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Dried blood spot (DBS) is a blood collecting and storage method, which has been widely applied for newborn screening, therapeutic drug monitoring (TDM), and others. The concept of DBS involves the use a porous filter paper to absorb blood drops and preserve them as dried blood samples for further analysis. However, there are some disadvantages of the DBS process, such as, unequal distribution of the analyte of interest, haematocrit effects and difficulties in accurate analyte extraction, all of which have limited their applications for highly sensitive analysis. In addressing these issues, the spreading/imbibition of blood during the DBS sampling process has been investigated from both experimental and theoretical points of view in this work. DBS is described as a spreading process of a non-Newtonian drop (blood) over porous substrates (filter paper). Porcine blood with different haematocrit levels of spreading/penetrating over different porous substrates, including, commercial DBS filter papers and nitrocellulose membranes, have been used for the investigation via our spreading experiment. The time evolution of spreading parameters of DBS sampling process, such as, contact angles, droplet base radius and wetted region radius, were recorded and analysed to describe the spreading/imbibition behaviour of DBS sampling. The experimental results have shown that the spreading parameters of DBS sampling on each porous substrate fall into a universal curve under the dimensionless scale. According to our observations, the spreading behaviour of blood can be described as a complete wetting on the commercial DBS cards (Whatman 903) and partial wetting on the nitrocellulose membrane. Simulating models have been developed to describe the spreading/imbibition behaviours of DBS sampling process over porous substrate in complete wetting case. In the case of complete wetting, a system of two differential equations is derived, which describes the time evolution of radii of both the drop base and the wetted region inside the porous medium. The results show a good agreement while validating the spreading parameter, known as, droplet base radius, wetting region radius and contact angle with experimental data. These models provide simple and reliable methods to describe the blood spreading behaviour during DBS sampling process. In this project, the investigation of spreading behaviour of DBS analysis has been developed and presented. Reliable theoretical prediction of DBS sampling result can improve the defect of current detecting method and enhance the accuracy of the DBS analysis. In the consideration of the practical application of DBS analysis, the theoretical investigation of spreading/imbibition behaviour of DBS sampling should allow us to better control and predict the performance and outcome of pharmaceutical and analytical studies while using DBS method.

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.

Vitamin D is an essential micronutrient required for maintenance of the skeletal system and its deficiency leads to diminished availability of calcium for maintaining bone mineralization. Vitamin D deficiency (VDD) during pregnancy and/or in early infancy can lead to rickets and a fragile bone state resulting in fractures with minimal forces. Such multiple unexplained fractures (MUF) present in infants at less than 6 months of age are often misdiagnosed as cases of child abuse. Therefore, it is important to evaluate the status of vitamin D in early infancy. The analysis of dried blood spots (DBS) provides a unique screening method for early and late identification of diseases. The objective of this study was to develop a mass spectrometry (MS) method to analyze vitamin D levels in newborn DBS. We used a methanol-hexane solvent system for the extraction of vitamin D analytes from filter paper spots. The use of methanol-hexane solvent system was evaluated using desipramine HCl as a positive control. We were able to extract desipramine HCl from standard spots spiked with concentrations 8 -5000 ng/ml. We applied this extraction method to control and test DBS with slight modification involving preincubation of DBS in methanol and sodium hydroxide. Our MS results have shown qualitative separation of 25 (OH) D2 (m/z - 413.2) and 25 (OH) D3 (m/z - 401.2) with SNR > 3. The identity of vitamin D analytes was confirmed using MS/MS analysis showing the presence of respective fragment peaks (m/z - 336.2 and 365.2). For quantitative estimation i.e. SNR > 10, we derivatized vitamin D analytes in DBS using 4-Phenyl-1,2,4- triazoline-3,5-dione (PTAD) in a Diels-Alder conjugation reaction. However, both 25 (OH) D2 and 25 (OH) D3 show the formation of identical product ion (m/z- 298.0) making it difficult to quantitate them separately. In addition to MS method development, we also tried to evaluate genetic mutations in vitamin D metabolizing enzymes and vitamin D receptor as a possible cause of VDD in these infants. We extracted genomic DNA and RNA from DBS for mutation scanning using melt curve analysis However, the outcome of such analysis was not successful owing to lack of integrity in genetic material extracted from DBS. In summary we developed a MS method for qualitative detection of vitamin D and also attempted to test genetic mutations as a cause of VDD in infants. Further experiments are necessary to evaluate the use of alternative options of liquid chromatography/gas chromatography (LC/GC) coupled with MS and/or use of atmospheric pressure chemical ionization (APCI) as source of ionization for quantitative assessment of vitamin D.