Abstract

A study was undertaken to demonstrate the use of Liquid Chromatography-Quadrupole Time of Flight (LC-QTOF) Mass Spectrometry to screen urine and serum samples for compounds covering a variety of drug classes relevant to post-mortem and routine forensic drug screening. To assess the robustness of this methodology, three independent laboratories used identical hardware configurations to screen for 61 compounds in spiked and authentic forensic case samples. False positive detections were practically eliminated by applying enhanced confirmation criteria based on the concept of ‘diagnostic’ qualifier ions, resulting in a sensitive and robust toxicological screening method with a low detection threshold.

Forensics: The need for certainty

In addition to the common drugs of abuse, such as cocaine and cannabis, new psychoactive substances, (also known as ‘designer drugs’ or ‘legal highs’) are constantly emerging on the international recreational drug scene. The UN Office on Drugs and Crime (UNODC) reported that 348 new ‘legal highs’ emerged worldwide from 2009 to 2013.[1]

In order to confidently identify the increasing number of substances present in samples, forensic toxicologists require methods that can provide rapid screening over a wide range of compound classes.

Authors

Petra Decker, Tony Drury, Carsten Baessmann, Bruker Daltonik GmbH, Bremen, Germany1

Jürgen Kempf, Laura M Huppertz, Volker AuwärterInstitute of Forensic Medicine, Department of Forensic Toxicology, Medical Center - University Freiburg, Germany2

Anna Pelander, Mira Sundstroem, Ilkka OjanperaDepartment of Forensic Medicine, University of Helsinki, Finland3

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Application Note LCMS-106

Efficacy of Enhanced Confirmation Criteria to Reduce False Positive Detections in Forensic Screening Using LC-QTOF Mass Spectrometry

Keywords Instrumentation and Software

Forensic Bruker impact QTOF

Screening Ultimate 3000 Rapid Separations LC

Accurate mass

Toxicology

Diagnostic ion

False positive

For research use only. Not for use in diagnostic procedures.

Moreover, eliminating false positives and false negatives is a key priority, so as to ensure the completeness and accuracy of data such that it can stand up to scrutiny in legal proceedings.

High resolution LC-QTOF mass spectrometry has been successfully used in forensic toxicological screening for several years.[2] Inherent characteristics of accurate mass make it an ideal tool for this sector, as it provides sensitive wide scope screening with retrospective and general unknown analysis capabilities. In this application note, a recently developed method using LC-QTOF mass spectrometry was tested for its suitability for routine forensic toxicology screening.

The approach described herein incorporates tools for enhanced results confirmation using the ´diagnostic ion´ concept. The diagnostic ion concept was tested in urine and serum with spiked and authentic forensic samples to assess the efficiency of removing false positive findings whilst maintaining a low detection threshold.

Methods

To illustrate the reliability of the screening setup and methods identical hardware configurations were set up to screen urine and serum samples at Bruker Daltonics, Bremen and the forensic laboratories located in 3Helsinki and 2Freiburg. Sixty one target compounds were selected for this study as shown in Table 1. These compounds included a variety of compound classes and were selected based on their practical relevance in post-mortem and routine drug screening and their varied physiochemical properties, including exact mass, retention time and fragmentation energy.

Instrumentation

UPLC: Ultimate 3000 Rapid Separations LC Column: Acclaim RSLC C18 2.1 x 100 mm, 2.2 µm Temperature: 30°CMobile phase: A = H2O/MeOH 90:10, B = MeOH (both 5 mM NH4 formate / 0.01% HCOOH)Gradient: Multistep gradient 5 – 99.9% in 14 minFlow rate: Flow gradient 0.2 - 0.48 mL/minInjection: 1 µLMS: Bruker impact QTOF mass spectrometerScan mode: Full scan TOF MS with broadband Collision Induced Dissociation (bbCID)*Scan range: m/z 30 – 1000.Ionization: ESI +ve 2,500V

*Broadband Collision Induced Dissociation (bbCID)

Broadband Collision Induced Dissociation (bbCID) is a data acquisition process where both TOF MS full scan data and MS/MS fragments are continuously generated in two independent, rapidly alternating data channels, (cycle time 1Hz) without either precursor ion or threshold selection.Channel 1 TOF MS (low collision energy): Precursor ions are not isolated or fragmented, producing full scan TOF MS spectra across every LC peak.

