Solid-phase extraction coupled in-line to capillary electrophoresis: determination of drugs of abuse in biological samples
- Autores: Tatiana Baciu
- Directores de la Tesis: Maria del Carme Aguilar Anguera (dir. tes.), Marta Calull Blanch (codir. tes.)
- Lectura: En la Universitat Rovira i Virgili ( España ) en 2017
- Idioma: español
- Tribunal Calificador de la Tesis: Francesc Borrull Ballarin (presid.), Fernando Julián Benavente Moreno (secret.), Rawi Ramautar (voc.)
- Programa de doctorado: Programa de Doctorado en Ciencia y Tecnología Química por la Universidad Rovira i Virgili
- Materias:
- Enlaces
- Tesis en acceso abierto en: hdl.handle.net TDX
- Resumen
ABSTRACT The (ab)use of drugs of abuse (DOAs) represents important social and health problem worldwide. Detection and quantification of these substances in biological samples of drug (ab)users have important clinical and forensic applications, from diagnosis of acute/lethal intoxication, monitoring of compliance/abstinence in detoxification centres to identification of drugs affecting performance at work (workplace drug testing), while driving (driving under the influence of drugs) and in sports (doping control). Within this context, there is an emerging need for the continuous development of analytical methods suitable to determine these compounds in biological samples.
Capillary electrophoresis (CE), with its high efficiency and resolution, possibility of automation, minimal consumption of reagents and low sample volume for analysis, as well as it low cost, is a well-established analytical separation technique that has been successfully applied for drug testing purposes. However, despite these great benefits, CE suffers from poor concentration sensitivity, especially when ultraviolet detection (UV) is used. To overcome this problem, several strategies have been developed, including the in-line coupling between solid-phase extraction (SPE) and CE (in-line SPE-CE), which has proven to be highly useful for improving the sensitivity of CE in the analysis of different kinds of compounds, including DOAs. Traditionally, in in-line SPE-CE, a hand-made SPE microcartridge containing an appropriate sorbent material is inserted near the injection end of the capillary.
In view of the above, the research included in this Doctoral Thesis aimed to develop inexpensive, green and easily performed methods suitable for determining DOAs in human biological samples by means of in-line SPE-CE-UV. Classic DOAs, such as cocaine, codeine, methadone and morphine, were investigated since these substances are frequently consumed. The major metabolite of cocaine, benzoylecgonine, and a heroin-specific metabolite, 6-acetylmorphine, were also included. Meanwhile, new psychoactive substances, such as mephedrone and 3,4-methylenedioxypyrovalerone, have gained popularity among young people and teenagers in recent years. The abuse of these drugs has soared to reach epidemic levels. For this reason, these compounds, as well as 4-methylephedrine (mephedrone metabolite), were also analysed here. With respect to the biological samples analysed, the research particularly focused on hair and urine. Urine represents one of the most conventional matrices used to detect (ab)use of DOAs, while hair has emerged over the past years as an alternative and/or complementary matrix due to its extended detection window, allowing retrospective monitoring of consumption for several months and even up to years before sample collection.
The experimental work presented in this Doctoral Thesis has been structured into two parts. In the first part, the potential of in-line SPE-CE-UV was explored for the quantitative determination of different DOAs in hair samples. Three scientific articles, published in different international journals, have resulted from the obtained data here. In all cases, a 2 mm length segment of a capillary of 150 µm internal diameter (i.d.) packed with 60 µm particle size Oasis HLB (reversed-phase) was used for the construction of the in-line SPE-CE microcartridge. Particularly, the first paper deals with the determination of cocaine and benzoylecgonine in hair samples from drug abusers. To extract the analytes from the hair matrix, an overnight acidic digestion procedure was followed from the literature and successfully combined with the in-line SPE-CE system.
