Aula 2 28 de Fevereiro de 2018

Transcrição

1 Mestrado em Química, Análise Química Forense 2017/2018 Aula 2 28 de Fevereiro de 2018 Helena Gaspar hmgaspar@ciencias.ulisboa.pt Gab , Lab

2 Identifications of substances Visual inspections Sampling Botanical identification (if applicable) Sample preparation Chemical screening (colour test) Confirmatory examination Classical drugs/nps Classical drugs all procedures are well established

3 Confirmatory Examination TLC or GC-FID (comparison with standarts) GC-MS or LC-MS, GC-MS/MS or LC-MS/MS (Retentions Time and Mass Spectrum) Comparison with MS spectra in databases Interpertation of MS and MS/MS patterns FTIR (pure compounds comparison with IR spectra in databases) GC-IR (Retention Time and IR spectrum) RMN ( 1 H, 13 C, H H COSY, H C ghsqc, H C HMBC, pure compounds or mixtures) HRMS (pure compounds or simetimes mixtures, molecular formula) Most of substances analysed by GC-MS

4 Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) Website 1) SWGDRUG Recommendations Version 7.1 was approved on June 9, ) Supplemental Document (SD-6) was approved on June 9, 2016 and was posted on October 31, This document provides examples of estimating measurement uncertainty for extrapolations of net weights and unit counts. 3) Drug Monographs (being updated regularly) 4) Searchable Mass Spectral Library Version 3.1 (dated November 29, 2016). 5) Searchable Infrared Library Version 1.2 (dated May 26, 2015) 6) ENFSI-DWG has published a new Guideline on Sampling of Illicit Drugs for Quantitative Analysis and a corresponding Sampling Calculator, both of which have been posted to this website. 7) To keep up with the latest updates (including new monographs, libraries, etc.) from SWGDRUG, follow us on Twitter

5 Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG)

6 MassBank

7 Cayman - GC-MS Library

8 RESPONSE

9 Bruker Topsin

10

11 Choice of analytical technique Matrix (blood, urine, human tissue, plant, extracts..) Sample amount Number of samples Time of response Analyse cost Available instruments Search of know/unknow substance Available standards Sampling Sample preparations

12 Identifications of synthetic NPS in smartshop produts PRODUCTS PROBABLY WITH KNOWN COMPOUNDS Plants Extracts Dried plant leaves

13 Natural Products Salvia divitorium From Mexico (Mazatec Indians) Hallucinogenic plant (Botanical identification if possible) Leaves Extracts (5x-60x) Salvinorine A Active Substance Chemotaxonomic marker Major compounds (80%) Salvinorine B Inactive Substance

14 GC-MS fingerprint of Salvia Products Extraction with filtration mg powder/ ml MeOH 1mg/mL 60x 1mg/mL 50x 10mg/mL 15x 10mg/mL 5x 10mg/mL Leaves can be used as standard for identification Salvia GC HP5 60ºC (2min); rampa 30ºC/min até 300ºC; 300ºC (15min), split 1/9

15 Natural Products Kraton (Mitragyna speciosa) plant used in tradicional medicine (Asia) Kratom has some opiate and stimulant like properties Botanical identification if possible Extracts15x Dried plant leaves 25 Alkaloids % depend on geographic origin Mytragynine principal psychoactive compounds

16 GC-MS identification of MG with standard Mestrado em Química TR= 13,1min Mitragynina Standard TR= 13,1min Kratom HP5 200ºC/300ºC (20ºC/min); 300ºC (15min); split 1/239 MS

17 HPLC profile - fingerprint Mitragynina Kratom TR: 9,88min TR: 10,18min Phenomenex Luna RP18 (3µm, 150x4,60mm) ; 35ºC; 20µL; Gradiente MeOH/ 0,1%TFA UV UV

18 Sample Mitraginina %(m/m) * Kr01 Kratom Bali ± Folhas secas Kr02 Kratom Bali 15x 2.67 ± 0.10 Kr03 Kratom Thai ± Folhas secas Kr04 Kratom Thai 15x ± Kr05 Kratom Thai ± Folhas cortadas Kr06 Kratom Thai ± Folhas em pó Kr ± Kr08 Kratom (euphoria) ± Folhas secas Kr09 Kratom Balinese ± Folhas em pó Chromadex Kr10 Kratom ± óleo essencial Kr11 Fenix Black (Líquido) Kratom Bali ± Kr12 Resina ± Kratom Bali Kr13 Kraton Bali 15x ± Kr14 Kratom Gold 27.6 ± 1.2 Kr15 Kratom red 15x ± The identification is correct but the quantification? Why we cannot use NMR?