Channel 2 broadband CID (high collision energy): Again, there is no precursor ion isolation but in this instance all ions are fragmented in the collision cell resulting in the generation of bbCID MS/MS spectra across every LC peak.Since the two data channels are continuously acquired across all chromatographic peaks, there is little chance of missing important spectral information in complex matrices. Sample Analysis

After protein precipitation with acetonitrile, urine and serum samples were spiked with the compound mixes (10/11 compounds per mix) at four levels (10, 50, 100 and 500 ng/ml) and analyzed on the impact QTOF system in Bremen in TOF-MS and bbCID modes. For automated processing the intensity threshold for compound detection was set to allow detection on all relevant traces in a 10 ng/mL sample in solvent. The total numbers of findings in all samples were collected and compared to the number of expected findings. For the runs acquired in bbCID mode, additional enhanced detection confirmation criteria were applied. In this instance, compounds were only considered as detected when at least one diagnostic ion with RT difference <0.05 min of the main compound ion was detected in either the full scan (TOF-MS) or bbCID channel.

The same processing method and detection criteria were applied on data acquired from authentic samples from routine cases. These included 11 urine samples from Helsinki autopsy cases and 8 urine / serum samples from

6-Monoacetylmorphine (MAM), 7-Aminoflunitrazepam, 9-Hydroxyrisperidone (Paliperidone), Alprazolam, Amiodarone, Amisulpride, Amitriptyline, Amphetamine, Benzoylecgonine, Bromazepam, Buprenorphine, Citalopram, Cocaethylene, Cocaine, Codeine, Caffeine, Cotinine, Diazepam, Dihydrocodeine, Diphenhydramine, Doxepin, Ecgonine methyl ester, EDDP, Fentanyl, Flunitrazepam, Lamotrigine, MDA, MDMA (Ecstasy), Methadone, Methamphetamine, Midazolam, Mirtazapine, Morphine, Norbuprenorphine, Norcitalopram, Nordazepam, Nordoxepin, Norfentanyl, Nortilidine, Nortrimipramine, Nortriptyline, O-Desmethyltramadol, O-Desmethylvenlafaxine, Olanzapine, Oxazepam, Oxycodone, Paracetamol (Acetaminophen), Paroxetine, Pregabalin, Promethazine, Quetiapine, Risperidone, Strychnine, Temazepam, THC-COOH, Tilidine, Tramadol, Trimipramine, Venlafaxine, Zolpidem, Zopiclone

Table 1: Key forensic compounds investigated

a combination of post mortem & roadside testing cases from Freiburg. The results were compared to findings from established routine mass spectrometric screening methods.

Results

LC Method Suitability

In the test mixtures analysed at all three sites, compounds eluted evenly spread across the chromatogram, with good peak shape fidelity for early eluting compounds including those commonly known for chromatographic issues e.g. morphine (Figure 1).

For spiked urine and serum samples, the RT reproducibility between the three sites was better than 0.2 min (0.35 min for olanzapine). RT values were stable over the complete sequence and independent from matrix interference (see Figure 2).

MS Database

The database (DB) was created for the processing of the TOF MS full scan data and contained 73 entries for the 61 compound set. An almost exclusive [M+H]+ ionization was observed, with only diphenhydramine, amphetamine and related compounds presenting significant ”in-source” fragmentation whilst for temazepam and pregabalin some sodium adduct formation takes place.

