Hair is considered an alternative specimen for the identification of drugs. It is routinely used as a tool for the detection of xenobiotics (drugs of abuse, pharmaceuticals, environmental contaminants, doping agents, etc.) in forensic science, traffic medicine, occupational medicine and clinical toxicology. Analysis of drugs and their metabolites which are incorporated in the hair shaft, can provide information about the quantity and historical pattern of individual drug use. This type of information is not available from any other specimens. The Toxicological Chemistry Analysis laboratory co-ordinated by Prof. Veniero Gambaro, where this research doctorate was carried out, analyses hair in cases of poisoning or investigation of dependency on substances of abuse. As a result, participation in an external quality control project on the search for substances of abuse in hair was decided for improving the identification of critical passages in the context of this matrix. This project, known as HAIRVEQ, is promoted by the Senior Health Institute. Some reported research describing various operational procedures for the search for substances of abuse in hair was taken into consideration, the critical points analysed further and new hypotheses of work to improve or innovate the control procedures were assessed. The traditional procedure consists of several steps: • sampling: hair is best collected from the area at the back of the head; • washing: this is required to rule out surface contamination; • hydrolysis: this is required to enhance drug solubility; • extraction: liquid-liquid or SPE extraction were applied for drug purification; • derivatization; • analysis by GC/MS. Initially, this procedure was applied on drug-free control hair spiked with a standard solution of cocaine, opiates and cannabinoids. Then the method developed was used to analyse hair samples, including pubic hair, collected from drug abusers. The washing stage is particularly important to avoid any external contamination. It is an operation which could already lead to a preliminary, unwanted extraction of analyte from the biological sample. The next, equally important, step is the amount of material to subject to analysis. This choice has a considerable effect of the final result expressed, in this field of application, as nanograms of analyte over milligrams of hair weighed. As a result, the sensitivity of the method can be augmented by increasing the amount of material to examine. This is possible because the hair matrix does not contain high amounts of interfering endogen substances and a correct assessment of samples with concentrations near to the cut-off level can be made. The cut-off levels are concentration values below which a sample is considered negative. This parameter is used in diagnostic comparison in Forensic Medicine or checks carried out for suitability to drive motor vehicles. Hydrolysis of the keratin matrix was carried out chemically, both in basic or acid conditions depending on the substance to be looked for. Enzymatic-type hydrolysis is also being experimented for use in the preparation of the sample for the application of immune-enzymatic tests. This technique is faster as it does not require ‘overnight’ incubation of the sample and is, therefore, more suitable for routine analyses. Today, chromatographic procedures, especially those coupled with mass spectrometry, are the gold standard for the identification and quantification of drugs in hair owing to their separation ability and sensitivity of detection. In this work, procedures for the detection of opiates, cocaine and its metabolite benzoylecgonine, amphetamines and cannabinoids were carried out. GC/MS in the electron impact mode (EI) and chemical ionization mode (CI) were used for the identification and quantitative determination of these compounds. As a result, some molecules need a derivatization stage. The polar functions of the molecules need to be protected in opiates while the limited fragmentation of amphetamines does not allow differentiation. Therefore, the derivatization stage is useful for obtaining mass spectra which can be differentiated from molecule to molecule. As a result, the applicability of the procedure carried out for the detection of methamphetamine, Methylenedioxyethylamphetamine (MDE) and methylenedioxymethamfetamine (MDMA-Ecstasy) was checked. Encouraging results were obtained but the derivatization step is more difficult for these compounds. Several common derivatizing reagents such as, bis-(trimethylsilyl)trifluoroacetamide (BSTFA), pentafluoropropionic anhydride (PFPA), N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and trifluoroacetic anhydride (TFA) were tested in standard solutions and drug-free control hair spiked with standard solutions. PFPA was the most suitable derivate. Condensation of amphetamines with cycloexanone was also carried out. The method was simple: the dried residue of extraction was mixed with cyclohexanone and injected into the GC/MS without incubation at 70°C for 30 minutes. Positive results were only obtained for primary amine. An important step in this experimentation was the assessment of the precision and accuracy of the methods using the isotopic dilutions technique which was applied to the analysis of acid compounds such as those in the cannabinoid class: Tetrahydrocannabinol (THC) and its metabolite l-9-Carboxy-11-nor- Tetrahydrocannabinol (THC-COOH). Use of the deuterated internal standard in the context of research was very difficult as it is very