UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE...
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UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE CIÊNCIAS MÉDICAS KELLY FRANCISCO DA CUNHA ESTUDO DA ESTABILIDADE DE NOVAS SUBSTÂNCIAS PSICOATIVAS (NPS) EM AMOSTRAS DE SANGUE SECO EM PAPEL (DBS), E SUA APLICAÇÃO EM TOXICOLOGIA FORENSE STABILITY OF NEW PSYCHOACTIVE SUBSTANCES (NPS) IN DRIED BLOOD SPOTS (DBS), AND ITS APPLICATION IN FORENSIC TOXICOLOGY CAMPINAS 2018
Transcript of UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE...
UNIVERSIDADE ESTADUAL DE CAMPINAS
FACULDADE DE CIÊNCIAS MÉDICAS
KELLY FRANCISCO DA CUNHA
ESTUDO DA ESTABILIDADE DE NOVAS SUBSTÂNCIAS PSICOATIVAS (NPS) EM
AMOSTRAS DE SANGUE SECO EM PAPEL (DBS), E SUA APLICAÇÃO EM
TOXICOLOGIA FORENSE
STABILITY OF NEW PSYCHOACTIVE SUBSTANCES (NPS) IN DRIED BLOOD
SPOTS (DBS), AND ITS APPLICATION IN FORENSIC TOXICOLOGY
CAMPINAS
2018
KELLY FRANCISCO DA CUNHA
ESTUDO DA ESTABILIDADE DE NOVAS SUBSTÂNCIAS PSICOATIVAS (NSP) EM
AMOSTRAS DE SANGUE SECO EM PAPEL (DBS), E SUA APLICAÇÃO EM
TOXICOLOGIA FORENSE
STABILITY OF NEW PSYCHOACTIVE SUBSTANCES (NPS) IN DRIED BLOOD
SPOTS (DBS), AND ITS APPLICATION IN FORENSIC TOXICOLOGY
Dissertação apresentada à Faculdade de Ciências Médicas da
Universidade Estadual de Campinas como parte dos requisitos exigidos
para a obtenção do título de Mestra em Ciências, na área de
concentração Patologia Clínica.
Dissertation presented to the Faculty of Medical Sciences, University of
Campinas, in partial fulfillment of the requirements for the Master degree
in Sciences, in the concentration area of Clinical Pathology.
ORIENTADOR: Prof. Dr. José Luiz da Costa
ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA DISSERTAÇÃO DEFENDIDA
PELA ALUNA KELLY FRANCISCO DA CUNHA, E ORIENTADA PELO PROF. DR. JOSÉ
LUIZ DA COSTA.
CAMPINAS
2018
BANCA EXAMINADORA DA DEFESA DE MESTRADO
KELLY FRANCISCO DA CUNHA
ORIENTADOR: PROF. DR. JOSÉ LUIZ DA COSTA
MEMBROS:
1. PROF. DR. JOSÉ LUIZ DA COSTA
2. PROFA. DRA. MARY ANN FOGLIO
3. PROF. DR. ÁLVARO JOSÉ DOS SANTOS NETO
Programa de Pós-Graduação em Ciências Médicas da Faculdade de Ciências
Médicas da Universidade Estadual de Campinas.
A ata de defesa com as respectivas assinaturas dos membros da banca
examinadora encontra-se no processo de vida acadêmica do aluno.
Data: 23/02/2018
AGRADECIMENTOS
À Faculdade de Ciências Médicas da Universidade Estadual de Campinas e às
agências de fomento (FAPESP, CNPq e FAEPEX) por viabilizarem este trabalho.
Aos meus pais, Regina e Jorge, e minha irmã, Larissa, por toda compreensão e
apoio.
Ao meu orientador, Prof. Dr. José Luiz da Costa, por todo o ensinamento, orientação
e confiança depositados em mim em todos os momentos.
Ao farmacêutico, MSc Rafael Lanaro, por ter atuado diretamente no estabelecimento
da minha orientação, além de constantemente me co-orientar.
Aos demais funcionários e colegas do Centro de Informação e Assistência
Toxicológica de Campinas (CIATox)/Faculdade de Ciências Médicas, por tornarem o
trabalho diário menos exaustivo.
Aos colegas do Laboratório Thomson de Espectrometria de Massas do Instituto de
Química, por auxiliarem na execução desse trabalho.
RESUMO
Introdução: A classe de drogas de abuso conhecida como novas substâncias
psicoativas ou, ainda, drogas desenhadas (do inglês new psychoactive substances
(NPS) ou designer drugs) é composta por substâncias químicas obtidas a partir de
alterações estruturais de substâncias ou mistura de substâncias psicoativas já
existentes (e ilícitas), a fim de mimetizar e/ou potencializar os efeitos proporcionados
por estas, com a vantagem de circundar a legislação antidrogas vigente. Dentro da
classe das NPS, os grupos de maior destaque atualmente são das catinonas
sintéticas, dos canabinóides sintéticos e dos NBOMes (feniletilaminas N-2-
metoxibenzil substituídas). As catinonas sintéticas, vendidas pela internet como “sais
de banho” ou bath salts, são substâncias estruturalmente relacionadas a catinona, um
alcalóide presente no Catha edulis (Khat), com propriedades estimulantes. A classe
dos NBOMes é derivada da classe dos alucinógenos 2C, a partir da adição de um
grupo N-2-metoxibenzil na amina primária. Os NBOMes possuem ação agonista nos
receptores de serotonina, especialmente do subtipo 5-HT2A, o que confere os efeitos
alucinógenos. Objetivos: Desenvolver e validar métodos para identificação e
quantificação de algumas das NPS de maior relevância em amostras de manchas de
sangue seco em papel (do inglês dried blood spots, DBS) e comparar a estabilidade
entre dois tipos de amostras (DBS e sangue total), em três diferentes temperaturas
(ambiente, 4 °C e -20 °C) e períodos de armazenamento. Metodologia: As amostras
de DBS e sangue total foram analisadas em cromatógrafo líquido acoplado à
espectrômetro de massas com analisador de massas triplo quadrupolar (LC-MS/MS),
utilizando os métodos primeiramente desenvolvidos e validados para cada classe de
NPS (catinonas sintéticas e NBOMes). Resultados: A técnica de DBS apresentou um
aumento significativo da estabilidade das NPS em comparação à técnica convencional
de armazenamento de sangue total. Para os NBOMes, observou-se que os compostos
eram estáveis no DBS por período de tempo de até 6 meses, nas três temperaturas
estudadas, enquanto que no sangue total, os analitos tiveram uma queda maior que
20% da concentração em 15 ou 30 dias em temperatura ambiente e 180 dias para 4
°C. Para as catinonas, em temperatura ambiente, observou-se baixa estabilidade nas
duas matrizes. Porém, para armazenamento à 4 °C, observou-se uma queda na
concentração inicial maior que 20% com 60 e 90 dias para mefedrona e benzedrona
em DBS, respetivamente, contra 45 e 25 dias. Catinonas que possuem grupamento
metilenodioxi em sua estrutura, como a butilona e a pentilona, foram mais estáveis,
independente da matriz, em comparação àquelas com grupamento alquila no anel
aromático, como mefedrona e benzedrona. Conclusão: A técnica de DBS se mostrou
vantajosa para análises forenses em comparação à técnica convencional de
armazenamento de sangue total. Além de facilitar o armazenamento (por ocupar
menos espaço), sua extração se torna mais rápida e facilitada, já que envolve menos
etapas, além de tornar possível a identificação dos analitos de interesse por um maior
período de tempo, podendo facilmente ser aplicada na rotina laboratorial.
Palavras-chave: teste em amostras de sangue seco; drogas desenhadas; toxicologia
forense; cromatografia líquida; espectrometria de massas.
ABSTRACT
Introduction: New psychoactive substances (NPS) or designer drugs is a class of
drugs of abuse composed of chemical substances obtained from structural alterations
of substances or mixture of existing psychoactive substances (and illicit), in order to
mimic and/or maximize their effects, with the advantage of circumventing existing anti-
drug legislation. Within the NPS class, the most prominent groups currently are
synthetic cathinones, synthetic cannabinoids and NBOMes (phenylethylamines N-2-
methoxybenzyl substituted). Synthetic cathinones, sold by the internet as "bath salts",
are substances structurally related to cathinone, an alkaloid present in Catha edulis
(Khat), with stimulant properties. The class of NBOMes is derived from the
hallucinogen class 2C, from the addition of an N-2-methoxybenzyl group to the primary
amine. NBOMes have agonist action at serotonin receptors, especially the 5-HT2A
subtype, which confers the hallucinogenic effects. Objectives: To develop and
validate methods to identify and quantify some of the most relevant NPS in dried blood
spots (DBS) samples and to compare the stability between two matrixes (DBS and
whole blood), at three different temperatures (ambient, 4 °C and -20 °C) and storage
periods. Method: DBS and whole blood samples were analyzed using a liquid
chromatography tandem mass spectrometer with a triple quadrupole analyzer (LC-
MS/MS), using the developed and validated methods for each class of NPS (synthetic
cathinones and NBOMes). Results: The DBS technique showed a significant increase
in the NPS stability compared to the conventional whole blood storage technique. For
the NBOMes, the compounds were found to be stable in DBS for a time period of up
to 6 months, at the three temperatures studied, whereas in whole blood, the analytes
had a decreased higher than 20% of the initial concentration in 15 or 30 days at room
temperature and 180 days at 4 °C. For the cathinones, at room temperature, low
stability was observed in both matrixes. However, for storage at 4 °C, the initial
concentration decreased more than 20% at 60 and 90 days for mephedrone and
benzedrone in DBS, respectively, against 45 and 25 days. Cathinones having
methylenedioxy group in their structure, such as butylone and pentylone, were more
stable, independent of the matrix, compared to those with alkyl group on the aromatic
ring, such as mephedrone and benzedrone. Conclusion: The DBS technique proved
to be advantageous for forensic analysis compared to the conventional storage
technique. In addition to occupying less storage space, DBS extraction becomes faster
and easier, since it involves fewer steps, besides to make possible to identify the
analytes of interest for a longer period of time, which can easily be applied in the
laboratory routine.
Key words: dried blood spot testing; designer drugs; liquid chromatography; mass
spectrometry; forensic toxicology.
LISTA DE ABREVIATURAS E SIGLAS
4-MBC – Mefedrona (do inglês, mephedrone)
4-MMC – Benzedrona (do inglês, benzedrone)
ANOVA – Análise de variância (do inglês, one-way analysis of variance)
Ar – Argônio
bk-MBDB – Butilona (do inglês, butylone)
bk-MBDP – Pentilona (do inglês, pentylone)
CE – Energia de Colisão (do inglês, collision energy)
CEP – Comitê de Ética em Pesquisa da UNICAMP
CXP – Potencial de saída da cela de colisão (do inglês, collision cell exit potential)
DBS – Mancha de sangue seco em papel (do inglês, dried blood spots)
DEA – United States Drug Enforcement Administration
DP – Potencial do orifício (do inglês, declustering potential)
EMCDDA – European Monitoring Centre for Drugs and Drug Addiction
EP – Potencial de entrada (do inglês, entrance potential)
ESI – Fonte de ionização por electrospray
EWS – Early Warning System
FIA – Análise por injeção direta (do inglês, flow injection analysis)
GS1 – Pressão do gás de nebulização (do inglês, nebulizer gas)
GS2 – Pressão do gás de secagem (do inglês, auxiliary gas)
IS – Padrão interno (do inglês, internal standard)
LC-MS – Cromatógrafo líquido acoplado à espectrômetro de massas
LC-MS/MS - Cromatógrafo líquido acoplado à espectrômetro de massas sequencial
LLE – Extração líquido-líquido (do inglês, liquid-liquid extraction)
LOD – Limite de detecção (do inglês, limit of detection)
LOQ – Limite de quantificação (do inglês, limit of quantification)
LSD-d3 – Padrão deuterado da dietilamida do ácido lisérgico
MDMA-d5 – Padrão deuterado de metilenodioximetanfetamina
MRM – Monitoramento de reações múltiplas (do inglês, multiple reaction monitoring)
MTBE – Éter butil t-metílico
m/z – massa/carga
N2 – Nitrogênio
NA – Não informado (do inglês, not available), para o artigo dos NBOMes; Não
avaliado (do inglês, not evaluated) para o artigo das catinonas sintéticas
NPS – Novas substâncias psicoativas (do inglês, new psychoactive substances)
PP – Precipitação de proteínas (do inglês, protein precipitation)
Q1 – Quadrupolo 1
Q3 – Quadrupolo 3
QC – Controle de qualidade (do inglês, quality control)
RSD – Desvio padrão relativo (do inglês, relative standard deviation)
SPE – Extração em fase sólida (do inglês, solid-phase extraction)
SWGTOX – Scientific Working Group for Forensic Toxicology
UNODC – United Nations Office on Drugs and Crime
SUMÁRIO
1. INTRODUÇÃO .................................................................................................................... 13
1.1 Novas Substâncias Psicoativas (NPS) ........................................................................... 13
1.1.1 Feniletilaminas .................................................................................................................... 15
1.1.2 Catinonas sintéticas ........................................................................................................... 15
1.2 Estabilidade em análises toxicológicas.........................................................................16
1.3 Mancha de sangue seco em papel (DBS) ...................................................................... 17
2. OBJETIVOS ........................................................................................................................ 20
3. METODOLOGIA ................................................................................................................. 21
3.1 Análise de NBOMes ........................................................................................................... 21
3.1.1 Reagentes ........................................................................................................................... 21
3.1.2 Instrumentação ................................................................................................................... 21
3.1.3 Extração das amostras de sangue total para análise de NBOMes ............................ 22
3.1.4 Preparo e extração do DBS ................................................................................................ 22
3.1.4.1 Teste de saturação dos discos de DBS..................................................................23
3.1.4.2 Otimização de misturas...........................................................................................24
3.2 Catinonas sintéticas ........................................................................................................... 25
3.2.1 Reagentes ........................................................................................................................... 25
3.2.2 Instrumentação ................................................................................................................... 25
3.2.3 Extração das amostras de sangue total para análise de catinonas sintéticas ......... 26
3.2.4 Preparo e extração do DBS .............................................................................................. 27
3.2.4.1 Otimização de misturas...........................................................................................27
3.3 Validação ............................................................................................................................. 28
4. RESULTADOS .................................................................................................................... 29
4.1 Artigo 1 ................................................................................................................................. 29
4.2 Artigo 2 ................................................................................................................................. 53
4.3 Artigo 3 ................................................................................................................................. 70
5. DISCUSSÃO GERAL ......................................................................................................... 99
6. CONCLUSÃO ................................................................................................................... 102
7. REFERÊNCIAS ................................................................................................................ 103
8. ANEXOS ............................................................................................................................ 107
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1. INTRODUÇÃO
1.1 Novas Substâncias Psicoativas (NPS)
A classe de drogas de abuso conhecida como novas substâncias
psicoativas (do inglês new psychoactive substances (NPS) ou ainda designer drugs)
é composta por substâncias químicas obtidas a partir de alterações estruturais de
substâncias ou mistura de substâncias psicoativas já existentes (e ilícitas), a fim de
mimetizar e/ou potencializar os efeitos proporcionados por estas, com a vantagem de
circundar a legislação antidrogas vigente (já que não são listadas nas convenções
antidrogas mundiais - 1961 Single Convention on Narcotic Drugs e 1971 Convention
on Psychotropic Substances), mas que podem representar um problema de saúde
pública [1].