19 Solução de Mitraginina (ChromaDex ) 5ug/mL + PI MG PI Kr09 Kraton Balinese (Mitragyna speciosa) Folhas da ChromaDex (moidas 10 ml MeOH, diluição 1/10, + PI) MG PI Kr01Kraton Bali (Folhas) (moidas 10 ml MeOH, diluição 1/10, + PI) MG PI

20 MITRAGYNINA SPECIOGYNINA PAYNANTHEINA 7-HIDROXIMITRAGYNINA SPECIOCILIATINA

21 LC-MS Better resolution than LC-DAD Whitou standards we cannot identify all Isomeric alkaloids LC-MS fingerprint

22 Identifications of synthetic NPS in seized produts TYPE OF PRODUCTS WHITE POWDERS CRISTAL HERBAL MATRIX OLEUM

23 Identification of Synthetic NPS Samples Absence of Standards Tentative Identification GC-MS MS Libraries Standards Identification GC-MS Mass Spectrum + R t NMR Identification SAMPLES STANDARDS

24 Analytical Standards Supply (+8 days) Synthesised in the Lab (+8 days) But the fastest way is to performe NMR of the seized product! 5 to 20 mg (400MHz, 6h) Drawback - only for pure compouns or simple mixture

25 Why Synthesised cathinones in the Lab? Synthetic cathinones R1 R2 R3 R4 Composto Methyl H Methyl H Methcatinone Ethyl H Methyl H Buphedrone Propyl H Methyl H Pentedrone Methyl Pyrrolidine H α-ppp Ethyl Pyrrolidine H α-pbp Propyl Pyrrolidine H α-pvp Methyl H Methyl Methyl 4-MMC Ethyl H Ethyl Methyl 4-MEC Synthesis is only necessary for: Supply standarts for the creation on in house datebases Supply compounds for toxicological evaluation

26 Methodology simple matrix Powder/Oleum Identification GC-MS R t, EI fragmentation NMR 1 H, 13 C, APT, HSQC, HMBC, COSY 1mg/mL MeOH 10-20mg, 500μL First choice DMSO why? Other solvents (D 2 O, MeOD or CDCl 3 ) for data comparison Qualitative Standards In-house EI-MS and NMR libraries 26

27 27 Mestrado em Química Methodology herbal matrix Herbal matrix Isolation LC N-Hexane/AcOEt Identification GC-MS R t, EI fragmentation NMR 1 H, 13 C, APT, HSQC, HMBC, COSY 100mg/mL MeOH 10-20mg, 500μL First choice CDCl 3 why? Other solvents (DMSO, MeOD) for data comparison Qualitative Standards In-house EI-MS and NMR libraries

28 Supply Cannabinoids Isolated from seized products 5F- AKB-48 5F- PB22 STS-135 Seized Products Liquid Chromatography Pure NPS

29 Methodology LPC/PJ Fulcral Structure Identification FCUL Quantitative standards GC-MS NMR Qualitative standards Getting the Right Structure >95% Purity FTIR HRMS Ion Chromatography Elementar Analysis Toxicity Evaluation Complementary Structure Identification

30 Identifications of NPS whithout standarts LPC/PJ FCUL GC-MS NMR NMR allows the identification of new compounds NMR allows the identifications of isomeric compounds not distinguishable by GC-MS NMR alows the identification of compounds not detected in routine GC-MS NMR allows to determine the purity of compounds

