Mass Spectrometry Application Strategies of Dried Blood Spots Analysis

El análisis de manchas de sangre seca (DBS) es una tecnología de análisis bien conocida, cuyas primeras aplicaciones de rutina se remontan a la década de 1960. Los avances en los instrumentos de espectrometría de masas durante el siglo pasado, permitieron alcanzar la sensibilidad necesaria para abrir nuevos mercados. Sin embargo, algunas incertidumbres y la falta de comprensión de los métodos han impedido hasta el momento que la tecnología tenga una amplia aceptación en el mercado.En esta tesis doctoral se han desarrollado y validado diversas técnicas, condiciones y flujos de trabajo de análisis DBS, que demuestran la viabilidad y el potencial de aplicación de la tecnología DBS-LC-MS.Se han desarrollado métodos avanzados para su aplicación en el campo de la pediatría neonatal, en particular la prueba del talón en recién nacidos, donde se ha ampliado y estandarizado el panel de análisis.También se han investigado nuevos campos de aplicación, como la vigilancia de medicamentos terapéuticos y la toxicología forense. Se describe la aplicación de la farmacovigilancia remota de antirretrovirales en regiones de escasos recursos y se presentan nuevos enfoques analíticos para la vigilancia del abuso de alcohol. Finalmente, en esta tesis se ha introducido un método innovador con el que se pueden detectar más de 1.200 drogas ilícitas a partir de una sola gota de sangre.La investigación científica realizada se presenta en forma de compendio de publicaciones (6), que son incluidas en esta Memoria. Se adjuntan a modo de apéndice otros dos trabajos del candidato que no constan oficialmente en dicho compendio. Todos los trabajos constituyen una unidad temática coherente sobre la técnica de la DBS y su acoplamiento a la espectrometría de masas.This doctoral thesis is a compendium of dried blood spot (DBS) applications in the fields of newborn screening, forensic toxicology and therapeutic drug monitoring. DBS is a well-known analysis technology, which first routine applications date back to the 1960s. Advancements in mass spectrometry instruments during the last century, enabled to reach the required sensitivity to open up new markets. Some uncertainties and missing method understanding remain and this is holding back the technology from wide spread market acceptance. For the general scientific acceptance of this technology, several methods have been developed and validated within this work. Advanced methods for the field of newborn screening were developed, where the analysis panel has been extended and standardized. Goal of the first study was to transfer the amino acids and acyl carnitines analysis onto the automated DBS-MS 500 platform. Also, a steroid panel of 17OHP, cortisol and androstenedione was defined to exclude the 17OHP from the immune assay panel and to transfer this as well onto the fully automated DBS-LC-MS/MS. The conventional 17OHP enzyme-linked immunosorbent assay (ELISA) based on a manually punched DBS disc leads to a high percentage of false positive. First, cortisol increases when the babies are stressed, which also leads to elevated 17OHP concentrations and secondly, the ELISA has significant cross reaction potential with steroid sulfate which can be monitored with androstenedione. Both, the amino acid and acyl carnitine detection plus the integration of a steroid panel into the DBS-LC-MS/MS workflow was successfully achieved [1]. Newborn screening panels are not unified across borders and sometimes even differ within countries and laboratories. The implementation of the fully automated DBS platform could be a good starting point of standardization and unification of those programs. Here, a method based on an official newborn screening kit was introduced within this thesis. The analysis takes only 2 minutes per sample; however it is limited to amino acids and acyl carnitines only. The DBS extract is directly guided to the mass spectrometer without any column. This is a well-known procedure in newborn screening and allows speeding up the process to its optimum [2]. Therapeutic drug monitoring, especially in remote and rural areas is another upcoming application field of DBS. Several methods have been supported and co-authored, whereas the first method focuses of the three antiretroviral drugs nevirapine, efavirenz and lopinavir. The study highlights the transportation advantages of DBS, without any biohazard labeling neither requiring cooling chains. DBS were drawn in Tanzania, Africa, and sent to Switzerland where the samples went through different climate zones. Nonetheless, the samples showed very good results and stability of the monitored drugs [3]. We ran a follow up study on some of the samples approximately one year after the publication, where still all analytes could be recovered in the same concentration as published. Also the DBS sampling itself was investigated in a rural area of Tanzania [4] and the technique proved to be suitable. Another therapeutic drug, where a more efficient sampling process is required for mass drug administration (MDA) campaigns, is Ivermectin. This drug lowers the incidence of river blindness and lymphatic filariasis infections. Further, recent studies demonstrated that Ivermectin is also active against several other parasites and even against certain mosquitoes. As one of these mosquitoes is Anopheles gambiae, the major vector of malaria in Africa, Ivermectin could be mass administered to fight malaria. In other words, the drug makes the human blood lethal for the Anopheles gambiae and therefore reduces the number of vectors in an area. Still, more safety data is required from a larger population. Here, an according fully automated DBS method has been developed and validated. In addition, a comparison study to plasma samples, stability and hematocrit impact has been studied [5]. In forensic toxicology, it was shown that Dried Matrix Spot (DMS) is a suitable technology for large scale DBS applications. A fully automated method, where either 1200 drugs can be screened from a DBS and a quantitative follow up method focusing on the 28 most abundant drugs of abuse, was developed. This was documented within two publications, where the first publication was a proof of concept study to determine if the detection limits can be reached using the DBS-LC-MS/MS methodology [6]. The second, follow up publication was a specific method development and implementation. More than 1200 illicit drugs can be screened from a single DBS or Dried Urine Spots (DUS) within 20 minutes per sample using a Forensic Toxicology Database. The criteria of bio-analytical method validation guidelines were fulfilled, and the method was transferred into a routine laboratory successfully [7]. Furthermore, the discovery of phosphatidylethanol (PEth) as direct alcohol marker prolonged the window of detection for alcohol consumption to several weeks. PEth proved to be instable during storage of liquid blood samples. By using DBS sampling, this biomarker can be stabilized due to the inactivation of enzymatic activity. Also, for PEth, a fully automated DBS-LC-MS/MS was established for the determination of the two most abundant PEth homologs in a range from 20–1500 ng/mL. Automated DBS card handling and online solid phase extraction LC-MS/MS permits baseline separation and detection of PEth 16:0/18:1 and PEth 16:0/18:2 within 7 minutes per sample [8]. The methods from the various fields of application were presented at several conferences as oral presentations and posters, showing their interest from the scientific blood analysis community.<br /

