scientific background: chemistry student
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Scientific background+447523356417 | [email protected]: federico.floris | LinkedIN: Federico Floris
Federico FlorisMSc
Education Bachelor of Science in Chemistry
from the University of Cagliari110/110 cum laude (first class honours).2007/2010
Master of Science in Chemistry: Analysis and Characterization from Alma Mater Studiorum – University of Bologna110/110 cum laude (first class honours).2010-2013
Karolinska Institutet (Stockholm, Sweden)Abroad thesis, analytical method development2012
Thesis title: “Development of a GC/MS method for the quantification of 7α-hydroxy-3-oxo-4-cholestenoic acid (BAX) in cerebrospinal fluid (CSF) as possible biomarker for neurodegeneration and blood-brain barrier dysfunction.”
CAGLIARI
BOLOGNA
Research activity in Bologna Laboratory of Analytical and Bioanalytical
Chemistry in Sant’Orsola Hospital (Bologna, Italy)
Prof. Aldo Roda (Department of Chemistry G. Ciamician, University of Bologna, Italy)
6 months
Aldo Roda, full professor of Analytical Chemistry, Faculty of Farmacy, University of Bologna.
Quantification of bile acids (BA) and cholesterol in complex matrixes (human and animal blood, serum,
plasma) through HPLC-ESI-MS/MS.
Determination of physical-chemical properties of newly synthesised bile acids (HP/LP, CMC, permeability).
Utilisation of animal models to test the variation of cholesterol and bile acids induced by exogenous BAs.
Methabolism and biodistribution of exogenous BAs in different animal species.
Monitoring of “intermediate” biomarkers for the synthesis of bile acids (BAs) and cholesterol in insulin-resistant subjects and comparison to healthy matched
controls
• Correlation between insuline-resistance and the synthesis of cholesterol and bile acids.
Hypothesis
• Quantification of lathosterol (intermediate in the synthesis of cholesterol) through GC-EI-MS.
• Quantification of 7α-hydroxy-4-cholesten-3-one (C4, intermediate in the synthesis of bile acids).
Claim support
• Bile acids (BA) and cholesterol profiles of n=160 patients (HPLC-ESI-MS/MS) showed increased levels of BAs for subjects with insulin-resistance.
Premises
BAs in serumSPE with C18 reverse phase
chromatographyHPLC
ESI-MS/MS• Triple quadrupole• MRM
Lathosterol in serum
Liquid/liquid extraction Derivatization GC
EI-MS• Quadrupole• SIM
Quantification of BAs and Lathosterol in human serum
• Healthy and diabetic patients (n=286)
• Internal standard method
• Calibration curve
Method specifics
Monitoring of “intermediate” biomarkers for the synthesis of bile acids (BAs) and cholesterol in insulin-resistant subjects and comparison to healthy matched controls
Lathosterol A typical bile acid (cholic), and areas in which conjugation may occur.
Matched by sex, BMI and serum triglycerides.
Data about BAs, cholesterol, lathosterol and C4 of both insulin resistant and healthy subject were compared.
Monitoring of “intermediate” biomarkers for the synthesis of bile acids (BAs) and cholesterol in insulin-resistant subjects and comparison to healthy matched controls
Chemiometric study in progress.
Further analysis required.
•Improved manual skills.•Enhanced skills and competences with SPE, HPLC, GC, MSn.•Improved organisation of time.
Personal achievements
Research activity in Stockholm Division of Laboratory Medicine, Karolinska
Institutet (Stockholm, Sweden)
Prof. Ingemar Björkhem (Karolinska Institutet, Stokholm, Sweden)
3 monthsIngemar Björkhem Full professor of Bioanalytical Arteriosclerosis, Karolinska Institutet (Stockholm).
Development of a method for the quantification of
7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA) in cerebrospinal fluid (CSF)
27-hydroxycholesterol (27OHC) 7α-hydroxy-3-oxo-4-cholestenoic acid(7-HOCA)
High levels in patients with AD and in CSF of patients with BBB dysfunction1. Possible metabolic path for
the elimination of 27-hydroxilated sterols from brain2.
1. Leoni, V., C. Caccia. 2002. Chemistry and Physics of Lipids. 164: 515–524.2. Maney, S., M. Heverin, U. Panzenboeck, L. Ekström, M. Axelson, U.
Andersson, U. Diczfalusy, I. Pikuleva, J. Wahren, W. Sattler, I. Björkhem. 2007. J. Lipid Res. 48: 944-951.
Development of a method for the quantification of7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA) in cerebrospinal fluid (CSF)
Aims of the work
Development of a GC/MS method for the quantification of 7α-hydroxy-3-oxo-4-cholestenoic acid in CSF.
Requirements: The method has to be suitable for the clinical analysis for
diagnostic and prognostic purposes; Short working times, limited costs, reliability.
Determination of the average concentration of 7-HOCA in CSF of healthy subjects and patients affected by neurodegenerative diseases or blood-brain barrier (BBB) dysfunction.
