Aula TP 15 de Fevereiro de 2017

Transcrição

1 Mestrado em Química, Análise Química Forense 2016/2017 Aula TP 15 de Fevereiro de 2017 Helena Gaspar Gab , Lab

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

3 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

4 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

5 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

6 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 6

7 7 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

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

9 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

10 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

11 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

12 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

13 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

14 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

15 (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

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

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

18 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

19 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

20 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

21 3MMC 4MMC Pentedrone SAMPLE in D 2 O why? 4MMC?

22 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

23 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

24 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

25 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

26 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)

27 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

28 4F-PBP: A Novel NPS Mestrado em Química seized white powders Comparison with MS Libraries - MDPBP But.

29 29 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

30 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

31 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

32 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

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

34 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

35 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

36 White powders NMR allows the absolute quantification D 2 O Purity > 99% 1 H NMR Sample 1 IS MDPBP Sample 3 Sample 1 IS myo-inositol 4F-PBP 1 H qnmr Sample 1 61% 38% Sample 3 57% 42% 36

37 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

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

39 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

40 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

41 Caso 2 4F-PBP: Uma nova NSP

42 Público 28 May 2015

43 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

44 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

45 ?2xCH 2

46 Yes 2xCH 2

47

48 4MPHP 4MPHP and 4MMC?

49 4MPHP 2H 3H 2H 2H 3H

50 CH 2 CH 2 CH 2 CH 3

51 HBr, Br 2?CH 2 CH 2 CH 2 CH 3

52 4MPHP? 2H 3H 2H 2H 3H

53 4MPHP? 2H 3H 2H 2H 3H

54 HBr, Br 2

55

56 4MPHP 2H 3H 2H 2H 3H

57 4MPHP 2H 3H 2H 2H 3H