Department of Medicine

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Browsing Department of Medicine by Subject "Acute toxicity testing"

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    Development of LC-MS/MS toxicology screening methods for common poisoning agents in South Africa
    (Stellenbosch : Stellenbosch University, 2020-12) Tiya, Luthando Lukhanyo; Stander, Marietjie; Kellermann, Tracy; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine: Clinical Pharmacology.
    ENGLISH ABSTRACT: Background: Hospitals admit many patients suffering from acute poisoning on a regular basis. Over a single year period the Tygerberg Poison Information Centre (TPIC) dealt with 4 771 consultations related to human exposure to various poisonous substances. Of these consultations, exposures to medicines and pesticides accounted for the majority of cases. As a response to this medical burden, this project aims to develop effective screening tools for both medicines and pesticides in human plasma. It is believed that such a tool could enhance how medical practitioners accurately treat and respond to serious poisonings. There are many toxicological screening methods employed today. In the past immunoassays (IAs) and gas chromatography coupled to mass spectrometry (GC-MS) were seen to be the gold standard for the identification of compounds. Due to false positive results associated with immunoassays and the labour intensive sample preparation associated with GC-MS, it is clear that there is a need for faster, more robust and more sensitive bioanalytical methods. This study’s objective therefore is to develop a cost effective, fast and robust liquid chromatography tandem mass spectrometry (LC-MS/MS) screening tools for the identification of drugs and pesticides. Relevant drug and pesticide selection was done by consulting with the TPIC, a centre which deals with poisoning cases on a daily basis, as well as clinicians in the Division of Clinical Pharmacology who deal with patients admitted at Tygerberg Hospital daily and CropLife SA, an organisation that has the knowledge of which pesticides are currently in use in South Africa. A total of 37 drugs and 10 pesticides were identified as those often suspected to be involved in poisoning/overdose cases. Two methods were developed: An untargeted liquid chromatography quadrupole time-of-flight mass spectrometry (LC-TOFMS) method for the determination of therapeutic drugs; and a targeted LC-MS/MS method for the determination of pesticides. Method: Analytes were extracted from CPD plasma using protein precipitation (PP). For the untargeted method, a Waters Synapt G2 Quadrupole Time-of-flight (QTOF) mass spectrometer (MS) fitted with an ultra-high performance liquid chromatography (UHPLC) was used. The separation was done using a Waters HSS T3 (1.8 μm, 2.1 ×150 mm) column. A gradient consisting of a mobile phase of 0.1% formic acid in both water (A) and in acetonitrile (B) at a flow rate of 0.25 mL/min. Ionisation mode was set for both positive and negative (ESI +/-); Nitrogen as the desolvation gas was set at a flow rate of 650L/h; Desolvation temperature was set at 275˚C. The instrument was operated in the MSE mode. The total run time was 37 min. Four concentration levels (10, 50, 100, 1000 ng/mL) were determined to cover the pharmacokinetic peak concentrations of all drugs. This range was determined according to sub-therapeutic, therapeutic and overdose type concentrations. Furthermore, the data generated from the untargeted method development process was used to accurately identify analytes using online search libraries. For the targeted method, two liquid chromatographic separation methods were developed on a Shimadzu 8040 LC-MS/MS instrument. One gradient method using the Agilent Poroshell 120 EC-C18 (2.7 μm, 4.6 ×100 mm) column was developed for atrazine, MCPA, fipronil, methomyl and aldicarb, while a second isocratic method utilizing of the Restek Raptor Biphenyl (2.7 μm, 2.1 x 100 mm) column was also developed for imidacloprid. Both methods used mobile phase consisting of 0.1% formic acid in water (A) and in acetonitrile (B) with a flow rate of 0.5 mL/min. The LC program on the Poroshell method starts at 30% (B) for 2 min followed by a 0.5 minute ramp to 90% (B); 2.5 min at 90% (B); 0.5 min to 30% (B), equilibrate at 30% (B) for 2.5 min. The total run time is 9 min. The biphenyl method isocratic run comprises of (65%:35%, v/v) for a run time of 1.75 min. A semi-quantitative method with a quadratic curve fit with 1/x regression over the range of 20 to 600 ng/mL was chosen. The two targeted methods were subjected to a partial validation to ensure that the methods were accurate, robust and reliable. Results: The untargeted method was able to detect and identify 92% of the therapeutic drugs included in the screen. Due to matrix effects and differences in compound ionisation some could not be detected at lower concentrations. Polarity and thus lack of retention on the columns, difficulty in ionisation, and the inability to produce stable fragments made the detection of valproate, levetiracetam and metformin not possible on the method. A screening tool for simultaneous determination of pesticides was optimized. Due to several challenges associated with the LC-MS/MS determination of glyphosate, paraquat and deltamethrin, the study resorted to excluding these three pesticides from the current methods. The targeted method was developed for the determination of seven pesticides. The USA Food and Drug Administration (FDA) and the European Medicines Agency (EMA) guidelines were followed for a partial validation of the semi-quantitative method. Three independent runs were assessed to measure accuracy and precision. The calibration curve was linear over the ranges 20 to 600 ng/mL. Matrix effects, recovery, process efficiency, on-bench stability, freeze/thaw stability, storage stability, and whole blood stability for the methods was successfully determined for the seven pesticides. Conclusion: To respond to the medical burden of acute poisoning cases faced by our hospitals, this study has developed efficient screening tools for both relevant medicines and relevant pesticides in human plasma. The latter method was subjected to a partial validation, ensuring accuracy and reliability of results that will be applied to the clinical management of poisoning patients.