As NPS se iniciam, primeiramente, na União Europeia e EUA, e vem sido
vendidas através da internet ou lojas físicas em embalagens com dizeres “impróprio
para consumo humano” e com finalidade fictícia como sais de banho, incensos,
desodorizador de ambiente ou adubo de plantas, etc, circundando assim as
legislações de drogas de abuso. Atualmente, também podem ser comercializadas
virtualmente com a insígnia fictícia de padrões analíticos, com finalidade exclusiva de
pesquisa [2].
Dentro da classe das NPS, os grupos mais expressivos (em número de
substâncias) são os canabinóides sintéticos, as catinonas sintéticas e os derivados
das feniletilaminas. Além destes grupos, atualmente as NPS já se expandiram aos
análogos da triptamina, derivados dos alucinógenos psilocibina e fenilpiperazinas,
análogos dos fentanil e análogos dos benzodiazepínicos, os dois últimos grupos ainda
agrupados genericamente como “outras substâncias” pela classificação da United
Nations Office on Drugs and Crime (UNODC). Essas substâncias são usualmente
sintetizadas na Ásia, principalmente na China [3].
Uma das maiores preocupações associadas ao uso das NPS está
relacionado aos diversos casos de intoxicações agudas e/ou fatais já reportados em
diversos países [4-9]. Além disso, seu potencial de causar intoxicações mesmo em
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baixas doses exigem o desenvolvimento de métodos que utilizam técnicas analíticas
sensíveis, além de alta qualificação por parte dos toxicologistas para interpretar os
resultados obtidos.
Outros fatores de risco associados ao uso de NPS incluem a grande
variabilidade de concentração entre e/ou dos produtos, mais de um composto
psicoativo em um único produto, diferentes produtos vendidos sob o mesmo nome
comercial, falta de informação confiável relacionado aos efeitos adversos. Além disso,
pouco se conhece do seu metabolismo e interação com outras drogas e/ou fármacos
[10].
Diante dessas preocupações, acrescido do constante avanço e
surgimentos praticamente semanal de novas substâncias, países de todo mundo
estão tentando adequar seus sistemas de saúde e de repressão às drogas ao
problema das NPS. A título de exemplo, viu-se a necessidade de alterar na União
Europeia o seu recorrente Early-Warning System (EWS) [11]. Até 2005, o European
Monitoring Centre for Drugs and Drug Addiction (EMCDDA) recolhia dados sobre um
restrito número de medicamentos, a maioria controlados pelas convenções antidrogas
mundiais. A partir de 2005, o principal objetivo é divulgar rapidamente informações a
respeito das NPS – que não são controladas pelas leis antidrogas, seus possíveis
riscos sociais e de saúde, além de tentar controlar e identificar novos compostos em
circulação na Europa. Relatórios anuais são publicados pelo EMCDDCA. Em 2014,
101 novas substâncias foram notificadas ao EWS, entre elas 31 catinonas sintéticas,
30 canabinoides sintéticos e 9 feniletilaminas [12].
De acordo com o relatório mundial de drogas publicado pela UNODC de
2017, 739 NPS foram identificadas no mundo entre 2009 e 2016; apenas em 2015,
500 NPS estavam disponíveis no mercado mundial. Os grupos com maiores
quantidades de representantes ainda são os das catinonas sintéticas e canabinóides
sintéticos que apresentam uma proporção de 36% e 33%, respectivamente, frente aos
demais grupos das NPS [13].
O expressivo número de NPS se deve ao constante lançamento de
moléculas que possuam o efeito psicoativo desejado, mas ainda não são enquadradas
como ilegais dentro das legislações de drogas vigentes em cada país. Uma vez que a
grande maioria dos países mantem uma legislação baseada na substância, a
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alteração estrutural mínima pode, muitas vezes, manter o efeito psicoativo, mas tornar
legalizada essa nova molécula, até que uma atualização do instrumento legal do país
seja feita. Assim, na mesma rapidez com que essas moléculas surgem, acabam
desaparecendo do mercado em poucos meses [14].
1.1.1 Feniletilaminas
O grupo das feniletilaminas são de substâncias derivadas dos alucinógenos
2C, bastante consumidos na década de 80. Os primeiros derivados do 2C foram os
NBOMes, feniletilaminas N-benzil substituída, o que aumenta sua ação agonista sob
o receptor 5-HT2A, responsável pela propriedade alucinógena. Além disso, são
farmacologicamente ativos em baixas doses (microgramas). Efeitos alucinógenos
podem ser alcançados com doses que vão de 50 µg até 800 µg. A duração dos efeitos,
dependente da via de administração, pode variar de 3 a 10 horas. Podem ser
consumidos na forma de comprimidos, pó, líquido, spray ou, mais comumente, em
papéis conhecidos como “selo” (forma comumente utilizada para venda/uso do
alucinógeno LSD). Além dos efeitos alucinógenos, outros efeitos já reportados incluem
euforia, aumento da sociabilidade, aumento das sensações visuais, auditivas, olfativas
e táteis, além de efeitos de despersonalização. Já os efeitos adversos incluem
náusea, vômito, dor de cabeça, sudorese, ansiedade, pânico, agitação, agressividade,
insônia, psicose aguda, rabdomiólise, taquicardia, hipertensão e hipertermia [15].
Diversos casos de morte relacionados ao uso de NBOMes já foram descritos na
literatura [15-17].
1.1.2 Catinonas sintéticas
As Catinonas sintéticas derivam da catinona, molécula naturalmente
presente na planta Khat (Catha edulis), que apresenta efeitos semelhantes ao da
anfetamina. O ato de mascar folhas do Khat, principalmente na região da Península
Arábica e Leste da África, é antigo e tem como benefícios reportados por seus
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usuários o de aumentar a sociabilidade, aumento de energia e bem-estar, melhorando
a capacidade de trabalho e estado de alerta [18].
Usualmente, as catinonas sintéticas são vendidas sob o rótulo de sais de
banho, mas também podem ser encontradas em comprimidos e vendidos como 3,4-
metilenodioximetilanfetamina (MDMA, ecstasy), substância com ação estimulante do
sistema nervoso central, aumentando os níveis de noradrenalina, dopamina e
serotonina extracelularmente, além de inibir sua receptação. As catinonas sintéticas
apresentam entre si diferentes seletividades para esses três neurotransmissores [19].
Até março de 2015, 77 catinonas sintéticas foram reportadas ao EWS. Só em 2014,
foram 31 catinonas reportadas, o que mostra o crescimento exponencial dessas
substâncias. Vale ressaltar que esse número se restringe apenas aos casos
reportados ao EWS, o que provavelmente não condiz com o número de catinonas
sintéticas disponíveis no mercado ilícito global [20].
A dose usual das catinonas sintéticas pode variar entre 25 e 250 mg e sua
forma de uso mais usualmente se encontra na administração oral, na forma de
comprimidos (semelhante aos de ecstasy) ou cápsulas, ou nasal e intravenosa [21].
Assim como os derivados da feniletilaminas, diversos casos de
intoxicações são reportados na literatura em associação ao uso de catinonas
sintéticas, nem sempre sendo possível determinar como a causa da morte,
principalmente devido a associação de outras drogas de abuso (ou até mesmo outras
NPS) [22-24].
1.2 Estabilidade em análises toxicológicas
Em análises toxicológicas, resultados falsos negativos podem levar a
interpretações erradas, assim como dados quantitativos sem conhecimento das
condições de transporte e armazenamento das amostras podem levar à super- ou
subestimativa dos efeitos. Conhecer a estabilidade dos analitos de interesse na matriz
biológica de estudo é importante para a interpretação dos resultados obtidos na
análise e, juntamente com o histórico do caso, fundamentar a causa mortis [25].
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Estudos de estabilidade das substâncias em diferentes matrizes se tornou
parte do desenvolvimento e validação de novos métodos de análise. A ideia principal
é manter as amostras de estudo dentro das condições reais de armazenamento que
uma amostra verdadeira estaria sujeita até a análise ou mesmo para uma reanálise e,
assim, determinar o tempo que o analito se mantém com a concentração inicial do
estudo. A maior parte dos guias de validação aceitam uma variação de ±15 ou ±20%
para considerarem o analito estável naquela matriz e condição de estocagem [26].
Durante o período de estocagem pode ocorrer degradação da droga bruta,
por ação enzimática e/ou pela instabilidade química da substância. Dependendo da
velocidade da degradação, é possível não mais detectar o produto, já completamente
convertido no seu produto de degradação. A velocidade de degradação também será
dependente de fatores como temperatura, pH, exposição à luz e tipo de matriz. Soh e
Elliott avaliaram a estabilidade de 13 NPS em sangue total e plasma, de diferentes
classes, em temperatura ambiente e por até 37 dias. 4-methylethcathinone (4-MEC) e
α-methyltryptamine (AMT) se mostraram instáveis após 21 dias nas duas matrizes
[27]. O fato de diversas NPS serem detectadas em ordem de concentração abaixo de
ng/mL exige não só equipamentos sensíveis, como maior cuidado e conhecimento da
estabilidade desses analitos, para chegar a interpretações corretas.
1.3 Mancha de sangue seco em papel (DBS)
O sangue (e seus derivados soro e plasma) é o fluido biológico mais
utilizado em análises toxicológicas, isto porque a concentração do xenobiótico neste
material frequentemente apresenta boa correlação com seu efeito (tóxico). Contudo,
uma vez que a coleta desta matriz biológica é sempre invasiva, vários cuidados devem
ser tomados para garantir a segurança do paciente e a qualidade da amostra coletada,
tais como assepsia do local da coleta, a qualidade e a quantidade de anticoagulante
empregado (escolhido de acordo com a análise que será realizada), o tipo de
armazenagem (temperatura ambiente, sob refrigeração ou congelado) e o tempo
decorrido entre a coleta e a análise [28].
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A técnica de DBS (dried blood spots ou manchas de sangue seco em papel)
é utilizada desde a década de 60 na determinação de casos de fenilcetonúria em
recém-nascidos. Crescente é o número de publicações mostrando estudos nas áreas
de monitorização terapêutica de fármacos, diagnóstico de doenças, identificação de
drogas de abuso em eventos esportivos (controle antidopagem) e na toxicologia
ocupacional para a monitorização da exposição de trabalhadores, já que possuem
vantagens em comparação com as amostras convencionais. As técnicas que
empregam o conceito de manchas secas (como a DBS), são exemplos de técnicas de
microamostragem, que pode ser definida como a utilização de uma quantidade inferior
a 50 μL de amostra biológica para realização as análises (toxicológicas) [29].
A técnica de DBS oferece inúmeras vantagens em relação a outras formas
de obtenção de amostras de sangue que se têm disponíveis, que dependem da
punção da veia do paciente por pessoa especializada, são invasivas, com risco de
surgimento de edemas ou flebites, além de necessitarem de um volume maior de
amostra (mínimo de 0,5 mL para neonatos), o que leva a maior desconforto do doador,
principalmente crianças ou animais. Já o DBS necessita de um pequeno volume de
amostra, resultando em menores custos de transporte e armazenamento, e torna
menos invasiva para pacientes ou para animais de experimentação, podendo ser
treinado para coleta na própria residência. E, ainda, há estudos que têm demonstrado
que, por ter sido submetida a processo de secagem, há redução da degradação de
compostos contendo ésteres e carbonatos, por hidrólise química e enzimática,
oferecendo maior estabilidade aos analitos ali presentes [30].
Na área de monitoramento terapêutico, onde se estuda a possibilidade de
o paciente realizar a coleta da amostra de DBS em sua residência e enviar o cartão
por correio, Silva et al analisaram a estabilidade da fluoxetina e norfluoxetina em
amostra de DBS mantidas a 25 e 45 °C por 14 dias e ambos analitos foram estáveis
nessas condições [31], ou seja, mantiveram suas concentrações dentro de ±20% da
concentração inicial, critério de estabilidade adotado pela Scientific Working Group for
Forensic Toxicology (SWGTOX) [32].
Linder et al também avaliaram a estabilidade de amostras de DBS para os
fármacos carbamazepina, lamotrigina, levetiracetam e ácido valpróico por um ano em
19
temperatura ambiente, 48 horas para 50 °C e 3 meses para -80 °C e todos analitos se
mantiveram estáveis nas três condições estudadas [33].
Atualmente, devido o baixo custo da coleta e um menor espaço de
armazenamento para replicatas de uma mesma amostra, é discutido o uso de DBS
para construção de biobancos de proteínas que poderiam se tornar futuros
biomarcadores de doenças. Björkesten et al estudou a estabilidade de noventa e duas
proteínas com relevância na área de oncologia a temperaturas de 4 e -24 °C por 30
anos e mostrou que não houve prejuízo na detecção da maioria das proteínas
estudadas, principalmente quando mantidas a -24 °C [34].
20
2. OBJETIVOS
Estudar a estabilidade de algumas das mais relevantes NPS, pertencentes
aos grupos dos NBOMes e das catinonas sintéticas, em amostras de manchas de
sangue seco em papel.
Para atingir este objetivo, foram delineados os seguintes objetivos
específicos:
• Desenvolver e validar método analítico para identificação e quantificação
de NPS em amostras de DBS;
• Desenvolver e validar método analítico para identificação e quantificação
de NPS em amostras de sangue total;
• Comparar a estabilidade das NPS nas duas matrizes, quando
armazenadas em diferentes condições de temperatura (ambiente, 4 °C e -
20 °C) por até 6 meses.
21
3. METODOLOGIA
3.1 Análise de NBOMes
3.1.1 Reagentes
Os padrões analíticos certificados do 25B 100%, 25C 100%, 25D, 25E,
25G, 25H 100% e 25I-NBOMe 100% foram adquiridos da Cayman Chemical (Ann
Arbor, MI, EUA) e do LSD-d3 99,7%, usado como padrão interno, da Cerilliant®
(Round Rock, TX, EUA). Acetonitrila, metanol e ácido fórmico, grau HPLC, foram
adquiridos da Scharlau (Barcelona, Espanha). Éter butil t-metílico (MTBE) foi obtido
da Sigma-Aldrich® (St Louis, MO, EUA). Tetraborato de sódio foi adquirido da LS
Chemicals (Ribeirão Preto, SP, Brasil). Água ultra-pura foi obtida de equipamento Milli-
Q® RG da Millipore (Billerica, MA, USA).