Development of a method for the quantification of7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA) in cerebrospinal fluid (CSF)
Liquid/liquid extraction Derivatisation GC/MS
Ether
CSF 1 mL
5 mL
+ 20 μL HCl 0.1 M
1. TMS-diazomethane
2. TMS reagent
7-HOCA 7-HOCA trimethylsilil methyl ester derivative
EI Quadrupole Detector
Experimental Method
Development of a method for the quantification of7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA) in cerebrospinal fluid (CSF)
Method characteristics
0 10 20 30 40 50 60 70 80 900
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
7-HOCA Concentration [ng/mL]
Rela
tive
resp
onse
y = (0,017 ± 0,005)x + (0,010 ± 0,002)R2 = 0,993 ± 0,005
Analyte C(7-HOCA) [ng/mL]
Precision (CV) Accuracy Recovery LOD
[ng/mL]LOQ
[ng/mL]
7-HOCA 13.9 ± 0.7 5 % 2.1 % 103 % 0.5 2
Name Measured ion m/z
Retention Time [min]
7-HOCA 426 21.8
d4-7-HOCA 430 21.7
Quantification: Internal standard methodInternal standard (IS): d4-7-HOCA
Calibration curve: y = (a ± SD)x + (b ± SD)
1 2 3 4 5 60
20
40
60
80
100
120
140
Studied groups
Conc
entr
ation
of 7
-HO
CA in
CSF
[ng/
mL]
• Studied groups (n = 9 subjects per group):1. Healthy subjects2. Headache without objective findings3. Memory problems not associated to neurodegeneration4. Memory problems associated to AD5. Blood-brain barrier dysfunction6. CYP7B1 gene mutation
< LOQ
Comparison of 7-HOCA concentration in cerebrospinal fluid of different subjects.
Application of the method
Development of a method for the quantification of7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA) in cerebrospinal fluid (CSF)
Development of a method for the quantification of7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA) in cerebrospinal fluid (CSF)
Conclusions• The newly developed GC/MS method showed satisfactory accuracy and precision, and
confirmed that 7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA) is the most abundant compound in cerebrospinal fluid (CSF)
• 7-HOCA did not show any apparent correlation with neurodegeneration due to the Alzheimer’s Disease (AD), but it is possible to use it as a biomarker for blood-brain barrier dysfunction.
• It has been confirmed that CYP7B1 is a critical enzyme for the formation of 7-HOCA in the brain.
• The method is suitable for clinical analysis with diagnostic and prognostic purposes, because of the low LOQ which allows to determine the physiological concentrations of 7-HOCA.
•Familiarity with Method development.•Article on the Journal of Lipids Research
Personal achievements
J Lipid Res, 2014 Feb; 55(2): 313-8There is a continuous flux of the oxysterol 27-hydroxycholesterol (27-OHC) from the circulation across the blood-brain barrier (BBB) into the brain. The major metabolite of 27-OHC in the brain is 7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA). We confirm a recent report describing the presence of this metabolite in cerebrospinal fluid (CSF) at a relatively high concentration. A simple and accurate method was developed for assay of 7-HOCA in CSF based on isotope dilution-mass spectrometry and use of (2)H4-labeled internal standard. The concentration of this metabolite was found to be markedly increased in CSF from patients with a dysfunctional BBB. There was a high correlation between the levels of 7-HOCA in CSF and the CSF/serum albumin ratio. The concentration of 7-HOCA in CSF was not significantly affected by neurodegeneration. Our findings suggest that 7-HOCA could be used as a diagnostic marker for conditions with a dysfunctional BBB.
Research activity at Unilever R&D PS
Oral Care Discover, Personal Care, Unilever R&D (Port Sunlight, UK)
Andrew Joiner, Oral Care project leader (Unilever R&D PS)
6 months
Andrew Joiner, Oral Care project leader at Unilever R&D, Port Sunlight.
Preparation of various substrates, like teeth surrogates, for analytical purposes.
Measures of Physical-Chemical properties of new materials, in order to see if they could be suitable systems for new products’ goals.
Testing the effect of new raw and synthetic materials’ properties on the tooth surface, in order to verify if they are useful for new products (i.e. toothpastes).
• Improved time management (strict deadlines)• Improved problem solving• Experience in a multinational company
Personal achievements
Evaluation of new materials as possible promoters of remineralisation for dental
enamel Tooth wear
• Abrasion• Attrition• Caries• Erosion
Utilisation of fluoride containing toothpastes
• Helps with tooth sensitivity and demineralisation.
• Not sufficient for subjects with high degree of cavities.
New materials*
•Lower demineralisation•Higher remineralisation
*Unilever confidential
Technical skills and competences
Separation techniquesChromatography (GC, LC, HPLC); electrophoresis; familiar with Flow Field Fractionation.
Extraction techniquesSPE, SPME.
Qualification/Quantification techniques:Mass Spectrometry (MS) and tandem Mass Spectrometry (MSn), UV-Vis Spectrophotometry, IR Spectrophotometry, Atomic Absorption Spectroscopy (AAS), Inductively Coupled Plasma associated with Atomic Emission Spectroscopy (ICP-AES), conductivity meter, ion selective electrode.
Optical techniquesMicroHardness tester
Interests and ambitions Working in the Research field
PhD in Analytical Chemistry Research & Development
• Separative Science / Life Sciences• Method development• Application of Analytical Chemistry to Medicine
Chemistry, Life Sciences and or to clinical analysis for diagnostic and prognostic purposes.
• Mass Spectrometry• Degenerative/terminal diseases
Scientific Interests tagcloud
Thank you for your kind attention!
Any questions?