<figure><img src="image_1.png" /></figure>

HAL
open science

# Development of a method for quantitative profiling of the carnitine family by LC-HRMS

Océane Delos, Pauline Le Faouder, Floriant Bellvert, Jean-Charles Portais,
Justine Bertrand-Michel

► To cite this version:

Océane Delos, Pauline Le Faouder, Floriant Bellvert, Jean-Charles Portais, Justine Bertrand-Michel. Development of a method for quantitative profiling of the carnitine family by LC-HRMS. RFMF, May 2023, Perpignan, France. hal-04406276

HAL Id: hal-04406276

https://hal.science/hal-04406276v1

Submitted on 19 Jan 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

Public Domain
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

Océane Delos¹,², Pauline Le Faouder¹,², Floriant Bellvert¹,³, Jean-Charles Portais¹,⁴, Justine Bertrand Michel¹,²

¹MetaboHUB-Metatoul, National Infrastructure of Metabolomics and Fluxomics - INRAE, ²Institut des Maladies Métaboliques et Cardiovasculaires - Université Toulouse III - Paul Sabatier, Institut National de la Santé et de la Recherche Médicale, ³TBI - Toulouse Biotechnology InstituteINSA - Institut National des Sciences Appliquées, INRAE, CNRS: UMR5504, ⁴Geroscience and rejuvenation research center - Université Toulouse III - Paul Sabatier, EFS, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique

## β-oxidation cycle

<figure><img src="image_4.png" /></figure>

* Acylcarnitines production to transport fatty acids (FA) across mitochondrial inner membrane

* In the mitochondria, the acyl-CoA undergo β-oxidation

* Supply of ATP to organs with high energy needs

## Pathologies

Carnitine and its derivatives are key partners in physiology. Deregulations can generate pathologies.

* Free L-carnitine deficiency

* Mitochondrial β-oxidation anomalies

* Carnitine cycle anomaly

* β-oxidation cycle anomaly

<figure><img src="image_5.png" /><figcaption>Acylcarnitine structure</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Characteristics of mitochondrial β-oxidation deficiencies[1]</figcaption></figure>

## Why study acylcarnitines profile ?

Acylcarnitines have an important role in metabolism and are markers of several deficiencies : profiles are different depending on the anomaly. Measuring them in blood or plasma is essential to understand metabolism and could be a powerful diagnostic tool.

## Development of the analytical method[2]

Column : Acquity Premier HSS T3 1,8 µm 2,1*100 mm

A : H₂O, 5 mM Ammonium acetate, 0,1% formic acid

B : MeOH, 5 mM Ammonium acetate, 0,1% formic acid

Flow rate : 0.3 mL/min

T<sub>oven</sub> : 40°C

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>Acquity UPLC System (Waters) - Exploris 240 (ThermoScientific)</figcaption></figure>

### Source parameters

Positive ion = 3500 V

Sheath gas = 50

Auxiliary gas = 10

Sweep gas = 1

T<sub>Ion transfert tube</sub> = 325°C

T<sub>Vaporizer</sub> = 350°C

### Scan parameters : DDA acquisition

<figure><img src="image_11.png" /></figure>

* Carnitine and 30 acylcarnitines separated in 19 min

* Analyse of short and long acylcarnitines: from 2C to 18C, saturated, insaturated, hydroxylated and dicarboxylated

* 3 internal standards : Carnitine-d9, car C2:0-d3, car C18:1-d3

<figure><img src="image_12.png" /></figure>

## Samples preparation

**Extraction ①**
20 µL plasma
+ 20 µL ISTD
+ 100 µL cold MeOH

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

Supernatant evaporated and reconstituted in 20 µL MeOH

<figure><img src="image_15.png" /></figure>

Injection 1 µL

%

<figure><img src="image_16.png" /></figure>

**Extraction ② [3]**
20 µL plasma
+ 20 µL ISTD
+ 50 µL H₂O
+ 400 µL EtOH
+ 400 µL hexane

<figure><img src="image_17.png" /></figure>

② +
400 µL
hexane

<figure><img src="image_18.png" /></figure>

② evaporated and reconstituted in 20 µL MeOH

<figure><img src="image_19.png" /></figure>

→ Plasmatic composition : relative abundance = $\frac{\text{Compound area}}{\text{ISTD area}}$

<figure><img src="image_20.png" /><figcaption>Relative abundance of acylcarnitine in plasma</figcaption></figure>

## Conclusion and perspectives

* The method developed allows a good separation of the acylcarnitine family. → LOD, LOQ, linearity still need to be determined to validate the method.

* Extraction ② seems to offer better results than extraction ①. → Extraction steps could be simplified to reduce time extraction.

* This new profiling will be offered as a service on MetaToul. It will also be use to follow the 13C-labelled acylcarnitines in the context of fluxomic experiments. It will be an important complement to the profile of labelled fatty acids already proposed.

## References

[1] Wanders RJA, Ruiter JPN, Ijlst L, Waterham HR, Houten SM. The enzymology of mitochondrial fatty acid beta-oxidation and its application to follow-up analysis of positive neonatal screening results. J Inherit Metab Dis. Oct 2010;33(5):479-494

[2] C. Roy et al./J. Chromatogr. B 1027 (2016) 40-49

[3] Détermination du profil des acylcarnitines plasmatiques par spectrométrie de masse en tandem : développement de la technique et intérêt pour le diagnostic de maladies héréditaires du métabolisme. Sciences pharmaceutiques. 2011, ddumas-00640975

## Acknowledgments

MetaboHUB (MetaboHUB-ANR-11-INBS-0010)

Poster presented at RFMF 2023 Perpignan, France

<figure><img src="image_21.png" /></figure>

Inserm

<figure><img src="image_22.png" /></figure>

UNIVERSITÉ
TOULOUSE III
PAUL SABATIER

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

i2MC
Instituts Maladies Métaboliques et Cardiovasculaires