aula 2 28 de fevereiro de 2018 - fenix.ciencias.ulisboa.pt · helena gaspar análise química...

Mestrado em Química 2017-2019

Helena Gaspar Análise Química Forense 28 de Fevereiro de 2018

Aula 2 –28 de Fevereiro de 2018

Helena Gaspar

[email protected]

Gab 8.5.49, Lab 8.5.55

Mestrado em Química, Análise Química Forense 2017/2018

mailto:[email protected]



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



• 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 (1H, 13C, H–H COSY, H–C gHSQC, H–C HMBC, pure compounds or mixtures)

• HRMS (pure compounds or simetimes mixtures, molecular formula)

Confirmatory Examination

Most of substances analysed by GC-MS



1)SWGDRUG Recommendations Version 7.1 was approved on June 9, 2016.

2) Supplemental Document (SD-6) was approved on June 9, 2016 and was posted on October 31, 2016.

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 Follow @swgdrug

http://www.swgdrug.org/

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

http://www.swgdrug.org/approved.htm

http://www.swgdrug.org/Documents/SD-6_MU for Extrapolations.pdf

http://www.swgdrug.org/monographs.htm

http://www.swgdrug.org/ms.htm

http://www.swgdrug.org/ir.htm

http://www.swgdrug.org/references.htm

http://www.swgdrug.org/tools.htm

https://twitter.com/swgdrug



http://www.swgdrug.org/monographs.htm

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



http://www.massbank.jp/PeakSearch.html

MassBank



Cayman - GC-MS Library

https://www.caymanchem.com/forensics/overview



RESPONSE http://www.policija.si/apps/nfl_response_web/seznam.php



https://www.bruker.com/nc/pt/service/support-upgrades/software-downloads/nmr/free-topspin-processing/download-page.html

Bruker Topsin



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

SamplingSample preparations



Identifications of synthetic NPS in smartshop produts

PRODUCTS PROBABLY WITH KNOWN COMPOUNDS

Plants Extracts Dried plant leaves



Salvinorine AActive SubstanceChemotaxonomic markerMajor compounds (80%)

Salvinorine BInactive Substance

Salvia divitorium – From Mexico (Mazatec Indians)Hallucinogenic plant (Botanical identification if possible)

Leaves

Extracts (5x-60x)

Natural Products



Salvia GC HP5

1mg/mL 60x

1mg/mL 50x

10mg/mL 15x

10mg/mL 5x

10mg/mL Leavescan be used as standard for identification

60ºC (2min); rampa 30ºC/min até 300ºC; 300ºC (15min), split 1/9

GC-MS fingerprint of Salvia Products

Extraction with filtrationmg powder/ mL MeOH



Extracts15xDried plant leaves

25 Alkaloids% depend on geographic originMytragynine principal psychoactive compounds

Natural Products

Kraton (Mitragyna speciosa) – plant used in tradicional medicine (Asia)Kratom has some opiate and stimulant like propertiesBotanical identification if possible



GC-MS identification of MG with standard

MitragyninaStandard

Kratom

HP5 200ºC/300ºC (20ºC/min); 300ºC (15min); split 1/239

TR= 13,1minTR= 13,1min

MS



HPLC profile - fingerprint

Mitragynina

TR: 9,88min

UVUV

Kratom

TR: 10,18min

Phenomenex Luna RP18 (3µm, 150x4,60mm) ; 35ºC; 20µL; Gradiente MeOH/ 0,1%TFA



Sample Mitraginina %(m/m) *

Kr01 Kratom Bali

Folhas secas1.551 ± 0.057

Kr02 Kratom Bali 15x 2.67 ± 0.10

Kr03 Kratom Thai


Kr04 Kratom Thai 15x 1.036 ± 0.041

Kr05 Kratom Thai

Folhas cortadas1.601 ± 0.059

Kr06 Kratom Thai

Folhas em pó1.436 ± 0.054

Kr07 1.189 ± 0.045

Kr08 Kratom (euphoria)


Kr09 Kratom Balinese

Folhas em pó

Chromadex

1.381 ± 0.052

Kr10 Kratom

óleo essencial

0.01757 ± 0.00058

Kr11 Fenix Black (Líquido)

Kratom Bali

0.00937 ± 0.00033

Kr12 Resina

Kratom Bali0.1680 ± 0.0062

Kr13 Kraton Bali 15x 0.622 ± 0.030

Kr14 Kratom Gold 27.6 ± 1.2

Kr15 Kratom red 15x 2.246 ± 0.086

The identification is correctbut the quantification?