3.1.2 Instrumentação
As análises de NBOMes nas matrizes DBS e sangue total foram realizadas
em sistema de cromatografia líquida 1260 Infinity (Agilent Technologies, Santa Clara,
CA, USA) acoplado a espectrômetro de massas com analisador de massas híbrido
triplo quadrupolo-iontrap linear 5500-QTRAP® (ABSciex, Concord, ON, Canadá). Foi
utilizada fonte de ionização do tipo electrospray em modo positivo. Nitrogênio foi
usado como gás de secagem, de nebulização e de colisão. Os parâmetros otimizados
da fonte de ionização foram: temperatura de 700 °C; voltagem do spray de 3,5 kV;
potencial de entrada (EP), 10 V; pressão do gás de nebulização (GS1), 40 psi; pressão
do gás de secagem (GS2), 50 psi; pressão do gás de dessolvatação (curtain gas), 25
psi. A aquisição de dados foi realizada no modo de aquisição por monitoramento de
reações múltiplas (MRM), sendo monitorada duas transições para cada analito, a mais
intensa usada para a quantificação e a segunda como qualificadora (para
confirmação). A otimização das voltagens de transmissão e energias de fragmentação
de cada MRM foi realizada por infusão direta de soluções padrões de cada analito (20
ng/mL, em água/metanol, 1:1, v/v). Software Analyst® 1.6.2 foi usado para aquisição
22
dos dados e MultiQuant™ 3.0.1 para o processamento (ABSciex, Concord, ON,
Canadá).
Para a separação cromatográfica foi utilizada coluna de fase reversa C18
Atlantis® T3 (150 x 3 mm, 3 µm, Waters Dublin, Irlanda), termostatizada a 40 °C. A
fase móvel foi composta de água ultra-pura na fase A e acetonitrila na fase B, ambas
contendo ácido fórmico (0,1%, v/v) como aditivo, com vazão de 0,5 mL/min. O
gradiente de eluição iniciou-se com 10% de B, seguido do aumento linear até 95% em
15 min, mantido nesta condição por 2 min e retornando à condição inicial em 0,5 min
e resultando em um total de análise de 22 min.
3.1.3 Extração das amostras de sangue total para análise de NBOMes
Para a extração das amostras de sangue total para análise de NBOMes, foi
realizada extração líquido-líquido em tubo de polipropileno de 2 mL, para onde foram
transferidos inicialmente 300 µL de sangue total adicionado, seguido por 50 µL de
solução de LSD-d3 (10 ng/mL, utilizado como padrão interno), 300 µL de solução
saturada de tetraborato de sódio e 1,2 mL de MTBE. Essa mistura foi agitada com
auxílio de vórtex por 5 min, centrifugada a 8.000 rpm por 5 min e 900 µL da fase
orgânica foi transferida para outro tubo para ser evaporada completamente, sob fluxo
constante de N2 e aquecimento (40 °C). As amostras foram ressuspendidas com 500
µL de metanol e 5 µL foram injetados no sistema cromatográfico descrito
anteriormente.
3.1.4 Preparo e extração do DBS
Para preparo das amostras de sangue adicionado, às amostras de sangue
total branco foi adicionada solução de trabalho nas concentrações do calibrador ou
controle. Estas amostras foram então aplicadas, à volume fixo de 15 µL, nos discos
de DBS previamente cortados (6 mm) e mantidos em temperatura ambiente (25 °C)
para completa secagem (3 h). A Figura 1 mostra um esquema do preparo das
amostras de DBS.
23
Fig. 1 Fluxograma do preparo da amostra de sangue total adicionado e sua
aplicação nos discos de DBS previamente cortados do cartão Whatman®.
Para a extração, cada disco foi transferido para tubo de polipropileno de 2
mL, onde foram adicionados 300 µL de solvente extrator (metanol/água, 75:25 v/v)
contendo o padrão interno LSD-d3 (0,5 ng/mL). As amostram foram agitadas com
auxílio de vórtex por 10 min, centrifugadas a 10.000 rpm por 5 min, 150 µL da solução
extratora sobrenadante foi transferida para vials e 20 µL foi injetado no sistema
analítico descrito no item 3.1.2.
3.1.4.1 Teste de saturação dos discos de DBS
Para que o máximo de sangue total fosse utilizado no preparo do DBS,
sem que houvesse seu extravasamento devido a saturação do papel, testou-se,
em duplicata, a aplicação volumes de amostra crescentes aos discos de DBS. O
volume de 15 µL foi adotado para os ensaios, por ser o maior volume de sangue
aplicado ao disco, sem que houvesse seu extravasamento para a bancada (Figura
2).
24
Fig. 2 Resultado do teste de saturação dos discos de DBS. Utilizando 20
µL de amostra de sangue total, observou-se o seu extravasamento. Dessa forma, foi
padronizado o uso de 15 µL de amostra por disco.
3.1.4.2 Otimização de misturas
Para determinar qual seria o melhor solvente extrator ou a mistura
destes, foi realizado um. Amostras de DBS foram preparadas na concentração de 10
ng/mL e, após secagem completa, foram extraídas, em duplicata, utilizando como
solvente extrator: metanol, acetonitrila, água, metanol-acetonitrila 1:1, metanol-água
1:1, acetonitrila-água 1:1 e metanol-acetonitrila-água 1:1:1. Essas amostras foram
injetadas e as áreas absolutas de cada analito foram avaliadas por planejamento
experimental do tipo centroide simplex, em software Unscrambler® X 10.3 (CAMO
Software AS, Oslo, Norway). Dessa forma, foi determinado que a melhor proporção
entre os solventes testados para obter máxima recuperação dos analitos foi metanol-
acetonitrila 75:25, v/v.
25
Fig. 3 Gráfico da melhor proporção de solventes a ser utilizado para conseguir máxima
extração dos NBOMes.
3.2 Catinonas sintéticas
3.2.1 Reagentes
Os padrões analíticos certificados de butilona 99,1%, pentilona 99,2%,
mefedrona 99,8% e benzedrona 100,0% foram adquiridos da Cayman Chemical (Ann
Arbor, MI, EUA) e do MDMA-d5 99,9%, usado como padrão interno, da Cerilliant®
(Round Rock, TX, EUA). Metanol, grau HPLC, foi adquirido da Scharlau (Barcelona,
Espanha). Éter butil t-metílico (MTBE) e ácido fórmico foram obtidos da Sigma-
Aldrich® (St Louis, MO, EUA). Tetraborato de sódio foi adquirido da LS Chemicals
(Ribeirão Preto, SP, Brasil). Água ultra-pura foi obtida de equipamento Milli-Q® RG da
Millipore (Billerica, MA, USA).
3.2.2 Instrumentação
As análises de catinonas sintéticas foram realizadas em sistema de
cromatografia líquida Nexera UHPLC, acoplado a espectrômetro de massas com
analisador de massas triplo quadrupolar LCMS8040 (Shimadzu, Kyoto, Japão).
26
Para as análises em DBS foi usada coluna Kinetex™ XB-C18 (50 x 4,6 mm,
2,6 µm, Phenomenex®, EUA), termostatizada a 40 °C. A fase móvel foi composta por
água ultra-pura (A) e metanol (B), tendo ácido fórmico (0,1%, v/v) como aditivo e vazão
de 0,35 mL/min. O gradiente de eluição inicia com 10% de B, seguido de aumento
linear até 95% de B em 3,9 min, mantido nesta condição por 2 min e retornando as
condições iniciais em 0,2 min, obtendo-se tempo total de análise de 8,5 min. Para as
análises de catinonas sintéticas em sangue total, foi utilizada coluna Force™ Biphenyl
(50 x 2,1 mm, 1,8 µm, Restek®, EUA), termostatizada a 50 °C, com mesma fase móvel
descrita para análises em DBS, porém vazão de 0,5 mL/min e gradiente de eluição
iniciando com 5% de B, seguida de aumento linear até 95% de B em 2,7 min, mantido
nesta condição por 1,4 min e retornando as condições iniciais em 0,1 min, obtendo-se
tempo total de análise de 6 min.
O espectrômetro de massas foi equipado com fonte de ionização por
electrospray em modo positivo para essas análises. Os parâmetros da fonte de
ionização foram otimizados, e as melhores condições obtidas foram: temperatura do
bloco, 400 °C; voltagem do capilar, 4,5 kV; fluxo do gás nebulizador (N2), 3 L/min;
temperatura da linha de dessolvatação, 250 °C; fluxo do gás de secagem (N2) 15 L/min
e pressão do gás de colisão (Ar), 230 kPa. A aquisição de dados foi realizada no modo
MRM. Para cada analito, duas transições foram monitoradas, sendo a mais intensa
usada para a quantificação e a segunda como qualificadora (para confirmação). Para
a otimização dessas transições, assim como das energias ótimas a serem usadas nos
quadrupolos (Q1 e Q2) e da energia de colisão (CE), foi feita infusão direta no LC (FIA,
flow injection analysis) de soluções padrão contendo cada analito na concentração de
1000 ng/mL em metanol. Os dados foram adquiridos e processados em software
LabSolutions 5.75 SP2.
3.2.3 Extração das amostras de sangue total para análise de catinonas
sintéticas
Em tubo de polipropileno de 2 mL foram adicionados 200 µL de sangue
total adicionado, 50 µL de solução estoque de MDMA-d5 (250 ng/mL, IS), 200 µL de
solução saturada de tetraborato de sódio e 800 µL de MTBE. Os tubos foram agitados
com auxílio de vórtex por 5 min, centrifugados a 8.000 rpm por 5 min e 600 µL da fase
27
orgânica transferida para outro tubo para completa evaporação do solvente, sob fluxo
constante de N2 e aquecimento (40 °C). As amostras foram ressuspendidas com 500
µL de metanol e 1 µL foi injetado no sistema analítico descrito no item 3.2.2.
3.2.4 Preparo e extração do DBS
Para preparo das amostras de sangue adicionado, às amostras de sangue
total branco foi adicionada solução de trabalho nas concentrações do calibrador ou
controle (Figura 1). Estas amostras foram então aplicadas, à volume fixo de 15 µL,
nos discos de DBS previamente cortados (6 mm) e mantidos em temperatura
ambiente (25 °C) para completa secagem (3 h).
Para a extração, cada disco foi transferido para tubo de polipropileno de 2
mL, onde foram adicionados 300 µL de solvente extrator (metanol) contendo o padrão
interno MDMA-d5 (10 ng/mL). As amostram foram agitadas com auxílio de vórtex por
10 min, centrifugadas a 10.000 rpm por 5 min, 150 µL da solução extratora
sobrenadante foi transferida para vials e 2 µL foi injetado no sistema analítico descrito
no item 3.2.2.
3.2.4.1 Otimização de misturas
Para determinar qual seria o melhor solvente extrator ou a mistura destes, foi
realizado um. Amostras de DBS foram preparadas na concentração de 1000 ng/mL e,
após secagem completa, foram extraídas, em duplicata, utilizando como solvente
extrator: metanol, acetonitrila, água, metanol-acetonitrila 1:1, metanol-água 1:1,
acetonitrila-água 1:1 e metanol-acetonitrila-água 1:1:1. Essas amostras foram
injetadas e as áreas absolutas de cada analito foram avaliadas por planejamento
experimental do tipo centroide simplex, em software Unscrambler® X 10.3 (CAMO
Software AS, Oslo, Norway). Dessa forma, foi determinado que o melhor solvente
testado para obter máxima recuperação dos analitos foi metanol.
28
Fig. 4 Gráfico da melhor proporção de solventes a ser utilizado para
conseguir máxima extração das catinonas sintéticas.
3.3 Validação
Todos os métodos foram validados de acordo com os parâmetros
recomendados pela SWGTOX para análises quantitativas em matrizes biológicas [32].
29
4. RESULTADOS
4.1 Artigo 1
Os resultados obtidos no estudo de estabilidade dos NBOMes na matriz
DBS foram publicados na revista Forensic Toxicology (Online ISSN 1860-8973, fator
de impacto 3,744), DOI 10.1007/s11419-017-0391-8. O texto do manuscrito aprovado
para publicação será apresentado a seguir.
30
Development and validation of a sensitive LC–MS/MS method to analyze NBOMes in
dried blood spots: evaluation of long-term stability
Kelly Francisco da Cunha1, Marcos Nogueira Eberlin2, Jose Luiz Costa1,3
1Campinas Poison Control Center, Faculty of Medical Sciences, University of
Campinas, Campinas, São Paulo 13083-859, Brazil
2ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of
Campinas, Campinas, São Paulo 13083-970, Brazil
3Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo
13083-859, Brazil
31
Abstract
Purpose We evaluated the use of dried blood spots (DBS) to determine seven
NBOMes by liquid chromatography–tandem mass spectrometry (LC–MS/MS), and
also evaluated the stability of these compounds in this dried matrix.
Methods An LC–MS/MS method was developed and fully validated to quantify seven
NBOMes (25C-, 25H-, 25I-, 25B-, 25G-, 25D- and 25E-NBOMe) in DBS samples. The
extraction procedure was optimized using mixture design experiment. Stability study
was performed in two different concentrations over 180 days at three different storage
temperatures.
Results Good linearity, and limits of detection and quantitation of 0.05 and 0.1 ng/mL,
respectively, were obtained. The interday imprecision (n = 15) and bias (n = 15) were
not higher than 11.4 and 10.3%, respectively, and no carryover was observed. All
analytes remained stable in DBS at − 20, 4 °C and even at room temperatures for 180
days, except 25B-NBOMe and 25I-NBOMe which experienced degradation (22 and
21%, respectively) of the initial concentration at room temperature after 180 days of
study. The method was applied to a DBS of an authentic postmortem blood from an
NBOMe user, and it was found to be reliable with good selectivity and specificity.
Conclusions DBS has been found to allow reliable, sensitive, accurate and robust
detection and quantification of seven NBOMes via LC–MS/MS. Also, DBS provided
great stability to most of the compounds at room temperature, and no degradation was
observed for DBS kept at 4 and − 20 °C. This is the first trial to analyze NBOMes in
DBS samples to our knowledge.
Keywords Dried blood spots (DBS), NBOMes, new psychoactive substances (NPS),
designer drugs, liquid chromatography–tandem mass spectrometry
32
Introduction
NBOMes (Fig. 1) are N-methoxybenzyl derivatives of the 2C hallucinogenic
compounds, and were initially synthesized for research purposes as a potent 5-HT2A
receptor agonist [1]. The presence of the N-methoxybenzyl moiety increases the
affinity of the molecule to serotonergic receptors [2]. For instance, 25I-NBOMe was
found to have at least ten times the affinity of its analog 2C-I, producing great behavior
responses in animals [3].
NBOMes are classified as new psychoactive substances (NPS), a class of
substances of abuse which was not controlled by the 1961 United Nations Single
Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substances,
but they pose serious public health threats [4]. NBOMes have recently become popular
as drugs of abuse, particularly because they are sold over the Internet as an alternative
to classical (and illegal) hallucinogenic lysergic acid diethylamide (LSD). Since 2013,
several compounds of this group were considered illegal in many countries such as in
Brazil since 2014.
Fig. 1 Structures of seven common NBOMes.
