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?

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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

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Extraction procedure

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Example chromatogram

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100%

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100%

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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

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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

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

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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

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NL: 8.34E9TIC F: FTMS + p ESI Full ms [100.00-650.00] MS urin_Amf_r70000

RT: 2.30 - 2.50

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NL: 6.59E9TIC F: FTMS + p ESI Full ms [100.00-650.00] MS Genesis urin_r70000

RT: 0.00 - 6.00

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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

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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

RT: 1.00 - 6.00

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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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136.06183

136.02176

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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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)

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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

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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

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

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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

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

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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

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

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The End

Thank You!