scientific background: chemistry student

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.


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


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



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


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


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?