The development and assessment of sustained release nevirapine tablets

The use of antiretroviral (ARV) agents in the management of HIV/AIDS has significantly improved the lifestyle and wellbeing of patients. Despite the success that has been achieved with the use of ARV therapy, the occurrence of adverse effects and unpredictable bioavailability associated with most of these drugs remains a major concern. Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) that is used in combination with other ARV compounds for the treatment of HIV-1 infections. It is also used for the prevention of mother to child transmission of the HIV-1 virus. NVP is a Biopharmaceutics Classification System (BCS) Class II compound. Although NVP exhibits good oral absorption, it induces self-metabolism leading to low and sometimes unpredictable bioavailability. NVP is commercially available as an immediate release and extended release dosage form, viz., Viramune® XR. Formulation of a generic sustained release (SR) dosage form for once daily dosing would result in delivery of constant amount of the drug to the circulation, reduce dose related adverse effects, improve patient compliance to medication and reduce the costs of therapy. A simple RP-HPLC method was developed and optimised using a central composite design approach. The method was validated using ICH guidelines and was found to be linear, precise, specific and accurate for the analysis of NVP both in bulk and dosage forms. Direct compression was used as the method of tablet manufacture. Different polymers were assessed for suitability as rate retarding polymers and included Methocel® K4M, Carbopol® 71G NF and Eudragit® RSPO. Powder blends were assessed for flow properties using the angle of repose, bulk and tapped density, Carr’s Compressibility index and Hausner’s ratio. The traditional approach of changing the amount of polymers and diluents systematically to achieve a desired NVP release profile was used for the development of a preliminary formulation. Response surface methodology was used for the optimisation of the formulation using a Box-Behnken quadratic design. Physical characteristics of the tablets such as thickness, weight, hardness, tensile strength and friability were assessed and the tablets passed Pharmacopoeial testing. NVP assay and content uniformity were assessed using a validated RP-HPLC method. Initially, USP Apparatus 2 was used to study NVP release over a 24 hour period and subsequently dissolution studies were performed using USP Apparatus 3 as it can be used to simulate GIT conditions. The dissolution profiles generated were used to determine the agitation rate for USP Apparatus 3 that would be equivalent to an agitation rate of 50 rpm when using USP Apparatus 2. The effect of the mesh screen pore size, buffer molarity strength and concentration of surfactant on NVP release were also investigated in order to select discriminatory dissolution test conditions for the test formulation. Dissolution profiles were compared to those of the commercially available Viramune® XR using the FDA recommended difference (f1) and similarity (f2) factors. The calculated values for f1 and f2 revealed that the dissolution profile for the optimised formulation that was identified was statistically similar to Viramune® XR. In vitro release data were fitted to different kinetic models to study the release kinetics of NVP. The overall mechanism of NVP release was best described using the Korsmeyer-Peppas diffusion exponent value, n. NVP release was found to be anomalous, implying that the release was influenced by a combination of diffusion, swelling and polymer chain relaxation. The Hixson-Crowell model revealed that there was constant change in surface area of the dosage form suggesting that erosion and swelling were significant factors affecting NVP release from the hydrophilic matrix technology. The release kinetics data were also used to design the optimised formulation. Tablets manufactured using the optimised formulation were subjected to water uptake and erosion studies and the results revealed that swelling and erosion occur simultaneously. The