Spelling suggestions: "subject:"tried blood spots"" "subject:"dried blood spots""
1 |
An investigation into the use of dried blood spot analysis in pharmacokinetic studiesPatel, Parul January 2011 (has links)
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.
|
2 |
Design of an LC-MS/MS method for measuring concentrations of Cyclosporine A and Tacrolimus from dried blood spotsHansson, Anna January 2015 (has links)
Patients that have undergone organ transplantation are life-long treated with immunosuppressant drugs and these have to be monitored regularly to get the desired effect of suppressing the immune system. To monitor the drug concentration normally a venous blood sample is collected at a clinic but the use of dried blood spots (DBS) as a matrix for drug monitoring for immunosuppressant drugs will make home sampling possible for this patient group. The aim of this study was to develop and validate a bioanalytical method for quantifying cyclosporine A and tacrolimus in dried blood spots. The method consist of punching out a 5 mm disc from a blood spot , followed by extracting the spot in a 96-well hydrophobic filter plate with 150 µL extraction solution containing internal standard (ascomycin and cyclosporine A d12) in a methanol water solution (80:20v/v%). The extract is then centrifuged through the filter plate down in a 96-deep well plate and injected on the LC-MS/MS, with an analysis time of 2.5min. The method will be validated in accordance with the guidelines set by the European Medicines Agency with additions specific to DBS. The method is not fully validated but will be in due time. The validated parameters show a robust and fast analysing method that has the prospects of being used for analysing DBS samples for patients and in the future can possibly be used by patients in home environment.
|
3 |
Analysis of Archived Dried Blood Spots by Mass Spectrometry for Vitamin D and Real-time PCR for its Enzymes and ReceptorJoshi, Amod N. 08 December 2011 (has links)
No description available.
|
4 |
Extraction of therapeutic proteins from dried blood spots and their analysis on GyrolabGarbergs, Hanna January 2011 (has links)
A method for extraction of therapeutic proteins from dried blood spots (DBS) followed by quantification on Gyrolab(TM) has been developed. The method makes it possible to measure the concentration of the analyte in the range 100-6000 ng/mL. The procedure can generate full analytical information from 15 μL blood originally sampled from a subject. The modest sample requirements allows for sampling a full pre-clinical pharmacokinetic profile from a single mouse. This may allow for reduced usage of animals during preclinical development of new therapeutic proteins in accordance with the 3R’s, replace, refine and reduce.
|
5 |
Development and evaluation of procedures and reagents for extraction of proteins from dried blood spots for analysis using ProseekBjörkesten, Johan January 2014 (has links)
A method for extraction of proteins from dried blood spots (DBS) for analysis using Proseek is developed and evaluated. DBS, as sample format, possesses a number of desirable advantages over for example plasma samples. These advantages include for example minimal patient invasiveness, sampling simplicity and non regulated sample transportation. Highly reproducible quantitative detection of 92 proteins is demonstrated from a 1.2 mm in diameter DBS disk. The DBS inter spot analysis precision (7% coefficient of variance) is comparable to plasma inter assay precision (6% coefficient of variance). Coefficient of variance is the ratio between standard deviation to mean value for the analysed replicates. Proseek analysis of DBS could possibly reveal a unique opportunity to examine health related issues in extremely premature infants hopefully resulting in increased survival rates in the future.
|
6 |
A NEW APPROACH TO DRIED BLOOD SPOT ANALYSIS FOR NEWBORN SCREENING USING HIGH RESOLUTION LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRYMiller, John H., IV 21 November 2012 (has links)
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.
|
7 |
Development of Field-adapted Analytical Methods for the Determination of New Antimalarial Drugs in Biological FluidsLindegårdh, Niklas January 2003 (has links)
<p>This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper.</p><p>Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated. </p><p>Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil).</p><p>Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE.</p><p>Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.</p>
|
8 |
Development of Field-adapted Analytical Methods for the Determination of New Antimalarial Drugs in Biological FluidsLindegårdh, Niklas January 2003 (has links)
This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper. Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated. Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil). Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE. Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.
