HPLCDADFD EM EL ANLISIS DE ANFETAMINAS CICLADAS

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HPLC-DAD/FD EM EL ANÁLISIS DE ANFETAMINAS CICLADAS
José Luiz da Costa Alice A da Matta Chasin
BRASIL
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DOB ou BromoDMA 4 Bromo 2,5 dimetoxianfetamina
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D O B
2,5-dimetoxi-4-bromoanfetamina Cápsula do medo ou
cápsula do vento
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Êxtase in São Paulo
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Number of tablet seizures in 2002 and 2003.
(source Núcleo de Entorpecentes IC/SP)
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Metilenodioxianfetaminas
MDMA (êxtase) 3,4-metilenodioximetanfetamina
MDEA (Eve) 3,4-metilenodioxietilanfetamina
MDA 3,4-metilenodioxianfetamina
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• HPLC/DAD
• V Advantages
• Absorption spectrum comparable to library
• Disadvanges
• MDMA, MDA, MDEA e MBDB have the same absorption
  spectrum
• At 200 nm is very near to the wave length at
  which the solvents like metanol and acetonitril
  absorb the UV light (High purity solvent)
• Isocratic conditions (gradients would cause drift
  of the background)
• The LD is not good enough for blood samples
  (130-200 ng/ml) with the HPLC-UV absorbance
  detection

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Spectrogram of MDA, MDMA and MDEA, 20µg/ml
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Chromatogram of MDMA, MDEA and MDA (10 ug/ml)
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Quantitative analysis HPLC/DAD

• HPLC LaChrom serie 7000 (Merck-Hitachi).
• Chromatographic column C18 LiChrospher 100
  250x4mm, 5?m 30ºC
• Mobile Phase phosphate bufferacetonitrilemetha
  noltrietilamine
  (60075251,5) flow rate 1,0 ml/min
• Detection 190 a 320 nm (quantification at 200
  nm)
• Calibration curve 20/15/10/5/2/1 ng/ml r2 gt
  0,9992

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Material e método

• comprimidos apreendidos que deram entrada no
  Núcleo de Entorpecentes do IC/SP (n31)
• Análise quantitativa ? HPLC/DAD
• Análise qualitativa (pesquisa de adulterantes)
  GC/FID

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Sample preparation
Comprimido
pesar triturar
Solução metanólica 0,2
diluir 1100
Pesquisa de adulterantes GC/FID
Quantificação do MDMA HPLC/DAD
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( B )
( A )

• Standard cromatogram of MDA, MDMA e MDEA.
• Seizure tablet.

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Figura 3. Distribuição dos comprimidos analisados
em classes de acordo com a concentração de MDMA.
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Tabela 01. Classificação dos comprimidos de
êxtase analisados de acordo com a quantidade de
MDMA.
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Análise qualitativa

• GC Shimadzu modelo 17A.
• Coluna DB-5, 30m X 0,25mm, 5?m.
• Temperatura da coluna
• 150ºC (5min) 8ºC/min 210ºC 15ºC/min
  260ºC (5 min).
• Temperatura do injetor 200ºC
• Temperatura do detector 280ºC
• Fluxo do gás de arraste (N2) 2,0 ml/min, split
  110

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( C )
( B )
( A )

• Figura 5. Pesquisa de adulterantes presentes em
  comprimidos de êxtase por GC/FID.
• Cromatograma de solução padrão de possíveis
  adulterantes.
• Cromatograma de amostra de êxtase adulterado com
  cafeína
• Cromatograma de amostra de êxtase adulterado com
  efedrina.

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• Advantages HPLC/FD
• The methylenedioxylated amphetamines, such as
  MDMA, MDEA, MDA and MBDB are naturally
  fluorescent
• There is high selectivity and sensitivity, which
  enables quantification at concentrations as low
  as 10 ng/ml
• No endogenous interferences were observed during
  the analysis of the blank urines. Selectivity of
  the method was verified, not only with common
  substances that can appear in seized tablets,
  such as caffeine, paracetamol, ketamine and
  ephedrine, but also with some drugs such as
  cocaine, amphetamine, methamphetamine
• A very simple chromatogram can be obtained
• Good linearity correlation coefficient (r2) was
  higher than 0.98 for the studied dynamic range
  (50-2,000 ng/ml).
• LD and LQ compatible with NIDA cut-off (200
  ng/ml)
• Disadvantages
• There are only a few substances that are
  detectable by fluorimetric detection.

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EXPERIMENTAL

• High performance liquid chromatograph series 1100
  HPLC coupled with a model 1046A programmable
  fluorescence detector (Hewlett-Packard ?, Little
  Falls, DE, USA). The excitation and emission
  wavelengths were set to 285 and 324 nm,
  respectively (?ex288 nm ?em324 nm)
• Spherisorb ODS2 C18 column (125 mm x 4 mm,
  5?m, Hewlett Packard), maintained at 40ºC during
  the analysis
• mobile phase, pumped at 1.0 ml/min, was composed
  of 50 mM solution of sodium dodecyl sulfate in
  HPLC-grade water, pH adjusted to 4.0 with HCl 1M
  (60) and acetonitrile (40), in isocratic mode.

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Experimental

• Chemicals
• - MDMA, MDEA, MDA e MBDB 1,0 mg/ml Cerilliant?
• - cafeína, cetamina, cocaína, dextrometorfano,
  dietilpropiona, efedrina e femproporex 1,0 mg/ml
  (Núcleo de Análise Instrumental/IC-SP).
• Mobile phase
• dodecil sulfato de sódio 50mM pH
  4,0acetonitrila (6040)

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Fluxograma do procedimento para extração de MDMA,
MDEA e MDA em sangue total.
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Fluxograma do procedimento para extração de MDMA,
MDEA e MDA em urina.
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Whole blood
(A)
(B)
Chromatogram of (A) blank blood (B) blank
blood spiked with 50 ng/ml MDMA, MDEA e MDA and
internal standard (MBDB 100 ng/ml).
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Urine
(A)
(B)
Chromatogram of (A) blank urine spiked with
500ng/ml of MBDB (internal standard) and (B)
blank urine sample spiked with 500 ng/ml of
MDMA, MDEA, MDA and MBDB.
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salting out effect
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Good linearity
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Confidence parameters

• Limit of Detection
• whole blood 3 ng/ml
• urine MDA (10 ng/ml) MDMA (15 ng/ml) MDEA (20
  ng/ml)
• Limit of Quantification
• whole blood 5 ng/ml
• urine MDA (15 ng/ml) MDMA (20 ng/ml) MDEA (30
  ng/ml)

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Confidence parameters of the validated method for
MDMA, MDEA and MDA in whole blood and urine by
HPLC/FD

• Precision

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Confidence parameters of the validated method for
MDMA, MDEA and MDA in whole blood and urine by
HPLC/FD

• Acuracy and Recovery

.
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Chromatogram of urine sample from the Forensic
Toxicology Laboratory, MDA (69 ng/ml) and MDMA
(1804 ng/ml).
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Conclusions

•   Methodology can be carried out in any
  average-sized Forensic Toxicology Laboratory
• Adequate specificity and safe analytical
  parameters for verification of the use of Ecstasy
  e Eve
• Can be used as a method of confirmation.

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José Luiz da Costa
XIV BRAZILIAN CONGRESS OF TOXICOLOGY- RECIFE
PE Oct 9-12
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MUCHAS GRACIAS!!!!
alimoily_at_usp.br