# Selective Isolation of Lipid Classes from cold-water corals via SPE for Quantitative Fatty Acid Profiling

Audrey M. Pruski, Gilles Vétion

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## To cite this version:

Audrey M. Pruski, Gilles Vétion. Selective Isolation of Lipid Classes from cold-water corals via SPE for Quantitative Fatty Acid Profiling. 2025, 10.17504/protocols.io.5qpvodx37g4o/v1. hal-05214372

HAL Id: hal-05214372

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Distributed under a Creative Commons Attribution 4.0 International License
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# Selective Isolation of Lipid Classes from cold-water corals via SPE for Quantitative Fatty Acid Profiling

DOI

dx.doi.org/10.17504/protocols.io.5qpvodx37g4o/v1

Audrey M. Pruski¹, Gilles Vétion¹

¹Laboratoire d'Ecogéochimie des environnements benthiques, Sorbonne Université

Biomarker group

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Audrey M. PRUSKI

Sorbonne University

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OPEN

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ACCESS

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DOI: dx.doi.org/10.17504/protocols.io.5qpvodx37g4o/v1

**Protocol Citation:** Audrey M. Pruski, Gilles Vétion 2025. Selective Isolation of Lipid Classes from cold-water corals via SPE for Quantitative Fatty Acid Profiling. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvodx37g4o/v1

**Manuscript citation:**

*In situ* diet patterns and health status of cold-water corals in the Lacaze-Duthiers canyon (NW Mediterranean Sea): insights from fatty acid biomarkers on lipid classes. Audrey M. Pruski, Gilles Vétion, Franck Lartaud, Erwan Peru, and Nadine Le Bris. Deep Sea Research Part I, accepted with minor modifications

**License:** This is an open access protocol distributed under the terms of the **Creative Commons Attribution License**, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

**Protocol status:** Working
We use this protocol and it's working

**Created:** June 30, 2025

**Last Modified:** August 18, 2025

**Protocol Integer ID:** 221340
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**Keywords:** Lipids, Fatty acids, Solid Phase Extraction, Corals, extraction of total lipid, water corals via spe, coral polyp sample, selective isolation of lipid class, separation of lipid class, quantitative fatty acid profiling, quantitative fatty acid profiling this protocol, fatty acid methyl ester, water coral, total lipid, lipid class, extraction, solid phase extraction, step acidic transesterification

## Abstract

This protocol describes the extraction of total lipids from coral polyp samples using the method of Bligh and Dyer (1959), followed by the separation of lipid classes via solid phase extraction (SPE) on aminopropyl (NH₂) silica cartridges as described by Kaluzny et al. (1985). A one-step acidic transesterification, adapted from Indarti et al. (2005) and Christie (2003), is then applied to produce fatty acid methyl esters (FAMEs) for GC-MS analysis.

<figure><img src="image_9.png" /><figcaption>Protocol flowchart</figcaption></figure>

## Image Attribution

Pruski Audrey M.

## Guidelines

**Sample Handling:** Handle sample quickly after collection to prevent lipolysis

**Equipment:** Ensure all glassware, tubes, and Pasteur pipettes are pre-burnt (ignition 450 °C) to avoid contamination

**Chemicals and solvents:** Use GC-MS or HPLC reagent grade products

**Internal Standard:** Nonadecanoic acid (C19:0) is used to correct for extraction losses during transesterification

**Temperature Control:** Maintain precise heating and cooling conditions to ensure consistent results

**Storage:** Keep eluates and FAME extracts at (-20 °C) if not analyzed immediately
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# Materials

## Equipments:

- Freeze dryer (Christ Alpha 1-4)

- TissueLyser II (QIAGEN)

- Solid phase extraction manifold (Phenomenex®)

- Hand-operated vacuum pump

- Rotary shaker (Bioblock)

- Ultracentrifuge (Sigma 6K-15)

- Speed-Vac concentrator (Thermo Scientific™ Savant™)

- GC-MS system (Agilent)