Overlay of three chromatograms

Figure 1: Overlay of three chromatograms acquired at all three sites for the same compound mix, demonstrating very good system-to-system reproducibility of retention times.

Overlay of three chromatograms

Figure 2: Retention time stability over time and in spiked matrices: Overlay of four chromatograms for a mix of all 61 compounds in solvent (2 times from start and end of sequence) for urine and serum. a) complete chromatogram, b) expanded chromatogram range (5.5 – 9.0 min).

Conversely, in bbCID mode sufficient fragmentation was achieved by raising the collision energy to 30 eV with +/-6V stepping without precursor selection resulting in simultaneous fragmentation of all ions present. From the bbCID spectra ‘diagnostic’ qualifier ions (QIs) could be assigned for most compounds as shown in Table 3. Only buprenorphine, norbuprenorphine and strychnine did not show sufficient fragmentation even when using the optimized collision energy settings, so that no QIs could be assigned for these compounds. For 41 compounds three QIs were assigned, two QIs were available for 11 compounds and for the last 6 compounds only one reasonable QI could be assigned. This extended bbCID database contained 136 entries including the M+1 or M+2 isotope trace definitions and bbCID fragment ions.

Discussion

For the spiked samples all compounds were detected in full scan TOF MS analyses at all concentration levels, with no false negatives being observed. However, if the threshold level for detection was lowered, then many compounds would be detectable even at significantly lower levels. In

m/z RT sum formula name

300.1594 3.40 C18H21NO3 Codeine

195.0877 4.35 C8H10N4O2 Caffeine

177.1022 3.75 C9^13CH12N2O Cotinine (^13C)

285.0789 9.53 C16H13ClN2O Diazepam

302.1751 3.36 C18H23NO3 Dihydrocodeine

256.1696 6.63 C17H21NO Diphenhydramine

167.0855 6.63 C13H11^1+ Diphenhydr. Fragm 167

280.1696 6.88 C19H21NO Doxepin

Table 2: Example of extract from the full scan TOF-MS database

m/z RT sum formula name QI 1 QI 2 QI 3

316.0080 7.28 C14N3H10OBr Bromazepam 182.0839 209.0947 288.0131

316.0080 7.28 C14N3H10O^81Br Bromazepam (^81Br)

300.1594 3.40 C18H21NO3 Codeine 58.0651 215.1067 243.1016

300.1594 3.40 C17^13CH21NO3 Codeine (^13C)

177.1022 3.75 C10H12N2O Cotinine 70.0651 80.0495 98.0600

177.1022 3.75 C9^13CH12N2O Cotinine (^13C)

285.0789 9.53 C16H13ClN2O Diazepam 154.0418 228.0575 257.0840

285.0789 9.53 C16H13^37ClN2O Diazepam (^37Cl)

256.1696 6.63 C17H21NO Diphenhydramine 152.0621 165.0699 167.0855

256.1696 6.63 C16^13CH21NO Diphenhydramine (^13C)

167.0855 6.63 C13H11^1+ Diphenhydr. Fragm 167 152.0621 165.0699 167.0855

Table 3: Extract from the full scan bbCID database, detailing additional diagnostic qualifier ions (QIs).

addition to the expected compounds, a few additional plausible compounds were also detected such as caffeine in blank matrix or analytical artifacts (e.g. cocaine detection in a mix that contains cocaethylene and methanol). However, without applying the enhanced diagnostic confirmation criteria, the number of false positive detections in TOF-MS mode is significantly more than the number of expected findings. In sum for all TOF-MS analyses 333 false positives were detected in serum. This is greater than the 274 expected findings. For the detailed numbers of total, expected and plausible findings, as well as the number of false positives, (see Table 4).

In the bbCID channel, all compounds were detected in at least on one of their traces, but after applying the enhanced diagnostic confirmation detection criteria, zopiclone and paracetamol were missed at the 10ng/mL spike level due to the absence of a confirmatory diagnostic qualifier ion. However both zopiclone and paracetamol would have been detected if a lower data processing threshold was used.