effects of pH and molarity on the swelling and erosion of the tablets were also investigated. The data suggest that increase in pH resulted in a slight increase in swelling while an increase in molarity did not have a significant effect on swelling. The change in pH did not have a significant effect on erosion while an increase in molarity strength resulted in a decrease in matrix erosion. The effect of HPMC grade on swelling, erosion and NVP release revealed that the grade of HPMC used had a significant effect on NVP release, with the release rate decreasing, swelling increasing and erosion decreasing as the viscosity of the HPMC grade increased. The effect of the particle size of MCC on NVP release was also studied by manufacturing tablets containing different grades of MCC and these studies revealed that particle size did not appear to have a significant effect on NVP release. Similarly the use of different types of lactose did not appear to have a significant impact on NVP release. In conclusion a sustained release NVP tablet formulation that has the potential for further development and optimisation has been developed, assessed and manufactured successfully and has been shown to exhibit similar dissolution behaviour to Viramune® XR, a commercially available NVP extended release product

1H-MRS neurometabolite profiles and motor development in school-aged children who are HIV-exposed uninfected: a birth cohort study

ObjectiveAlterations in regional neurometabolite levels as well as impaired neurodevelopmental outcomes have previously been observed in children who are HIV-exposed uninfected (CHEU). However, little is known about how neurometabolite profiles may relate to their developmental impairment. This study aimed to compare neurometabolite concentrations in school-aged CHEU and children who are HIV-unexposed (CHU) and to explore associations of neurometabolite profiles with functional neurodevelopment in the context of perinatal HIV exposure.MethodsWe used 3 T single voxel proton magnetic resonance spectroscopy (1H-MRS) to quantify absolute and relative neurometabolites in the parietal gray and parietal white matter in school-aged CHEU and aged- and community-matched CHU. Functional neurodevelopmental outcomes were assessed using the early learning outcome measure (ELOM) tool at 6 years of age.ResultsOur study included 152 school-aged children (50% males), 110 CHEU and 42 CHU, with an average age of 74 months at the neuroimaging visit. In an adjusted multiple linear regression analysis, significantly lower glutamate (Glu) concentrations were found in CHEU as compared to CHU in the parietal gray matter (absolute Glu, p = 0.046; Glu/total creatine (Cr+PCr) ratios, p = 0.035) and lower total choline to creatine ratios (GPC+PCh/Cr+PCr) in the parietal white matter (p = 0.039). Using factor analysis and adjusted logistic regression analysis, a parietal gray matter Glu and myo-inositol (Ins) dominated factor was associated with HIV exposure status in both unadjusted (OR 0.55, 95% CI 0.17–0.45, p = 0.013) and adjusted analyses (OR 0.59, 95% CI 0.35–0.94, p = 0.031). With Ins as one of the dominating metabolites, this neurometabolic factor was similar to that found at the age of two years. Furthermore, this factor was also found to be correlated with ELOM scores of gross motor development in CHEU (Pearson’s r = −0.48, p = 0.044). In addition, in CHEU, there was a significant association between Ins/Cr+PCr ratios in the parietal white matter and ELOM scores of fine motor coordination and visual motor integration in CHEU (Pearson’s r = 0.51, p = 0.032).ConclusionReduced Glu concentrations in the parietal gray matter may suggest regional alterations in excitatory glutamatergic transmission pathways in the context of perinatal HIV and/or antiretroviral therapy (ART) exposure, while reduced Cho ratios in the parietal white matter suggest regional myelin loss. Identified associations between neurometabolite profiles and gross and fine motor developmental outcomes in CHEU are suggestive of a neurometabolic mechanism that may underlie impaired motor neurodevelopmental outcomes observed in CHEU

