The application of dried blood spots in toxicokinetic and pharmacokinetic studies

Dried Blood Spot (DBS) sampling is a microsampling technique used throughout the World for neonatal screening. The work set out in this thesis shows the development and implementation of DBS in the area of preclinical and clinical pharmaceutical drug development, specifically in support of Toxicokinetics and Pharmacokinetics. The advantages of the technique are explored along with the issues faced. The papers discussed in this commentary, include in papers 1 and 2, the concept of supporting both Toxicokinetics and Pharmacokinetics studies and the validation of bioanalytical assays utilising DBS. Commentary paper 3 further explores the practicalities of DBS in the Clinical environment and commentary paper 4 the transferability of DBS technology between laboratories. Commentary paper 5 uses Incurred Sample Reanalysis data to answer questions around specific DBS issues and commentary paper 6 looks at indicating papers for Dried Plasma Spots. Commentary papers 7 and 8 explore the use of consortia to investigate hematocrit and homogeneity when using DBS and finally commentary paper 9 explores the training required to ensure quality DBS samples. The impact and contributions to this field of research are demonstrated through discussion and critical examination of selected examples of the author’s peerreviewed publications in this area. Developments of scientific practices, where the author has contributed intellectual, leadership and practical insight to achieve significant improvements in the generation of knowledge, are highlighted throughout the commentary

A NEW APPROACH TO DRIED BLOOD SPOT ANALYSIS FOR NEWBORN SCREENING USING HIGH RESOLUTION LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY

The primary purpose of newborn screening is to quickly identify children that are at risk of having a specific disorder in order to start treatment, prevent early death and reduce the chances of permanent physical or mental damage. The current and widely accepted approach used for identification of metabolism disorders involves a flow injection analysis with mass spectrometry detection of acylcarnitines and amino acids. Although this approach is widely accepted and has shown to be sufficient for identification of multiple metabolism disorders the method is not fully quantitative and results often have to be confirmed by second-tier tests. The primary focus of this research was to improve the accuracy and selectivity of this screening method by employing a high resolution chromatographic separation for the combined analysis of twelve acylcarnitines and seven amino acids. This method is an improvement over the current methodology allowing for separation of key isomers that are diagnostic for different metabolism disorders, reducing the need for multiple second-tier tests to confirm results and shortening the time to diagnosis. In order to further improve the efficiency of newborn screening we developed an in-line desorption device, which allows for direct analysis of DBS eliminating the need for punching disks from the filter paper cards. Our device was the first published paper that demonstrated the ability to directly analyze dried blood spots, without the need for any offline sample processing. Using this device, we validated a method to quantify biomarkers related to Maple Syrup Urine Disease, a disorder that requires a second-tier test for confirmation. To further improve the accuracy of dried blood spot analysis we evaluated a technique to correct the sample volume in low and high hematocrit samples. The level of hematocrit in blood spotted on filter paper cards affects the volume of sample analyzed, leading to errors in accuracy. Diffuse reflectance was used to relate differences in sample hematocrit on dried blood spots. We validated our technique with eighteen donor samples at various levels of hematocrit. Correcting sample volume for hematocrit showed improved precision and accuracy over the standard approach, ultimately reducing the potential to misidentify samples

An Investigation into the Use of Dried Blood Spot Analysis in Pharmacokinetic Studies

The ethical and practical issues of obtaining a blood sample pose a significant challenge to performing pharmacokinetic studies in children, infants and neonates. Dried blood spot analysis, based on the collection of a micro blood sample has potential to overcome these difficulties. There are at present a limited number of reports on the utility of dried blood spot analysis in clinical pharmacokinetic studies. The studies described in this thesis were undertaken to investigate the accuracy and precision of dried blood spot sampling coupled with mass spectrometry detection for drug quantification, and clinically validate the robustness and feasibility of this technique for pharmacokinetic studies in preterm neonates. Dried blood spot methods were developed for application to pharmacokinetic studies of test drugs dexamethasone and caffeine. Investigations were focused on the blood collection system, analyte recovery and optimisation of the detection system. In-vitro validation results indicated developed methods were precise, accurate and selective in accordance with the Food and Drug Administration regulatory guidelines on the assessment of bioanalytical methods. Results were not significantly affected by small variations in the blood volume spotted or the presence of petroleum jelly, which is often used on the sampling site during capillary blood collection in neonates. Variability in haematocrit was determined to be the single most important factor affecting assay accuracy. Stability assessments by comparison with freshly prepared samples verified the suitability of sample drying, storage and post sample extraction conditions. An investigation of method transferability between different analytical instruments was undertaken with caffeine to provide an assessment of the robustness of dried blood spot analysis. Results generated from a single and triple quadrupole mass spectrometer were comparable with an expected lower limit of quantification with the latter technique most likely due to a greater ionisation and detection efficiency. Intravenous dexamethasone pharmacokinetics was determined in 5 preterm neonates receiving treatment for chronic lung disease. Individual pharmacokinetic analyses were performed using a one compartment model to estimate primary pharmacokinetic parameters, clearance (mean, 0.18 l/h/kg) and volume of distribution (mean, 1.33 l/kg). The whole blood derived mean estimates were similar to previous plasma clearance and volume estimates of 0.14 l/h/kg and 1.91 l/kg, respectively reported in neonates (n=7). This highlights the potential for dried blood spot analysis as an alternative to conventional plasma based methods for dexamethasone dose optimisation studies in neonates. The population pharmacokinetics of oral / intravenous caffeine was determined in 67 preterm neonates. A one compartment model was used to describe the blood concentration-time data. Model evaluation using a bootstrapping technique confirmed the robustness and stability of the developed model. Pharmacokinetic parameters derived from dried blood spot drug measurements were estimated with precision (relative standard error < 10%) and were comparable to estimates of plasma clearance (mean, 7.3 vs. 7.0 ml/h/kg) and volume of distribution (mean, 593 vs. 851 ml/kg) from a previous population study in neonates (n=110). Weight and postnatal age were the most influential covariates in the clearance model which is in agreement with previous population studies. These results demonstrate that dried blood spot analysis is a practical technique, with significant potential as a robust method for use in clinical pharmacokinetic studies in vulnerable populations such as preterms. Haematocrit related effects on paper will need to be accounted for if this potential is to be realised. Further investigations to determine the reproducibility of capillary blood sampling in neonates and the impact of using blood drug measurements on pharmacokinetic parameter estimation will be necessary before widespread use of the technique is possible