These drugs are powerful and are consumed orally, and many cases of
intoxications were reported in the last few years. Most effects are related to serotonin
syndrome, and include hallucination, aggression, self-harm, agitation, mydriasis,
33
tachycardia, hypertension, fever, seizures and rhabdomyolysis [1, 5–11]. Besides the
serotonergic effects, NBOMes are expected to interact with α-adrenergic receptors,
because they exhibit stimulant effects [8, 10]. Many cases of fatal intoxications have
been also reported [1, 4–6, 12].
Due to their high potency, few milligrams of NBOMes are sufficient to
produce prolonged effects. Poklis et al. [13] analyzed blotter paper containing different
NBOMes, and found concentrations between 510 and 1500 μg of the main constituent,
and also traces of other NBOMes in same samples (ranging from 7 to 15 μg/blotter).
As a consequence of such low effective dose, the concentration of NBOMes in
biological fluids are normally very low, typically in the order of pg/mL (Table 1). All
published methods to quantify NBOMes in biological samples have employed liquid
chromatography–tandem mass spectrometry (LC–MS/MS) [14, 15].
Dried blood spot (DBS) technique has been reevaluated recently and is
gaining growing popularity as a faster, less invasive and simpler sampling procedure
in the bioanalytical, clinical and pharmaceutical fields [16–19]. The DBS sample
process also requires no anticoagulants or plasma separations, and the samples can
be often stored for a long time at room temperature without any appreciable analyte
loss. The DBS technique also leads to a minimum risk of analyst infection by pathogens
such as HIV or hepatitis [17, 20, 21].
In preclinical pharmacokinetics studies, the DBS technique also improves
data quality, because it enables serial samples from a single animal, significantly
reduces the number of animals required and offers significant ethical and cost benefits.
These benefits are possible because of very low blood volume sampled at each time
point [22].
Although the DBS technique is nowadays widely used in clinical diagnostics
and therapeutic drug monitoring, not many methods have been reported dealing with
DBS use in forensic toxicology and analysis of abused drugs [20, 21, 23–29], and no
NBOMe analytical protocols using DBS have been reported, to the best of our
knowledge. We have therefore tested the use of DBS to determine seven common
NBOMes by LC–MS/MS. We also evaluated the stability of these compounds in this
matrix over 180 days at three different storage temperatures.
34
Table 1 Literature review on the analysis of NBOMes in biological samples.
Analyte Matrix Sample
preparation
LOD
(ng/mL)
LOQ
(ng/mL)
Linearity
(ng/mL)
Detected
blood
concentration
(ng/mL)
Reference
25B-NBOMe Blood
(200 µL) LLE 0.0053 0.0159 0.02 – 2.0 38.4 - 661 [39]
25C-NBOMe
Blood, urine, tissue
homogenates
(2 mL)
SPE NA NA 0.05 – 10 0.5 – 2.1 [12]
25B-NBOMe Plasma, urine NA 0.1 0.1 (plasma)
1.0 (urine) 0.1 - 10 0.7 – 10.7 [11]
25I-NBOMe
25B-NBOMe
Blood
(250 µL) LLE 0.2 0.5 0.5 - 20 1.6 [31]
25I-NBOMe Blood PP 0.2 0.5 0.5 - 20 0.76 [32]
35
25C-NBOMe
Blood, urine,
vitreous humor, liver
and gastric content
(500 µL)
LLE NA 0.1 0.1 - 10 0.6 [6]
25B-NBOMe Serum, urine
(500 µL) LLE 0.01 0.025 0.025 – 2.0 0.18 [42]
25I-NBOMe Blood
(1 mL) SPE NA 0.025 0.025 – 2.0 0.41 [33]
25H-NBOMe, 25D-
NBOMe, 25B-NBOMe,
25G-NBOMe, 25I-
NBOMe
Urine
(500 µL) SPE 0.1 1.0 1.0 - 100 [17]
25B-NBOMe, 25C-
NBOMe, 25D-NBOMe,
25H-NBOMe, 25I-
NBOMe, 25T2-NBOMe
Blood, plasma, urine
(500 µL) SPE
0.005 -
0.01 0.01 - 0.02 0.01 - 20 [41]
25I-NBOMe Serum
(1 mL) LLE 0.1 0.1 0.1 - 10 0.76 [7]
25C-NBOMe, 25I-
NBOMe
Serum
(1 mL) SPE 0.01 0.03 0.03 – 2.0 0.25 - 2.8 [14]
LOD limit of detection, LOQ limit of quantification, LLE liquid-liquid extraction, SPE solid-phase extraction, NA not available, PP protein
precipitation
36
Materials and methods
Standards and chemicals
Reference materials of 25B-NBOMe, 25C-NBOMe, 25H-NBOMe, 25I-
NBOMe, 25G-NBOMe, 25D-NBOMe and 25E-NBOMe were purchased from Cayman
Chemical (Ann Arbor, MI, USA); LSD-d3 (used as internal standard, IS) was purchased
from Cerilliant (Round Rock, TX, USA). Acetonitrile, methanol and formic acid (98–
100%) were purchased from Scharlau (Barcelona, Spain). Ultrapure deionized water
was supplied by a Milli-Q RG unit from Millipore (Billerica, MA, USA). All solvents
employed in the extraction were HPLC grade, and LC–MS grade for the
chromatographic system. Whatman 903 Protein Saver Cards™ were obtained from
GE Healthcare Bio-Sciences (Pittsburgh, PA, USA).
Standard solution preparation
Stock solutions of NBOMes were prepared by dilution of reference materials
in methanol. Calibrator working solutions at 1, 2, 5, 10, 50 and 100 ng/mL were
prepared by appropriate dilution of the stock solutions in methanol. Quality control (QC)
working solutions were prepared in methanol at low (3 ng/mL), medium (20 ng/mL) and
high (80 ng/mL) concentrations. IS solution (LSD-d3, 0.5 ng/mL) was prepared by
appropriate dilution in methanol/acetonitrile mixture (75:25, v/v). All solutions were
stored at − 20 °C.
DBS preparation
Fortified whole blood was prepared by dilution of calibrator and QC working
solutions in blank blood. For the DBS preparation, 15 μL of blank or fortified whole
blood was spotted onto a 6-mm-diameter pre-punched Whatman 903 DBS paper cards
disc. The blood spots were allowed to dry at room temperature for 3 h before extraction.
37
Extraction procedure
The 6-mm-diameter disc was placed in a 2-mL polypropylene (Eppendorf)
tube. The extraction was performed with 300 μL of the IS solution. The tubes were
capped, vortexed for 10 min and centrifuged at 10,000 rpm for 5 min (room
temperature). The supernatant (150 μL) was transferred to LC–MS/MS vials.
Instrumentation
The DBS analyses were performed on a 1260 Infinity liquid chromatography
system (Agilent Technologies, Santa Clara, CA, USA) coupled to a 5500-QTRAP®
hybrid triple quadrupole mass spectrometer (ABSciex, Concord, ON, Canada).
The chromatographic separation was performed with a C18 column (Atlantis
T3, 150 × 3 mm, particle size 3 μm, Waters, Dublin, Ireland), maintained at 40 °C. The
mobile phase consisted of ultra-pure water (A) and acetonitrile (B), both containing
formic acid (0.1%, v/v). The gradient elution was programmed as follows: 10% B,
followed by a linear change to 95% B over 15 min, held at 95% B for 2 min and returned
to initial conditions over 0.5 min (total run time of 22 min). The mobile phase flow rate
was 0.5 mL/min.
The 5500-QTRAP® mass spectrometer was equipped with a
TurboIonSpray™ interface using electrospray ionization in the positive ionization
mode. Nitrogen was used as curtain, collision and nebulizer gas. The source
parameters were: ion source temperature, 700 °C; ion spray voltage, 3.5 kV; entrance
potential (EP), 10 V; nebulizer gas (GS1) pressure, 40 psi; auxiliary gas (GS2)
pressure, 50 psi; and curtain gas pressure, 25 psi. The analyses were performed in
multiple reaction monitoring (MRM) mode. For each compound, two MRM transitions
were chosen for quantification and confirmation, and optimized by constant infusion of
working solutions of each analyte (20 ng/mL in water/methanol, 1:1 v/v). Table 2 shows
the optimized conditions of declustering potential (DP), collision energy (CE), collision
cell exit potential (CXP) and the retention time for NBOMes and LSD-d3. Analyst 1.6.2
software was used for data collection and MultiQuant 3.0.1 for data processing (both
ABSciex).
38
Table 2 Parameters for analysis of seven NBOMes and LSD-d3 by liquid
chromatography–tandem mass spectrometry (LC–MS/MS).
Analyte MRM transitions
(m/z) DP (V) CE (V) CXP (V)
Retention time
(min)
25B-NBOMe
380.2121.1
380.291.1
66
66
25
63
11
13
9.88
25C-NBOMe
336.2121.1
336.291.1
81
81
23
47
13
9
9.71
25H-NBOMe
302.2121.1
302.290.9
66
66
23
53
7
11
9.00
25I-NBOMe
428.2121.0
428.291.0
76
76
25
67
13
11
10.18
25G-NBOMe
330.0121.0
330.091.0
92
92
26
51
9
9
10.13
25D-NBOMe
316.0121.0
316.091.0
92
92
23
50
6
8
9.74
25E-NBOMe
330.2121.0
330.291.0
90
90
24
59
6
6
10.38
LSD-d3 (IS)
327.4226.2
327.4210.1
50
50
31
57
5
11
7.81
Quantifier transitions were underlined
MRM multiple reaction monitoring, DP declustering potential, CE collision energy, CXP
collision cell exit potential, IS internal standard
39
Validation of the method
The method was validated generally according to the guidelines of Scientific
Working Group for Forensic Toxicology (SWGTOX) published for quantitative analysis
[30]. Parameters evaluated were sensitivity and linearity, specificity, accuracy and
imprecision, matrix effects, carryover and stability.
Sensitivity and linearity
Analyte identification criteria were a symmetric peak eluting within ± 2%
relative standard deviation (RSD) of average calibrator retention times, a signal-to-
noise ratio of at least 3 and a qualifier/quantifier MRM peak area ratio within ± 20%
RSD of the mean ratio of the calibrators. The limit of detection (LOD) was defined as
the lowest concentration that reached the identification criteria. The limit of quantitation
(LOQ) was the lowest concentration fulfilling the identification criteria, but with signal-
to-noise ratio of at least 10 and quantifying within 20% RSD of each target
concentration. Calibration curves were constructed following literature data of real
NBOMe concentrations in acute poisoning and postmortem cases [6, 11, 31–33].
Linearity was assessed over 5 days and calibration curves were fit by linear least
squares regression (1/x2 weighting) with concentrations of 0.1, 0.2, 0.5, 1.0, 5.0 and
10 ng/mL over the dynamic range for each analyte. Calibrators were required to
quantify within ± 20% RSD of each target concentration.
Specificity
Endogenous interferences were assessed by analyzing blank whole blood
samples, from ten different individuals. Potential exogenous interferences by 28
commonly encountered drugs of abuse and medications were evaluated by fortifying
them at 500 ng/mL into LOQ and negative (only IS) samples. No interference was
noted; all analytes in the LOQ were quantified within ± 20% RSD of target with
acceptable qualifier/quantifier MRM ratios, and no peaks in the negative sample
satisfied LOD criteria.
40
Accuracy and imprecision
Intraday and interday accuracy and imprecision data for seven NBOMes
were evaluated with 3 replicates of each QC concentration over 5 days (ninter = 15).
Accuracy was defined as percent target concentration and was required to be within ±
20% RSD of the target concentration; imprecision was also assessed within ± 20%
RSD. One-way analysis of variance (ANOVA) was performed on each QC
concentration to assess potentially significant interday variability at p<0.05.
Matrix effects
Matrix effects were determined by fortifying two sets of samples at low and
high QC concentrations. In set A, 10 blank blood samples were spotted and carried
through the extraction procedure, and the extraction solvent was fortified with analytes
and IS at the QC concentrations. In set B, the same volume of solvent
(methanol/acetonitrile, 75:25, v/v) only was fortified with analytes and IS (neat sample).
The final samples of the sets A and B were transferred directly to autosampler vials.
Matrix effect was calculated as the peak area ratio of set A to set B, subtracted by 1,
and expressed as a percentage; a negative percentage represents peak suppression,
and a positive percentage represents peak enhancement.
Carryover
Blank blood was fortified with all analytes at the highest point of each
calibration curve; the extract was injected into the LC–MS/MS instrument, and then a
blank sample extract was injected. Carryover was absent if no analyte peak met LOD
criteria for any blank injection.
Stability
Stability of the samples was evaluated by fortifying blank blood with analytes
at low (0.3 ng/mL) and high (8.0 ng/mL) QC concentrations (n = 3) and analyzed after
being stored at ambient, refrigerated (4 °C) and freeze (− 20 °C) temperatures, for 7,
15, 30, 60, 90 and 180 days. Additionally, processed samples were stored on the 4 °C
41
autosampler for 24 h before being reinjected. Stability was observed if concentration
changes were ± 20% of the initial target concentrations.
Results and discussion
As mentioned before, NBOMes are found in biological fluids in trace levels.
Despite many advantages of DBS, one of its limitations is the low sample quantity,
because only 15 μL (almost one drop volume) of whole blood was spotted on paper.
The combination of these factors makes NBOMes-DBS analysis a challenge, requiring
the development of a highly sensitive method.
Prior to method validation, the DBS extraction procedure was optimized
using a mixture design experiment (The Unscrambler X, version 10.3, CAMO Software
AS, Oslo, Norway), in which different ratios of methanol, acetonitrile and water were
evaluated. Major parameters such as the presence of formic acid (0.1%, v/v) in
extraction solvent and type of shaking (vortex mixer or ultrasonic bath) were also
evaluated. We found that a methanol/acetonitrile mixture (75:25, v/v) without formic
acid and vortex mixing for 10 min provided the best extraction efficiency.
To avoid hematocrit interference, which is a well-documented DBS problem
[34–38], we decide to use a fixed blood volume (15 μL) and whole spot analysis.
The procedure led to the LOD and LOQ of 0.05 and 0.1 ng/mL, respectively
(Fig. 2), using very small blood volume (15 μL). Lower detection limits have been
reported but using large volumes of whole blood samples [39]. The method was also
found to be linear from 0.1 to 10 ng/mL for all the seven NBOMes, with 1/x2 weighted
linear regression. Fortunately, this range is within that typically found in human
intoxications [12, 32, 33].
As summarized in Table 3, the imprecisions (intraday and interday %RSD)
and accuracy biases (%) in 5 days were satisfactory at all three QC concentrations,
with no more than 20.0% RSD.
No interference was observed when the LOQ was verified in the presence
of 500 ng/mL of other drugs of abuse and pharmaceuticals, and no carryover was
observed even at 10 ng/mL (highest analyte concentration).
42
Fig. 2 Multiple reaction monitoring (MRM) chromatogram of a dried blood spot (DBS)
made of whole blood spiked with a mixture of the seven NBOMes. The concentration
of each NBOMe in the original blood was 0.1 ng/mL (limit of quantification). In 25G-
NBOMe (the former peak) and 25E-NBOMe (the latter peak) panels, two peaks
appeared because they are regioisomers. The higher and lower peaks appeared
overlapped; higher: quantifier ion; lower: qualifier ion. Sample preparation and
analytical conditions are described in the experimental section.
