jfr immunokemi 33

34 Final aspects We see a great future of LC-HRMS for drug screening Further development and validation of system for routine application in drug testing: Reliable and automated data evaluation Improved summary report forms Electronic transfer of results to LIMS 34

35 Det är möjligt att utmana immunokemiscreening med masspektrometri! 35

36 36

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