HIV drug resistance prediction with weighted categorical kernel functions

Background: Antiretroviral drugs are a very effective therapy against HIV infection. However, the high mutation rate of HIV permits the emergence of variants that can be resistant to the drug treatment. Predicting drug resistance to previously unobserved variants is therefore very important for an optimum medical treatment. In this paper, we propose the use of weighted categorical kernel functions to predict drug resistance from virus sequence data. These kernel functions are very simple to implement and are able to take into account HIV data particularities, such as allele mixtures, and to weigh the different importance of each protein residue, as it is known that not all positions contribute equally to the resistance. Results: We analyzed 21 drugs of four classes: protease inhibitors (PI), integrase inhibitors (INI), nucleoside reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI). We compared two categorical kernel functions, Overlap and Jaccard, against two well-known noncategorical kernel functions (Linear and RBF) and Random Forest (RF). Weighted versions of these kernels were also considered, where the weights were obtained from the RF decrease in node impurity. The Jaccard kernel was the best method, either in its weighted or unweighted form, for 20 out of the 21 drugs. Conclusions: Results show that kernels that take into account both the categorical nature of the data and the presence of mixtures consistently result in the best prediction model. The advantage of including weights depended on the protein targeted by the drug. In the case of reverse transcriptase, weights based in the relative importance of each position clearly increased the prediction performance, while the improvement in the protease was much smaller. This seems to be related to the distribution of weights, as measured by the Gini index. All methods described, together with documentation and examples, are freely available at https://bitbucket.org/elies_ramon/catkern.Peer ReviewedPostprint (published version

Immune-Mediated Drug Induced Liver Injury: A Multidisciplinary Approach

This thesis presents an approach to expose relationships between immune mediated drug induced liver injury (IMDILI) and the three-dimensional structural features of toxic drug molecules and their metabolites. The series of analyses test the hypothesis that drugs which produce similar patterns of toxicity interact with targets within common toxicological pathways and that activation of the underlying mechanisms depends on structural similarity among toxic molecules. Spontaneous adverse drug reaction (ADR) reports were used to identify cases of IMDILI. Network map tools were used to compare the known and predicted protein interactions with each of the probe drugs to explore the interactions that are common between the drugs. The IMDILI probe set was then used to develop a pharmacophore model which became the starting point for identifying potential toxicity targets for IMDILI. Pharmacophore screening results demonstrated similarities between the probe IMDILI set of drugs and Toll-Like Receptor 7 (TLR7) agonists, suggesting TLR7 as a potential toxicity target. This thesis highlights the potential for multidisciplinary approaches in the study of complex diseases. Such approaches are particularly helpful for rare diseases where little knowledge is available, and may provide key insights into mechanisms of toxicity that cannot be gleaned from a single disciplinary study