## Materials:

- Strata NH₂ cartridges (Phenomenex®)

- 20 and 30 ml Borosilicate glass tubes round bottom with PBT red screw cap & PTFE liner

- 20 ml Borosilicate glass tubes conical bottom with PBT red screw cap & PTFE liner

- 2ml GC vials with 200μl insert

- Polar capillary column (e.g. VF-Wax-MS or ZB-WAX-MS)

## Chemicals and reagents (HPLC or GC grade):

- Acetic acid (CH₃CO₂H)

- Butylated hydroxytoluene (BHT)

- Chloroform (CH₃Cl)

- Dichloromethane (CH₂Cl₂)

- Diethyl ether ((C₂H₅)₂O)

- Ethyl acetate (C₄H₈O₂)

- Hexane (C₆H₁₄)

- Hydrochloric acid (HCl)

- Methanol (CH₃OH)

- Nonadecanoic acid (C19:0= internal standard) (C₁₉H₃₈O₂)

- Phosphoric acid (H₃PO₄)

- Potassium bicarbonate (KHCO₃)

- 2-Propanol (C₃H₈O)

- Sodium chloride (NaCl)

- Sodium hydroxide (NaOH)

- Sulphuric acid (H₂SO₄)

- Water (ultrapure)
- Commercial FAME standards (Qualmix Fish Synthetic, Ladoran Fine Chemicals, INTERCHIM, France; Supelco 37, PUFA No. 1 and No. 3, SUPELCO France) for GC-MS calibration

**Note: use GC/HPLC grade reagents**

**Biological samples:**

- Coral nubbins

**Safety warnings**

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**Safety information**

- Always wear safety goggles and gloves

- Work in a fume hood when handling solvents

- Dispose of waste in appropriate containers
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# Lipid Extraction from the Biological Matrix (Cold-water coral nubbins)

1d 9h 6m

## 1 Sample Preparation

1.1 Flash-freeze 🧭 coral nubbins on board to prevent degradation

10m

1.2 Freeze-dry samples and store at 🧭 -80 °C until analysis

1d

1.3 Grind freeze-dried 🧭 coral nubbins into a fine powder using a bead mill homogenizer while keeping the chamber filled with liquid nitrogen to prevent heating

10m

## 2 Preparation of Working Solutions

4h

2.1 Chloroform/methanol/water (1:2:0.8) reagent: 5.265 mL CHCl₃, 10.525 mL CH₃OH and 4.21 mL H₂O. Keep at 🧭 4 °C in a flammable storage refrigerator

2.2 1M NaCl solution in ultrapure water (58g.L⁻¹). Keep at 🧭 4 °C in a refrigerator

2.3 0.3N NaOH solution (12 g.L⁻¹). Keep at 🧭 4 °C in a refrigerator

2.4 0.2M H₃PO₄ solution (19.6g.L⁻¹). Keep at 🧭 4 °C in a refrigerator

2.5 BHT (butylated hydroxytoluene) at 50 mg.mL⁻¹ in Chloroform. Keep at 🧭 4 °C in a flammable storage refrigerator

## 3 Total lipid extraction

4h
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Note

Total lipids are extracted using a modified version of the Bligh and Dyer method (1959), optimized to improve extraction efficiency.

3.1 Place the freeze-dried coral nubbins (~ 3 g) in a 30 mL glass tube (round bottom, screw tap with PTFE liner)

3.2 Add 20 mL of CHCl₃/CH₃OH/H₂O (1:2:0.8) with 1M NaCl + 0.2M H₃PO₄

Note

The solution of NaCl 1M-H₃PO₄ 0.2M increases protein unwinding and lipid recovery

3.3 Sonicate ( 00:05:00 On ice if needed ), then rotate ( 00:20:00 at Room temperature ) 25m

3.4 Add 2.5 mL CHCl₃, then 2.5 mL H₂O. Shake 00:05:00 5m

3.5 2500 rpm, 4°C, 00:05:00 . Recover the organic phase (bottom) using a Pasteur pipette 5m

