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Authors
Abstract(s)
O consumo de drogas remonta há inúmeros anos e continua, nos dias de hoje, a representar
um problema a nível global. A cocaína (COC) é umas das drogas mais consumidas a nível
mundial e atua como estimulante do sistema nervoso central (SNC), enquanto que os
opiáceos, lícitos ou ilícitos, atuam como depressores do mesmo. De forma a detetar e
quantificar estas drogas podem ser utilizadas várias amostras biológicas, destacando-se a
utilização do fluido oral, que nos últimos anos tem vindo a aumentar por se tratar de uma
amostra de recolha fácil e não invasiva. Uma das maiores limitações dos laboratórios de
toxicologia reside na dificuldade de armazenar amostras antes e após análise até a sua
destruição ou ordem devido aos sistemas clássicos de armazenamento. Além disso devido
ao elevado volume de amostras que alguns laboratórios recebem, tal obriga a que muitas
vezes estas amostras tenham de ser guardadas até à sua análise, podendo estar
comprometida a estabilidade dos analitos nelas presentes. Por este motivo o parâmetro da
estabilidade, é cada vez mais um parâmetro fundamental que deve ser estudado em
qualquer desenvolvimento e validação de um método bioanalítico. Existem atualmente
diferentes sistemas de tratamento de amostras, dentre eles os dried saliva spots (DSS). Os
DSS, uma variação dos dried blood spots (DBS), consistem em dispositivos que permitem a
recolha e o tratamento de amostras de fluido oral com a vantagem do rápido processamento
laboratorial e baixo custo.
Assim sendo, o presente estudo tem como principal objetivo avaliar a influência de
diferentes conservantes (ácido ascórbico, fluoreto de sódio e azida de sódio) na estabilidade
da COC e opiáceos, bem como dos seus metabolitos, em amostras de fluido oral utilizando
como técnica de extração os DSS e como técnica de identificação e quantificação a
cromatografia gasosa acoplada à espetrometria de massa em tandem (GC-MS/MS). Os
analitos englobados no estudo foram a COC, ecgonina metilester (EME), benzoilecgonina
(BEG), cocaetileno (COET), norcocaína (NCOC), morfina (MOR), codeína (COD) e 6-
monoacetilmorfina (6-MAM). O procedimento de extração previamente otimizado,
resultou na aplicação de 50 µL de fluido oral, com conservante, nos spots que foram
deixados a secar 12 horas. A extração foi realizada com a adição de 3 mL de metanol, seguido
de agitação durante 5 minutos, centrifugação, evaporação à secura do sobrenadante,
derivatização e posterior injeção no sistema de GC-MS/MS. Os parâmetros suscetíveis de
influenciar a estabilidade como a temperatura, luz, concentração do conservante e tempo
de armazenamento foram avaliados recorrendo a um desenho experimental (DOE, do inglês design of experiments), uma ferramenta estatística que permite avaliar de forma
multivariada estes parâmetros e o modo como interagem entre si.
No caso do ácido ascórbico, a BEG, a COD e a MOR apresentaram o fator tempo como
aquele com maior influência na estabilidade. Relativamente ao fluoreto de sódio nenhum
dos fatores teve influência estatisticamente significativa, e a azida de sódio foi descartada
uma vez que apresentava piores resultados relativamente à resolução cromatográfica dos
analitos em estudo. Assim, as melhores condições que permitem observar um aumento da
estabilidade, e por isso as escolhidas para realizar o estudo de estabilidade a longo prazo,
foram temperatura ambiente, presença de luz e 1% de fluoreto de sódio.
No que diz respeito à estabilidade a longo prazo os resultados revelaram que, nas condições
referidas, a COD foi estável apenas 1 dia, a EME manteve-se estável durante 3 dias, a COC,
o COET, a NCOC e a 6-MAM foram estáveis por 7 dias, a MOR apresentou estabilidade de
14 dias e a BEG manteve-se estável ao longo de todo o estudo (136 dias).
Este estudo permitiu avaliar quais as melhores condições para maximizar a estabilidade da
COC, opiáceos e respetivos metabolitos em amostras de fluido oral, permitindo ainda
avaliar a utilização dos DSS como dispositivos de recolha, armazenamento e tratamento das
amostras. É ainda importante salientar que este é o primeiro estudo que deteta estas drogas
de abuso em amostras de fluido oral com recurso à técnica de DSS e que avalia a sua
estabilidade.