Immune-Mediated Drug Induced Liver Injury: A Multidisciplinary Approach

This thesis presents an approach to expose relationships between immune mediated drug induced liver injury (IMDILI) and the three-dimensional structural features of toxic drug molecules and their metabolites. The series of analyses test the hypothesis that drugs which produce similar patterns of toxicity interact with targets within common toxicological pathways and that activation of the underlying mechanisms depends on structural similarity among toxic molecules. Spontaneous adverse drug reaction (ADR) reports were used to identify cases of IMDILI. Network map tools were used to compare the known and predicted protein interactions with each of the probe drugs to explore the interactions that are common between the drugs. The IMDILI probe set was then used to develop a pharmacophore model which became the starting point for identifying potential toxicity targets for IMDILI. Pharmacophore screening results demonstrated similarities between the probe IMDILI set of drugs and Toll-Like Receptor 7 (TLR7) agonists, suggesting TLR7 as a potential toxicity target. This thesis highlights the potential for multidisciplinary approaches in the study of complex diseases. Such approaches are particularly helpful for rare diseases where little knowledge is available, and may provide key insights into mechanisms of toxicity that cannot be gleaned from a single disciplinary study

PHENOTYPIC CHARACTERIZATION OF NOVEL ANTIVIRALS FOR THE TREATMENT OF MULTIDRUG RESISTANT HIV-1 AND EMERGING VIRUSES

Abstract Phenotypic characterization of novel antivirals for the treatment of multidrug resistant HIV-1 and emerging viruses Doctoral Research School of Medical Biotechnologies – Cycle XXXV Supervisor: Maurizio Zazzi; Candidate: Federica Giammarino Background The need for new antiviral drugs has increased overtime due to the worldwide circulation of different viruses together with the increased frequency and diversity of new outbreaks. The ideal option for a prompt response against both emerging and re-emerging viruses is represented by the use and the development of direct acting antiviral agents. During my PhD I was involved in several projects focused on the evaluation of the antiviral activity of licensed and investigational antiviral drugs against Human Immunodeficiency (HIV-1), West Nile (WNV), Dengue (DENV) and SARS-CoV-2 viruses. Results and discussion Doravirine The antiviral activity of the NNRTI doravirine was evaluated against viruses harbouring different patterns of NNRTI resistance mutations in two studies. Globally, our data confirmed that the antiviral activity of doravirine may be compromised by the presence of multiple NNRTI resistance mutations, even in the absence of specific doravirine mutations. A third study was focused on the role of the natural polymorphism of the reverse transcriptase V106I. Our results indicate that it minimally affects the susceptibility to doravirine in clinical isolates and that it does not impact the genetic barrier to resistance as compared to reference wild-type virus, while viruses including the NNRTI resistant mutation V106A or V106M rapidly showed viral breakthrough under doravirine pressure due to the reduced susceptibility. Islatravir Our study confirmed the decrease of susceptibility of the investigational NRTTI islatravir due to the presence of M184V mutation. The clinical impact of NRTI mutations in the activity of islatravir has still to be defined and the threshold of fold-change values associated to reduced activity in vivo remains to be established. Ibalizumab The combinatorial activity of ibalizumab together with other antivirals, both approved and investigational, was evaluated through a newly developed cell-based assay consisting in the infection of the MOLT4-R5 cell line with the wild-type strains NL4-3 and AD8, and by the analysis of the results using the innovative software SynergyFinderPlus. Our data suggest that ibalizumab positively interacts with other antivirals with possible synergistic effects in select cases. Further studies are needed to determine the impact of Env variability and viral tropism in combination with other entry inhibitors. Development of a Cell-Based Immunodetection Assay for Simultaneous Screening of Antiviral Compounds Inhibiting Zika and Dengue Virus Replication An easy-to-perform and fast flavivirus immunodetection assay (IA) was developed to determine antiviral activity of promising compounds against ZIKV and DENV. The system, validated with references compounds against both viruses, was able to distinguish between the inhibitory effect of molecules targeting the early and the post-budding phase of viral replication cycle. Evaluation of sofosbuvir activity and resistance profile against West Nile virus in vitro Since the activity of sofosbuvir has been documented against different flaviviruses, we investigated whether it may exert an activity also against WNV. In both cell-based and enzymatic assays sofosbuvir was able to inhibit WNV replication in the low micromolar range. Moreover, in vitro selection and molecular docking experiments indicated that HCV and WNV share a similar sofosbuvir resistance pattern. ORIGINALE CHEMIAE in Antiviral Strategy - Origin and Modernization of Multi-Component Chemistry as a Source of Innovative Broad Spectrum Antiviral Strategy The “ORIGINALE CHEMIAE in Antiviral Strategy” project aims to identify promising broad-spectrum antivirals by taking advantage of the Multi-Component Chemistry strategy. Following the synthetization of molecules, their antiviral activity was determined in in vitro standardized virus-cell systems against DENV, WNV, HIV-1 and SARS-CoV-2. We identified eight molecules able to inhibit at least one of the viruses tested. However, their low selectivity indexes indicate the need to further improve the design of these molecules to increase the antiviral activity and/or reduce the cell toxicity in order to identify candidates for preclinical testing in animal models. Monoclonal antibodies and antivirals vs. SARS-CoV-2 After the development of a quantitative live-virus microneutralization assay, we evaluated the efficacy of licensed monoclonal Antibodies (mAbs) and the antiviral drugs remdesivir, nirmaltrevir and molnupiravir against different circulating SARS-CoV-2 variants. Our results showed that these drugs, contrary to the mAbs, retained activity against all tested variants. Conclusions A continuous challenge for public health is represented by the control of viral infections. Both vaccines and antiviral drugs may synergistically help to reduce the spread and the fatality of acute viral diseases and chronic infections. All the studies described in this thesis emphasize the role of the laboratory of virology within all the steps of the in vitro investigation of antiviral drugs, from the identification of molecules endowed with antiviral activity to the definition of the mechanism of action