3.6 Repeat extraction 3 times with 5 mL CHCl₃

3.7 Pool all organic phases (final volume ≈ 22.765 mL) and vortex. ▲ Keep organic phase cold On ice

3.8 Wash organic phase with 2.5 mL cold 0.3N NaOH. Shake 00:01:00 1m

3.9 2500 rpm, 4°C, 00:05:00 and recover the organic phase 5m
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3.10 Take Ⓔ 20 mL or more into a new glass tube (conical bottom, screw tap with PTFE liner) and evaporate at Ⓒ 40 °C under vacuum (rotary evaporator)

Note

⚠️ Lipid extracts should never be allowed to dry out!

3.11 Cool at Ⓒ -20 °C and redissolve in Ⓒ 400 µL CHCl₃ + BHT (50 mg.L⁻¹)

Note

BHT is added optionally to chloroform to prevent lipid oxidation during extraction!

3.12 Centrifuge Ⓒ 2000 rpm, 4°C, 00:05:00

5m

Store at Ⓒ -20 °C

## Lipid Class Separation by Solid Phase Extraction (SPE)

1d

# 4 Solvent Preparation

4.1 Solvent A: Chloroform:2-propanol (2:1 v/v)

Solvent B: 2% Acetic acid in diethyl ether

Solvent C: Methanol

Solvent D: Hexane

Solvent E: 1% Diethyl ether + 10% Dichloromethane in hexane

Solvent F: 5% Ethyl acetate in hexane

Solvent G: 15% Ethyl acetate in hexane

Solvent H: Chloroform:Methanol (2:1 v/v)
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**Note**

Keep all solvents at ⌒ 4 °C in a flammable storage refrigerator!

5 **Separation of neutral lipids, fatty acids and phospholipids**

1d

**Note**

3 Strata NH₂ (aminopropyl silica) cartridges (Phenomenex) are needed per sample.
7 lipid fractions are efficiently recovered from the initial chloroform extract with minimal
impurities.

5.1 Condition the first cartridge (C1) with 2 × ⌧ 2 mL hexane, vacuum dry 2–5 min

5.2 Load ⌧ 390 µL lipid extract. Vacuum to dryness

5.3 Elute neutral lipids (E1): 4 × ⌧ 2 mL solvent A

5.4 Elute free fatty acids (E2): 4 × ⌧ 2 mL solvent B

5.5 Elute phospholipids (E3): 4 × ⌧ 2 mL solvent C

5.6 Store eluates at ⌒ -20 °C.

6 Neutral Lipid Fractionation

**Note**

In the original method by Kaluzny et al. (1985), the elution of steryl esters was
demonstrated using commercially available lipid standards, which did not include wax
esters. However, based on our experimental results, wax esters appear to co-elute with
steryl esters under the same solvent conditions used for fraction E4.
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6.1 Condition a second cartridge (C2) as above with hexane

6.2 Load ⬇ 390 µL from E1. No need to dry

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6.3 Elute wax esters and sterol esters (E4): 4 × ⬇ 2 mL solvent D

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Note

Note: Mono-, di-, and triglycerides and sterols are retained on C2.

6.4 Condition a third cartridge (C3) as above

6.5 Stack C2 onto C3 using an adapter

6.6 Elute triglycerides (E5): 6 × ⬇ 2 mL solvent E

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6.7 Elute sterols (E6): 9 × ⬇ 2 mL solvent F

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6.8 Remove and discard C3, place C2 back on manifold

6.9 Elute diglycerides (E7): 4 × ⬇ 2 mL solvent G

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6.10 Elute monoglycerides (E8): 4 × ⬇ 2 mL solvent H

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7 Evaporation of lipid fractions

7.1 Evaporate under vacuum all eluates ( ⬇ 45 °C )

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7.2 Redissolve in 100 µL CHCl₃ + BHT. Store at -20 °C

Note

Eluates for GC-MS analysis of fatty acids are E2 (Free Fatty Acids), E3 (Phospholipids), E4 (Wax esters and Sterol esters), E5 (Triglycerides), E7 (Diglycerides), and E8 (Monoglycerides). E6 may be used for sterol analysis!

