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

226.14@hcd50.00 [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