The false positive rate in urine is even higher due to the increased matrix load and low detection threshold of 750 cts, yielding 547 false positives compared to 276 expected (see Table 4).

All serum and urine sample mixes were then reprocessed, but this time with the diagnostic ion enhanced confirmation criteria being applied. Table 5 summarises the results.

After applying the enhanced confirmation criteria, with the exception of tramadol, (mix 2), the false positives were completely removed. Tramadol cannot be removed as a finding in the presence of o-desmethylvenlafaxine as they are isobaric, co-elute and have very similar fragmentation patterns (see Figure 5).

Example of hrXIC traces and bbCID spectrum for fentanyl

FENTANYL (337)

Mix50-1_bbCID30_80-120_GE2_01_5440.d: FENTANYL, 337.2274�0.005, C 22 H 28 N 2 O 1, 6.0min

105

Mix50-1_bbCID30_80-120_GE2_01_5440.d: FENTANYL, 105.0699�0.005 (bbCID), C 22 H 28 N 2 O 1, Qualifier

188

Mix50-1_bbCID30_80-120_GE2_01_5440.d: FENTANYL, 188.1434�0.005 (bbCID), C 22 H 28 N 2 O 1, Qualifier

216

Mix50-1_bbCID30_80-120_GE2_01_5440.d: FENTANYL, 216.1383�0.005 (bbCID), C 22 H 28 N 2 O 1, Qualifier

FENTANYL (^13C)

Mix50-1_bbCID30_80-120_GE2_01_5440.d: FENTANYL (^13C), 338.2308�0.005, C 21 H 28 N 2 O 1 ^13C

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QI 1

QI 2

13C

QI 3

105.0699

134.0963

188.1441

216.1388

337.2282

95. +bbCID MS

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bbCID hrXIC traces for tramadol and o-desmethylvenlafaxine

Figure 4:bbCID hrXIC traces for (a) tramadol and (b) o-desmethylvenlafaxine. Note, that they almost co-elute, have identical monoisotopic mass and a common fragment ion m/z=58

M+H

QI m/z 58

13C

O-Desmethylvenlafaxine (M+H)264

U_M2_10ppb_bbCID: O-DESMETHYLVENLAFAXINE, 264.1958, 4.8 min

58U_M2_10ppb_bbCID: O-DESMETHYLVENLAFAXINE, 58.0651QI1 4.8min

O-Desmethylvenlafaxine ^13C

U_M2_10ppb_bbCID: O-DESMETHYLVENLAFAXINE, 265.1992(^13C) 4.8min

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Tramadol (M+H) 264U_M1_10ppb_bbCID: TRAMADOL, 264.1958 , 4.8min

58U_M1_10ppb_bbCID: TRAMADOL, 58.0651 (bbCID), QI1, 4.8min

Tramadol ^13CU_M1_10ppb_bbCID: TRAMADOL (^13C), 265.1992, ^13C, 4.8min

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a) b)

a) b)

Figure 3: bbCID spectrum (a) and (b) hrXIC qualifier ion traces providing unambiguous identification for fentanyl

bbCID spectra for tramadol and o-desmethylvenlafaxine

Figure 5: bbCID spectra for tramadol and o-desmethylvenlafaxine. Note, that the common m/z = 58 fragment ion cannot be used to distinguish tramadol in the presence of o-desmethylvenlafaxine. However, the two QIs circled in green allow for unambiguous identification of o-desmethylvenlafaxine in the presence of tramadol

264.1964

264.1958

14. O-DESMETHYLVENLAFAXINE(C₁₆H₂₅N₁O₂, 263.1885, err[mDa]: -0.55, mSigma: 9.4)

58.0660

86.0960

107.0491

133.0646201.1275

107.0491 (-0.1), rel. I 0.21133.0646 (-0.2), rel. I 0.0914. +bbCID MS, 30.0eV, 4.74-4.79min #549-55558.0651 (0.9 mDa), 107.0491 (-0.1 mDa), 133.0648 (-0.2 mDa), : points : 0