Since the enantiomers of a given drug might have different pharmacological and toxicological effects, the development of analytical methods that could cope with the identification and quantification of each of them in biological samples can provide valuable assistance in interpreting drug testing results. Of the DOAs examined in this Doctoral Thesis, methadone occurs as a chiral molecule. In view of this, the second paper deals with the simultaneous determination of 6-acetylmorphine, benzoylecgonine, cocaine, codeine and morphine, all of which are non-chiral compounds, and the enantiomers of methadone, in hair samples from drug abusers. α- and β-cyclodextrin (CD) were evaluated as chiral selectors, of which α-CD proved to be the most appropriate for the intended purpose. To extract the analytes from the hair matrix, a procedure based on pressurized liquid extraction (PLE) was developed and satisfactorily combined with the in-line SPE-CE system. An acidic aqueous solution was used as the extracting medium. Compared to the overnight acidic digestion applied in our previous work (around 16 hours), the use of PLE instrument enabled us to extract the target drugs in a massively reduced time (around 15 minutes), representing an excellent achievement in the field of hair drug testing.
Mephedrone and its metabolite, and 3,4-methylenedioxypyrovalerone also exist as chiral molecules. Within this framework, the third paper focuses on the development of a new method based on the in-line coupling between SPE and CD-modified-CE with a previous sample pretreatment procedure based on PLE for the enantiomeric determination of these substances in hair samples. α-, β- and γ-CD, and highly sulphated-γ-CD were evaluated as chiral selectors, of which β-CD provided the best results in terms of resolution. The PLE was completed within 8 minutes using a basic aqueous solution as the extracting medium.
The three analytical methods established in the first part were validated in line with the guidelines of the Scientific Working Group for Forensic Toxicology and Society of Toxicological and Forensic Chemistry guidelines. Linearity, repeatability, reproducibility, limit of detection and quantification, and relative recoveries were evaluated using pooled drug-free hair samples spiked with the studied analytes. In all cases, the limits of detection (LODs) achieved with UV detection for the hair matrix were in the range 0.02-0.13 ng/mg, which are generally in accordance with the cut-offs recommended by the Society of Hair Testing, except for morphine (1.0 ng/mg). Quantitative precision in both intraday and day-to-day experiments was very satisfactory with relative standard deviations (RSDs) less than 13%. Relative recoveries greater than 80% were obtained. In addition, the hair pretreatment procedures proposed here based on acidic digestion and PLE allow a considerable simplification of the sample preparation process, especially when compared to procedures involving the use of organic solvents. This is because the hair extract solutions can be directly injected into the in-line SPE-CE system without the need for evaporation and reconstitution steps.
Recently, magnetic particles have emerged as a new generation of adsorbent materials used in sample preparation. Due to their magnetic properties, these can be easily manipulated inside a CE capillary just by the application of a magnetic field. Therefore, magnetic particles represent a very simple way to create an analyte concentrator inside the capillary. Taking advantage of this feature, the second part, which consists of a scientific article reported in an international journal, focuses on the ability to construct SPE microcartridges inside a 50-µm internal diameter CE capillary using magnetic particles as the adsorbents for the extraction of DOAs. Particularly, silica-coated iron oxide magnetic particles functionalized with octadecyl groups were synthesized, loaded into the capillary and used to preconcentrate cocaine, codeine, methadone and morphine in urine samples from drug abusers. For trapping the magnetic particles, two commercial neodymium iron boron magnets were used.
The analytical method established in the second part was also validated in accordance with the guidelines of the Scientific Working Group for Forensic Toxicology and Society of Toxicological and Forensic Chemistry guidelines. Linearity, repeatability, reproducibility, limit of detection and quantification, and relative recoveries were evaluated using pooled drug-free urine samples spiked with the studied analytes. The developed method provided LODs for urine samples in the range 20-50 ng/mL, which are below the cut-offs recommended by the European Workplace Drug Testing Society and Substance Abuse and Mental Health Services Administration. Quantitative precision in both intraday and day-to-day experiments was satisfactory with RSDs less than 20%. Relative recoveries greater than 76% were obtained.
Combining magnetic particles in-line with CE proved to offer advantages over the conventional in-line SPE-CE configuration used in the first part of the present Doctoral Thesis, resulting in an easier and faster way to create an analyte concentrator inside the capillary, which does not require time-consuming procedures for manufacture. The use of a very simple instrumentation and the achievement of satisfactory sensitivity make this strategy a reasonably attractive analytical tool for routine analysis of DOAs.
The use of both in-line SPE-CE configurations has proven to be valuable and promising approaches to detect DOAs in biological samples. The research results obtained here contribute to the overall knowledge of both techniques in the field of drug testing, proposing cheap, simple and environmentally friendly CE-based methodologies.