Comparison of Magnetic Resonance Spectroscopy (MRS) data in children with and without HIV at 11-12 years

Although HIV and antiretroviral drugs have been shown to cause damage in the brain, the long-term impacts of perinatal infection, early treatment and exposure in children at 11 years, remain unclear. The effects of HIV and antiretroviral therapy (ART), whilst indistinguishable, can be investigated at a chemical level through proton magnetic resonance spectroscopy (1H-MRS). Previous studies in children have largely focused on individual metabolite changes. However, several adult studies have now advanced beyond this to address patterns of metabolic activity that are altered with HIV infection. Using a 3T Skyra scanner, 136 children (76 HIV+, 30 HEU, 30 HU; 71 males) between the ages of 11.0- 12.5 years, and from a similar socioeconomic background, were scanned. In this study metabolite concentrations were quantified within the basal ganglia (BG), midfrontal gray matter (MFGM) and peritrigonal white matter (PWM). We utilised linear regression to investigate individual metabolite differences, comparing HIV-infected (HIV+) children from the Children with HIV Early Antiretroviral Therapy (CHER) trial, and HIV-exposed-uninfected (HEU) children, to HIV-unexposed (HU) children. Pearson's correlation analysis, factor analysis and logistic regression were then used to study alterations in metabolic patterns between HIV+ and HIV-uninfected (HIV-) children. Analysis of the data was carried out in R. We found elevated total choline in the BG (p = 0.03) and MFGM (p < 0.001) of HIV+ children, as well as reduced PWM total NAA (p = 0.03) and total creatine (p = 0.01). Altered metabolite concentrations were further observed in HEU children. Additionally, we identified a cross-regional coupling of choline which distinguishes HIV+ from HIV- children (p < 0.001). These findings indicate that multiregional inflammation and PWM axonal damage are occurring in HIV+ children at 11 years. Ultimately, the consequences of perinatal HIV acquisition, in spite of early treatment, continue to be seen at 11 years, as do the impacts of exposure

A Neurometabolic Pattern of Elevated Myo-Inositol in Children Who Are HIV-Exposed and Uninfected: A South African Birth Cohort Study.