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o-desmethylvenlafaxine, C16H25NO2bbCID spectrum

58.0657

+MS2(264.1960), 24.0-36.0eV, 14.51-14.90min #871-894

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Tramadol, C16H25NO2bbCID spectrum

o-desmethylvenlafaxine

SERUM: Full scan SERUM: Full Scan URINE: Full scan URINE: Full Scan(TOF MS) (bbCID) (TOF MS) (bbCID)

Conc.

[ng/ml] 500 100 50 10 500 100 50 10

Conc.

[ng/ml] 500 100 50 10 500 100 50 10

Mix 1

29 27 29 24 41 36 29 36

Mix 1

40 43 35 46 52 44 50 43

12 12 12 11 22 22 21 18 12 12 12 13 22 22 22 22

5 4 4 3 8 6 5 2 6 2 1 1 4 2 2 2

12 11 13 10 11 8 3 16 22 29 22 32 26 20 26 19

Mix 2

29 25 25 23 32 27 33 31

Mix 2

38 36 36 39 42 38 44 40

10 10 10 10 20 20 20 19 10 10 10 10 20 20 20 19

1 1 1 1 2 2 2 2 1 1 1 1 2 2 2 2

18 14 14 12 10 5 11 10 27 25 25 28 20 16 22 19

Mix 3

30 31 33 27 44 50 41 36

Mix 3

36 32 34 34 44 46 49 39

15 15 16 13 26 28 26 21 15 15 15 14 26 27 27 19

4 4 3 2 7 5 4 3 3 3 2 2 5 4 4 2

11 12 14 12 11 17 11 12 18 14 17 18 13 15 18 18

Mix 4

28 30 20 28 30 31 29 28

Mix 4

27 34 36 35 45 37 46 36

9 9 9 9 18 18 18 17 9 9 9 9 18 18 19 17

2 2 2 2 4 4 4 3 2 2 2 1 4 3 3 2

17 19 9 17 8 9 7 8 16 23 25 25 23 16 24 17

Mix 5

31 22 29 25 27 29 28 26

Mix 5

35 32 32 38 41 39 42 43

9 9 9 9 18 18 18 17 9 9 9 9 18 18 18 15

1 1 1 1 2 2 2 2 1 1 1 1 2 2 2 2

21 12 19 15 7 9 8 7 25 22 22 28 21 19 22 26

Mix 6

27 29 26 30 38 39 36 36

Mix 6

32 35 39 38 50 45 46 41

14 14 14 14 26 26 26 24 14 14 14 14 26 26 26 23

2 1 1 1 2 2 2 2 1 1 1 1 2 2 2 2

11 14 11 15 10 11 8 10 17 20 24 23 22 17 18 16

TOTALS 657 274 50 333 813 507 79 227 TOTALS 862 276 39 547 1042 508 61 473

Table 4: Detection statistics for spiked serum samples before applying the diagnostic ion confirmation criteria:

Black: total # of findings, Blue: # expected findings, Green: # plausible positive findings, Red: # remaining false positives

SERUM FALSE POSITIVE FINDINGS (bbCID)Conc. [ng/ml] 500 100 50 10

Mix 1 0 0 0 0Mix 2 1 1 1 1Mix 3 0 0 0 0Mix 4 0 0 0 0Mix 5 0 0 0 0Mix 6 0 0 0 0

URINE FALSE POSITIVE FINDINGS (bbCID)Conc. [ng/ml] 500 100 50 10

Mix 1 0 0 0 0Mix 2 1 1 1 1Mix 3 0 0 0 0Mix 4 0 0 0 0Mix 5 0 0 0 0Mix 6 0 0 0 0

Table 5: False Positive detection statistics for (a) spiked serum samples and (b) spiked urine samples after applying the diagnostic ion confirmation criteria:

Screening Results from Authentic Case Samples

Authentic case samples from Helsinki and Freiburg were analyzed using an identical analysis protocol and the findings using the accurate mass diagnostic ion concept (DI concept) were compared to results from other established analytical techniques.