43
Table 3 Method validation regarding imprecisions, accuracy biases and matrix effects
for the determination of seven NBOMes in dried blood spots by LC-MS/MS.
Analyte
Intraday imprecision
(%RSD) (n=3)
Low Med High
Interday imprecision
(%RSD) (n=15)
Low Med High
Bias (%) (n=15)
Low Med High
Matrix effect (%) (n = 10)
Low High
25B-NBOMe 20.0 4.5 4.8 10.7 6.2 3.2 2.0 -3.4 1.2 -11.4 14.0
25C-NBOMe 11.2 2.5 2.1 6.8 9.0 7.2 4.9 -10.3 -5.1 17.2 24.8
25H-NBOMe 5.1 3.7 2.3 6.5 7.6 4.0 6.0 -7.9 -3.5 50.5 64.6
25I-NBOMe 4.7 5.3 4.1 4.6 7.3 6.1 4.9 -5.5 -3.2 1.9 6.1
25G-NBOMe 4.4 6.7 5.8 10.7 7.4 6.4 0.4 -8.9 -5.3 -6.3 4.1
25D-NBOMe 7.1 3.5 3.8 8.1 8.0 5.1 0.9 -8.5 -3.9 16.0 20.8
25E-NBOMe 20.0 3.6 5.1 11.4 8.0 4.4 1.1 -6.2 -1.7 -1.3 5.6
The matrix effects showed great variation according to each analyte. The
highest enhancement effects were observed for 25H-NBOMe (50.5 and 64.6%).
Medium enhancement effects were observed for 25C-NBOMe and 25D-NBOMe. The
other analytes showed minimal matrix effects (Table 3).
The stability experiments showed that the analytes were generally stable in
DBS for 180 days at ambient, refrigerated and freeze temperatures, except for 25B-
NBOMe (low QC, 0.3 ng/mL) and 25I-NBOMe (high QC, 8 ng/mL), which had a
decrease more than 20% of each initial concentration at room temperature after 180
days (Figs. 3 and 4).
Data for the stability of NBOMes in biological samples are limited. Soh and
Elliot [40] investigated the stability of 13 NPS in blood and plasma (not for DBS) at
room temperature (20–23 °C) over 21 days, finding that 25C-NBOMe was stable under
the test conditions. However, Johnson and coworkers [41] studied the stability of these
substances in sample extracts, storing the vials at 4 °C for 7 days after sample
44
extraction, and observed discrepant results among sample extracts (urine, plasma and
whole blood), with major deterioration of analytes in whole blood extracts specially for
25I-NBOMe, 25T2-NBOMe, 25B-NBOMe and 25C-NBOMe, with decay of 50.8, 37.8,
35.2 and 7.6%, respectively.
Poklis and coworkers [42] had studied the auto-sampler stability of 25B-
NBOMe and 25H-NBOMe over 72 h of extraction of serum and urine samples, and
observed good stability.
As a proof of reliability for the developed DBS method, we tested a DBS
made of whole blood obtained from a cadaver of a drug user. We could detect distinct
peaks using transitions at m/z 428.2 > 121.0 and 428.2 > 91.0, showing the presence
of 25I-NBOMe (Fig. 5) and quantified it to be 0.5 ng/mL.
Fig. 5 MRM chromatogram obtained from an authentic DBS sample: a LSD-d3 and b
25I-NBOMe. Both quantifier and qualifier ions are shown overlapped.
45
Fig. 3 Stabilities of seven NBOMes on DBS at a low concentration (0.3 ng/mL of each in original whole blood) under three different
storage temperatures over 180 days. a 25B-NBOMe, b 25C-NBOMe, c 25H-NBOMe, d 25I-NBOMe, e 25G-NBOMe, f 25D-NBOMe
and g 25E-NBOMe.
46
Fig. 4 Stabilities of seven NBOMes on DBS at a high concentration (8 ng/mL of each in original whole blood) under three different
storage temperatures over 180 days. Identification of analytes is the same as specified in Fig. 3.
47
Conclusions
DBS have been found to allow for reliable, sensitive, accurate and robust
detection and quantitation of seven NBOMes via LC–MS/MS. The developed DBS–
LC–MS/MS method was successfully validated with proper figures of merits. Another
major benefit of DBS sampling was that almost all analytes were found to be stable in
the DBS for up to 180 days at room temperature, except for 25B-NBOMe and 25I-
NBOMe which showed slight degradation (22 and 21%, respectively). No degradation
was observed for DBS kept at 4 and − 20 °C.
Acknowledgements
The authors thank Fundação de Amparo à Pesquisa do Estado de São
Paulo – FAPESP (Process Number 2015/10650-8), Conselho Nacional de
Desenvolvimento Científico e Tecnológico – CNPq (Process Number 830525/1999-8),
Fundo de Apoio ao Ensino, à Pesquisa e à Extensão – FAEPEX (Agreement 519.292
MF 86739) and Superintendência da Polícia Técnico-Científica.
Compliance with ethical standards
Conflict of interest There are no financial or other relationships that could
lead to a conflict of interest.
Ethical approval All procedures performed in studies involving human
participants were in accordance with the ethical standards of the institutional research
committee (Comitê de Ética em Pesquisa da UNICAMP – CEP, CAAE
58452716.2.0000.5404 and Superintendência da Polícia Técnico-Científica, ofício No.
766/2015/ATS/SPTC-SSP) and with the 1964 Helsinki Declaration.
48
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4.2 Artigo 2
Os resultados obtidos no estudo de estabilidade das NBOMes na matriz
sangue total será submetido para publicação na revista Journal of Analytical
Toxicology (Online ISSN 1945-2403, fator de impacto 2.409). O texto do manuscrito
em preparação para submissão será apresentado a seguir.
54
Long-term stability of NBOMes in whole blood
Kelly Francisco da Cunha1, Marcos Nogueira Eberlin2, Marilyn A. Huestis3, José Luiz
Costa1,4*
1Campinas Poison Control Center, Faculty of Medical Sciences, University of
Campinas, Campinas, São Paulo 13083‑859, Brazil
2ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of
Campinas, Campinas, São Paulo 13083‑970, Brazil
3Institute of Emerging Health Professions, Thomas Jefferson University, Philadelphia,
PA, USA
4*Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo
13083‑859, Brazil, [email protected], +55 019 3521 7232
55
Abstract
NBOMes - a potent group of designer drugs that requires small doses to reach
recreational and also toxic effects. A new method was developed and fully validated to
quantify 7 NBOMes (25B, 25C, 25D, 25E, 25G, 25H and 25I) in whole blood, using
LSD-d3 as internal standard. Then, a stability study of 6 months, at two concentration
levels and at three storage temperatures (room, 4 °C and -20 °C) was developed. The
calibration curve was found to be linear between 0.1 to 10 ng/mL, with administrative
LOQ of 0.1 ng/mL. Intraday and interday imprecisions (%RSD) and bias (%) was up to
around 8% and 7%, respectively. At room temperature and low concentration, some
analytes were unstable over 15 days and undetectable over 30 days. All NBOMes were
undetectable at 60 days. At 4 °C, some of them were undetectable at 180 days. At high
concentration, same pattern of concentration decreased was observed. All analytes
were stable at -20 °C. NBOMes are stable at -20 °C over 6 months, but at 4 °C and
room temperature, depending on the initial concentration, the analytes were unstable
at 180 days, except 25H-NBOMe; and 25B, 25C, 25I and 25E-NBOMes at 15 days,
respectively.
Keywords NBOMes, new psychoactive substances (NPS), stability, liquid
chromatography- tandem mass spectrometry (LC-MS/MS), whole blood, designer
drugs.
56
Introduction
NBOMes are a class of new psychoactive substances (NPS) that acting on
central nervous system (CNS), more specifically at serotonin receptors.
Phenethylamines derivate from 2C compounds, there N-methoxybenzyl moiety confer
a higher affinity to 5-HT2A receptors, being capable to produce stronger hallucinogenic
effects in small doses – usually between 0.5 and 1,5 mg. NBOMes are available at
illicit market since 2011 with 25I-NBOMe, followed by others derivatives (1).
Sold, in many cases, in blotter papers such as lysergic acid diethylamide
(LSD), NBOMes have been related to several intoxications and deaths around the
world since 2013 (2-9). Most common effects includes tachycardia, hypertension,
agitation, visual and auditory hallucinations and rhabdomyolysis (10). Blood
concentrations founded in intoxications cases are very low, in order of pg/mL, requiring
sensitive techniques to quantify these analytes.
In forensics, blood samples are frequently preferred by their capability to
better correspond concentrations values and effects, but analytes stability is a
recurrent concern. Same sample analyzed in different times or sampling many hours
after the exposition, it can provide different quantitation or false negative results (11).
The aim of this work is to evaluate the long-term stability of NBOMes in
whole blood. The stability study was performed over 6 months, at two concentration
levels and at three storage temperatures (room, 4 °C and -20 °C) was developed.
Experimental
Standards and chemicals
Reference materials of 25B-NBOMe, 25C-NBOMe, 25D-NBOMe, 25E-
NBOMe, 25H-NBOMe, 25I-NBOMe and 25G-NBOMe were purchased from Cayman
Chemical (Ann Arbor, MI, USA); LSD-d3 (used as internal standard, IS) was purchased
from Cerilliant (Round Rock, TX, USA). Acetonitrile and formic acid (98-100%) were
purchased from Merck (Darmstadt, Germany) and Scharlau (Barcelona, Spain),
respectively. MTBE (t-butyl methyl ether) was obtained from Sigma Aldrich (St Louis,
MO, EUA). Sodium tetraborate was purchased from LS Chemicals (Ribeirao Preto,
57
SP, Brazil). Ultra-pure deionized water was supplied by a Milli-Q RG unit from Millipore
(MA, USA). All solvents employed in the extraction were HPLC grade.
Ethical approval
All procedures performed in studies involving human samples were in
accordance with the ethical standards of the University of Campinas committee
(Comitê de Ética em Pesquisa da UNICAMP – CEP, CAAE 58452716.2.0000.5404)
Standard solutions preparation
Stock solutions of NBOMes were prepared by dilution of reference materials
in methanol. Calibrator working solutions at 1, 2, 5, 10, 50 and 100 ng/mL were
prepared by appropriate dilution of stock solutions in methanol. QC working solutions
were prepared in methanol at low (3 ng/mL), medium (20 ng/mL) and high (80 ng/mL)
concentrations. Internal standard solution (LSD-d3, 10 ng/mL) was prepared by
appropriate dilution in methanol. All solutions were stored at -20 oC.
Extraction procedure
Liquid-liquid extraction was performed in a 2 mL polypropylene tube with
300 µL of fortified whole blood, 50 µL of internal standard solution (LSD-d3 10 ng/mL),
300 µL of sodium tetraborate (saturated solution) and 1.2 mL of MTBE. The tubes were
capped, homogenized by inversion 10x, vortexed for 5 min and centrifuged at 8.000
rpm x 5 min (room temperature). The organic phase (900 µL) was transferred to
another polypropylene tube, evaporated to dryness under nitrogen (40 °C) and
resuspended in 500 µL of methanol to LC-MS/MS vials and 5 µL of injection.
Instrumentation
Samples analysis were performed in a 1260 Infinity liquid chromatograph
system (Agilent Technologies, CA, USA) coupled to a 5500-QTRAP® triple quadrupole
mass spectrometer (ABSciex, ON, Canada).
58
The chromatographic separation was performed with a C18 column (Atlantis
T3, 150 x 3 mm, 3 µm, Waters, Ireland), maintained at 40°C. The mobile phase
consisted of ultra-pure water (A) and acetonitrile (B), both of containing formic acid
(0.1%, v/v). The gradient elution was programmed as follows: a linear change of B,
initialing in 10% until 95% over 15 min, held at 95% B for 2 min and returned to 10% B
over 0.5 min, keeping it to reach the initial condition of pressure (22 min run time). The
mobile phase flow rate of 0.5 mL/min.
The 5500-QTRAP mass spectrometer was equipped with a
TurboIonSprayTM interface, operated in positive ionization mode (electrospray).
Nitrogen was used as curtain, collision and nebulizer gas. The source parameters were
optimized as set: temperature of 700 °C; ion spray voltage of 3.5 kV, entrance potential
(EP) of 10 V, nebulizer gas (GS1) pressure of 40 psi, auxiliary gas (GS2) pressure of
50 psi and curtain gas pressure of 25 psi. The analyses were performed in multiple
reaction monitoring mode (MRM). For each compound, two MRM transitions were
chosen, one for quantitation and one for confirmation. Analyst 1.6.2 software was used
for data collection and MultiQuant 3.0.1 for processing.
Validation of the method
The method was validated according to the guideline for quantitative
method validation published by the Scientific Working Group for Forensic Toxicology
(SWGTOX) (12).
Bias
Bias study was applied at three different concentrations, which one with
three replicates over five days. The highest average acceptable from the nominal
concentration were ± 20%. Results were presented in percentage.
Imprecision
Intraday and interday imprecisions were evaluated as the coefficient of
variation (%RSD) of results from replicates (low, medium and high QC) in a day (n= 3)
59
and over 5 days (n=15), respectively. Twenty percent was accepted as the limit of
variation. One-way analysis of variance (ANOVA) was performed on each QC
concentration to assess potentially significant interday variability at p < 0.05.
Table 1 Mass spectrometer parameters for analysis of seven NBOMes and LSD-d3 in
whole blood by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Analyte MRM
transitions (m/z)
DP (V) CE (V) CXP (V)
25B-NBOMe 380.2121.1
380.291.1
66
66
25
63
11
13
25C-NBOMe 336.2121.1
336.291.1
81
81
23
47
13
9
25H-NBOMe 302.2121.1
302.290.9
66
66
23
53
7
11
25I-NBOMe 428.2121.0
428.291.0
76
76
25
67
13
11
25G-NBOMe 330.0121.0
330.091.0
92
92
26
51
9
9
25D-NBOMe 316.0121.0
316.091.0
92
92
23
50
6
8
25E-NBOMe 330.2121.0
330.291.0
90
90
24
59
6
6
LSD-d3 (IS) 327.4226.2
327.4210.1
50
50
31
57
5
11
Quantifier transitions were underlined, MRM multiple reaction monitoring DP
declustering potential CE collision energy CXP collision cell exit potential IS internal
standard
60
Sensitivity and linearity
The limit of detection (LOD) was defined as the lowest concentration that
reached the identification criteria - symmetric peak eluting within ± 2% relative standard
deviation (RSD) of average calibrator retention times, a signal-to-noise ratio of at least
3 and a qualifier/quantifier MRM peak area ratio within ± 20% RSD of the mean ratio
of the calibrators. The limit of quantitation (LOQ) was the lowest concentration fulfilling
the identification criteria, but with signal-to-noise ratio of at least 10 and quantifying
within 20% RSD of each target concentration. Calibration curves were constructed
following literature data of real blood concentration at NBOMes intoxications. Linearity
was assessed over 5 days and calibration curves with six points of calibration (0.1, 0.2,
0.5, 1.0, 5.0 and 10 ng/mL). Calibrators were required to quantify within ± 20% RSD of
each target concentration.
