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Title: Solid phase extraction techniques for the analysis of pesticides and drugs in biological specimens
Author: Asri, Kamarruddin
ISNI:       0000 0001 3431 0400
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2001
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The aims of the project were to investigate the potential of solid phase extraction techniques for the detection and quantification of organophosphate pesticides in human biological specimens. Achievement criteria which were applied were that analytical methods should be sufficiently sensitive, specific, robust and cost effective for use in routine forensic toxicology, and, if possible, were inexpensive and within the economic and technical means of laboratories in developing countries. The procedures developed were based on established chromatographic and spectrometric techniques, drawing on analytical methods for environmental samples such as foodstuffs (which resemble target matrices such as autopsy blood and tissue) but also incorporated the more recently available technique of solid phase micro extraction (SPME). The project evaluated the application of these methods to real specimens from forensic cases, which occurred in Malaysia and elsewhere during the course of the study, as the incidence of pesticide poisoning cases in the United Kingdom is very low. The SPME technique was also evaluated for analysis of benzodiazepines in urine. An overview is given of pesticides and organophosphates in particular, with respect to their chemistry, usage, metabolism and biological effects. Problems encountered in sample pretreatment and preparation for analysis are reviewed, with emphasis on techniques, which aim to reduce or avoid the use of environmentally damaging solvents. This review also focuses on the history and theory of SPME. The SPME process has two steps, equilibrium partitioning of analytes between the SPME fibre coating and sample matrix and then transfer of absorbed analytes to the analytical instrument. In step 1, the extraction is considered to be complete after equilibrium is reached, i.e. the stage at which absorption is constant within the limits of experimental error and independent of further increase in extraction time. However, SPME is an equilibrium, but not an exhaustive, extraction. Parameters that contribute to extraction efficiency include sampling time, temperature, pH and ionic strength. Application of SPME under non-equilibrium conditions requires adequate control of these parameters. In the initial method development study, SPME in combination with capillary column gas chromatography and nitrogen-phosphorus detection were evaluated as a method sequence for the analysis of organophosphate pesticides in whole blood and urine. Eleven pesticides were used initially in the study as model compounds representing the range of commonly-used substances and also representing different functional groups found in these phosphates. Two types of fiber were evaluated, with coatings of polydimethylsiloxane (PDMS) or polyacrylate (PA). Parameters affecting the adsorption of analytes on the SPME fibre were examined, including fiber pretreatment, sampling time, temperature, the use of sample agitation, and modification of the ionic strength and pH of the samples. The best conditions for extraction involved a PDMS fiber along with a sample temperature of 90°C, with a conditioning time of 10 minutes, exposure time of 30 minutes and desorption time of 5 minutes. The addition of sodium chloride and hydrochloric acid increased the recovery of the organophosphates. All samples were agitated during the extraction process. The technique was later employed in urine as well as whole blood specimens. The primary detection and quantification technique was GC-NPD. The Limit of Detection and Precision was measured for validation purposes and were found to be in the range 1-50 ng/0.5 ml and 3.5-14 % RSD respectively. The developed method was subsequently applied to real cases involving fenthion and chlorpyrifos. The method developed was tested in the Department of Chemistry in Malaysia to assess its viability and ruggedness. The proposed extraction procedure was maintained but modifications to the instrumentation involved dual column gas chromatography with twin nitrogen phosphorus detectors. Twenty-five specimens of post-mortem blood were examined using HS-SPME as a routine screening procedure for pesticides. Of these samples, 3 positive cases of malathion poisoning were identified and quantified. A comparison was made of SPME and Solid Phase Extraction (SPE), another alternative technique for sample preparation which has become popular, for the extraction of organophosphates from blood and urine. Two type of SPE cartridge were evaluated: Ci8-substituted silica and mixed-mode Bond-Elut® Certify. In the development of an SPE method, parameters affecting the extraction capabilities such as solubility, pH, salt effect were examined. An overall method of extraction was devised and validated including a limit of detection and precision study. The method was applied to biological specimens from forensic autopsy cases. It was concluded that the two techniques are complementary and that SPME is particularly useful for dirty specimens containing many interfering substances. An investigation was carried out to elucidate which factors contributing toward biodegradation of pesticides in aqueous matrices including water and blood in order to address the problem of specimen preservation between the medico-legal autopsy and the toxicological analysis. The HS-SPME technique was used in this study, which was carried out by exposing samples containing malathion under different conditions of pH and temperature and in the presence or absence of preservatives. The conclusion was that the rate of degradation of malathion can be reduced by lowering the storage temperature and adjusting the sample to a slightly acidic pH in the range 5-7. Fluoride was found to increase the rate of degradation. Finally, the potential in forensic toxicology of analytical techniques based on direct immersion SPME was investigated using, as a representative system, the determination of benzodiazepines, especially diazepam, desmethyldiazepam, temazepam and oxazepam in urine. The technique was shown to be capable of producing reproducible and robust analytical methods and was applied successfully to urine specimens from different types of forensic case.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Toxicology & poisons