Drug use dates back many years and continues to be a global problem. Cocaine (COC) is one of the most consumed drugs in the world and acts as a central nervous system (SNC) stimulant, while opioids, legal or illegal, act as depressants of the same. In order to detect and quantify these drugs, several biological samples can be used and in recent years, the use of oral fluid, has been increasing due to its easy and non-invasive collection. One of the biggest limitations of toxicology laboratories is the difficulty of storing samples before and after analysis, which is due to the classic storage systems. This is even more important for heavily busy laboratories, and may have consequences in the stability of the analytes. For this reason, the stability is increasingly an essential parameter that must be studied in any development and validation of a bioanalytical method. Different sample treatment systems exist, and dried saliva spots (DSS) is one of them. DSS, a variation of dried blood spots (DBS), consist of a device that allows the collection and treatment of oral fluid samples with the advantage of rapid sample processing with low cost associated. Therefore, the present study aims to evaluate the influence of different preservatives (ascorbic acid, sodium fluoride and sodium azide) on the stability of COC and opiates, as well as their metabolites in oral fluid samples using DSS as extraction technique and gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) for identification and quantification of the selected analytes. The chosen substances were COC, ecgonine methylester (EME), benzoylecgonine (BEG), cocaethylene (COET), norcocaine (NCOC), morphine (MOR), codeine (COD) and 6-monoacetylmorphine (6-MAM). The previously optimized extraction procedure resulted in the application of 50 µL of oral fluid with preservative to the spots after which were left to dry for 12 hours. The extraction was performed by adding 3 mL of methanol, followed by agitation for 5 minutes, centrifugation, evaporation of the supernatant to dryness, derivatization and subsequent injection into the GC-MS/MS system. The parameters likely to influence stability such as temperature, light, preservative concentration and time of storage were evaluated using the design of experiments (DOE), a statistical tool that allows multivariate evaluation of these parameters and the way they interact with each other. For ascorbic acid, BEG, COD and MOR presented the time factor as the one with greatest influence on stability. Regarding sodium fluoride, none of the studied factors had a statistical influence, and sodium azide was rejected, as it presented the worst results regarding the chromatographic resolution of the analytes under study. This way, the best conditions that increased stability, and therefore those chosen for the long-term stability study, were room temperature, presence of light and 1% sodium fluoride. Regarding long-term stability, the results revealed that under the mentioned conditions, COD was only stable for 1 day, EME was stable for 3 days, COC, COET, NCOC and 6-MAM were stable for 7 days, MOR was stable for 14 days and BEG remained stable throughout the study period of time (136 days). This study allowed us to assess which were the best conditions to maximize the stability of COC, opiates and their metabolites in oral fluid samples, allowing, also, the evaluation of DSS as devices for storage and sample treatment. It is also important to note that this is the first study that detects these drugs of abuse in oral fluid samples using DSS technique and evaluates their stability.
Drug use dates back many years and continues to be a global problem. Cocaine (COC) is one of the most consumed drugs in the world and acts as a central nervous system (SNC) stimulant, while opioids, legal or illegal, act as depressants of the same. In order to detect and quantify these drugs, several biological samples can be used and in recent years, the use of oral fluid, has been increasing due to its easy and non-invasive collection. One of the biggest limitations of toxicology laboratories is the difficulty of storing samples before and after analysis, which is due to the classic storage systems. This is even more important for heavily busy laboratories, and may have consequences in the stability of the analytes. For this reason, the stability is increasingly an essential parameter that must be studied in any development and validation of a bioanalytical method. Different sample treatment systems exist, and dried saliva spots (DSS) is one of them. DSS, a variation of dried blood spots (DBS), consist of a device that allows the collection and treatment of oral fluid samples with the advantage of rapid sample processing with low cost associated. Therefore, the present study aims to evaluate the influence of different preservatives (ascorbic acid, sodium fluoride and sodium azide) on the stability of COC and opiates, as well as their metabolites in oral fluid samples using DSS as extraction technique and gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) for identification and quantification of the selected analytes. The chosen substances were COC, ecgonine methylester (EME), benzoylecgonine (BEG), cocaethylene (COET), norcocaine (NCOC), morphine (MOR), codeine (COD) and 6-monoacetylmorphine (6-MAM). The previously optimized extraction procedure resulted in the application of 50 µL of oral fluid with preservative to the spots after which were left to dry for 12 hours. The extraction was performed by adding 3 mL of methanol, followed by agitation for 5 minutes, centrifugation, evaporation of the supernatant to dryness, derivatization and subsequent injection into the GC-MS/MS system. The parameters likely to influence stability such as temperature, light, preservative concentration and time of storage were evaluated using the design of experiments (DOE), a statistical tool that allows multivariate evaluation of these parameters and the way they interact with each other. For ascorbic acid, BEG, COD and MOR presented the time factor as the one with greatest influence on stability. Regarding sodium fluoride, none of the studied factors had a statistical influence, and sodium azide was rejected, as it presented the worst results regarding the chromatographic resolution of the analytes under study. This way, the best conditions that increased stability, and therefore those chosen for the long-term stability study, were room temperature, presence of light and 1% sodium fluoride. Regarding long-term stability, the results revealed that under the mentioned conditions, COD was only stable for 1 day, EME was stable for 3 days, COC, COET, NCOC and 6-MAM were stable for 7 days, MOR was stable for 14 days and BEG remained stable throughout the study period of time (136 days). This study allowed us to assess which were the best conditions to maximize the stability of COC, opiates and their metabolites in oral fluid samples, allowing, also, the evaluation of DSS as devices for storage and sample treatment. It is also important to note that this is the first study that detects these drugs of abuse in oral fluid samples using DSS technique and evaluates their stability.
Description
Keywords
Cocaína Dried Saliva Spots Estabilidade Fluido Oral Opiáceos