Using LC-MS/MS for Exhaled Breath Drug Testing and LC-HRMS...
Transcript of Using LC-MS/MS for Exhaled Breath Drug Testing and LC-HRMS...
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Olof Beck Karolinska Institutet Stockholm, Sweden
Using LC-MS/MS for exhaled breath drug
testing and LC-HRMS for drug screening
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Drug testing in exhaled breath
- A new possibility for toxicology?
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First publication 2010
Substances in breath can be detected after sampling on filter
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• 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
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Sampling device
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Extraction procedure
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Example chromatogram
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100%
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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)
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20 pg/filter
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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).
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Tramadol in breath after 50 mg oral intake
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LC-HRMS
For drug screening
application
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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
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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
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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
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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.
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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
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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)
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d:\tracefinderdata\...\data\urin_r70000 08/21/15 13:00:59neg amfRT: 0.00 - 6.00 SM: 9G
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NL: 8.34E9TIC F: FTMS + p ESI Full ms [100.00-650.00] MS urin_Amf_r70000
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NL: 6.59E9TIC F: FTMS + p ESI Full ms [100.00-650.00] MS Genesis urin_r70000
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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
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NL: 2.67E5Base Peak m/z= 136.11139-136.11275 F: FTMS + p ESI Full ms [100.00-650.00] MS Genesis urin_r70000
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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
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136.11222136.03994
RT: 1.00 - 6.00
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Z:\TraceFinderData\...\Data\Linj_250 02/11/15 00:18:08
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Resolution vs. Scanspeed (Full MS)
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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)
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Resolution Aquisition time 17500 64 ms 35000 128 ms 70000 256 ms 140000 512 ms
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Orbitrap_ULMVXT_201508110705_1 08/11/15 15:54:24 K_1000
RT: 2.60 - 3.60
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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
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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
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False negative results: Mass accuracy fail within a batch (Hardware problems)
orbitrap_ulmvxt_201501220653_1 01/22/15 14:31:31 1000
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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
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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
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False positive/negative results within a low concentration levels integration difficulties
Spike False positve findings (integration difficulties)
Pitfalls
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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
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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
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Method comparison
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LC-HRMS
LC-MS/MS
Neg Pos
Neg 57 2*
Pos 0 153
*O-desmethyltramadol, interference in LC-MS/MS qualifier
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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
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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
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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
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The End
Thank You!