Specificity
Twenty-eight possible exogenous interferences (drugs of abuse and
pharmaceuticals) were used to fortified negative and LOQ whole blood samples at
1000 ng/mL and extracted concomitantly. To characterize no interference was
stablished that all analytes in the LOQ should quantified within ± 20% RSD of target
with acceptable qualifier/quantifier MRM ratios, and no peaks in the negative sample
satisfied LOD criteria.
Matrix effects
Matrix effects were evaluated by the average of peak areas of a set of 10
blank blood samples fortified with all analytes after the extraction against the average
of peak areas of a set of a methanolic solvent fortified with the analytes. The result was
expressed as percentage and a negative result represents peak suppression, and a
positive result represents peak enhancement.
61
Carryover
After the injection of extract from fortified blood with all analytes at the
highest point of each calibration curve – 10 ng/mL, a blank sample extract was injected
over 5 days. No carryover was determined if none analyte peak met LOD criteria for
blank injections.
Stability
Stability study was applied at low and high QC samples (n=3). Whole blood
was fortified with stock solutions at day zero, aliquoted at 2 mL propylene tubes and
storage at room temperature (25 °C), 4 °C and -20 °C. At 15, 30, 60, 90 and 180 days,
replicates of each concentration and temperatures were extracted and analyzed
against a freshly curve. The analyte was assigned as stable if it concentration varied
up to ±20% of initial concentration. Auto-sampler stability was also evaluated.
Results and discussion
One of the concerns about NPS refer to constant release of very similar new
analytes, within isobaric compounds that can be wrongly identified by mass
spectrometry. Due their similar structure, the fragmentation patterns for NBOMes by
ESI+ are the same, resulting as the two most intense fragment ions for m/z=121 and
m/z=91 (Fig 1). Different parent ions generate these fragments. Thus, at MRM mode,
the analytes are accurately identified. Adding 25G (m/z=330) and 25E-NBOMe
(m/z=330) as possible results, the chromatography optimization become fundamental
to guarantee a selective method. Baseline resolution of analytes was only achieved
using acetonitrile as mobile phase and adjusting the gradient increment.
Fig. 1 Fragmentation pattern for NBOMes in mass spectrometer (ESI+).
62
Linearity was obtained from 0.1 to 10 ng/mL for all the seven NBOMes, with
1/x2 weighted linear regression (r>0.98 for a set of five calibration curves). Method
limits of detection (LOD) and quantitation (LOQ) administrated were 0.05 and 0.1
ng/mL (Fig 2), respectively. Angular coefficients %RSD were up to 6.8%.
Fig. 2 Extracted chromatogram from 7 NBOMes in whole blood at 0.1 ng/mL (LOQ)
by LC-MS/MS
Low matrix effect was obtained against ten replicates from different sources,
varying up to 9.2% for 25C-NBOMe. No carryover was observed at a negative sample
was injected after the highest sample concentration (10 ng/mL). There was no
interference when a mix of other drugs and pharmaceuticals at 1000 ng/mL were
extracted concomitantly with negative and LOQ samples.
Intraday and interday imprecisions (%RSD) were up to 7.8% for 25I-NBOMe
and 8.1% for 25C-NBOMe, respectively. Bias were up to 6.8% for 25C-NBOMe (Table
2).
Lewis et al (2014) (13) found out low stability for 25I (39.0% RSD), 25T2
(34.5% RSD) and 25B-NBOME (31.2% RSD) whole blood extracts, kept at 4 °C for 7
days. Poklis et al (2013) (5) studied the stability of 25C and 25I-NBOMes in serum
63
samples after freeze-thaw cycles, bench-top at room temperature (48 h), one month at
-20 °C and extract stability in auto-sampler for 72 h at room temperature. In all these
conditions, the analytes were stable. Poklis et al (2014) (3) still reported 25B and 25H-
NBOMe serum extracts stability up to 72h at room temperature (auto sampler stability).
There are no more stability studies involving longer time of storage or with this number
of N-methoxybenzyl derivatives.
Table 2 Validation results regarding imprecisions, bias and matrix effects for the
determination of seven NBOMes in whole blood by LC-MS/MS.
Analyte
Intraday
imprecision
(%RSD) (n=3)
Low Med High
Interday
imprecision
(%RSD) (n=15)
Low Med High
Bias (%) (n=15)
Low Med High
Matrix effect
(%) (n = 10)
Low High
25B-NBOMe 6.9 2.6 2.8 6.1 3.5 5.3 -5.3 2.7 -2.0 2.4 -2.1
25C-NBOMe 7.6 2.9 3.1 8.1 5.9 5.3 -1.3 6.8 -6.2 9.2 1.8
25H-NBOMe 4.6 3.4 6.1 5.7 6.2 6.4 -2.2 3.4 -3.2 4.7 0.1
25I-NBOMe 7.8 3.3 4.0 7.5 4.0 4.7 -2.4 3.5 -3.0 -2.5 -6.6
25G-NBOMe 6.7 4.1 3.9 6.8 4.9 5.3 -4.2 5.3 -1.8 -1.0 3.3
25D-NBOMe 5.6 3.6 3.6 5.7 5.4 4.4 -4.4 2.9 -3.0 -0.9 -1.8
25E-NBOMe 5.7 4.3 2.3 6.1 4.7 3.9 -2.9 3.4 -4.6 -0.6 -0.5
As stability results, at room temperature and in low concentration, 25B, 25C,
25I and 25E-NBOMe presented a decreased in concentration higher than 20% in the
first 15 days, up to 43%. All NBOMes were undetectable at 60 days.
Only 25B, 25C and 25I-NBOMe were not more detectable at 4 °C, at 180
days. The other NBOMes showed a decreased of concentration higher than 20%,
expect for 25H-NBOMe (-12%). All NBOMes were stable at -20 °C during the 6 months
studied – % difference of initial concentration up to ±20% (Fig 3).
64
At high concentration, all NBOMes had a decreased of concentration higher
than 20% at room temperature at 15 days, except 25H-NBOMe. Despite this, all of
them were detectable during the 180 days of analysis.
25B, 25C and 25I-NBOMe had their initial concentration values decreased
higher than 20% at 4 °C in 6 months. All Analytes were stable at -20 °C, same results
as obtained for low concentration (Fig 4).
65
Fig. 3 Average concentration and %RSD obtained at three storage temperatures for 7 NBOMes at 0.3 ng/mL (Low QC)
for 6 months. All concentrations were calculated as percentage of initial concentration at day 0. a 25B, b 25C, c 25H, d 25I, e 25G, f
25D and g 25E-NBOMe.
66
Fig. 4 Average concentration and %RSD obtained at three storage temperatures for 7 NBOMes at 8 ng/mL (High QC) for 6 months.
All concentrations were calculated as percentage of initial concentration at day 0. a 25B, b 25C, c 25H, d 25I, e 25G, f 25D and g
25E-NBOMe.
67
To evaluate the auto-sampler stability, QC samples (low, medium and high)
were extracted, analyzed, kept in auto-sampler (15 °C) and re-injected after 32 h
against a freshly curve. The analytes were considered stable as the concentrations of
re-injections were up to 15.6% from initial concentration.
As proof of concept, two real postmortem samples were extracted and injected
following this descripted method. 25B-NBOMe (0.2 ng/mL) was identified in one
sample and 25I-NBOMe (0.5 ng/mL) in the other (Figure 5), the same identification
obtained at Toxicology Laboratory from Sao Paulo Legal Medical Institute.
Fig. 5 Extracted chromatogram from two real postmortem samples, positive to 25B-
NBOMe (A) and 25I-NBOMe (B), respectively.
Conclusions
At room temperature, most of the analytes showed low stability over 15 days.
Moreover, most of them had their initial concentration decreased higher than 20% at 4
°C, at 180 days. At -20 °C, the analytes are stable up to 180 days. 25I-NBOMe had the
worst stability. In 15 days, it initial concentration decreased more than 40% in both
concentration values studied.
Once most of the intoxication cases associated with NBOMes quantify low
concentrations of these analytes in blood, more careful must be given involving storage
of samples with clinical finds suggesting for these group of analytes, mainly in forensic
cases.
68
Acknowledgements
The authors thank Fundação de Amparo à Pesquisa do Estado de São
Paulo – FAPESP (Process Number 2015/10650-8) and Conselho Nacional de
Desenvolvimento Científico e Tecnológico – CNPq (Process Number 131780/2017-4),
and Superintendência da Polícia Técnico-Científica.
References
1. Poklis, J. L., Raso, S. A., Alford, K. N., Poklis, A., Peace, M. R. (2015) Analysis of
25I-NBOMe, 25B-NBOMe, 25C-NBOMe and other dimethoxyphenyl-N-[(2-
methoxyphenyl) methyl]ethanamine derivatives on blotter paper. Journal of Analytical
Toxicology, 39, 617-623.
2. Suzuki, J., Dekker, M. A., Valenti, E. S., Cruz, F. A. A., Correa, A. M., Poklis, J. L.,
Poklis, A. (2015) Toxicities associated with NBOMe ingestion - a novel class of potent
hallucinogens: a review of the literature. Psychosomatics, 56, 129-139.
3. Poklis, J. L., Nanco, C. R., Troendle, M. M., Wolf, C. E., Poklis, A. (2014)
Determination of 4-bromo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-
benzeneethanamine (25B-NBOMe) in serum and urine by high performance liquid
chromatography with tandem mass spectrometry in a case of severe intoxication. Drug
Testing and Analysis, 6, 764-769.
4. Rose, S. R., Poklis, J. L., Poklis, A. (2013) A case of 25I-NBOMe (25-I) intoxication:
a new potent 5-HT2A agonist designer drug. Clinical Toxicology, 51, 174-177.
5. Poklis, J. L., Charles, J., Wolf, C. E., Poklis, A. (2013) High-performance liquid
chromatography tandem mass spectrometry method for the determination of 2CC-
NBOMe and 25I-NBOMe in human serum. Biomedical Chromatography, 27, 1794-
1800.
6. Suzuki, J., Poklis, J. L., Poklis, A. (2014) “My Friend Said it was Good LSD”: A
Suicide Attempt Following Analytically Confirmed 25I-NBOMe Ingestion. Journal of
psychoactive drugs, 46, 379-382.
7. Hermanns-Clausen, M., Angerer, V., Kithinji, J., Grumann, C., Auwärter, V. (2017)
Bad trip due to 25I-NBOMe: a case report from the EU project SPICE II plus. Clinical
Toxicology, 55, 922-924.
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8. Morini, L., Bernini, M., Vezzoli, S., Restori, M., Moretti, M., Crenna, S., Papa, P.,
Locatelli, C., Osculati, A. M. M., Vignali, C., Groppi, A. (2017) Death after 25C-NBOMe
and 25H-NBOMe consumption. Forensic Science International, 279, e1-e6.
9. Ameline, A., Kintz, P., Blettner, C., Bayle, E., Raul, J. S. (2017) Identification of 25I-
NBOMe in two intoxications cases with severe hallucinations. Journal of Analytical
Toxicology, 29, 117-122.
10. Locatelli, C. A., Buscaglia, E., Valli, A., Rocchi, L., Rolandi, L., Di Tuccio, M.,
Scaravaggi, G., Crevani, M., Papa, P. (2017) Clinical features of severe intoxications
associated with analytically confirmed use of NBOMe. Clinical Toxicology, 55, 444-
445.
11. Soh, Y. N. A., Elliott, S. (2014) An investigation of the stability of emerging new
psychoactive substances. Drug Testing and Analysis, 6, 696-704.
12. SWGTOX (2013) Standard Practices for Method Validation in Forensic Toxicology.
Journal of Analytical Toxicology, 37, 452-474.
13. Johnson, R. D., Botch-Jones, S. R., Flowers, T., Lewis, C. A. (2014) An evaluation
of 25B-, 25C-, 25D-, 25H-, 25I-and 25T2-NBOMe via LC-MS-MS: method validation
and analyte stability. Journal of Analytical Toxicology, 38, 479-484.
70
4.3 Artigo 3
Os resultados obtidos no estudo de estabilidade das catinonas sintéticas
nas matrizes DBS e sangue total foram publicados na revista Forensic Toxicology
(Online ISSN 1860-8973, fator de impacto 3,744), DOI 10.1007/s11419-018-0418-9.
O texto do manuscrito aprovado para publicação será apresentado a seguir.
71
Long-term stability of synthetic cathinones in dried blood spots and whole blood
samples: a comparative study
Kelly Francisco da Cunha1, Marcos Nogueira Eberlin2, Jose Luiz Costa1,3*
1Campinas Poison Control Center, Faculty of Medical Sciences, University of
Campinas, Campinas, São Paulo 13083-859, Brazil
2ThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of
Campinas, Campinas, São Paulo 13083-970, Brazil
3Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo
13083-859, Brazil
*Corresponding author: Prof. Dr. Jose Luiz Costa [email protected] Phone: +55 19 3521 7232
Fax: +55 19 3521 7592
72
Abstract
Purpose The long-term stabilities of four synthetic cathinones in dried blood spots
(DBS) were tested and compared with those of whole blood samples.
Method A method to determine four cathinones (pentylone, butylone, mephedrone and
benzedrone) in DBS was developed and validated. For comparison, the traditional
liquid-liquid extraction for the same analytes in whole blood was also performed.
Results The calibration curve was found to be linear over 25 – 1000 ng/mL for DBS
samples, with a limit of quantification at 25 ng/mL. The interday imprecisions and bias
results [up to 7.1 % relative standard deviation (RSD) and 5.8 %, respectively] were
much lower than the maximum data recommended by SWGTOX forensic guidelines.
The validation results were similar to those of whole blood samples, which showed
interday imprecisions and bias results of up to 8.1%RSD and 12.3 %, respectively, with
a linear calibration curve between 10 and 1000 ng/mL and LOQ of 10 ng/mL. A stability
study to compare the degradation rate between both matrixes at three storage
temperatures [room (25 °C), 4 °C and -20 °C]] and during 90 days proved the poor
stability of cathinones in whole blood, where the methylenedioxy cathinones displayed
better stability than those with ring substituents. DBS allowed detection of synthetic
cathinones after much longer periods than in whole blood samples.
Conclusions DBS proved, therefore, to be an useful alternative technique to store
blood samples. Extraction of DBS is easy and analytes remain stable for much longer
periods.
Keywords Synthetic cathinones, Dried blood spots (DBS), New psychoactive
substances (NPS), Liquid chromatography–tandem mass spectrometry (LC–MS/MS)
73
Introduction
Cathinone is a natural alkaloid, structurally related to amphetamine. In many
African countries, the chewing the leaves of Khat (Catha edulis) to obtain stimulating
and euphoric effects is a traditional habit [1].
