Erfarenheter från att drogtesta med LC-HRMS Olof Beck Klinisk farmakologi Karolinska Universitetssjukhuset Stockholm, Sweden
Mass spectrometry For high throughput drug screening application 2
Analytisk teknikutveckling 196-217 GC-MS/MS UPLC-MS/MS LC-MS/MS LC-MS EMIT, FPIA HPLC GC-MS LC-HRMS RIA TLC GC
Drogtestning Immunokemisk screening PROBLEM: För osäkert falska positiva resultat (1-15%) Begränsat sortiment Höga gränsvärden (tex. amfetamin) 4
24 Designer drugs - NPS 5
6 Enkel provberedning
Arbetet med NPS STRIDA-projektet LC-MS/MS 7
8 LC-TOFMS
9 Resultat (LC-TOFMS)
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
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 (1.5 scan/sec) Scan speed Max scan rate up to 12 Hz at resolution setting of 17,5 @ 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
Resolution Resolution vs. scan speed 16 14 12 1 8 Resolution (m/z 2) Max. scan speed (Hz) 17.5 12 35. 7 7. 3 14. 1.5 6 4 2 2 4 6 8 1 12 14 Scan speed (scan/s) Resolution Aquisition time 175 64 ms 35 128 ms 7 256 ms 14 512 ms
Spectral resolution (R) R = M/ΔM (5%) ΔM = Peak width @5% (FWHM Full width half maximum) Isotope A+3 för M3G: 465.1835 Internal standard M3G-d3: 465.1939 R = 465.1835/.6 = ~ 8 1 8 6 4 ΔM =.6 465.1835 465.1939 2 1 465.1837 1 8 6 A+3 of M3G 465.1835 465.1939 Relative Abundance D3-M3G 8 6 4 2 14, R for m/z 2 NL: 1.75E6 Karolinska_Opiates14k_16#8-87 RT: 465.1962.76-.82 AV: 8 SB: 2 4.1, 4.1 T: FTMS + p ESI Full lock ms [1.-5.] 4 1 465.1874 2 8 1 8 6 4 465.1837 465.1962 6 4 2 7, R for m/z 2 NL: 4.8E6 karolinska_opiates_16#165-174 RT:.73-.77 AV: 1 SB: 2 4., 4. T: FTMS + p ESI Full lock ms [1.-5.] 465.1 465.12 465.14 465.16 465.18 465.2 465.22 465.24 465.26 465.28 465 m/z 2 1 8 6 465.1874 35, R for m/z 2 NL: 2.74E6 karolinska_opiates14k_16_11111 713339#411-438 RT:.95-1.2 AV: 28 SB: 2 4., 4. T: FTMS + p ESI Full lock ms [1.-5.] 4 2 465.1 465.12 465.14 465.16 465.18 465.2 465.22 465.24 465.26 465.28 465.3 m/z
Use of LC HRMS in full scan-xic mode for multi-component urine drug testing a step towards a black-box solution? 15
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 198.68 Unknown 1. 2. 3. 4. 5. Retention time (min)
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.
Selectivity at high mass resolution Relative Abundance d:\tracefinderdata\...\data\urin_r7 8/21/15 13::59 neg amf RT:. - 6. SM: 9G 1 8 6 4 2..5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. Time (min) Relative Abundance NL: 8.34E9 TIC F: FTMS + p ESI Full ms [1.-65.] MS urin_amf_r7 RT: 2.3-2.5 1 8 6 4 2 2.3 2.32 2.34 2.36 2.38 2.4 2.42 2.44 2.46 2.48 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:. - 6. 1 8 6 4 2 RT: 1. - 6. 1 8 6 4 2.51 2.4 1.5 1.78 1.88 2.88 3.6 4.7 4.67 5.2 5.23 5.41 1 2 3 4 5 6 Time (min) 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. Time (min) RT: 1. - 6. 1 8 6 4 2 RT: 1.89 AA: 1497245 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. Time (min) NL: 4.25E7 Base Peak m/z= 136.11139-136.11275 F: FTMS + p ESI Full ms [1.-65.] MS urin_amf_r7 Relative Abundance NL: 8.15E7 Base Peak m/z= 135.6127-136.6127 F: FTMS + p ESI Full ms [1.-65.] MS Genesis urin_r7 Relative Abundance NL: 1.71E6 Base Peak m/z= 136.11139-136.11275 F: FTMS + p ESI Full ms [1.-65.] MS Genesis urin_r7 Relative Abundance RT: 2.3-2.5 1 8 6 4 2 2.3 2.35 2.4 2.45 2.5 Time (min) urin_r7 #276 RT: 2.4 AV: 1 SB: 484.5-4.3, 4.6-5. NL: 3.7E6 T: FTMS + p ESI Full lock ms [1.-65.] 136.6183 1 8 6 4 136.2176 2 136.3994 136.11222 136. 136.5 136.1 136.15 m/z urin_r7 #276 RT: 2.4 AV: 1 SB: 484.5-4.3, 4.6-5. NL: 3.7E6 T: FTMS + p ESI Full lock ms [1.-65.] 1..8.6.4.2 136.3994. 136. 136.5 136.1 136.15 m/z NL: 2.67E5 Base Peak m/z= 136.11139-136.11275 F: FTMS + p ESI Full ms [1.-65.] MS Genesis urin_r7