Paediatric food allergy and the role of vitamin D

Vitamin D plays a significant role in multiple physiological functions in humans. The prevalence of paediatric food allergy (FA) has been tentatively linked to vitamin D insufficiency. In Victoria, Australia, the frequency of both vitamin D insufficiency and paediatric FA is high.Vitamin D conventionally is measured in serum / plasma and the interpretation cut-offs for adults are well-defined. However, the interest in the use of dried blood spots (DBS) as an alternative matrix for vitamin D assessment has increased or large-scale population studies. we aimed to develop a sensitive and robust local DBS vitamin D method, to to provide a description of vitamin D levels at birth in the Melbournian infant population and investigate the association between vitamin D status and prevalence of paediatric FA and eczema in early childhood. We developed a sensitive liquid chromatography tandem mass spectrometery method and measured vitamin D from 2700 newborns&#039; DBS samples. Our research demonstrated that, vitamin D levels in newborn babies is influenced by season of birth and maternal vitamin D supplementation. Lower vitamin D levels do not influence the risk of challenge-proven FA or eczema in infancy. Further studies are required to define age appropriate decision limits for vitamin D

Diagnostic technologies for wound monitoring

Chronic wound infections represent a worldwide problem, generating high morbidity and medical expense. Failure to control infections such as MRSA in the reparative process of a wound can cause disruption of normal anatomical structure and function, resulting in a chronic wound. Existing approaches to identifying infection largely involve surveying a range of physical parameters, and a limited use of non-invasive technologies. Evaluation is time consuming, and often results in inconsistencies in patient care. This project researches three possible alternative methodologies/technologies for the monitoring of wounds, by measuring components of wound fluid. Two of the three technologies are designed to be used by physicians and patients, similarly to commercially available home blood glucose test kits, and are based on the measurement of three biomarkers: glucose, ethanol and H2O2 in PBS, and in serum as surrogate wound fluid. The first is a voltammetric technique known as dual pulse staircase voltametry (DPSV), which produces peaks characteristic of particular analytes at an electrode. The second is an amperometric biosensor array, based on screen printed three electrode assembies of carbon, rhodinised carbon (glucose biosensor only) and Ag/AgCl reference. The glucose biosensor uses glucose oxidase enzyme as the biorecognition agent, the H2O2 biosensor is a mediated system using horseradish peroxidase enzyme and dimethylferrocene mediator, and the ethanol biosensor is a bienzyme mediated system utilising alcohol oxidase enzyme horseradish peroxidase enzyme and coupled dimethylferrocene mediator. Wounds are known to produce characteristic odours, therefore the third technology studied is a single sensor odour analyser with advanced data analysis to detect five commonly occuring wound bacteria, S.aureus, K.pneumoniae, S.pyogenes, E.coli and P.aeruginosa in growth media and surrogate wound fluid. This technology would be used as a 'near patient' monitoring system and is based on machine olfaction similar to that of a commercial electronic nose, but uses a single metal oxide sensor in combination with principle components analysis. DPSV scans of the individual analytes demonstrated distinctive peaks, exhibiting nonlinear relationships with concentration. A great deal of useful information was generated using this technique, however, limitations were discovered regarding repeatability and inter-analyte interference in mixtures. Limits of detection in surrogate wound fluid with the glucose biosensor, hydrogen peroxide biosensor, and ethanol biosensor were as follows: 169.5 µM glucose, 8.43 µM hydrogen peroxide, and 7.94 µM ethanol respectively (all at 99.7% confidence). Direct detection of ethanol from metabolically active S.aureus in surrogate wound fluid yielded a limit of detection of 1.23 x 108 CFU/ml at 99.7% confidence, and 19 µM in terms of ethanol specific response. The single sensor odour analyser demonstrated the ability to detect and discriminate between the three biomarkers, between five bacteria individually, and partial discrimination of paired bacteria (in broth and surrogate wound fluid). It was also found that S.aureus could be detected down to a cell density of 5x106CFU/ml in surrogate wound fluid, lower than that found for the biosensor concept.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Innovaciones en técnicas de microextracción con agitación integrada