Authentic urine case

ALPRAZOLAM 309

954 dil x 10_RB1_01_609.d: ALPRAZOLAM, 309.0902�0.005, 8.4min

274

954 dil x 10_RB1_01_609.d: ALPRAZOLAM, 274.1213�0.005 (bbCID), Qualifier, 8.4min

281

954 dil x 10_RB1_01_609.d: ALPRAZOLAM, 281.0714�0.005 (bbCID,) Qualifier, 8.4min

ALPRAZOLAM (^37Cl)

954 dil x 10_RB1_01_609.d: ALPRAZOLAM (^37Cl), 311.0872�0.005, 8.4min

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QI 1

QI 2

13C

269.0474274.1207

281.0716

309.0904274.1207 (-0.6), rel. I 0.07281.0716 (0.2), rel. I 0.32309. +bbCID MS, 25.0eV, 8.20-8.25min #965-971274.1213 (-0.6 mDa), 281.0714 (0.2 mDa), : points : 0

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bbCID spectrum, sample

274.1215

281.0720

309.0908

+bbCID MS, 24.0-36.0eV,

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bbCID spectrum, standard

Figure 6: Authentic urine case number 954 at Helsinki. Alprazolam is confirmed as an additional finding by QI traces and bbCID spectrum.

Authentic case: post-mortem serum and urine samples

TRAMADOL VENLAFAXINE (^13C)

TRAMADOL (^13C)

Amphetamine Fragm 91Amphetamine Fragm 119BENZOYLECGONINEAmphetamine (^13C)BENZOYLECGONINE (^13C)

COTININECaffeine

O-DESMETHYLVENLAFAXINE METHADONE (^13C)

METHADONEEDDP (METHADONE METABOLITE)

O-DESMETHYLVENLAFAXINE (^13C)

EDDP (METHADONE METABOLITE) (^13C)Caffeine (^13C)

COTININE (^13C)

ECGONINE METHYL ESTERAmphetamineECGONINE METHYL ESTER (^13C)

GS250-12 HeSe_RA6_01_447.d

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Amphetamine QI m/z = 91Amphetamine

Amphetamine QI m/z 119

BENZOYLECGONINE

BENZOYLECGONINE (^13C)Amphetamine (^13C)

DESMETHYLVENLAFAXINE

AmphetamineCoffein (^13C)- TRAMADOL

O- METHADONE

BENZOYLECGONINE (^13C)

Caffeine

COTININE (^13C)ECGONINE METHYL ESTER Amphetamine Fragm 91 CARBOXY -THC

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CARBOXY-THC (345)

CARBOXY-THC, 345.2060�0.005, C 21 H 28

299

CARBOXY-THC, 299.2006�0.005 (bbCID), C 21 H 28 O 4, Qual

327CARBOXY-THC, 327.1955�0.005 (bbCID), C 21 H 28 O 4, Qual

CARBOXY-THC (^13C)

CARBOXY-THC (^13C), 346.2094�0.005, C 20 H 28 O 4 ^13

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13C

Figure 7: Authentic case (Freiburg case number GS250). a) post-mortem serum, b) post-mortem urine for same case. hrXIC’s showing confirmation of trace findings for amphetamine and carboxy-THC using bbCID qualifier ion traces.

a)

b)

Table 6: Findings in authentic post-mortem urine samples (Helsinki). Samples were extracted using mixed-mode SPE [3]. Reconstituted samples were diluted 1:10 prior to analysis