delicate and must be reconstituted frequently; it deteriorates in the non-deuterated one giving rise to false positives. Preparation of the sample by the method developed required several hours; therefore, the extraction step was eliminated using a new system, called Chromate-Probe, for intra-injector thermal extraction. This direct sample introduction device transforms a conventional GC injector in a GC-MS system, into a very cost effective alternative to the standard direct insertion probe. Sampling was in a small vial (capacity 30µL) which, by retaining the harmful and non-volatile matrix residue, eliminated the need for extraction. Each analysis began with gentle evaporation followed by brief heating of the injector required for achieving intra-injector thermal extraction and vaporisation of the sample compound. The sample semi-volatile compounds were focused on the early portion of the separation column and GC analysed, as normal. This method was based on compound identification by mass spectra and found to have the following advantages: reduction or elimination of sample preparation, efficient thermal extraction, and smaller sample size etc. Initially, standard solutions and dried residues of extraction were placed directly in the vial and then analysed by Chromato-Probe to performing instrument conditions. The EI and CI modes were compared in these experiments and the same results obtained. Afterwards a single hair and pulverized hair samples collected from drug abusers were transferred to a chromate-probe vial and analysed by the method carried out. The best results were obtained from the second one. Furthermore, extraction was found to be more efficient by adding 10µl methanol to the vial. Positive results were obtained for codeine. The system proved to be suitable for the study of this type of matrix and analysis times were notable reduced as no preparation of the sample is required. However, the procedure was found not to be applicable as the very small amount of material subjected to direct extraction did not allow the predetermined cut-off levels to be respected. Determination of the substance at the cut-off levels set out in legislation is important for legal purposes in the analysis of the keratin matrix. There must be appropriate working standard samples for the methods to be adequately substantiated and it must be possible to reproduce the effective conditions of the real sample, something which is particularly difficult for a biological matrix of this type. The HAIRVEQ protocol provides for the use of hair from babies with parents who are not drug addicts as blanks in quality control and, as a positive sample, hair made homogeneous through pulverisation from more than one person making use of substances of abuse. The discordant results obtained from all the laboratories taking part in the project underline the difficulty of fine-tuning these procedures. The work carried out has enabled application of the ball mill previously used for the measurement of coloured pigments in the plumage of birds to be hypothesised. Therefore, pulverisation a previously washed sample of hair from a cocaine addict in an appropriate solvent inside a jar was considered. The suspension obtained was filtered and analysis of the supernatant in GC-MS and GC/MS-MS highlight the presence of cocaine. Therefore, it was believed that pulverising a ‘blank’ sample in an appropriate solvent, to which a standard solution of analyte and an internal standard are added, could recreate a situation similar to that obtained through the pulverisation of a positive sample in the same solvent. The experimentation was carried out taking cocaine, one of the most widespread and extensively examined substances in the hair matrix, into consideration. The linearity of the method was assessed for this molecule in a range of concentrations from 0.1 µg/mg to 10 µg/mg, obtaining a correlation coefficient higher than 0.99. Both the traditional procedure and the latest technique were subsequently applied to ‘blank’ samples spiked with standard solution of analyte and to real samples. The second method was found to be more precise than the traditional one from this comparison. The traditional procedures applied for hair analysis have some significant problems which have been critically assessed in this thesis. The innovative techniques suggested have been tested with the aim of improving some relevant aspects such as the extraction stage and the importance of having work standards suitable for a more correct assessment of real samples. Encouraging results have been obtained with the ball-mill technique which is fast and simple and enables us to have work standards similar to real samples and, as a result, more adapted for assessing the effective yield of the extraction. This technique has proved to be worthy of further investigation and experimentation.
|Titolo:||Rilievo di sostanze esogene o xenobiotiche in matrici biologiche alternative quali peli e capelli|
|Supervisori e coordinatori interni:||CARINI, MARINA|
|Data di pubblicazione:||2007|
|Settore Scientifico Disciplinare:||Settore CHIM/08 - Chimica Farmaceutica|
|Citazione:||Rilievo di sostanze esogene o xenobiotiche in matrici biologiche alternative quali peli e capelli ; Tutor: Veniero Gambaro ; Coordinatore: Marina Carini. - Milano : Università degli studi di Milano. DIPARTIMENTO DI SCIENZE FARMACEUTICHE "PIETRO PRATESI", 2007. ((20. ciclo, Anno Accademico 2004/2005.|
|Appare nelle tipologie:||13 - Tesi di dottorato discussa entro ottobre 2010|