31 GC-EI-MS Fragmentation of Synthetic Cathinones +. Base Peak Acylium ion Iminium ion D. Zuba, Identification ofcathinones and other active components of 'legal highs' by mass spectrometric methods. Trends in Analytical Chemistry, 32, pp

32 EI-MS of Cathinones (Text File) #17: 4-MEC; 2-ethylamino-1-(4-methylphenyl)propan-1-one; 4-methylcathinone; methyl-N-cathinone; ????? (Text File) #17: 4-MEC; 2-ethylamino-1-(4-methylphenyl)propan-1-one; 58 4-methylcathinone; 4-methyl-N-cathinone; 2-????? Drawbacks molecular ion absence low intensity Acylium ions low intensity (Text File) #4: Methedrone; (4-methoxyphenyl)-2-(methylamino)propan-1-one; para-methoxymethcathinone; 4-methoxym????? (Text File) #6: Pentedrone; 1-phenyl-2-(methylamino)pentan-1-one; alpha-methylaminovalerophenone

33 EI Fragmentation of Synthetic Cathinones +. Acylium Ions m/z 135 m/z 119 Iminium Ions (Base peak) m/z 149 m/z 123 m/z 58 m/z 72 m/z 86 m/z 105 m/z 133 m/z 112 m/z

34 7,005 7,105 7,205 7,305 7,405 7,505 7,604 7,704 7,804 7,904 8,004 8,104 8,204 8,304 8,404 8,503 8,603 8,703 8,803 8,903 9,003 9,103 9,203 9,302 9,402 9,502 9,602 9,702 9,802 9,902 10,002 10,102 10,201 10,301 10,401 10,501 10,601 10,701 10,801 10,901 Mestrado em Química Charlie Buphedrone vs N-ethylcathinone

35 (Text File) #5: N-ethylcathinone; 2-ethylamino-1-phenyl-propan-1-one; ethcathinone; ethylpropion; ETH-CAT; Ethyl????? (Text File) #8: Buphedrone; 2-(methylamino)-1-phenylbutan-1-one; alpha-methylamino-butyrophenone; MABP

36 NMR identification 1 H NMR 400 MHz, MeOD N-ethylcathinone Buphedrone

37 NMR identification 1 H NMR 400 MHz, MeOD Crabby 3,4-DMCC Bliss methedrone Pentedrone -NET Pentedrone

38 NMR identification a Proton ( 5,0-5,5 ppm) 3,4-DMCC 1 H NMR 400 MHz, MeOD quartet q Crabby Bliss a Integration =1 Multiplicity methedrone quartet q Pentedrona -NET Pentedrone Triplet t quartet R2= Methyl triplet R2 Alkyl

39 NMR identification Alkyl groups ( 4,0-0,5 ppm) 1 H NMR 400 MHz, MeOD 2 x ar-ch 3 Crabby 3,4-DMCC O-CH 3 N-CH 3 CH 3 Bliss Value Integration Multiplicity metedrona CH 3 Pentedrona -NET CH 3 N-R3 R1, R2 Pentedrona CH 2 CH 2

40 NMR allows the differentiation of positional isomers 1971 UN Convention PS NPS Decreto-Lei 15/1993 Decreto-Lei 54/ ppm ppm Crucial to assign the substituent position in the aromatic ring

41 3MMC 4MMC Pentedrone SAMPLE in D 2 O why? 4MMC?

42 Identification of compounds not detected in routine GC-MS Detection of Methylamine by NMR, a contaminant Pentedrone Isopentedrone Caffeine Methylamine 1 H NMR 400 MHz, MeOD 2`/6` 8 4` 3`/5` 2CH 7Me N-Me 1Me 3Me N-Me 5Me 3-CH 2 4-CH 2 1CH 5Me ppm

43 Quantificação relativa por 1 H NMR A integração (área dos sinais) é proporcional ao número de moles de protões que originam o sinal de ressonância I composto 1 = n composto 1 n composto 1 I composto 2 n H composto 2 n = m (g)/m (mol/g) H n composto 2 m 1 = I 1 n H 2 m 2 I H M 2 2 n C1 M 1

