Liquid-liquid Extraction of Everolimus an Immunosuppressant from Human Whole Blood and its Sensitive Determination by UHPLC-MS/MS

A sensitive and precise ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method has been developed and fully validated for therapeutic drug monitoring of everolimus in human whole blood. Sample preparation involved liquid-liquid extraction of everolimus and its deuterated internal standard (IS, everolimus-d4) from 100 µL blood sample using diethyl ether: ethyl acetate (30:70, v/v) solvent system under alkaline conditions. The chromatography was conducted on a COSMOSIL 2.5C18-MS-II (50 mm × 2.0 mm, 2.5 µm) analytical column. The analyte and IS were eluted within 2.5 min under isocratic conditions using 10mM ammonium acetate, pH 6.00 adjusted with formic acid and acetonitrile (20:80, v/v). Multiple reaction monitoring was used for the quantitation of everolimus (m/z 975.5 → 908.5) and everolimus-d4 (m/z 979.6 → 912.6) in the positive ionization mode. The method was shown to be linear over the entire concentration range from 0.10-50.0 ng/mL. The recovery ranged from 90.9-94.8 % for everolimus and 91.4-95.6 % for everolimus-d4. The selectivity of the method is demonstrated in six different sources of blank human blood. The method is free from matrix effect as apparent from the post-column analyte infusion experiment, absolute and relative matrix effect results. The stability of everolimus in whole blood was thoroughly established under different storage conditions

Nuevos elementos de reconocimiento molecular selectivo y métodos de amplificación para el desarrollo de (bio)sensores ópticos

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, leída el 01-04-2022Mycotoxins are low molecular weight substances produced as secondary metabolites by a wide variety of filamentous fungi that can be found as natural contaminants in many foods and feeds. The number of toxic fungal metabolites currently known exceeds the thousand units, but only a few of them are considered a threat to humans and animal health. Exposure to mycotoxins can be due to the consumption of contaminated foodstuff, but also by inhalation of dust containing mycotoxigenic fungal spores. It has been estimated that nearly 40% of the global crops can be contaminated with mycotoxins. Hence, the development of analytical methods that can detect these mycotoxins in foodstuff are essential. On the other hand, despite their elevated risk infood quality, several mycotoxins present medical applications, for example as immunosuppressant drugs for organ transplantation. However, a limitation for this application is the narrow therapeutic window presented by these drugs, e.g., mycophenolic acid. High doses of these compounds can cause serious adverse health effects in humans, but little doses could not be sufficient to prevent organ rejection in transplanted patients. Thus, it is also of utmost importance to monitor the levels of these compounds in blood to improve the clinical efficacy of the immunosuppressant...Las micotoxinas son sustancias de bajo peso molecular producidas como metabolitos secundarios por una gran variedad de hongos filamentosos que pueden encontrarse como contaminantes naturales en muchos alimentos y piensos. El número de metabolitos fúngicos tóxicos que se conocen en la actualidad supera las mil unidades, pero sólo unas pocas docenas se consideran una amenaza para la salud humana y animal. La exposición a las micotoxinas puede deberse al consumo de alimentos contaminados, pero también a la inhalación de polvo que contiene esporas de hongos micotoxigénicos. Se calcula que aproximadamente el 40% de los cultivos del mundo pueden estar contaminados con micotoxinas, de ahí que sea esencial el desarrollo de métodos analíticos que permitan detectar estas micotoxinas en los alimentos. Por otro lado, a pesar de su elevado riesgo en la calidad de los alimentos, varias micotoxinas presentan aplicaciones médicas, por ejemplo, como fármacos inmunosupresores para el trasplante de órganos. Sin embargo, una limitación para esta aplicación es la estrecha ventana terapéutica que presentan estos fármacos, por ejemplo, el ácido micofenólico. Altas dosis de estos compuestos pueden causar graves efectos adversos para la salud delos seres humanos, pero pequeñas dosis pueden provocar el rechazo de órganos en pacientes trasplantados. Por ello, también es de suma importancia controlar los niveles de estos compuestos en sangre para mejorar la eficacia clínica del inmunosupresor...Fac. de Ciencias QuímicasTRUEunpu

Development and Study of the Microfluidic Open Interface Coupled with Solid-phase Microextraction for Rapid Analysis