La etapa de tratamiento de muestra es el cuello de botella de la mayoría de procedimientos analíticos y es la que más influencia tiene en la calidad de la información obtenida. Esto se debe fundamentalmente a que hay que adaptarla a la muestra que se analiza, a los analitos que se determinan y a la técnica instrumental seleccionada para tal efecto. En general, las técnicas de tratamiento de muestra juegan un papel importante en la mejora de la sensibilidad y selectividad de las determinaciones a través de la preconcentración de los analitos y de su aislamiento de la matriz de la muestra. Uno de los retos que presenta la Química Analítica, es el avance e innovación en las técnicas de tratamiento de muestra, y su evolución hacia metodologías más sencillas, automatizadas y miniaturizadas. Esta evolución ha permitido la aparición de las técnicas de microextracción, tanto en fase sólida como en fase líquida. La eficiencia de una técnica de microextracción viene definida por factores termodinámicos y cinéticos. Desde un punto de vista termodinámico, una extracción es un proceso de equilibrio químico entre fases, estando dicho equilibrio regido por una constante de distribución. Por otro lado, la cinética de la extracción influye en el tiempo que tarda en alcanzarse dicho equilibrio y viene determinada por la difusión de los analitos desde la muestra hacia una fase extractiva. La agitación desempeña un papel fundamental en la mejora de la cinética de la extracción, ya que favorece la difusión de los compuestos. Esta agitación puede llevarse a cabo mediante la utilización de un elemento externo o mediante la integración de este en la unidad de extracción. La presente Tesis Doctoral está enmarcada dentro del tratamiento de muestra y tiene como denominador común la integración de la agitación en la extracción para mejorar la cinética de los procesos microextractivos

Extraction and analysis of pharmaceutical residues in environmental samples using SPE with LC-MS/MS

Pharmaceuticals and personal care products (PPCPs) have recently emerged as a significant new class of organic micro-contaminants. Of recent years a number of reports detailing the presence of PPCPs in a variety of environmental matrices and compartments have been published in the peer-reviewed literature. However, in Ireland very little research has been conducted to determine the level of environmental contamination due to the presence of drug residues. The primary focus of this research is to develop suitably sensitive analytical methods for the determination of residual PPCP contamination based upon solid phase extraction (SPE) and liquid chromatography mass spectrometry (LC-MS). Monolithic silica based stationary phases were used for the development of high performance liquid phase separations of common pharmaceuticals, the antifouling and anti-dandruff agent zinc pyrithione and a range of illicit drugs and abused pharmaceuticals. As a pre-requisite to all the developed methods, a SPE sample enrichment procedure was also developed focusing upon either off-line formats using modern hydrophilic lipophilic balanced polymeric phases or the use of column switching, whereby short reversed-phase monolithic micro-columns were applied as suitable traps for on-line preconcentration. Method performance data for all the developed methods were also determined and analytical detection limits were found to lie in the ngL-1 range. The developed methods were applied for the determination of the selected analytes in environmental aquatic samples

Investigation of Volatile Organic Compounds (VOCs) released as a result of spoilage in whole broccoli, carrots, onions and potatoes with HS-SPME and GC-MS

Vegetable spoilage renders a product undesirable due to changes in sensory characteristics. The aim of this study was to investigate the change in the fingerprint of VOC composition that occur as a result of spoilage in broccoli, carrots, onions and potatoes. SPME and GC-MS techniques were used to identify and determine the relative abundance of VOC associated with both fresh and spoilt vegetables. Although a number of similar compounds were detected in varying quantities in the headspace of fresh and spoilt samples, certain compounds which were detected in the headspace of spoilt vegetables were however absent in fresh samples. Analysis of the headspace of fresh vegetables indicated the presence of a variety of alkanes, alkenes and terpenes. Among VOCs identified in the spoilt samples were dimethyl disulphide and dimethyl sulphide in broccoli; Ethyl propanoate and Butyl acetate in carrots; 1-Propanethioland 2-Hexyl-5-methyl-3(2H)-furanone in onions; and 2, 3-Butanediol in potatoes. The overall results of this study indicate the presence of VOCs that can serve as potential biomarkers for early detection of quality deterioration and in turn enhance operational and quality control decisions in the vegetable industry