Case # CompoundDetected Additional False

Findings Positives

934 Tramadol YESO-desmethyltramadol YES NONECaffeine YES

936 Methadone YESEDDP YES Cotinine NONECaffeine YES

943 Zopiclone YESParacetamol YES NONECaffeine YES

945 Cocaine YESBenzoylecgonine YES Alprazolam NONEEcgonine methyl ester YES CaffeineCocaethylene YESCitalopram YESNorcitalopram YESZopiclone YESTemazepam YESCotinine YES

946 Codeine YESMorphine YES NONETramadol YESO-desmethyltramadol YESParacetamol YES

954 Pregabalin YES AlprazolamCarboxy-THC YES CaffieneEDDP YESQuetiapine YESHydroxyquetiapine YESParacetamol YESTemazepam YESOxazepam YESCotinine YES

Case # CompoundAdditional False Findings Positives

956 Pregabalin YESCitalopram YES Caffeine NONENorcitalopram YES CotinineMirtazapine YES ParacetamolDiazepam YESTemazepam YESOxazepam YES

962 Citalopram YESNorcitalopram YES Caffeine NONECodeine YESMorphine YESParacetamol YESLamotrigine YESAlprazolam YES

970 Fentanyl YESNorfentanyl YESParacetamol YES

974 Zopiclone YESDiazepam YES Cotinine NONENordazepam YESTemazepam YESOxazepam YES

H382 Fentanyl YESOxycodone YES Norfentanyl NONEMirtazapine YESCotinine YESMidazolam YESNordazepam YESOxazepam YESCaffeine YES

(DI concept)

Detected(DI concept)

Table 7: Summary for serum and urine authentic case samples (Freiburg). Serum samples were extracted using alkaline liquid-liquid extraction. Urine samples were precipitated with ACN and reconstituted in eluent 50/50.[4]

Case# Compound Result typeAdditional Findings

False Positives

T1106562 Serum, roadside testingAmphetamine 147 ng/ml GC-MS YES Cotinine NONECocaine 1,1 ng/ml GC-MS YES CaffeineBenzoylecgonine 148 ng/ml GC-MS YESEcgonine methyl ester 4,0 ng/ml GC-MS YES

T1200778 Serum roadside testingCocaine 2,0 ng/ml GC-MS YES Cocaethylene NONEBenzoylecgonine 482 ng/ml GC-MS YES CotinineEcgonine methyl ester 48 ng/ml GC-MS YES CaffeineTHC 3,1 ng/ml GC-MS (not in DB)11-OH-THC 2,2 ng/ml GC-MS (not in DB)THC-COOH 36 ng/ml GC-MS ND *

T1210160 Serum roadside testingQuetiapine 110 ng/ml LC-MS/MS YES Cotinine TramadolVenlafaxine 100 ng/ml LC-MS/MS YES CaffeineO-desmethylvenlafaxine ca. 500 ng/ml LC-MS/MS YES Zopiclone

T1210408 Serum roadside testingMorphine 91 ng/ml LC-MS/MS YES 6-MAM NONECodeine 17 ng/ml LC-MS/MS YES CotinineDiazepam 410 ng/ml LC-MS/MS YES CaffeineNordazepam >1000 ng/ml LC-MS/MS YES ParacetamolTemazepam 220 ng/ml LC-MS/MS YESOxazepam 280 ng/ml LC-MS/MS YESFlunitrazepam 30 ng/ml LC-MS/MS YES7-Aminoflunitrazepam 32 ng/ml LC-MS/MS YES

GS 250 Se Post mortem serumTHC / Metabolites LC-MS/MS ND * AmphetamineBenzoylecgonine LC-MS/MS YES CotinineVenlafaxine LC-MS/MS YES Ecgonine-methylesterO-desmethylvenlafaxine LC-MS/MS YES TramadolNicotine LC-MS/MS (not in DB)Caffeine LC-MS/MS YESMethadone LC-MS/MS YESEDDP LC-MS/MS YES