Analytical chemistry mainly aims to identify and quantify analytes in matrices of interest. Analytical chemistry is an indispensable part of chemistry (and other scientific fields) because identification and quantification rely on repeatable and accurate measurements. Today's gold-standard systems for trace analysis are mainly chromatographic systems (liquid chromatography or gas chromatography) coupled to mass spectrometry. These methods attained the gold-standard status because of the overall method sensitivity and selectivity abilities. However, the main weakness of these systems is that they often require extensive sample preparation to reach outstanding performance regarding selectivity and sensitivity. Nevertheless, the chromatography step can be lengthy when selectivity is needed. Mass spectrometry has improved over the years, encouraging researchers to introduce samples directly to mass spectrometry, thus circumventing chromatographic separation. This field became known as direct-to-mass spectrometry. One of the main aims of this field is to avoid lengthy chromatography time and practically have real-time monitoring or high-throughput/rapid analysis of analytes at trace levels with little or no (laborious) sample preparation. However, these approaches serve only as the first line of study (rapid screening step), after which positive samples are submitted for thorough analysis using a gold-standard method. One of the main disadvantages of direct-to-mass spectrometry analysis is susceptibility to matrix effects (due to lack of separation), which internal standards can somewhat mitigate. Another disadvantage is instrument contamination resulting from inadequate or no sample preparation that is sacrificed for the overall rapid analysis. The most suitable sample preparation for direct-to-mass spectrometry analysis is solid-phase microextraction. It is a method developed to perform sampling and sample preparation in a single cohesive step. One of the advantages of this method is the non-exhaustive enrichment. The main advantages of this sample preparation method include using matrix-compatible coating, which offers analyte enrichment with minimal or no matrix interference co-enrichment. Additionally, the open-bed enrichment of solid-phase microextraction offers avenues for high-throughput sample preparation steps that minimize the overall analytical workflow time. When such analysis is used for the direct-to-mass spectrometry analysis, the general analytical workflow time is reduced by avoiding chromatography. Solid-phase microextraction has been coupled directly to mass spectrometry in many ways. One of the ways solid-phase microextraction can be coupled directly to the mass spectrometry is via a microfluidic open interface. The microfluidic open interface system coupled to mass spectrometry comprises a suction component (created by the ionization source of a mass spectrometer), an inflow component (pumping system), and a desorption chamber. All three components connect to a three-way chromatographic tee. The main requirement is that the suction component is constant. The liquid level in the desorption chamber can be introduced to the mass spectrometer by changing the inflow. Solid-phase microextraction devices (after the enrichment step) are then introduced to the very low-volume desorption chamber for analysis. The inflow stops after a short desorption time (e.g., 5 s). The suction component aspirates the volume of the desorption chamber, thus injecting the solution into the mass spectrometer for the analysis. The main highlights of the microfluidic open interface coupled to a mass spectrometer are desorption in a flow-isolated system and desorption into a low volume, which provides a tall Gaussian peak. The overall objective of this thesis is to redesign the microfluidic open interface system to demonstrate automation of the analysis workflow and make it suitable for rapid analysis. Firstly, Chapter 2 identifies the disadvantages of the existing microfluidic open interface system, which are used to improve the next design. The new system contains commonly available material to make the system approachable. Additionally, all system components are automated (by writing a homemade program from scratch), reducing the error from the manual operation. Nevertheless, this chapter depicts how the solid-phase microextraction coating design is essential for optimal desorption and analysis sensitivity. Chapter 3 expands on the fundamental idea raised towards the end of Chapter 2 by quantifying the mass transfer resistance in separation media. The effect of sorbent in matrix-compatible binders is crucial to understand for some applications. The extraction (or the desorption) of analytes will be controlled by the effective diffusion coefficient in the coating rather than at the interface or boundary layer, as it was a most common assumption before. The following two chapters, Chapter 4 and Chapter 5, contain applications that utilize the two designs of the microfluidic open interface for the analysis of immunosuppressive drugs and fentanyl analytes from the whole blood, respectively. For both works, the sample preparation is done in a high-throughput fashion. After the analysis with a microfluidic open interface, the overall method times (per sample) are substantially lower than gold-standard methods reported in the literature while maintaining similar detection and quantification limits compared to state-of-the-art reported methods. Therefore, solidphase microextraction coupled directly to mass spectrometry via a microfluidic open interface offers a suitable replacement for a gold-standard method. Chapter 6 contains an alternative and simplified use of the microfluidic open interface coupled to the homemade ultraviolet-visible light detection system. Finally, Chapter 7 encompasses the main findings and offers a future perspective on using solid-phase microextraction with a microfluidic open interface coupled directly to mass spectrometry or an alternative detector, such as ultraviolet-visible light detection

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

Improved methods in the collection of biological samples for complex occupational and environmental settings