Introduction: Exposure to maternal HIV in pregnancy may be a risk factor for impaired child neurodevelopment during the first years of life. Altered neurometabolites have been associated with HIV exposure in older children and may help explain the mechanisms underlying this risk. For the first time, we explored neurometabolic profiles of children who are HIV-exposed and uninfected (CHEU) compared to children who are HIV-unexposed (CHU) at 2-3 years of age. Methods: The South African Drakenstein Child Health Study enrolled women during pregnancy and is following mother-child pairs through childhood. MRI scans were acquired on a sub-group of children at 2-3 years. We used single voxel magnetic resonance spectroscopy to measure brain metabolite ratios to total creatine in the parietal grey matter, and left and right parietal white matter of 83 children (36 CHEU; 47 CHU). Using factor analysis, we explored brain metabolite patterns in predefined parietal voxels in these groups using logistic regression models. Differences in relative concentrations of individual metabolites (n-acetyl-aspartate, myo-inositol, total choline, and glutamate) to total creatine between CHEU and CHU groups were also examined. Results: Factor analysis revealed four different metabolite patterns, each one characterized by covarying ratios of a single metabolite in parietal grey and white matter. The cross-regional pattern dominated by myo-inositol, a marker for glial reactivity and inflammation, was associated with HIV exposure status (OR 1.63; 95% CI 1.11-2.50) which held after adjusting for child age, sex, and maternal alcohol use during pregnancy (OR 1.59; 95% CI 1.07 -2.47). Additionally, higher relative concentrations of myo-inositol to total creatine were found in left and right parietal white matter of CHEU compared to CHU (p=0.025 and p=0.001 respectively). Discussion: Increased ratios of myo-inositol to total creatine in parietal brain regions at age 2-3 years in CHEU are suggestive of early and ongoing neuroinflammatory processes. Altered relative concentrations of neurometabolites were found predominantly in the white matter, which is sensitive to neuroinflammation, and may contribute to developmental risk in this population. Future work on the trajectory of myo-inositol over time in CHEU, alongside markers of neurocognitive development, and the potential for specific neurodevelopmental interventions will be useful

Applications of Mass Spectrometry in Proteomics and Pharmacokinetics

Tremendous technology improvements of the last decades has given mass spectrometry a more and more expanding role in the study of a wide range of molecules: from the identification and quantification of small molecular weight molecules to the structural determination of biomacromolecules. Many are the fields of application for this technique and the various versions of it. In the present study three different applications have been explored. The first application is a pharmacokinetics study of anticancer drug Gemcitabine and its principal metabolite, where the role of the LC-MS/MS is essential both for the selectivity of the detection of the small analytes and the sensitivity enhanced by multi-reaction monitoring experiments. The design of the study involved the collection of several blood samples at selected times and from patients that would have met certain eligibility criteria. The ESI demonstrated to be the most suitable approach and it provided the necessary data to conclude that toxicity of Gemcitabine did not increase when administered at FDR (Fixed Dose Rate) infusion in patients with impaired hepatic function. The second application describes an example of how MS represents a powerful tool in cancer research, from serum profiling study with high resolution MALDITOF and bioinformatic analysis, to the identification of potential biomarker through peak identification. Almost 400 serum sample – homogeneously distributed between biopsy confirmed ovarian cancer and high risk serum samples – were analyzed on a high resolution MALDI-TOF instrument after automated reverse phase magnetic beads separation. The high throughput data have undergone sophisticated bioinformatic procedures that lead to a list of upand down-regulated peaks, although identification studies were possible only for those peaks that showed a good reproducibility. One down-regolated peak has been identified using the LC-MS/MS technique. The identified peak confirmed a basic role of fibrinogen in the ovarian cancer; the other four peaks that have been identified as down-regulated showed an absolutely not satisfactory ionization in electro-spray, therefore further analysis will be performed on these analytes in order to determinate their amino acidic sequence. The most suitable technique seems to be MALDI-TOF/TOF mass spectrometry, since the peptides already showed a good degree of ionization in MALDI. The third and last study belongs to a quite new field, which is the combination of immuno precipitation assays with MALDI-TOF (Immuno Precipitation Mass Spectrometry, IPMS) experiments in order to evaluate the specificity of a series of monoclonal antibodies to specific antigen. The automated assay that has been developed provides structural information about the antigen that binds the monoclonal antibody to be tested and previously conjugated to the surface of magnetic beads, ideal support for robotic automation. IPMS showed its potential as a complementary tool of crucial importance in the selection of the monoclonal antibody for the development of ELISA based assay to be applied in the screening of a consistent number of human specimens for the clinical validation of proteins indicated in literature as potential biomarkers. Mass spectrometry in association with fractionation techniques, such as liquid or magnetic beads chromatography, is a very flexible tool in the cancer research field. Further improvement in the instrumentation and in the technology will bring always more and more results to be confident in