Escaline. XIC and PRM (time 2.6 2.9) Z:\TraceFinderData\...\Data\Linj_25 2/11/15 :18:8 1 5 RT: 2.83 AA: 47861282 NL: 1.67E7 Base Peak m/z= 226.14264-226.1449 F: FTMS + p ESI Full ms [1.-65.] MS Genesis Linj_25 2.5 2.55 2.6 2.65 2.7 2.75 2.8 2.85 2.9 2.95 3. Time (min) 1 226.14372 5 226.1738 226.8392 226.11882 226. 226.2 226.4 226.6 226.8 226.1 226.12 226.14 226.16 226.18 226.2 226.22 226.24 m/z 1 5 226.14386 R 32 to resolve 226.587 226.1764 226. 226.2 226.4 226.6 226.8 226.1 226.12 226.14 226.16 226.18 226.2 226.22 226.24 m/z 1 5 RT: 2.83 AA: 2554363 NL: 1.13E6 Base Peak m/z= 181.8429-181.8791 F: FTMS + p ESI Full ms2 226.14@hcd5. [5.-25.] MS Genesis Linj_25 2.5 2.55 2.6 2.65 2.7 2.75 2.8 2.85 2.9 2.95 3. Time (min) 1 5 86.972 181.8598 84.4499 68.527 93.743 121.651 79.5491 69.762 95.4967 13.52 166.6257 114.9167 147.442 153.13868 7.6588 13.5468 15.732 138.6688 162.9134 182.8932 58.6596 67.55 18.7828 195.8772 228.6863 29.1285 219.919 234.93935 249.52481 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 m/z
Pitfalls EIC, Mass accuracy (R=7) Orbitrap_ULMVXT_21581175_1 8/11/15 15:54:24 K_1 RT: 2.6-3.6 1 9 8 7 6 Harmaline: m/z=215.11789 Extracted: ± 2 mda m/z: 215.11589 215.11989 RT: 3.13 AA: 823311996 NL: 3.2E8 Base Peak m/z= 215.11589-215.11989 F: FTMS + p ESI Full ms [1.-65.] MS Genesis Orbitrap_ULMVXT_215811 75_1 5 4 3 2 1 2.6 2.7 2.8 2.9 3. 3.1 3.2 3.3 3.4 3.5 3.6 Time (min) RT: 2.6-3.6 1 9 8 7 Harmaline: m/z=215.11789 Extracted: ±.1 mda m/z: 215.11779 215.11799 RT: 3.14 MA: 2887774 NL: 2.54E8 Base Peak m/z= 215.11779-215.11799 F: FTMS + p ESI Full ms [1.-65.] MS Orbitrap_ULMVXT_21581 175_1 6 5 4 3 2 1 2.6 2.7 2.8 2.9 3. 3.1 3.2 3.3 3.4 3.5 3.6 Time (min)
Pitfalls False negative results: Mass accuracy fail within a batch (Hardware problems) orbitrap_ulmvxt_215122653_1 1/22/15 14:31:31 1 RT:. - 6.1 1 8 6 4 Mass accuracy for dextromethorphane is outside ±1 ppm False neg. sample NL: 7.8E4 Base Peak m/z= 272.19817-272.2361 F: FTMS + p ESI Full ms [1.-65.] MS OM_Orbitrap_ULMVXT_215 3251443_1 2 1 8 6 The same sample reinjected: Mass accuracy is within ±1 ppm RT: 3.74 AA: 17914498 NL: 5.51E8 Base Peak m/z= 272.19817-272.2361 F: FTMS + p ESI Full ms [1.-65.] MS ICIS orbitrap_ulmvxt_21512265 3_1 4 2..5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. Time (min)
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
Pitfalls False positive/negative results within a low concentration levels integration difficulties False positve findings (integration difficulties) Spike
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
QC-prover i drift Nominal concentration ng/ml Mean measured concentration CV% N Nominal concentration ng/ml Mean measured concentration CV% N DMT 5 49 1,5 139 15 154 6,9 187 251-NBOMe 5 44 8,7 139 15 141 6,9 187 alfa-pvp 5 51 6,6 139 15 151 5,5 187 Atropine 5 48 8,1 334 15 153 7,4 397 Hydrocodone 5 49 6, 334 15 154 5,8 397 Hydromorphone 5 49 7,4 334 15 16 7,6 397 Mitraphylline 5 46 7,8 334 15 145 7,2 397 Zolpidem-COOH 5 45 6,5 334 15 145 4,8 397
Method comparison LC-HRMS Neg Pos LC-MS/MS Neg 57 2* Pos 153 *O-desmethyltramadol, interference in LC-MS/MS qualifier 27
Erfarenhet Metoden används i rutin >18. prover analyserade Turbopumpen går ibland sönder 28
Felresultat 3371Prover = 187 66 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
Nuvarande möjliga strategi LC-HRMS Negativt 3
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
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
Ekonomi 18 h per dygn tillgänglig analystid 6 h för underhåll 18 injektioner 15 okända prover 22 dgr per månad x 12 4. prover per år Kostnad ~ 2 SEK/prov Jfr immunokemi 33
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
Det är möjligt att utmana immunokemiscreening med masspektrometri! 35
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