Problem Statement: In the collection of traditional biological samples, such as liquid venous whole blood, plasma, and serum, the need for phlebotomy and cold chain often constrains their use in complex occupational and environmental settings. Less invasive methods that do not require phlebotomy or cold chain, such as dried blood spots (DBS), provide a potential alternative to traditional samples. Despite the advantages of DBS, scientific questions remain as to the range of potential applications of the method, as do technical challenges associated with field collection. Among these challenges, the requirement of open-air drying, which is not currently standardized and exposes DBS samples to potential contaminants while creating logistical hurdles in collection and storage, continues to hinder wider adoption of DBS. Methods: In the first of three related manuscripts, we conducted a review of the current state of the science in DBS sampling using a scoping review of reviews methodology. In the second manuscript, we designed and demonstrated proof-of-concept for novel methods in field collection and storage of DBS samples. This study measured drying rates of DBS samples collected under novel methods through use of resistance sensors designed specifically for the study. In the third manuscript, we conducted a validation of assay protocol for comparing RNA measurements in DBS samples collected under our novel methods with those of the current methods recommended by the United States Centers for Disease Control and Prevention (CDC). Results: In our first study, we identified approximately 2,000 (n=1,947) analytes that have been measured by one of more than 150 (n=169) different analytic methods. In our second study, we found that DBS samples collected under our novel methods in conditions of moderate and high humidity dried, on average, 30% and 50% faster respectively than DBS samples allowed to open-air dry under similar conditions as reported in the scientific literature. In our third study, our findings suggest that our novel methods demonstrated an overall improvement in performance on detection and quantification of RNA from DBS samples as compared with current methods. Conclusions: DBS provide researchers and practitioners a wide-ranging tool with potential applications for biosampling in complex occupational and environmental settings. Our novel methods in DBS collection and storage provide several advantages over current methodologies, including removal of the requirement for open-air drying of samples, reduced risk of sample contamination, reduced variability in environmental conditions incurred by samples, and overall improvements in measurements derived from DBS. Our findings support adoption of our novel methods in the collection and storage of DBS samples

African traditional medicine-antiretroviral interactions : effects of Sutherlandia frutescens on the pharmacokinetics of Atazanavir

In response to the urgent call for investigations into antiretroviral (ARV)-African traditional medicine (ATM) interactions, this research was undertaken to ascertain whether chronic administration of the ATM, Sutherlandia frutescens (SF) may alter the bioavailability of the protease inhibitor (PI), atazanavir (ATV), which may impact on the safety or efficacy of the ARV. Prior to investigating a potential interaction between ATV and SF in vitro and in vivo, a high performance liquid chromatography method with ultraviolet detection (HPLC-UV) was developed and validated for the bioanalysis of ATV in human plasma and liver microsomes. An improved and efficient analytical method with minimal use of solvents and short run time was achieved in comparison to methods published in the literature. In addition, the method was selective, linear, accurate and precise for quantitative analysis of ATV in these studies. Molecular docking studies were conducted to compare the binding modes and affinities of ATV and two major SF constituents, Sutherlandioside B and Sutherlandin C, with the efflux transporter, P-glycoprotein (P-gp) and the CYP450 isoenzyme, CYP3A4 to determine the potential for these phytochemicals to competitively inhibit the binding of ATV to these two proteins, which are mediators of absorption and metabolism. These studies revealed that modulation of P-gp transport of ATV by Sutherlandioside B and Sutherlandin C was not likely to occur via competitive inhibition. The results further indicated that weak competitive inhibition of CYP3A4 may possibly occur in the presence of either of these two SF constituents. The Caco-2 cell line was used as an in vitro model of human intestinal absorption. Accumulation studies in these cells were conducted to ascertain whether extracts and constituents of SF have the ability to alter the absorption of ATV. The results showed that the aqueous extract of SF significantly reduced ATV accumulation, suggesting decreased ATV absorption, whilst a triterpenoid glycoside fraction isolated from SF exhibited an opposing effect. Analogous responses were elicited by the aqueous extract and a triterpenoid glycoside fraction in similar accumulation studies in P-gp overexpressing Madin–Darby Canine Kidney Strain II cells (MDCKII-MDR1), which signified that the effects of this extract and component on ATV transport in the Caco-2 cells were P-gp-mediated. The quantitative analysis of ATV in human liver microsomes after co-incubation with extracts and components of SF was conducted to determine the effects of SF on the metabolism of ATV. The aqueous and methanolic extracts of SF inhibited ATV metabolism, whilst the triterpenoid glycoside fraction had a converse effect. Analogous effects by the extracts were demonstrated in experiments conducted in CYP3A4-transfected microsomes, suggesting that the inhibition of ATV metabolism in the liver microsomes by these SF extracts was CYP3A4-mediated. A combination of Sutherlandiosides C and D also inhibited CYP3A4-mediated ATV metabolism, which was in contrast to the response elicited by the triterpenoid fraction in the liver microsomes, where other unidentified compounds, shown to be present therein, may have contributed to the activation of ATV metabolism. The in vitro studies revealed the potential for SF to alter the bioavailability of ATV, therefore a clinical study in which the effect of a multiple dose regimen of SF on the pharmacokinetics (PK) of a single dose of ATV was conducted in healthy male volunteers. The statistical analysis showed that the 90 % confidence intervals around the geometric mean ratios (ATV + SF/ATV alone) for both Cmax and AUC0-24 hours, fell well below the lower limit of the "no-effect" boundary of 0.8 – 1.25, implying that the bioavailability of ATV was significantly reduced in this cohort of subjects. It may thus be concluded that if the reduction in bioavailability observed in this clinical study is found to be clinically relevant, co-administration of SF commercial dosage forms and ATV in HIV/AIDS patients may potentially result in subtherapeutic ATV levels, which may in turn contribute to ATV resistance and/or treatment failure. This research has therefore highlighted the potential risk for toxicity or lack of efficacy of ARV regimens which may result when ATMs and PIs are used concurrently and that patients and health care practitioners alike should be aware of these perils