Synthetic cathinones - chemical derivatives of cathinone - are categorized as
new psychoactive substances (NPS), a group of synthetic drugs of abuse, that could
lead to health problems, but was not controlled by the 1961 Single Convention on
Narcotic Drugs and the 1971 Convention on Psychotropic Substances. Synthetized
mostly in East Asia and distributed throughout the world, the first appearance of
synthetic cathinones on the market was in early 2000s, but new analogues have been
constantly released. In 2011, the United States Drug Enforcement Administration
(DEA) reported the necessity to control three of these synthetic cathinones:
mephedrone, 3,4-methylenedioxypyrovalerone (MDPV) and methylone [2]. According
to 2017 World Drug Report, stimulant drugs including synthetic cathinones, correspond
to 36% of NPS identified until December 2016; the second largest was the synthetic
cannabinoids group (33%) [3]. The administrating routes of synthetic cathinones are
usually by snorting the powder, oral for pills or capsules, by smoking or by intravenous
injection after dissolving the solid forms in water [4].
Synthetic cathinones are phenylalkylamine derivatives and are divided into four
main groups. Most of them are included in group 1 encompassing the N-alkyl
derivatives or those with an alkyl or halogen at the aromatic ring (e.g. benzedrone and
mephedrone). Group 2 encompasses those with a methylenedioxy substituent at the
aromatic ring (e.g. butylone and pentylone). Group 3 are for the cathinones with a N-
pyrrolidinyl substituent. Cathinones with substituents from both groups 2 and 3 are
74
included in group 4 [5]. Their mechanisms of action have been related to their
interaction with monoamine transporters such as dopamine, noradrenaline and
serotonin. Generally, users report similar effects for different cathinones: excitation,
euphoria, increased alertness, empathy and libido. The side effects include vomiting,
sweating, confusion, self-destructive behavior, headaches, dizziness, increased heart
rate and muscle tremors. In overdoses, panic, violent behavior, hallucinations,
tachycardia, rhabdomyolysis and even fatal cases have been reported [6-10].
Mephedrone (Fig. 1) was one of the first generation of synthetic cathinones
apprehended on a recreational market in Europe 2007. Since then, due to its spread
all over the world, severe intoxication and fatal cases have been associated - and
analytically confirmed - with mephedrone use [11,12]. Butylone was also associated
with fatal intoxications [7,13]. A couple of studies have confirmed the presence of
pentylone in biological matrices of intoxication patients, but when associated with
others substances [14,15]. Until now, benzedrone was not associated with intoxication
cases, but is frequently reported in the mixtures of other synthetic cathinones sold on
illegal market [16,17].
Fig. 1 Structures of four synthetic cathinones dealt with this study
75
In blood samples, the concern about degradation of synthetic cathinones exists,
since enzymatic reactions keep occurring in this medium after sampling. Tertiary
cathinones usually show better stabilities as compared to secondary amines [18,19].
In forensics, quantitative results sometimes give a clue to consider this problem,
because even at adequate storage temperatures, the reanalysis of cases at different
times can lead to different concentration results, even false negatives. Ambach et al.
[20] reported stabilities of 64 NPS in DBS at room temperature and 4 °C, but for up to
only 14 days.
The aims of this work was therefore to develop a new method to determine four
synthetic cathinones (benzedrone, butylone, mephedrone and pentylone) in both dried
blood spots (DBS) and whole blood, and also to study the long-term stability of these
compounds in both matrices up to 90 days.
Materials and method
Standards and chemicals
Reference standards of butylone, mephedrone, pentylone and MDMA-d5 (used
as internal standard: IS) were purchased from Cerilliant (Round Rock, TX, USA);
benzedrone from Cayman Chemical (Ann Arbor, MI, USA); acetonitrile, methanol,
formic acid (98–100%) and t-butyl methyl ether from Merck (Darmstadt, Germany),
Scharlau (Barcelona, Spain) and Sigma-Aldrich (St Louis, MO, USA), respectively;
sodium tetraborate from LS Chemicals (Ribeirao Preto, SP, Brazil). Ultrapure water
was supplied by a Milli-Q RG unit from Millipore (Billerica, MA, USA). All solvents
employed in the extraction were HPLC grade. Whatman 903 Protein Saver Cards™
were obtained from GE Healthcare Bio-Sciences (Pittsburgh, PA, USA).
76
Standard solution preparation
Stock solutions of synthetic cathinones were prepared by dilution of reference
standards in methanol. Working methanol solutions for constructing calibrators at 100,
250, 500, 1000, 2500, 5000 and 10,000 ng/mL were prepared by appropriate dilution
of the stock solutions.
Quality control (QC) samples were prepared with methanol stock solutions at
low (final concentration of 75 ng/mL and 30 ng/mL for DBS and whole blood,
respectively), medium (400 ng/mL) and high (750 ng/mL) concentrations. IS solutions
were prepared by appropriate dilution of MDMA-d5 in methanol at 10 and 250 ng/mL
for DBS and whole blood, respectively. All solutions were stored at -20 °C.
DBS samples preparation
For the DBS preparation, 15 μL of blank or fortified whole blood was spotted
onto a 6-mm diameter pre punched Whatman 903 DBS paper cards disc. The blood
spots were allowed to dry at room temperature (25 °C) for 3 h before extraction.
Whole blood samples preparation
Fortified whole blood samples were prepared by spiking each reference
standard to blank blood, to give 200 µL each of samples at the above final
concentrations.
77
Extraction procedure for DBS
The 6-mm diameter disc was placed in a 2-mL polypropylene tube. The
extraction was performed with 300 μL of the IS solution (MDMA-d5 10 ng/mL). The
tubes were capped, vortexed for 10 min and centrifuged at 10,000 rpm for 10 min (room
temperature). The supernatant (150 μL) was transferred to liquid chromatography–
tandem mass spectrometry (LC–MS/MS) vials (2 µL of injection).
Liquid-liquid extraction of whole blood samples
For whole blood analysis, 200 µL of sample were pipette to a 2-mL
polypropylene tube, followed by 50 µL of IS solution (MDMA-d5 250 ng/mL), 200 µL of
sodium tetraborate saturated solution and 800 µL of tert-butyl methyl ether. The
samples were vortex for 5 min and centrifuged at 8000 rpm for 5 min. The supernatant
(600 µL) was evaporated at 40 °C under nitrogen stream and resuspended in 500 µL
of methanol, and 1 µL was analyzed by LC–MS/MS.
Instrumentation
The DBS analyzes were performed in a Nexera UHPLC chromatographic
system coupled to a LCMS8040 triple quadrupole mass spectrometer (Shimadzu,
Kyoto, Japan).
The chromatographic separation for DBS analysis was performed with a Kinetex
XB-C18 column (50 x 4.6 mm i.d., particle size 2.6 µm; Phenomenex, Torrance, CA,
USA), maintained at 40 °C. The mobile phase consisted of ultra-pure water (A) and
methanol (B), both containing formic acid (0.1%, v/v). The gradient elution was
78
programmed as follows: 10% B, followed by a linear increase to 95% B over 4 min,
held at 95% B for 2 min and returned to initial conditions over 0.2 min (total run time of
8.5 min). The mobile phase flow rate was 0.35 mL/min. To confirm the selectivity of
chromatographic system for DBS analysis, the whole blood method development and
validation, a Force Biphenyl (50 x 2.1 mm i.d., particle size 1.8 µm; Restek, Bellefonte,
PA, USA) was used to for analyte separation. The column was maintained at 50 °C.
Because of a column that support a higher pressure, the same mobile phase was used,
but with a flow rate at 0.5 mL/min and a gradient elution initiating at 5% B, followed by
a linear increase to 95% B over 2.8 min with hold for more 1.4 min, and it returned to
initial conditions over 0.1 min (total run time: 6 min).
The mass spectrometer was equipped with an electrospray ionization source
and was performed in the positive ionization mode. The source parameters optimized
were: heat block temperature 400 °C; capillary voltage, 4.5 kV; nebulizer gas (N2) flow,
3 L/min; desolvation line temperature, 250 °C; drying gas (N2) flow, 15 L/min; and
collision induced dissociation gas pressure (Ar), 230 kPa. The analyses were
performed in multiple reaction monitoring (MRM) mode. For each compound, two MRM
transitions were chosen for quantification and qualification. Table 1 shows the MRM
conditions optimized to the method. Data were acquired and processed by
LabSolutions 5.75 SP2 software (Shimadzu).
79
Table 1 Optimized multiple reaction monitoring (MRM) conditions to analyze four
cathinones and MDMA-d5 (internal standard: IS) using liquid chromatography–tandem
mass spectrometry (LC–MS/MS) in positive electrospray ionization mode
Analyte MRM transitions
(m/z)
Q1Pre
bias (V) CE (V)
Q3Pre
bias (V)
Mephedrone 178.20→160.15
178.20→145.10
19
-9
14
21
19
27
Pentylone 236.20→188.10
236.20→218.15
16
16
18
13
21
17
Butylone 221.90→174.35
221.90→204.30
15
15
18
13
21
24
Benzedrone 254.25→91.15
254.25→65.15
12
12
22
55
18
27
MDMA-d5 (IS) 198.95→165.10
198.95→107.15
20
20
14
25
19
21
Underlined transition used for quantification CE: collision energy
Validation of the method
Both methods were validated according to the guidelines of Scientific Working
Group for Forensic Toxicology (SWGTOX) recommendations for quantitative analysis
[21].
Linearity
Six or seven point calibration were used to acquire a linear curve that ranged
the common blood concentrations [22] and evaluated over 5 days. Calibrators should
appear as symmetric peaks, eluting within ±2% relative standard deviation (RSD) of
80
average retention times, and as peak areas within ±20% RSD of each nominal
concentration.
Limits of detection and quantitation
Limit of detection (LOD) were defined as the lowest concentration to reach
positive criteria of identification and response of three times the signal-noise of the
blank sample. To be included as positive criteria for identification, the analyte peak
retention time should be as that the same established with the working solution (±2 %),
and the second chosen transition peak (qualifier ion) should have the same ion ratio
(between 20 and 30% of allowance, according to relative ion intensity). For limit of
quantification (LOQ), the response should be ten times the signal-noise in comparison
to blank samples and be include in the above mentioned positive criteria of
identification.
Imprecision
Intraday and interday imprecisions were evaluated with three QC
concentrations (triplicate of each concentration) and with each group over 5 days of
analysis (ninter= 15). The target concentration was required to be within ±20%RSD.
Bias
The variation between real and nominal concentration is defined as bias. With
5 days of analysis, at three QC levels in triplicate, the bias was required to be lower
than ±20%.
81
Matrix effect
The matrix effects were evaluated using ten blank blood samples from different
sources. For low and high QC concentrations, blank samples were extracted following
the original procedure, and the extraction solvent was fortified with analytes and IS.
Additionally, other neat solutions were prepared with same solvent volume and final
concentration, and analyzed at the same batch. The peak area ratio of the mean of
fortified blank samples to that of neat solutions was used to calculate the percentage
of the matrix effect.
Carryover and interferences
The extract of a negative sample was injected into instrument after the highest
point of each calibration curve. If no analyte peak met LOD criteria, carryover was
absent.
To evaluated interferences, a mixture of 35 pharmaceuticals and other drugs of
abuse (Supplementary material Table 1), at stock concentration of 10,000 ng/mL each,
was added to the final DBS extract or to the fortified whole blood to be extracted, at
negative and LOQ levels, which were injected into the LC–MS/MS system.
Stability
Stability study was performed after complete method validation. It was delimited
a period of 90 days of study, with two concentrations (low and high QC) and three
different storage temperatures [25 °C (room), 4 °C and -20 °C]. At the first day (day 0),
82
all samples that would be used in whole period were prepared in triplicate and stocked
with desiccant until the day of their analysis. The samples analyses were performed
against the calibration curve prepared on that day. The analyte was considered stable
if the concentration differences were within ±20% of initial concentration.
Supplementary material Table 1 Third-five pharmaceuticals and other drugs of abuse
used to evaluate selectivity of a method for synthetic cathinones using dried blood
spots (DBS) and whole blood matrices.
Group Analytes
Pharmaceuticals
Flunitrazepam, Midazolam, Clonazepam, Diazepam,
Nitrazepam, Alprazolam, Bromazepam, Acetaminophen,
Sildenafil, Piperazine, Fluoxetine, Fentanyl, Phenobarbital,
Phenytoin, Carbamazepine, Secobarbital, Butalbital,
Pentobarbital, Valproic acid
Drugs of abuse
Ketamine, Thiofentanyl, Acrilfentanyl, Valerylfentanyl,
Pentedrone, PMA, 2C-E, 2C-B, Cocaine, MDMA, α-PVP,
Methilone, 4-fluoro-metcathinone, MDPV, Naphirone, N-
ethylpentylone
Results and discussion
DBS analysis
To avoid the hematocrit effect [23,24], fixed volume of fortified whole blood was
used in pre-cut DBS cards. A linear curve with six calibration points from 25 to 1000
ng/mL was performed, using a 1/x² weight linear regression (r> 0.990, for the four
cathinones). Angular coefficients of imprecisions for 5 days of analysis ranged from
4.2% for butylone up to 5.3% for benzedrone. LOD and LOQ were 10 and 25 ng/mL
(Fig. 2), respectively.
83
Fig. 2 Individual multiple reaction monitoring chromatograms obtained from DBS made
of whole blood spiked with a mixture of four synthetic cathinones at 25 ng/mL each
(limit of quantification)
Intraday and interday imprecisions were calculated by one-way analysis of
variance (ANOVA, p< 0.05) and showed low values of %RSD for all analytes at three
concentrations of QC (75, 400 and 750 ng/mL) as well as low bias values (Table 2).
84
Table 2 Method validation for the quantification of four cathinones in dried blood
spots (DBS) by LC–MS/MS
Analyte
Intraday
imprecision
(%RSD) (n=3)
Interday
imprecision
(%RSD) (n=15)
Bias (%)
(n=15)
Matrix effect
(%) (n=10)
Benzedrone
Low QC
(75 ng/mL) 2.7 3.6 1.2 3.2
Medium QC
(400 ng/mL) 2.7 5.3 -3.8 NA
High QC
(750 ng/mL) 2.2 3.3 -3.8 -5.7
Butylone
Low QC
(75 ng/mL) 2.6 5.7 5.1 8.9
Medium QC
(400 ng/mL) 3.3 5.1 0.5 NA
High QC
(750 ng/mL) 3.1 3.9 2.8 -8.7
Mephedrone
Low QC
(75 ng/mL) 2.0 7.1 3.5 -1.1
Medium QC
(400 ng/mL) 2.7 4.0 -3.0 NA
High QC
(750 ng/mL) 2.1 2.5 -1.0 -16.6
Pentylone
Low QC
(75 ng/mL) 4.4 4.5 1.8 2.1
Medium QC
(400 ng/mL) 2.3 3.1 5.8 NA
High QC
(750 ng/mL) 2.8 2.7 -1.5 -9.0
%RSD percent relative standard deviation, NA not evaluated, QC quality control
85
Matrix effects, evaluated at low and high QC, was lower than 9.0%, except for
mephedrone with ionization suppression at 16.6% (Table 2).
