Browsing by Author "Rosado, Tiago Alexandre Pires"
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- Miniaturized approaches for sample preparation in hair testing for drugs of abuse and their application in clinical and forensic scenariosPublication . Rosado, Tiago Alexandre Pires; Gallardo Alba, Maria Eugénia; Barroso, Mário Jorge Dinis; Vieira, Duarte Nuno PessoaHair is nowadays one of the most important alternative matrices that have attracted attention for the analysis of various drugs. The fact that it can be collected under supervision, the lower probability of tampering with, and the greater stability are cited as major advantages. As with conventional matrices, the preparation of hair samples is an important step for clean-up and pre-concentration of analytes, which significantly affects the reliability and accuracy of the analysis. The use of miniaturized pre-concentration techniques, driven by the concept of “green chemistry”, has minimized the waste usually associated to classical techniques, and microextraction techniques are known for using lower solvent volumes and for saving time; however, their applicability to hair samples is still poorly explored. The aim of this dissertation is to discuss the status of miniaturized clean-up approaches for hair samples. In addition, the advantages of microextraction by packed sorbent (MEPS) in different analytical fields are addressed, and the practical applicability of this technique in hair samples is demonstrated by three different works using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS): determination of selected opioids; determination methadone and EDDP; determination of cocaine and metabolites. These novel methods were optimized and validated according to internationally accepted guidelines. Regarding the current status of the application of miniaturized approaches to hair samples, an increased research has been observed in both solid-phase (SPME) and liquid phase microextraction (LPME), with SPME showing higher representativeness. In this last approach, dispersive micro-solid phase extraction (D-μ-SPE) emerges as the most used in the last 5 years, if we do not include the different variants of fibre microextraction, namely the direct immersion (DI-SPME), headspace (HS-SPME) or in tube (IT-SPME) approaches. Moreover, D-μ-SPE was the one that showed more innovations in terms of solid sorbent material, driven by developments with carbon nanotubes, graphene, graphene oxide (GO), and the use of modified magnetic nanoparticles (MNPs) and ion-imprinted polymers (IIPs). As for LPME, the use of hollow fibre (HF)-LPME has been extensively explored for hair samples, showing great versatility for target analytes. Improvements were also observed by functionalization with GO and by the use of ionic liquids (IL). However, when all variants of dispersive liquid-liquid microextraction (DLLME) are considered, this is undoubtedly the most researched approach. The inclusion of methods involving solidification of a floating organic drop (DLLME-SFO), supramolecular solvents (SM-DLLME), and temperature-controlled ionic liquids (TIL-DLLME) were considered very beneficial due to their lower toxicity. Of all the microextraction techniques applied to hair samples, MEPS seems to be little explored. In fact, before this project was initialized , there was only one paper that had applied MEPS to pre-concentrate analytes from hair samples. Nonetheless, MEPS has been successfully used to extract a variety of compounds from different matrices, and its applicability has been demonstrated in a number of areas, including therapeutic monitoring, forensic toxicology, and food and environmental analysis. This technique is considered as a miniaturization of the classical solid phase extraction (SPE) and was developed in 2004 by Abdel-Rehim with the aim of reducing the volumes of both the sample and the solvents. It also became very attractive as it allows the reuse of the sorbent material and offers an automated procedure by easy coupling to chromatographic systems. In MEPS, the sorbent is reduced (1-4 mg) and is located in a micro-syringe instead of a cartridge. In turn, the sample flows bidirectionally through the sorbent (aspirations), improving the efficiency of the process due to the increased interaction with the sorbent. Considering the potential of MEPS in the context of sample preparation, we decided to test its usefulness for hair samples in three applications. In the first work, we present an analytical method which was developed and validated for the determination of tramadol (TRM), codeine (COD), morphine (MOR), 6- acetylcodeine (6-AC), 6-monoacetylmorphine (6-MAM) and fentanyl (FNT) using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). Using an M1 sorbent (4 mg; 80% C8 and 20% SCX), the procedure included the following steps: (i) conditioning (3 x 250 μL of methanol and 3 x 250 μL of 2% formic acid); (ii) sample load (15 x 150 μL); (iii) washing (150 μL of 3.36% formic acid); and (iv) elution (8 x 100 μL of 2.36% ammonium hydroxide in methanol). Linearity was obtained for all compounds between the lower limit of quantification (LLOQ) and 5 ng/mg, with determination coefficients higher than 0.99. The obtained LLOQs were 0.01 ng/mg for TRM, COD and 6-AC and 0.025 ng/mg for MOR, 6-MAM and FNT. The recoveries ranged from 74 to 90% (TRM), 51 to 59% (COD), 22 to 36% (MOR), 69 to 99% (6-AC), 53 to 61% (6-MAM) and 75 to 86% (FNT). The method proved to be precise and accurate with coefficients of variation typically below 15% and relative errors within a range of ± 15%, respectively. In the second work, a procedure was developed for the rapid concentration of methadone and its main metabolite (EDDP). The miniaturized approach was coupled to GC-MS/MS. MEPS was performed with an M1 (4 mg; 80% C8 and 20% SCX) sorbent conditioned with three cycles of methanol (250 μL) and three cycles of 2% formic acid (250 μL). Subsequently, the sample was loaded through nine cycles of 150 μL followed by a washing step that involved three cycles of 50 μL with 3.36% formic acid. For the elution of the compounds, six cycles of 100 μL with 2.36% ammonium hydroxide in methanol were used. The method was linear from 0.01 to 5 ng/mg for both compounds, with determination coefficients greater than 0.99. The recoveries ranged from 73 to 109% for methadone and from 84 to 110% for EDDP. Finally, precision and accuracy were in accordance with the international guidelines for analytical method validation. In the third work, a MEPS technique, with M1 (4 mg; 80% C8 and 20% SCX) sorbent, was developed for the pre-concentration of cocaine (COC), benzoylecgonine (BEG), ecgonine methyl ester (EME), norcocaine (NCOC), cocaethylene (COET) and anhydroecgonine methyl ester (AEME). The determination of the compounds was carried out using GC-MS/MS. The final procedure consisted of the following steps: (i) conditioning (250 μL of methanol and 250 μL of deionized water); (ii) sample load (21 x 150 μL); (iii) washing (50 μL of deionized water and 50 μL of acetate buffer pH 4); and (iv) elution (3 x 100 μL of 2% ammonium hydroxide in methanol). The obtained recoveries were considered acceptable for most compounds, namely 44-64% for COC, 63-73% for COET, 21-28% for BEG and 36-44% for NCOC. Lower recoveries were obtained for AEME (4-6%) and EME (1-3%). The method was linear between the LLOQs (0.010 ng/mg for COC and COET, 0.025 ng/mg for EME, BEG and NCOC and 0.150 ng/mg for AEME) and 5 ng/mg. In turn, the method was considered precise and accurate with coefficients of variation below 15%, and with an average relative error within ± 15% for all compounds, except for LLOQ (20%). With the successful application of MEPS it has been demonstrated that this miniaturized technique is an excellent alternative for toxicological analysis in hair samples. MEPS has the advantage of reducing solvent use, and the sorbent may be reused (> 100 extractions), which can be economically attractive to laboratories. Although there has been considerable academic interest in miniaturized clean-up approaches over the past five years, few implementations have been observed in routine laboratories. With the relevance and applicability herein described, we expect this panorama to change in the near future.