Why we cannot use NMR?



Solução de Mitraginina (ChromaDex ) 5ug/mL + PI

Kr09 – Kraton Balinese (Mitragyna speciosa) – Folhas da ChromaDex(moidas 10 mL MeOH, diluição 1/10, + PI)

PIMG

PIMG

PIMG

Kr01Kraton Bali (Folhas)(moidas 10 mL MeOH, diluição 1/10, + PI)



MITRAGYNINA PAYNANTHEINA

7-HIDROXIMITRAGYNINA

SPECIOGYNINA

SPECIOCILIATINA



LC-MSBetter resolution than LC-DAD

Whitou standardswe cannot identify all Isomeric alkaloids

LC-MS fingerprint



Identifications of synthetic NPS in seized produts

TYPE OF PRODUCTS

WHITE POWDERS OLEUMCRISTAL HERBAL MATRIX



Samples

Absence ofStandards

Tentative Identification – GC-MS

MS Libraries

StandardsIdentification – GC-MS

Mass Spectrum + Rt

Identification of Synthetic NPS

NMR Identification

SAMPLES STANDARDS



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



Why Synthesised cathinones in the Lab?

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 datebasesSupply compounds for toxicological evaluation

Synthetic cathinones


Helena Gaspar Análise Química Forense 28 de Fevereiro de 2018 26

Powder/Oleum

IdentificationGC-MS

Rt, EI fragmentation

NMR1H, 13C, APT, HSQC,

HMBC, COSY

QualitativeStandards

In-house EI-MS and NMR libraries

1mg/mL MeOH

10-20mg, 500μL First choice DMSO why?Other solvents (D2O, MeOD or CDCl3)for data comparison

Methodology – simple matrix



27

Methodology – herbal matrix

100mg/mL MeOH

Herbal matrix

IdentificationGC-MS

Rt, EI fragmentation

NMR1H, 13C, APT, HSQC,

HMBC, COSY

QualitativeStandards

In-house EI-MS and NMR libraries

10-20mg, 500μL First choice CDCl3 why?Other solvents (DMSO, MeOD)for data comparison

Isolation LC N-Hexane/AcOEt



Supply Cannabinoids – Isolated from seized products

5F- AKB-48 5F- PB22 STS-135

Liquid Chromatography Pure NPSSeized

Products



GC-MS NMR

HRMSFTIR

Elementar Analysis

Getting the Right Structure

LPC/PJ FCULFulcral Structure Identification

Complementary Structure Identification

Ion Chromatography

Qualitative standards

>95% Purity

ToxicityEvaluation

Methodology

Quantitative standards



Identifications of NPS whithout standarts

GC-MS NMR

LPC/PJ FCUL

• 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



Base Peak

D. Zuba, 2015. Identification of cathinones and other active components of 'legal highs' by mass spectrometric methods. Trends in Analytical Chemistry, 32, pp. 15-30

GC-EI-MS Fragmentation of Synthetic Cathinones

Acylium ion Iminium ion

+.



(Text File) #17: 4-MEC; 2-ethylamino-1-(4-methylphenyl)propan-1-one; 4-methylcathinone; 4-methyl-N-cathinone; 2-?????

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2800

50

100

44

5665

72

91

103119

128 146 176 191

(Text File) #17: 4-MEC; 2-ethylamino-1-(4-methylphenyl)propan-1-one; 4-methylcathinone; 4-methyl-N-cathinone; 2-?????

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2800

50

100

44

5665

72

91

103119

128 146 176 191

(Text File) #4: Methedrone; (4-methoxyphenyl)-2-(methylamino)propan-1-one; para-methoxymethcathinone; 4-methoxym?????

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2800

50

100

42 50

58

64 77 92107 120

135150 163 178

(Text File) #6: Pentedrone; 1-phenyl-2-(methylamino)pentan-1-one; alpha-methylaminovalerophenone

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2800

50

100

44

5157

70

77

86

91105

115 131 148

EI-MS of Cathinones

Drawbacks• molecular ion

absencelow intensity

• Acylium ions low intensity

191

193

191



Iminium Ions(Base peak)

Acylium Ions

m/z 58 m/z 72 m/z 86

EI Fragmentation of Synthetic Cathinones

m/z 135 m/z 119

m/z 149 m/z 123

m/z 105 m/z 133

m/z 112 m/z 126

+.