44 1 H NMR 400 MHz, CDCl 3 Mestrado em Química 3CH ``CH 3 2`,6` N-etilcationa 4CH 3 3`,5` N-CH 3 m 1 = I 1 n H 2 m 2 I H M 2 2 n C1 M 1 m 1 = I Me = 4,7418 m 2 I NMe 2,9789 N-etilcationa = 61,4% Bufedrona = 38,6% 4` CHCl 3 2CH Bufedrona Peak for integration? NH NH NH H 2 O N-CH 2 3CH 2

45 Quantificação absoluta por 1 H NMR (método do padrão interno) I Composto I PI = n Composto n PI n H Composto H n PI Comparando o integral de um sinal de ressonância do analíto com o integral de um sinal de um composto de concentração conhecida é possivel determinar a sua concentração w m composto = I Composto I PI H n PI H n Composto M Composto M PI m W PI P PI 100 VANTAGENS Não é necessário ter padrão do analíto Não necessita de ser estruturalmente semelhante ao analíto O mesmo composto permite determinar a concentração de vários componentes da mesma solução

46 Quantification absoluta 1 H NMR Carateristicas de um bom PI Contenha o núcleo de interesse ( 1 H) Sinal de ressonância diferente do analíto De pureza conhecida Químicamente inerte MA Ácido maleico Baixa volatilidade Solubilidade semelhante ao analíto T 1 (tempos de relaxação relativamente pequenos)

47 Solução mg/10 ml D 2 O C07Rush 1 H qnmr 400 MHz, D 2 O, 11.56mg/500mL Ácido Maleico (MA) C07Rush qnmr Pentedrona.HCl : 70% Metilamina.HCl: 19% Cafeína: 8% Isopentedrona.HCl: 3% PI= 100 Pentedrona MeNH 2 Isopentedrona

48 4F-PBP: A Novel NPS Mestrado em Química seized white powders Comparison with MS Libraries - MDPBP But.

49 49 4,001 4,117 4,233 4,349 4,465 4,581 4,697 4,813 4,929 5,045 5,161 5,277 5,393 5,509 5,626 5,742 5,858 5,974 6,09 6,206 6,322 6,438 6,554 6,67 6,786 6,902 7,018 7,134 7,25 7,366 7,482 7,599 7,715 7,831 7,947 8,063 8,179 8,295 8,411 8,527 8,643 8,759 8,875 8,991 Mestrado em Química CASE 1 4F-PBP a Novel NSP R t = 4.979min R t = 8.289min Sample 3 Sample 2 Sample 1 Different R t at same conditions different compounds

50 MS spectra fragmentation pattern analysed (Text File) Scan 466 (4.796 min): D\ data.ms Sample (Text File) Scan 1069 (8.292 min): D\ data.ms Sample (Text File) Scan 466 (4.795 min): D\ data.ms Sample 3

51 MS spectra fragmentation pattern analysed Sample 1 Sample ? (Text File) Scan 466 (4.796 min): D\ data.ms m/z =19 F (Text File) #2: Flephedrone; (4-fluorophenyl)-2-methylaminopropan-1-one; 4-FMC; fluoromethcathinone

52 NMR is crucial to assign the substituent position in the aromatic ring Aromatic pattern 1 H NMR Spectra Sample 1 Sample 3 MDPBP 4F-PBP 4-MEC Sample 1 Sample 3 Flefedrona Confirmation 19 F NMR

53 NMR allows the differentiation of positional isomers sample 1 sample 3 Crucial to assign the substituent position in the aromatic ring

54 NMR alows the identification of compounds not detected in routine GC-MS Is there another compound present not detected in GC-MS? Yes, the mio-inositol 1 H NMR in D 2 O, why this solvent? sample 1 sample 3 mio-inositol 4F-PBP MDPBP

55 H-1,3 ax H-4,6 ax H-5 ax Mestrado em Química NMR allows the identification of NPS also in mixtures 2D 13 C/ 1 H HSQC 1 H NMR 400 MHz, D 2 O Amostra 1 H a -1 H b -1 H a -4 H b -4 Padrão do mio-inositol H-2 eq

