Upload
others
View
5
Download
0
Embed Size (px)
Citation preview
Olof Beck Karolinska Institutet Stockholm, Sweden
Using LC-MS/MS for exhaled breath drug
testing and LC-HRMS for drug screening
Drug testing in exhaled breath
- A new possibility for toxicology?
2
First publication 2010
Substances in breath can be detected after sampling on filter
• Amphetamine • Methamphetamine • MDMA • Methadone • EDDP • THC • Buprenorphine • Cocaine • Benzoyl ecgonine • Hydromorphone • Tramadol • O-desmethyltramadol • 6-Acetylmorphine • Morphine • Codeine
• Diazepam • Oxazepam • Alprazolam • Zolpidem • Nicotine • Cotinine • Methylphenidate • Ritalinic acid • MDPV • Budesonide • Ceftazidime • Piperacillin • Meropenem • Phosholipids • PEth
Detected substances in breath
Sampling device
5
Extraction procedure
6
Example chromatogram
0.0 0.5 1.0 1.5 2.0 2.5 3.0
50%
100%
50%
100%
50%
100%
50%
100%
Breath RT: 1.05
Benzoylecgonine
m/z 290.1/105.0
m/z 290.1/168.1
Cocaine m/z 304.2/105.1
m/z 304.2/182.2
RT: 2.60
Time (min)
Inte
nsity
In
tens
ity
Inte
nsity
In
tens
ity 56 pg/filter
20 pg/filter
Substance Breath (n=2295)
Oral fluid (n=18095)
Urine (n=35142)
Amphetamine/methamphetamine 0.17 0.11 0.27
Cannabis 0.09 0.34 1.27
Cocaine/benzoylecgonine 0.09 0.03 0.05
Opiates 0.01 0.24 1.10
Benzodiazepines 0.13 0.01 1.55
∑ 0.52 0.73 3.55
Analytical findings in routine WDT application of exhaled breath drug testing as compared with oral fluid and urine during the same time period (15 months).
Tramadol in breath after 50 mg oral intake
LC-HRMS
For drug screening
application
10
11
Using LC-HRMS for urine drug screening Presented at poster 106
• Simple design in order to obtain a system to replace immunoassay screening • Attraction of HRMS to collect universial data
• To apply HRSIM - XIC
• More general screening method with improved sensitivity and accuracy
LC-MS Thermo Scientific™ 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
Method design Method: Postive ESI Full MS: Mass range 100-650 Da R=70000 Targeted MS/MS (Escaline): 2.6-3.0 R=35000 Total analysis time: 6 min ”Lock” mass Sample preparation: Dilution 1:5 with internal standards Number of analytes: Plant alkaloids and drugs = 20 (Validated and Analyzed since 2014.11) NPS= 122 (Validated) Total number: 142 analytes Reporting limit: Routine: 100 ng/mL Research: appr. 5 ng/mL
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.
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
465.10 465.12 465.14 465.16 465.18 465.20 465.22 465.24 465.26 465.28 465.30m/z
0
20
40
60
80
1000
20
40
60
80
100
Rela
tive
Abun
danc
e
0
20
40
60
80
100465.1835
465.1939
465.1837
465.1962
465.1874
NL: 1.75E6Karolinska_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: 4.08E6karolinska_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: 2.74E6karolinska_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
465.1835
465.1939
465 1837
ΔM = Peak width @50%
(FWHM – Full width half maximum)
d:\tracefinderdata\...\data\urin_r70000 08/21/15 13:00:59neg amfRT: 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 6.0Time (min)
0
20
40
60
80
100
Rela
tive
Abun
danc
e
NL: 8.34E9TIC 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 2.48Time (min)
0
20
40
60
80
100
Rela
tive
Abun
danc
e
NL: 6.59E9TIC 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 6Time (min)
0
20
40
60
80
100
Rela
tive
Abun
danc
e
2.40
1.88 2.881.78 5.235.02 5.413.60 4.07 4.671.500.51
NL: 4.25E7Base 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 2.50Time (min)
0
20
40
60
80
100
Rela
tive
Abun
danc
e
NL: 2.67E5Base 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 6.0Time (min)
0
20
40
60
80
100
Rela
tive
Abun
danc
e
NL: 8.15E7Base 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: 3.07E6T: FTMS + p ESI Full lock ms [100.00-650.00]
136.00 136.05 136.10 136.15m/z
0
20
40
60
80
100
Rela
tive
Abun
danc
e
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 6.0Time (min)
0
20
40
60
80
100
Rela
tive
Abun
danc
e
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 3.00Time (min)
0
50
100
Rela
tive
Abun
danc
e
Resolution vs. Scanspeed (Full MS)
0
20000
40000
60000
80000
100000
120000
140000
160000
0 2 4 6 8 10 12 14
Q-Exactive
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)
Res
olut
ion
Resolution Aquisition time 17500 64 ms 35000 128 ms 70000 256 ms 140000 512 ms
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 3.6Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abun
danc
e
RT: 3.13AA: 823311996
NL: 3.02E8Base 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 3.6Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rela
tive
Abun
danc
e
RT: 3.14MA: 288007774
NL: 2.54E8Base 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 6.0Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
Relat
ive A
bund
ance
RT: 3.74AA: 1790144980
NL: 7.80E4Base Peak m/z= 272.19817-272.20361 F: FTMS + p ESI Full ms [100.00-650.00] MS OM_Orbitrap_ULMVXT_201503251443_1
NL: 5.51E8Base Peak m/z= 272.19817-272.20361 F: FTMS + p ESI Full ms [100.00-650.00] MS ICIS orbitrap_ulmvxt_201501220653_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
Spike False 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, 50 ng/mL (Values during one month period – April 2015)
46,1 47,7 1,6 4,3 3,5 9,0 35 35
Method comparison
25
LC-HRMS
LC-MS/MS
Neg Pos
Neg 57 2*
Pos 0 153
*O-desmethyltramadol, interference in LC-MS/MS qualifier
Results from routine application
Plant alkaloids and drugs (20 compounds) Start: Nov-2014 (10 månader): Number of samples: ~ 500 samples/month = 5000 Positive rate ~ 17% Pregabaline ~ 12% Dextromethorphane ~ 3% Ketamine ~ 2% Hydromorphone Hydrocodone Mitragynine Pethidine Dihydrocodeine Psilocin Atropine
Maintenance
Clean: S-lens and ion source once/1-1.5 months (after 700 – 1500 samples)
Mass calibration Every second day
Clean: sweep cone and Ion transfer tube once/1-2 week
Final aspects
We see a great potential of LC-HRMS
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
28
The End
Thank You!