“Charlie” – Buphedrone vs N-ethylcathinone7

,00

5

7,1

05

7,2

05

7,3

05

7,4

05

7,5

05

7,6

04

7,7

04

7,8

04

7,9

04

8,0

04

8,1

04

8,2

04

8,3

04

8,4

04

8,5

03

8,6

03

8,7

03

8,8

03

8,9

03

9,0

03

9,1

03

9,2

03

9,3

02

9,4

02

9,5

02

9,6

02

9,7

02

9,8

02

9,9

02

10

,00

2

10

,10

2

10

,20

1

10

,30

1

10

,40

1

10

,50

1

10

,60

1

10

,70

1

10

,80

1

10

,90

1

8.593

8.475



(Text File) #5: N-ethylcathinone; 2-ethylamino-1-phenyl-propan-1-one; ethcathinone; ethylpropion; ETH-CAT; Ethyl?????

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2800

50

100

44

51

72

77

91105

132

(Text File) #8: Buphedrone; 2-(methylamino)-1-phenylbutan-1-one; alpha-methylamino-butyrophenone; MABP

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2800

50

100

44 51 57

63

72

77

89105

131 148



Buphedrone

N-ethylcathinone

1H NMR 400 MHz, MeOD

NMR identification



Pentedrone

3,4-DMCC

methedrone

Crabby

Bliss

Pentedrone -NET


NMR identification



Integration =1

quartet R2= Methyltriplet R2 Alkyl

Multiplicity

a Proton ( 5,0-5,5 ppm)

Pentedrone

3,4-DMCC

methedrone

Crabby

Bliss

Pentedrona -NET


quartet q

quartet q

Triplet t

a

NMR identification



Pentedrona

3,4-DMCC

metedrona

Crabby

Bliss

Pentedrona -NET

N-CH3O-CH3

2 x ar-CH3

CH3

Alkyl groups ( 4,0-0,5 ppm)

Value IntegrationMultiplicity

N-R3R1, R2

CH3

CH3

CH2 CH2


NMR identification



7.37.47.57.67.77.87.9 ppm7.37.47.57.67.77.87.9 ppm

NMR allows the differentiation of positional isomers

Crucial to assign the substituent position in the aromatic ring

1971 UN Convention PS

Decreto-Lei 15/1993 Decreto-Lei 54/2013

NPS



SAMPLE in D2O why?4MMC?

3MMC

4MMC

Pentedrone




Pentedrone

3Me7Me

8

1Me3`/5`2`/6`

N-Me

2CH

4-CH23-CH2

4`

5MeN-Me

1CH 5Me

Methylamine

Detection of Methylamine by NMR, a contaminant

Isopentedrone Caffeine

ppm

Identification of compounds not detected in routine GC-MS



Quantificação relativa por 1H NMR

𝑛 = m (g)/M (mol/g)

𝑚1

𝑚2=𝐼1𝐼2×𝑛2𝐻

𝑛𝐶1𝐻 ×

𝑀2

𝑀1

A integração (área dos sinais) é proporcional ao número de moles de protões que originam o sinal de ressonância

𝐼𝑐𝑜𝑚𝑝𝑜𝑠𝑡𝑜 1

𝐼𝑐𝑜𝑚𝑝𝑜𝑠𝑡𝑜 2=𝑛𝑐𝑜𝑚𝑝𝑜𝑠𝑡𝑜 1

𝑛𝑐𝑜𝑚𝑝𝑜𝑠𝑡𝑜 2×𝑛𝑐𝑜𝑚𝑝𝑜𝑠𝑡𝑜 1𝐻

𝑛𝑐𝑜𝑚𝑝𝑜𝑠𝑡𝑜 2𝐻



CHCl3

H2ONH

NH

NH

2`,6`

3`,5`

4`

2CH

3CH2

N-CH2

N-CH3

3CH3 2``CH3

4CH3

1H NMR 400 MHz, CDCl3

𝑚1

𝑚2=𝐼1𝐼2×𝑛2𝐻

𝑛𝐶1𝐻 ×

𝑀2

𝑀1

𝑚1

𝑚2=

𝐼𝑀𝑒𝐼𝑁𝑀𝑒

=4,7418

2,9789

N-etilcationa = 61,4%Bufedrona = 38,6%

Bufedrona

N-etilcationa

Peak for integration?



