Olof Beck Klinisk farmakologi Karolinska Universitetssjukhuset Stockholm, Sweden Erfarenheter från att drogtesta med LC-HRMS
Olof Beck
Klinisk farmakologi
Karolinska
Universitetssjukhuset
Stockholm, Sweden
Erfarenheter från att drogtesta
med LC-HRMS
Mass spectrometry
For high throughput drug
screening application
2
Analytisk teknikutveckling1960-2017
GC
RIA
GC-MS
HPLC
EMIT, FPIA
LC-MS
LC-MS/MS
UPLC-MS/MS
LC-HRMS
TLC
GC-MS/MS
Drogtestning
Immunokemisk screening
PROBLEM:
• För osäkert – falska positiva resultat (10-15%)
• Begränsat sortiment
• Höga gränsvärden (tex. amfetamin)
4
5
2004
Designer drugs - NPS
Enkel provberedning
6
Arbetet med NPS – STRIDA-projektet
7
LC-MS/MS
LC-TOFMS
8
Resultat (LC-TOFMS)
9
Selektivitet
10
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.
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
LC-HRMS Q Exactive™
Resolving power Max 140,000 @ m/z 200
(1.5 scan/sec)
Scan speed Max scan rate up to 12
Hz at resolution setting
of 17,500 @ m/z 200
Mass accuracy Internal: < 1 ppm RMS
External: < 5 ppm RMS
Dynamic range > 5000:1
Polarity switching One full cycle in < 1 sec
(one full scan positive
mode and one full scan
negative mode at
resolution setting of
17,500)
Multiplexing capability up to 10 precursors/scan
Resolution vs. scan speed
0
20000
40000
60000
80000
100000
120000
140000
160000
0 2 4 6 8 10 12 14
Resolution (m/z 200) Max. scan speed (Hz)
17.500 12
35.000 7
70.000 3
140.000 1.5
Scan speed (scan/s)
Resolu
tion
Resolution Aquisition time
17500 64 ms
35000 128 ms
70000 256 ms
140000 512 ms
Spectral resolution (R)
R = M/ΔM (50%)
Isotope A+3 för M3G: 465.1835
Internal standard M3G-d3: 465.1939
R = 465.1835/0.006 = ~ 80 000
465.10 465.12 465.14 465.16 465.18 465.20 465.22 465.24 465.26 465.28 465.30
m/z
0
20
40
60
80
100
0
20
40
60
80
100
Re
lativ
e A
bu
nd
an
ce
0
20
40
60
80
100465.1835
465.1939
465.1837
465.1962
465.1874
NL: 1.75E6
Karolinska_Opiates140k_16#80-87 RT: 0.76-0.82 AV: 8 SB: 2 4.01 , 4.01 T: FTMS + p ESI Full lock ms [100.00-500.00]
NL: 4.08E6
karolinska_opiates_16#165-174 RT: 0.73-0.77 AV: 10 SB: 2 4.00 , 4.00 T: FTMS + p ESI Full lock ms [100.00-500.00]
NL: 2.74E6
karolinska_opiates140k_16_111117133039#411-438 RT: 0.95-1.02 AV: 28 SB: 2 4.00 , 4.00 T: FTMS + p ESI Full lock ms [100.00-500.00]
A+3 of
M3G D3-M3G
140,000 R for m/z 200
70,000 R for m/z 200
35,000 R for m/z 200
ΔM = 0.006
465.10 465.12 465.14 465.16 465.18 465.20 465.22 465.24 465.26 465.28 465.30
m/z
0
20
40
60
80
100
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100465.1835
465.1939
465.1837
465.1962
465.1874
NL: 1.75E6
Karolinska_Opiates140k_16#80-87 RT: 0.76-0.82 AV: 8 SB: 2 4.01 , 4.01 T: FTMS + p ESI Full lock ms [100.00-500.00]
NL: 4.08E6
karolinska_opiates_16#165-174 RT: 0.73-0.77 AV: 10 SB: 2 4.00 , 4.00 T: FTMS + p ESI Full lock ms [100.00-500.00]
NL: 2.74E6
karolinska_opiates140k_16_111117133039#411-438 RT: 0.95-1.02 AV: 28 SB: 2 4.00 , 4.00 T: FTMS + p ESI Full lock ms [100.00-500.00]
ΔM = Peak width @50%
(FWHM – Full width half maximum)
15
Use of LC–HRMS in full scan-XIC mode for
multi-component urine drug testing – a step
towards a black-box solution?