56 White powders NMR allows the absolute quantification D 2 O Purity > 99% 1 H NMR Sample 1 Sample 3 Sample 1 IS IS myo-inositol MDPBP 4F-PBP 1 H qnmr Sample 1 61% 38% Sample 3 57% 42% 56

57 NMR after purifications the with CDCl 3 1 H COSY H-H 19 F 13 C APT HMBC H-C HSQC H-C H. Gaspar et al, 4F-PBP, a new substance of abuse: structural characterization and purity NMR profiling, Forensic. Science International 2015, (252) 168

58 NMR Allows the differentiation free base/sal NMR after purifications 1 H NMR CDCl 3 + NH

59 IC allows the identification of the salt form Ion Exchange Chromatography 4F-PBP Hydrochloride Crucial to distinguish hydroclhorides or bromide salts 4F-PBP Cloreto Nitrito Fluoreto Brometo Cl - Standard Nitrato ortofosfato fosfato

60 4F-PBP: A novel NPS Buy in the Net White powder Risks and effects 4F-PBP Illegal in Portugal 4F-α-PVP 2 deaths in Sweden 2014 Novel NPS In Europe α-pbp Portaria nº154/2013 DL nº54/2013 artigo 3º

61 Caso 2 4F-PBP: Uma nova NSP

62 Público 28 May 2015

63 Which NMR technique? 7.96d 6.93d 7.60d 7.12d 7.45s 7.96d 6.93d 7.45s 7.60d 7.45s 7.12d 6.93d 7.96d 7.60d 7.12d

64 6.93d 7.60d 7.12d 7.96d 6.93d 7.60d 7.45s 7.12s 7.96d NOESY 6-APB 7.45s 6.93d 7.45s 7.96d 5-APB 7.60d 7.12d

65 ?2xCH 2

66 Yes 2xCH 2

67

68 4MPHP 4MPHP and 4MMC?

69 4MPHP 2H 3H 2H 2H 3H

70 CH 2 CH 2 CH 2 CH 3

71 HBr, Br 2?CH 2 CH 2 CH 2 CH 3

72 4MPHP? 2H 3H 2H 2H 3H

73 4MPHP? 2H 3H 2H 2H 3H

74 HBr, Br 2

75

76 4MPHP 2H 3H 2H 2H 3H

77 4MPHP 2H 3H 2H 2H 3H

78 78 Mestrado em Química Caso 1 Canabinoide em Microselos Análise de GC-MS AMB-FUBINACA AB-FUBINACA Isomero 5 - AMB-FUBINACA 4 estruturas possíveis! Isomero 5 - AB-FUBINACA

79 79 Mestrado em Química Caso 1 Canabinoide em Microselos M = 383 M = 368 M = 383 M = 368

80 80 Mestrado em Química Caso 1 Canabinoide em Microselos Análise de GC-MS m/z = 109 M = 383 AMB-FUBINACA m/z = 253 M = 368 AB-FUBINACA m/z = 324 M = 383 Isomero 5 - AMB-FUBINACA m/z = 324 Fragmentos de massas iguais Dificil de distinguir sem padrões! M = 368 Isomero 5 - AB-FUBINACA

81 81 Mestrado em Química Caso 1 Canabinoide em Microselos AMB-FUBINACA Isomero 5 - AMB-FUBINACA AB-FUBINACA Isomero 5 - AB-FUBINACA 1 H RMN, 400 MHz, DMSO-d 6 OCH 3

82 82 Mestrado em Química Caso 1 Canabinoide em Microselos Análise de RMN 1 H RMN, 400 MHz, DMSO-d 6 AMB-FUBINACA AB-FUBINACA Isomero 5 - AMB-FUBINACA Isomero 5 - AB-FUBINACA RMN permitiu identificar o AMB-FUBINACA sem padrão!

83 Identificar sem ter pistas? Pescar um composto numa mistura? Cafeina

84

85

86 Referência d d d d d d

87 Referência d d d d d d

88 Referência d d d d d d X X X X

89 Referência d d d d d d X X X X X X X X X

90 HMBC X X X X X X X X X

91 19F=