Quantificação absoluta por 1H NMR(método do padrão interno)

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

𝑚𝑐𝑜𝑚𝑝𝑜𝑠𝑡𝑜𝑤 =

𝐼𝐶𝑜𝑚𝑝𝑜𝑠𝑡𝑜

𝐼𝑃𝐼×

𝑛𝑃𝐼𝐻

𝑛𝐶𝑜𝑚𝑝𝑜𝑠𝑡𝑜𝐻 ×

𝑀𝐶𝑜𝑚𝑝𝑜𝑠𝑡𝑜

𝑀𝑃𝐼×𝑚𝑃𝐼

𝑊 × 𝑃𝑃𝐼 × 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

𝐼𝐶𝑜𝑚𝑝𝑜𝑠𝑡𝑜

𝐼𝑃𝐼=𝑛𝐶𝑜𝑚𝑝𝑜𝑠𝑡𝑜

𝑛𝑃𝐼×𝑛𝐶𝑜𝑚𝑝𝑜𝑠𝑡𝑜𝐻

𝑛𝑃𝐼𝐻



Quantification absoluta 1H NMR

Carateristicas de um bom PI

➢ Baixa volatilidade

➢ T1 (tempos de relaxação relativamente pequenos)

➢ Solubilidade semelhante ao analíto

➢ Químicamente inerte

➢ Contenha o núcleo de interesse (1H)➢ Sinal de ressonância diferente do analíto➢ De pureza conhecida

MA

Ácido maleico



Solução 108.17 mg/10 mL D2O

Ácido Maleico (MA)

C07Rush qNMRPentedrona.HCl : 70%

Metilamina.HCl: 19%

Cafeína: 8%

Isopentedrona.HCl: 3%

C07Rush 1H qNMR 400 MHz, D2O, 11.56mg/500mL

Pentedrona

Isopentedrona

MeNH2

PI= 100



4F-PBP: A Novel NPS

• 3 seized white powders

• Comparison with MS Libraries - MDPBP

• But….



49

4,0

01

4,1

17

4,2

33

4,3

49

4,4

65

4,5

81

4,6

97

4,8

13

4,9

29

5,0

45

5,1

61

5,2

77

5,3

93

5,5

09

5,6

26

5,7

42

5,8

58

5,9

74

6,0

9

6,2

06

6,3

22

6,4

38

6,5

54

6,6

7

6,7

86

6,9

02

7,0

18

7,1

34

7,2

5

7,3

66

7,4

82

7,5

99

7,7

15

7,8

31

7,9

47

8,0

63

8,1

79

8,2

95

8,4

11

8,5

27

8,6

43

8,7

59

8,8

75

8,9

91

Rt= 4.979min

Rt= 8.289min

Sample 1

Sample 2

Sample 3

Different Rt at same conditions different compounds

CASE 1 – 4F-PBP a Novel NSP



(Text File) Scan 466 (4.796 min): 18530-1.D\ data.ms

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2800

50

100

42 55 70

82

95

112

123

133 146 161 177 190 206 220 233 Sample 1

(Text File) Scan 1069 (8.292 min): 18823.D\ data.ms

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2800

50

100

4255 65

70

77 84

96

105

112

121

127 135

149

154 162 175 190 201 215232

241 253 261 Sample 2


0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2800

50

100

42

55

70

75

82

95

112

123

133 149 161 171 179 187 198206

217 233

Sample 3

MS spectra fragmentation pattern analysed



(Text File) #2: Flephedrone; (4-fluorophenyl)-2-methylaminopropan-1-one; 4-FMC; fluoromethcathinone

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2800

50

100

4250

58

62 69

75

81

95

101 109123

150 166


0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2800

50

100

42 55 70

82

95

112

123

133 146 161 177 190 206 220 233

Sample 1

?Sample 3

MS spectra fragmentation pattern analysed

m/z 105

123-104=19 F



Sample 1Sample 3

MDPBP

Flefedrona

4-MEC

1H NMR Spectra Aromatic pattern

4F-PBP

Sample 1Sample 3

Confirmation 19F NMR

NMR is crucial to assign the substituent position in the aromatic ring



NMR allows the differentiation of positional isomers

sample 1sample 3

Crucial to assign the substituent position in the aromatic ring



sample 1

sample 3

MDPBP

1H NMR in D2O, why this solvent?