No carryover was observed in blank samples after analysis of a 1000 ng/mL
sample (higher calibrator) and no interferences were observed at negative and/or LOQ
samples in the presence of 35 others drugs and pharmaceuticals at 1000 ng/mL.
Whole blood analysis
Seven calibrators were used to obtain a linear curve ranging from 10 to 1000
ng/mL (1/x² weighted and r> 0.994). Both LOD and LOQ were 10 ng/mL.
Imprecisions (%RSD) and bias (%) were low, similar to those obtained with DBS
samples (Table 3).
Differently with DBS samples, matrix effects for whole blood were not greater
than 3.9%. Quantitative results for all analytes were not significantly influenced by
these matrix effects.
No carryover and interferences were observed using the same criteria
mentioned above.
86
Table 3 Method validation for the quantification of four cathinones in whole blood by
LC–MS/MS
Analyte
Intraday
imprecision
(%RSD) (n=5)
Interday
imprecision
(%RSD) (n=25)
Bias (%)
(n=25)
Matrix effect
(%) (n=10)
Benzedrone
Low QC
(30 ng/mL) 4.1 7.0 6.0 -2.2
Medium QC
(400 ng/mL) 3.4 6.2 0.2 NA
High QC
(750 ng/mL) 2.6 3.6 3.4 -0.3
Butylone
Low QC
(30 ng/mL) 4.0 5.5 8.3 1.7
Medium QC
(400 ng/mL) 3.2 4.7 0.4 NA
High QC
(750 ng/mL) 4.7 4.4 4.2 1.1
Mephedrone
Low QC
(30 ng/mL) 5.1 6.0 6.6 -2.6
Medium QC
(400 ng/mL) 3.0 4.4 1.4 NA
High QC
(750 ng/mL) 2.3 2.4 7.1 -2.8
Pentylone
Low QC
(30 ng/mL) 3.7 8.1 12.3 -3.9
Medium QC
(400 ng/mL) 4.2 5.8 10.2 NA
High QC
(750 ng/mL) 3.5 3.6 5.1 0.1
For abbreviations, see the footnote of Table 2
87
Effects of separation columns
A test was performed to evaluate C18 and biphenyl stationary phase columns
from the same DBS samples. Equally satisfactory results were obtained for both
columns; all of them were in accordance to the maximum of ±20% of variation criteria
(Table 4); both C18 and biphenyl stationary phase columns are applicable to analyze
synthetic cathinones.
Stability study
Because low stability of cathinones in whole blood has been reported [25,26],
90 days was stablished as a sufficient period after which differences between the
samples should be noted. As recently shown by Glicksberg and Kerrigan [27],
cathinones stability are temperature- and structure-dependent. Regardless the matrix,
butylone and pentylone showed the best stability, probably influenced by the
methylenedioxy group. For pentylone in whole blood, it was possible to detect this
molecule in the sample kept at room temperature up to 7 or 45 days, representing low
or high QC, respectively. In DBS samples detection was possible after 90 days (Fig.
3). Butylone showed a similar pattern; detection in whole blood were possible up to 25
or 60 days, and in DBS samples was possible up to 90 days. At either 4 or -20°C, no
significant degradation was noted for both DBS and whole blood samples (Fig. 4).
88
Table 4 Measured concentration and bias obtained from five replicates of DBS samples at 25 ng/mL (limit of quantification) when
analyzed using Force Biphenyl (Biphenyl) and Kinetex XB-C18 (C18) by LC–MS/MS
Butylone Pentylone Mephedrone Benzedrone
Column DBS
sample
Concentration
(ng/mL)
Bias
(%)
Concentration
(ng/mL)
Bias
(%)
Concentration
(ng/mL)
Bias
(%)
Concentration
(ng/mL)
Bias
(%)
Biphenyl A 23.7 -5.0 25.5 1.9 26.6 6.5 25.3 1.3
B 22.9 -8.6 28.1 12.3 24.5 -2.0 26.4 5.3
C 22.9 -8.4 26.8 7.1 25.6 2.3 24.1 -3.6
D 22.4 -10.2 26.1 4.3 24.2 -3.2 24.9 -0.3
E 23.7 -5.0 25.6 2.3 26.3 5.2 24.9 -0.3
C18 A 24.4 -2.2 25.1 0.4 25.4 1.5 29.6 18.5
B 25.7 2.8 24.5 -1.8 24.4 -2.2 26.8 7.1
C 24.7 -1.3 25.6 2.6 24.0 -4.0 26.0 3.9
D 27.0 8.0 26.8 7.2 25.4 1.8 29.0 16.0
E 26.6 6.2 27.6 10.3 24.3 -2.9 25.9 3.5
89
Fig. 3 Pentylone stability for 90 days, at three temperatures: a low quality control (QC)
of DBS (75 ng/mL), b high QC of DBS (750 ng/mL), c low QC of whole blood (30
ng/mL), d high QC of whole blood (750 ng/mL)
90
Fig. 4 Butylone stability for 90 days, at three temperatures: a low QC of DBS, b high
QC of DBS, c low QC of whole blood, d high QC of whole blood
Mephedrone and benzedrone, both with ring-substituted, presented the worst
stabilities. Quantitative analyses of mephedrone in whole blood samples kept at room
temperature were possible up to 7 days, both at low and high initial concentrations. At
4 °C, the initial mephedrone concentration decreased to about 60% after 90 days. For
DBS samples, stability at room temperature was also low (1 and 25 days), but at 4 °C,
decreased rate was much less (about 40%) when compared to whole blood (Fig. 5).
Benzedrone detection was possible in whole blood samples up to 1 and 7 days at room
temperature for low and high QC, respectively, and up to 60 days for low QC at 4 °C.
At -20 °C, a 23% decrease from the initial concentration was noted for high QC. For
DBS, detection at room temperature was possible for low and high QC samples up to
91
60 and 90 days, respectively. At 4 °C, quantification could be done up to 90 days, as
well as at -20 °C (Fig. 6).
Fig. 5 Mephedrone stability for 90 days, at three temperatures: a low QC of DBS, b
high QC of DBS, c low QC of whole blood, d high QC of whole blood
92
Fig. 6 Benzedrone stability for 90 days, at three temperatures: a low QC of DBS, b
high QC of DBS, c low QC of whole blood, d high QC of whole blood
Although the instability of cathinones is already known, few papers discuss
the possible products formed in this degradation. Tsujikawa et al [28] shows that once
more alkaline the solution, the degradation of 4-methylmethcathinone is higher. In
addition, the presence of antioxidant agents with same pH values tends to retard this
degradation. The degradation products formed come from the breakdown of the bond
with the secondary nitrogen, forming its corresponding alcohol.
The metabolism studies of Pedersen et al and Zaitsu et al [29,30] show that
different structures of the cathinones will undergo different reactions, possibly due to
their structural affinity with enzymes. Structures beta-keto amphetamines like tend to
reduce the carbonyl, forming the corresponding amino alcohol. Even in non-biological
93
matrices the stabilization of the molecule is observed, from the formation of an
intramolecular bond of enol hydrogen formed [31].
All these studies corroborate with the knowledge that the stability of the
synthetic cathinones are pH- and structure-dependent (e.g. tertiary amines present
greater stability, possibly due to the steric hindrance of this nitrogen). Possibly, in DBS
samples, enzymatic reactions were reduced, which conferred a higher stability.
Conclusions
A reliable and accurate DBS method to analyze cathinones using very small
volume of blood (15 µL) was developed. DBS showed great advantages as compared
to whole blood with liquid-liquid extraction, because of easy and rapid extraction
procedures, less space dedicated to sample storage and increase in stability for
cathinones (up to 83 days more stable in DBS as compared to whole blood).
Cathinones were unstable at room temperature both in DBS as well as in whole blood
samples, but its stability was much higher in DBS than in whole blood. We propose
that DBS should be used in forensic routine analysis of cathinones.
Acknowledgements
We thank the support by the Fundação de Amparo à Pesquisa do Estado de
São Paulo - FAPESP [grant numbers 2015/10650-8]; the Conselho Nacional de
Desenvolvimento Científico e Tecnológico - CNPq [grant number 131780/2017-4] and
the Superintendência da Polícia Técnico-Científica.
94
Compliance with ethical standards
Conflict of interest There are no financial or other relationships thatcould lead to
a conflict of interest.
Ethical approval All procedures performed in studies involving human
participants were in accordance with the ethical standards of the institutional research
committee (Comitê de Ética em Pesquisa da UNICAMP – CEP, CAAE
8452716.2.0000.5404 and Superintendência da Polícia Técnico-Científica, ofício No.
766/2015/ATS/SPTC-SSP).
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5. DISCUSSÃO GERAL
Durante o desenvolvimento do projeto, a fim de evitar diferenças na
dispersão das manchas obtidas no cartão de DBS devido a variações extremas nos
valores de hematócrito, foi estabelecido o corte inicial dos discos de tamanho
padronizado (6 mm) para posterior aplicação de volume fixo da amostra de sangue.
Foi padronizado também o volume máximo que o disco de papel poderia
absorver a amostra de sangue, sem que houvesse seu extravasamento e
consequente perda de amostra. A partir da aplicação de volumes crescentes de
sangue em diferentes discos, foi estabelecido o volume de 15 µL de amostra.
Para cada grupo de analito foi aplicado designer experimental do tipo
centroide simplex para otimização da mistura de solventes que levasse ao maior
rendimento de extração.
A separação cromatográfica mostrou sua importância principalmente na
análise de NBOMes, onde a presença de dois isômeros (25G-NBOMe e 25E-NBOMe,
m/z= 330) exigiu da cromatografia a separação completa dos picos. Na fase B foi
testado metanol e acetonitrila, mas só a acetonitrila forneceu a força cromatográfica
necessária para atingir resolução da linha de base entre eles.
Para conseguir alcançar a concentração em sangue total usualmente
reportada na literatura para intoxicações por NBOMes (entre pg/mL e pouco ng/mL) e
com um volume inicial de amostra tão baixo (15 µL), foi preciso um LC-MS/MS mais
sensível que o LCMS8040 (Shimadzu, Kyoto, Japão), tendo sido atingido com o 5500-
QTRAP (ABSciex, Ontário, Canadá).
Apesar de valores de imprecisão e inexatidão obtidos para as amostras de
NBOMes em DBS terem sido maiores que aqueles obtidos para sangue total,
possivelmente pelos baixos valores de concentração utilizados (0,1 a 10 ng/mL) e
volume de amostra (15 µL), ainda assim todos os analitos se mantiveram dentro da
faixa máxima de variação aceitável pelo guia de validação, mostrando que o método
é viável e preciso para ser aplicado na rotina laboratorial.
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Enquanto que as amostras de DBS apresentaram uma estabilidade de 6
meses para os NBOMes nas três temperaturas estudadas e em concentração baixa e
alta, excetuando o 25I-NBOMe (21% de perda da concentração inicial) e 25B-NBOMe
(22%), no sangue total, em temperatura ambiente, temos uma redução da
concentração para abaixo do limite de quantificação do método em 15 ou 30 dias,
dependendo do analito, para a concentração baixa e, pelo menos, 50% de redução
da concentração inicial das amostras em 6 meses de estudo. Em temperatura de 4
°C, na menor concentração, houve uma queda da concentração de, no mínimo, 20%
nos 6 meses, podendo ter sido quantificado por último no tempo de 90 dias (25B, 25C
e 25I-NBOMe). Esses resultados corroboram com a afirmação na literatura já
existente da capacidade do DBS em estabilizar alguns analitos [35-37].
Para as catinonas sintéticas, os resultados não foram tão expressivos em
um primeiro momento, já que, em temperatura ambiente, elas se mantiveram instáveis
assim como nas amostras de sangue total. Porém, foi possível observar que a taxa
de decaimento da concentração inicial das amostras foi muito mais expressiva no
sangue total do que no DBS. Desse modo, em altas concentrações, para analitos mais
instáveis, como a benzedrona, foi possível quantificar as amostras de sangue total em
no máximo 7 dias, enquanto que no DBS essa quantificação foi possível durante os
90 dias de estudo, 83 dias a mais. Nas amostras armazenadas em temperatura de 4
°C essa diferença na taxa de degradação também foi observada. Porém, para
temperaturas de -20 °C não foi possível estabelecer uma correlação dentro do período
de estudo.
Foi possível observar também que, assim como relatado na literatura, a
estabilidade das catinonas é dependente de sua estrutura, de modo que mesmo todas
sendo classificadas como aminas secundárias, a presença de um grupo metilenodioxi
na molécula tende a aumentar sua estabilidade quando comparadas as estruturas que
possuem um radical adicionado ao anel benzeno.
Na rotina forense, onde a possibilidade de reanálise da amostra decorrido
longo tempo da primeira não é incomum, a instabilidade dos analitos após a coleta da
amostra, mesmo que sob condições apropriadas de armazenamento, é uma das
grandes preocupações dos profissionais dessa área. Não só isso, a logística de
encaminhamento das amostras ao laboratório de toxicologia, o tempo decorrente
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desse trajeto, assim como o extenso número de casos por ano que os laboratórios
devem analisar, faz com que o período entre a coleta da amostra e sua análise não
seja imediato, aumentando a probabilidade de que analitos que possuam baixa
estabilidade no fluído biológico se degradem até a sua análise, chegando até a um
resultado falso negativo. Assim, resultados como os apresentados nessa dissertação
mostram uma possível alternativa para resolução deste problema.
Somado ao ganho na estabilidade, a coleta de amostras e armazenamento
nos discos de DBS, apresentam a vantagem de, em um mesmo espaço, ser possível
o armazenamento de um número maior de amostras, o que reduz o custo à longo
prazo, e, em curto prazo, mantem-se a estimativa de custo equivalente. Uma caixa de
cem unidades de tubos de coleta heparinizados, sai, em média, R$ 65,00 (site da
Labor import shop), um custo de R$ 0,65 por amostra; uma caixa com 100 cartões de
DBS, R$ 858,00 (site da Sigma-Aldrich®). Apesar do valor discrepante, cada cartão
equivale a R$ 8,60 e, em cada cartão, é possível gerar, no mínimo, 13 amostras em
triplicatas, levando a R$ 0,66 por amostra.
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6. CONCLUSÃO
Métodos analíticos sensíveis, precisos e exatos foram desenvolvidos para
determinação de substâncias pertencentes a duas importantes classes de NPS, em
amostras de DBS e sangue total.
A técnica de amostragem por DBS se mostrou vantajosa em comparação
ao armazenamento da amostra de sangue total convencional, por proporcionar melhor
estabilidade para NPS da classe dos NBOMes e das cationonas sintéticas. Além de
ocupar menos espaço de armazenamento, sua extração se torna mais rápida e
facilitada, já que necessita de menos etapas até obtenção do extrato a ser
efetivamente analisado, seu transporte não requer logística especial, além de tornar
possível a identificação de alguns analitos de interesse por um maior período de
tempo, mesmo, em alguns casos, quando armazenado em temperatura ambiente.
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8. ANEXOS
ANEXO 1: Parecer do Comitê de Ética em Pesquisa da Unicamp
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