Method designMethod:
Postive ESI Full MS: Mass range 100-650 Da
R=70000 XIC (±10 ppm)
Targeted MS/MS (10 analytes) R=17500
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: 100 ng/mL
Research: appr. 1 ng/mL
Exempel
1.0 2.0 3.0 4.0 5.0
Dete
cto
r re
sponse
Retention time (min)
m/z 198.0680
RT 2.81100
50
0
Unknown
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 ≤0.03 min.
d:\tracefinderdata\...\data\urin_r70000 08/21/15 13:00:59neg amf
RT: 0.00 - 6.00 SM: 9G
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
20
40
60
80
100
Rela
tive A
bundance
NL: 8.34E9
TIC F: FTMS + p ESI
Full ms
[100.00-650.00] MS
urin_Amf_r70000
RT: 2.30 - 2.50
2.30 2.32 2.34 2.36 2.38 2.40 2.42 2.44 2.46 2.48
Time (min)
0
20
40
60
80
100
Rela
tive A
bundance
NL: 6.59E9
TIC F: FTMS + p ESI
Full ms
[100.00-650.00] MS
Genesis urin_r70000
RT: 0.00 - 6.00
0 1 2 3 4 5 6
Time (min)
0
20
40
60
80
100
Rela
tive A
bundance
2.40
1.88 2.881.78 5.235.02 5.413.60 4.07 4.671.500.51
NL: 4.25E7
Base Peak m/z=
136.11139-136.11275 F:
FTMS + p ESI Full ms
[100.00-650.00] MS
urin_Amf_r70000
RT: 2.30 - 2.50
2.30 2.35 2.40 2.45 2.50
Time (min)
0
20
40
60
80
100
Rela
tive A
bundance
NL: 2.67E5
Base Peak m/z=
136.11139-136.11275 F:
FTMS + p ESI Full ms
[100.00-650.00] MS
Genesis urin_r70000
RT: 1.00 - 6.00
1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
20
40
60
80
100
Rela
tive A
bundance
NL: 8.15E7
Base Peak m/z=
135.61207-136.61207 F:
FTMS + p ESI Full ms
[100.00-650.00] MS
Genesis urin_r70000
urin_r70000 #276 RT: 2.40 AV: 1 SB: 484 0.50-4.30 , 4.60-5.00 NL: 3.07E6
T: FTMS + p ESI Full lock ms [100.00-650.00]
136.00 136.05 136.10 136.15
m/z
0
20
40
60
80
100
Rela
tive A
bundance
136.06183
136.02176
136.11222136.03994
RT: 1.00 - 6.00
1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
20
40
60
80
100
Rela
tive A
bundance
RT: 1.89
AA: 14972450
NL: 1.71E6
Base Peak m/z=
136.11139-136.11275 F:
FTMS + p ESI Full ms
[100.00-650.00] MS
Genesis urin_r70000
urin_r70000 #276 RT: 2.40 AV: 1 SB: 484 0.50-4.30 , 4.60-5.00 NL: 3.07E6
T: FTMS + p ESI Full lock ms [100.00-650.00]
136.00 136.05 136.10 136.15
m/z
0.0
0.2
0.4
0.6
0.8
1.0
Rela
tive A
bundance
136.03994
Selectivity at high mass resolution
Z:\TraceFinderData\...\Data\Linj_250 02/11/15 00:18:08
2.50 2.55 2.60 2.65 2.70 2.75 2.80 2.85 2.90 2.95 3.00
Time (min)
0
50
100
Rela
tive A
bundance
RT: 2.83
AA: 47861282
NL: 1.67E7
Base Peak m/z=
226.14264-226.14490 F: FTMS + p
ESI Full ms [100.00-650.00] MS
Genesis Linj_250
226.00 226.02 226.04 226.06 226.08 226.10 226.12 226.14 226.16 226.18 226.20 226.22 226.24
m/z
0
50
100
Rela
tive A
bundance
226.14372
226.10738
226.11882226.08392
226.00 226.02 226.04 226.06 226.08 226.10 226.12 226.14 226.16 226.18 226.20 226.22 226.24
m/z
0
50
100
Rela
tive A
bundance
226.14386
226.10764226.05087
2.50 2.55 2.60 2.65 2.70 2.75 2.80 2.85 2.90 2.95 3.00
Time (min)
0
50
100
Rela
tive A
bundance
RT: 2.83
AA: 2554363
NL: 1.13E6
Base Peak m/z= 181.08429-181.08791
F: FTMS + p ESI Full ms2
[email protected] [50.00-250.00] MS
Genesis Linj_250
50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250
m/z
0
50
100
Rela
tive A
bundance
181.0859886.09702
84.0449993.07043 121.06510
68.05027
130.05002 166.0625769.07062153.13868114.09167103.0546870.06588
79.0549195.04967
182.08932 228.06863138.06688 162.09134105.0703267.05500 180.07828 195.0877258.06596
147.04402
209.12850 234.93935 249.52481219.90190