4F-PBP

Is there another compound present not detected in GC-MS?

Yes, the mio-inositol

mio-inositol

NMR alows the identification of compounds not detected in routine GC-MS



H-1,3ax

H-4,6ax

H-5ax

Ha-1” Hb-1”

Ha-4”Hb-4”

H-2eq

1H NMR 400 MHz, D2O

Amostra 1

Padrão do mio-inositol

NMR allows the identification of NPS also in mixtures

2D 13C/1H HSQC



NMR allows the absolute quantification

Sample 1

Sample 3

Sample 1

4F-PBP

56

myo-inositol

Purity > 99%

White powders

D2O

Sample 1 61% 38%

Sample 3 57% 42%

IS

IS

1H NMR

1H qNMR

MDPBP



NMR after purifications the with CDCl3

1H COSY H-H

HSQC H-C13C APTHMBC H-C

19F

H. Gaspar et al, 4F-PBP , a new substance of abuse: structural characterization and purity NMR profiling, Forensic. Science International 2015, (252) 168



NMR Allows the differentiation free base/sal

1H NMR CDCl3

+NH

NMR after purifications



IC allows the identification of the salt form

Crucial to distinguish hydroclhorides or bromide salts

Nitrato

Brometo

fosfato

ortofosfato

Cloreto

FluoretoNitrito

Ion Exchange Chromatography

4F-PBP Hydrochloride

4F-PBP

Cl- Standard



4F-PBP: A novel NPS

4F-PBP

White powder

4F-α-PVP2 deaths in Sweden 2014

Risks and effects

Buy in the Net

α-PBPPortaria nº154/2013

DL nº54/2013 – artigo 3º

Illegal in Portugal

Novel NPS In Europe



Caso 2 4F-PBP: Uma nova NSP



Público 28 May 2015



7.96d

6.93d

7.60d

7.45s7.12d

7.96d

6.93d

7.96d

6.93d 7.60d7.45s

7.60d

7.12d

7.12d

7.45s

Which NMR technique?



7.96d 6.93d7.60d 7.45s 7.12s

NOESY

6-APB

5-APB

7.60d

7.96d

6.93d

7.96d

6.93d 7.45s

7.60d

7.12d

7.12d

7.45s



?2xCH2



Yes2xCH2



43.90

71.62

171.49176.39



4MPHP

4MPHP and 4MMC?



2H3H

2H2H

3H

4MPHP



CH2CH2CH2CH3



HBr, Br2

?CH2CH2CH2CH3



2H3H

2H2H

3H

4MPHP

?



HBr, Br2



2H3H

2H2H

3H

4MPHP



Caso 1 – Canabinoide em Microselos

78

Análise de GC-MS

4 estruturas possíveis!

AMB-FUBINACA

Isomero 5 - AMB-FUBINACA

AB-FUBINACA

Isomero 5 - AB-FUBINACA




79

M = 383

M = 383

M = 368

M = 368




80

Análise de GC-MS

Fragmentos de massas iguaisDificil de distinguir sem padrões!

AMB-FUBINACA


AB-FUBINACA


m/z = 109

m/z = 253

m/z = 324

m/z = 324

M = 383

M = 383

M = 368

M = 368



81


AMB-FUBINACA Isomero 5 - AMB-FUBINACA AB-FUBINACA Isomero 5 - AB-FUBINACA

OCH31H RMN, 400 MHz, DMSO-d6




82

Análise de RMN

AMB-FUBINACA


AB-FUBINACA


1H RMN, 400 MHz, DMSO-d6

RMN permitiu identificar o AMB-FUBINACA sem padrão!



CafeinaIdentificar sem ter pistas?Pescar um compostonuma mistura?



Referência

162.40 d140.31 d130.80 d124.20 d115.93 d114.70 d80.4363.6254.1342.9614.61



X

XX

X

Referência

162.40 d140.31 d130.80 d124.20 d115.93 d114.70 d80.4363.6254.1342.9614.61



X

XX

X

Referência

162.40 d140.31 d130.80 d124.20 d115.93 d114.70 d80.4363.6254.1342.9614.61

XX

X

X

X



X

XX

X

XX

X

X

X

HMBC



19F= -113.19