# One-Step Acidic Transesterification

1d

8 Lipid extraction and Transesterification

8.1 Preparation of Working Solutions

- Transmethylation reagent: 3.4 mL Methanol + 0.6 mL H₂SO₄ (98%), and 4.0 mL CHCl₃ with BHT (50 mg/L). Keep at 4 °C in a flammable storage refrigerator

Note

Reagent should be cold and freshly prepared

- C19:0 (internal standard) stock solution ~1 mg.mL⁻¹ in hexane. The solution is diluted before use (1/9). Keep at -20 °C

Note

Adjust the concentration of the internal standard so that its peak on the chromatogram falls within the range of peak intensities, ideally between the most abundant and the least abundant fatty acids. This ensures accurate quantification across the entire dynamic range

8.2 To the eluates: add 20 µL internal standard C19:0 + 8 mL of transmethylation reagent. Cap and shake

8.3 Incubate 01:30:00 at 90 °C. Shake occasionally (3-4 times)

1h 30m
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9 **Phase Separation**

9.1 Cool ( ⌶ On ice or ⌶ -20 °C ). Open carefully

9.2 Add ⌶ 2 mL cold H₂O. Vortex. 📶 300 rpm, 4°C, 00:05:00 Centrifuge 5 min, 3000 rpm, 4°C

9.3 Recover lower organic phase in a new glass tube

10 **Organic Phase Wash**

10.1 Re-extract aqueous phase 2 times with ⌶ 2 mL cold hexane:CHCl₃ (4:1)

10.2 Combine all organic phases (~8-9 mL)

11 **Final Purification**

11.1 Wash with ⌶ 4 mL cold 2% K₂CO₃. 📶 3000 rpm, 4°C, 00:05:00

11.2 Discard the upper polar layer and transfer ⌶ 6-9 mL of the lower organic phase to a clean screw-cap tube

12 **Drying and Dissolving FAMES**

12.1 Evaporate solvent in a SpeedVac concentrator at ⌶ Room temperature . Cool at
 ⌶ -20 °C

12.2 Redissolve in ⌶ 30-50 µL pure hexane. Vortex, 📶 3000 rpm, 4°C, 00:05:00

10m
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12.3 Transfer to GC vials with a 200 µl insert. Store at °C -80 °C

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12.4 Fatty acid methyl extracts are ready for GC-MS analysis

Note

In this study, the analyses were carried out using the instrumental conditions described in
a companion protocol developed by our group (Pruski & Vétion, 2024).

Protocol references

**Bligh, E.G., Dyer, W.J.,** 1959. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911-917.

**Christie, W.W., 2003.** Lipid analysis, volume 15 : isolation, separation, identification and structural analysis of lipids. Third edition. The Oily Press, Bridgawater, England.

**Indarti, E., Majid, M.I.A., Hashim, R., Chong, A., 2005.** Direct FAME synthesis for rapid total lipid analysis from fish oil and cod liver oil. Journal of Food Composition and Analysis, 18, 161-170.
https://doi.org/10.1016/j.jfca.2003.12.007

**Kaluzny, M. A., Duncan, L. A., Merritt, M. V., & Epps, D. E., 1985.** Rapid separation of lipid classes in high yield and purity using bonded phase columns. Journal of Lipid Research, 26, 135-140

**Pruski, A.M., Vétion, G. 2024.** Comparison of direct transesterification and conventional multi-step methods for fatty acid analysis in marine sediments. protocols.io.
https://dx.doi.org/10.17504/protocols.io.81wgbrz3ylpk/v1

**Simpson, N.J.K. 2000.** Solid-Phase Extraction: Principles, Techniques, and Applications. CRC Press.