Lung cancer biomarker discovery using proteomic techniques

Lung cancer has the highest mortality rate of any cancer, often due to the fact that it is detected at a late stage in its progression when it has already metastasised. The levels of certain biomarkers, such as proteins, metabolites and chemokines, in biological fluid or tissue could potentially detect cancer at an early stage, determine cancer subtype, or monitor the sensitivity to cancer treatment. Currently available lung cancer markers lack the sensitivity and specificity to be of great benefit and there is room for improvement. The research in this thesis aims to discover new biomarkers, using proteomic techniques, with the potential to improve or supersede those used at present for diagnosis and prognosis of lung cancer. Discovery phase was performed on conditioned media of lung cancer cell lines using 2D-DIGE in the hope it might mimic the serum/plasma environment of lung cancer patients. Further discovery phase was performed on serum using immunodepletion, proteominer, 2D-DIGE, label-free mass spectrometry followed by pathway analysis, metabolomic analysis, and multiplex assay analysis of cancer panels and a matrix-metalloproteinase panel. Validation in serum and plasma was performed using ELISAs, biochemical assays, and multiplex platforms, and in tissue using immunohistochemistry. Lung cancer subtypes examined in validation phase were squamous cell carcinoma, adenocarcinoma, and small cell lung cancer. There were insufficient serum/plasma samples to include a large cell carcinoma group. Potential biomarkers discovered include hnRNPA2B1, pyruvate kinase M2 (PKM2), HSC70-interacting protein (Hip), tenascin C, vascular endothelial growth factor alpha (VEGF-α), MMP-1, MMP-8, MMP-9, 12-HETE, and phenylalanine. In serum hnRNPA2B1, PKM2, Hip, tenascin C, VEGF-α, MMP-1, -8, and -9, and 12-HETE were increased in cancer compared to normal. Phenylalanine had similar levels in normal and small cell lung cancer but was decreased in non-small cell lung cancer. Of those examined in plasma hnRNPA2B1, VEGF-α, MMP-1 and -9, and 12-HETE were decreased in cancer compared to normal whereas PKM2 and tenascin C were increased. Where benign lung disease controls were used the markers were present at levels similar to lung cancer except for MMP-9 in plasma, VEGF-α in serum, and PKM2 in both serum and plasma. Immunohistochemistry of tissue showed an overall increase of expression of hnRNPA2B1 and HSC70-interacting protein in cancer tissue compared to normal. Functional assays were performed on hnRNPA2B1 showing its potential role in invasion and migration of lung cancer; its knockdown in the DLKP-M lung cancer cell line with two siRNA’s showed a 57% and 44% decrease in invasion and a 32% and 39% decrease in migration respectively. The research provided in this thesis demonstrates the importance for intensive validation before conclusions can be drawn on the overall usefulness of candidate lung cancer markers; a good serum marker does not necessarily make a good plasma marker and a marker that differentiates normal conditions from cancer conditions does not necessarily differentiate lung diseases/benign tumour from cancer