Escaline. XIC and PRM (time 2.6 – 2.9)
R 320 000 to resolve
Orbitrap_ULMVXT_201508110705_1 08/11/15 15:54:24 K_1000
RT: 2.60 - 3.60
2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
RT: 3.13AA: 823311996
NL: 3.02E8
Base Peak m/z= 215.11589-215.11989 F: FTMS + p ESI Full ms [100.00-650.00] MS Genesis Orbitrap_ULMVXT_201508110705_1
RT: 2.60 - 3.60
2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
RT: 3.14MA: 288007774
NL: 2.54E8
Base Peak m/z= 215.11779-215.11799 F: FTMS + p ESI Full ms [100.00-650.00] MS Orbitrap_ULMVXT_201508110705_1
Harmaline: m/z=215.11789
Extracted: ± 2 mDa
m/z: 215.11589 – 215.11989
Harmaline: m/z=215.11789
Extracted: ± 0.1 mDa
m/z: 215.11779 – 215.11799
EIC, Mass accuracy (R=70000)
Pitfalls
False negative results: Mass accuracy fail within a batch
(Hardware problems)
orbitrap_ulmvxt_201501220653_1 01/22/15 14:31:31 1000
RT: 0.00 - 6.01
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
RT: 3.74
AA: 1790144980
NL: 7.80E4
Base Peak m/z=
272.19817-272.20361 F:
FTMS + p ESI Full ms
[100.00-650.00] MS
OM_Orbitrap_ULMVXT_20150
3251443_1
NL: 5.51E8
Base Peak m/z=
272.19817-272.20361 F:
FTMS + p ESI Full ms
[100.00-650.00] MS ICIS
orbitrap_ulmvxt_20150122065
3_1
Mass accuracy for dextromethorphane is outside ±10 ppm
False neg. sample
The same sample reinjected:
Mass accuracy is within ±10 ppm
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
SpikeFalse positve findings (integration difficulties)
Pitfalls
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 measuredconcentration CV% N
Nominal
concentration
ng/mL
Mean measured
concentration CV% N
DMT 50 49 10,5 139 150 154 6,9 187
251-NBOMe 50 44 8,7 139 150 141 6,9 187
alfa-PVP 50 51 6,6 139 150 151 5,5 187
Atropine 50 48 8,1 334 150 153 7,4 397
Hydrocodone 50 49 6,0 334 150 154 5,8 397
Hydromorphone 50 49 7,4 334 150 160 7,6 397
Mitraphylline 50 46 7,8 334 150 145 7,2 397
Zolpidem-COOH 50 45 6,5 334 150 145 4,8 397
Method comparison
27
LC-HRMS
LC-MS/MS
Neg Pos
Neg 57 2*
Pos 0 153
*O-desmethyltramadol, interference in LC-MS/MS qualifier
Erfarenhet
• Metoden används i rutin
• >18.000 prover analyserade
• Turbopumpen går ibland sönder
28
Felresultat
3371Prover = 187 606 kromatogram
Missade toppar vid integrering i 3 fall
3 Fall av 813 möjliga positiva fall = 0.3 % falskt negativa
Numera korrigerat!
I 48 fall hittade systemet en topp som var fel 48/187606 = 0.02%
Men 48/3371 = 1.4% och 48/(810+48) = 5.6%
Falska positiva sorterades bort med manuell granskning!
29
Nuvarande möjliga strategi
30
LC-HRMS
Negativt
ResultatSubstance N Substance N
Pregabalin 1609 Metylon 6Ritalinsyra 444 Metedron 5Zopiklon 339 Hydrokodon 4
Gabapentin 308 3-MMC/4-MMC/6-APDB/2-MMC 3Metylfenidat 271 Akrylfentanyl 3Zolpidemsyra 107 Alfa-PVT 3
Dextrometorfan 67 Etylon 3
O-desmetyltramadol 39 MDPV 3Fentanyl 36 Metiopropamin 3Alfa-PVP 17 Mitragynin 3
Psilocin 15 4-CMC 2Hydromorfon 12 4-MEC 2Atropin 11 5-APDB/6-APDB 2
Efedrin 10 5-EAPB 2
MDPHP 10 5-MeO-NiPT 2
Etylfenidat 8 Alfa-PBP 2
Pyrovaleron 8 Bufedron/5-APDB/2-MMC/6-APDB/3-MMC/4-MMC
2
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 1500 – 2500samples)
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
• 180 injektioner – 150 okända prover
• 22 dgr per månad x 12 → 40.000 prover per år
• Kostnad ~ 20 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 immunokemi-
screening med masspektrometri!
35
36