GS 250 U Post mortem urineCotinine LC-MS/MS YES Amphetamine

Nicotine LC-MS/MS (not in DB) Carboxy-THC** TramadolCaffeine LC-MS/MS YES EDDP**Theobromine LC-MS/MS (not in DB) Methadone**Venlafaxine LC-MS/MS YESDesmethylvenlafaxine LC-MS/MS YESBenzoylecgonine LC-MS/MS YESEcgonine methyl ester LC-MS/MS YES

Detected(DI concept)

Table 7: continued

* ND = Not Detected, analyte shows low extraction yield with chlorobutane** Reported for serum

Case # Compound Result typeAdditional

Findings

False

Positives

GS 548 Se Post mortem serumPromethazine Toxtyper YES Cotinine NONEFentanyl Toxtyper YES CaffeineDiazepam Toxtyper YES OlanzapineMethadone Toxtyper YESNordazepam Toxtyper YESMirtazapine Toxtyper YESMethadone 32 ng/ml LC-MS/MS YESEDDP 10 ng/ml LC-MS/MS YESPregabalin 1.8 µg/ml LC-MS/MS ND*Promethazine 402 ng/ml LC-MS/MS YESFentanyl 74 ng/ml LC-MS/MS YESNorfentanyl 10 ng/ml LC-MS/MS YESPhenothiazine 27 ng/ml LC-MS/MS YESMirtazapine 48 ng/ml LC-MS/MS YESDiazepam LC-MS/MS YESNordazepam LC-MS/MS YESOxazepam LC-MS/MS YESTemazepam LC-MS/MS YESLorazepam LC-MS/MS (not in DB)

GS 548 U Post mortem urineEDDP Toxtyper YES Norfentanyl NONE

Methadone Toxtyper YESFentanyl Toxtyper YESMirtazapine Toxtyper YESNicotine Toxtyper (not in DB)Mirtazapine GC-MS YESPromethazine GC-MS YESPregabalin GC-MS YESCaffeine GC-MS YESMethadone GC-MS YESEDDP GC-MS YESNicotine GC-MS (not in DB)Cotinine GC-MS YESDiazepam GC-MS YESNordazepam GC-MS YESOxazepam GC-MS YESTemazepam GC-MS YES

Detected

(DI concept)

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References

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type stimulants and new psychoactive substances, UNODC

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mass spectrometry. Rapid Commun Mass Spectrom 2006

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[3] M. Sundström, A. Pelander, V. Angerer, M. Hutter, S.

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[4] L.M. Huppertz, S. Vogt, J. Kempf, Ein automatisiertes MSn-

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Discussion for Authentic Samples

Again applying the diagnostic ion concept to the data for all authentic case samples excellent agreement was found with findings from routine analysis, i.e. UPLC QTOF [5], GC-MS [6], LC-MS/MS [7] and Toxtyper [4], (see Tables 6 & 7). False positive findings were again completely removed, with the only exception again being tramadol in presence of o-desmethylvenlafaxine. Reliable detection was achieved for compounds included in the database at low and high levels.

A few additional findings were also observed compared to the routine methods. Many of them were commonly found compounds (e.g. cotinine, caffeine) or plausible trace findings (e.g. cocaethylene for a reported cocaine finding).

Conclusion

The forensic toxicology screening workflow and associated hardware has been shown to work reproducibly for serum and urine samples at three different sites. The efficacy of the ‘diagnostic ion’ concept using bbCID qualifier ions has proven to be a very powerful tool to eliminate false positive findings. Furthermore, the workflow provides a sensitive screening method exploiting a low detection threshold with optimum retention time windows to avoid false negatives. The ability to add new compounds to the accurate mass bbCID and TOF-MS database, without changing the acquisition method, provides considerable scope to easily extend the screening method to include additional compounds of forensic toxicological relevance as future needs arise.

Further work will center on applying the diagnostic ion concept to a larger forensic toxicology bbCID database to enable comprehensive, in-depth forensic investigations to be realized.