|
9 |
Development and Validation of Bioanalytical Methods : Application to Melatonin and Selected Anti-Infective DrugsRömsing, Susanne January 2010 (has links)
This thesis describes bioanalytical methods for measuring melatonin and some anti-infective drugs in biological fluids. Solid-phase extraction (SPE) or protein precipitation was used for enrichment and purification of the analytes and Liquid Chromatography (LC) was used to analyze the samples. Developed methods were validated according to international guidelines. Melatonin is a hormone secreted by the pineal gland with a robust circadian rhythm. Bioanalytical methods for determination of melatonin in plasma and saliva have been developed which were used for monitoring melatonin levels in volunteers and patients suffering from sleep related diseases. Eflornithine (DFMO) is a chiral drug used for the treatment of human African trypanosomiasis. A bioanalytical method for determination of the DFMO enantiomers in plasma, after precolumn derivatization with o-phtalaldehyde and N-acetyl-L-cystein has been developed. The method has been used to study the L- and D-DFMO pharmacokinetics, in order to investigate the possible development of an oral treatment of DFMO. A method for simultaneous determination of three antiretroviral drugs i.e. Lamivudine (3TC), Zidovudine (AZT) and Nevirapine (NVP) in dried blood spots (DBS) was developed. The method was used for drug determination in two subjects after receiving standard antiretroviral treatment. The method seemed well suitable for the determination of 3TC and NVP and in some extent for AZT. Lumefantrine (LF) is one of the active components in a new fixed drug combination recommended by the WHO as a replacement to older drugs that has lost their effect. A method for the determination of LF in DBS was developed. The method is suitable for monitoring of drug treatment in rural settings. Tafenoquine is a new promising antimalarial drug under development. A method for the determination of Tafenoquine in plasma and in DBS is described. The method may be useful in future clinical studies in laboratory environment as well as in rural settings. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 703
|
10 |
Triagem neonatal para mucopolissacaridose tipo VI (Síndrome de Maroteux-Lamy) em uma região com alta incidência da doençaBender, Fernanda January 2011 (has links)
A mucopolissacaridose tipo VI (MPS VI) ou Síndrome de Maroteaux-Lamy, é uma doença autossômica recessiva causada pela deficiência da enzima lisossomal Nacetilgalactosamina- 4-sulfatase (ARSB), a qual resulta no armazenamento lisossômico de dermatan sufato em vários tecidos e órgãos, dando origem a uma condição clínica de espectro variável, desde formas mais graves até formas mais atenuadas. O acúmulo de substrato não degradado causa um importante comprometimento ósseo, problemas respiratórios, baixa estatura e outros problemas, afetando os olhos, o coração e outros órgãos. Embora a síndrome de Maroteaux-Lamy não tenha uma incidência definida no Brasil, é reconhecido que, no nosso meio, é muito mais freqüente do que em outros países e regiões. Ela é particularmente freqüente no município de Monte Santo (Bahia), de aproximadamente 50.000 habitantes e onde já foram registrados 13 casos da doença. O diagnóstico é importante porque existe hoje um tratamento específico para a doença, a terapia de reposição enzimática (TRE), que vem mostrando bons resultados, especialmente quando iniciada em idade precoce. Descrevemos neste trabalho uma adaptação para microplacas da medida fluorimétrica da atividade de ARSB, e uma nova metodologia de análise molecular, ambas padronizados para sangue total impregnado em papel-filtro (STIPF). Essas técnicas foram desenvolvidas para incluir um teste de triagem neonatal para MPS VI, realizado nas amostras coletadas para o “teste do pezinho” nos neonatos do município de Monte Santo. Esses métodos permitem a detecção de pacientes com MPS VI e dos portadores da mutação específica que parece ser responsável pela alta incidência de MPS VI nessa localidade, uma vez que todos os pacientes lá diagnosticados apresentavam a mesma mutação (p.H178L) em homozigose. O trabalho foi desenvolvido em três etapas: na primeira foi realizada a padronização das técnicas em 100 amostras de STIPF; na segunda foi feito um teste-piloto com amostras de neonatos de Monte Santo, para avaliação das técnicas padronizadas e para o estudo de termoestabilidade em controles hígidos; na terceira foram analisadas amostras de STIPF de neonatos provenientes de Monte Santo pelos dois métodos (bioquímico e molecular). A padronização para realização da medida fluorimétrica da atividade enzimática de ARSB em microplacas indicou que o método é sensível, permitiu diferenciar os valores da população normal dos valores dos pacientes afetados e possibilitou a identificação segura de pacientes com MPS VI. Nas padronizações da análise molecular da mutação p.H178L em STIPF foi possível diferenciar os indivíduos normais, heterozigotos e homozigotos. Os resultados preliminares disponíveis indicam que o protocolo de triagem neonatal para MPS VI desenvolvido no presente trabalho poderá ser facilmente incorporado por laboratórios de referência, contribuindo para a detecção e tratamento precoce dos pacientes afetados por MPS VI. / Mucopolysaccharidosis type VI (MPS VI) or Maroteux-Lamy syndrome, is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme Nacetylgalactosamine- 4-sulfatase (ARSB), which results in lysosomal storage of dermatan sufate in various tissues and organs and leads to a variable clinical spectrum, including more severe and attenuated forms. The accumulation of undegraded substrate causes bone involvement, respiratory problems and short stature, among other signs and symptoms, affecting the eyes, heart and other organs. Although the Maroteaux-Lamy syndrome does not have a defined incidence in Brazil, it is recognized that in our environment it is much more frequent than in other countries and regions. It is particularly frequent in the municipality of Monte Santo (Bahia) approximately 50,000 inhabitants and where there have already been 13 cases of the disease. The diagnosis is important because today there is a specific treatment for the disease, enzyme replacement therapy (ERT) which has shown good results, especially when started at an early age. We describe herein the standardization of the microplate fluorometric method for the ARSB test and a new methodology of molecular analysis, both adapted for dried blood spots (DBS) samples. These techniques were developed for inclusion of MPS VI in the newborn screening program that already tests the neonates of the city of Monte Santo, Bahia, Brasil for metabolic diseases. The methods were developed to detect patients with MPS VI and also for carriers, once the disease seems to have a high incidence (around 1:5.000) at this location. Also, all patients that have already been diagnosed in this city presented the same mutation (p.H178L) in homozygosis. The study was conducted in three stages: in the first was performed in 100 DBS samples an standardization of the techniques; in the second was done a pilot test with samples of newborns of Monte Santo, for the evaluation of standardized techniques and for the thermostability study in healthy controls; in the third were analyzed newborns samples from Monte Santo for both biochemical and molecular methods. Standardization on microplate for fluorimetric enzyme activity of the ARSB showed the assay sensitivity, differentiating values between normal and affected and allowing a reliable detection of patients with MPS VI. On the standardization for molecular analysis in DBS it was possible to differentiate the results for normal individuals, heterozygous and affected for the mutation p.H178L. The preliminary results available indicate that the protocol of neonatal screening for MPS VI developed in this work can be easily incorporated by reference laboratories, contributing to the detection and premature treatment of MPS VI affected patients.
|
Page generated in 0.0496 seconds