Jul 21, 2025
Analysis of sphingosine, sphinganine and glucosylsphingosine from cells, animal tissues and plasma
- Danielle te Vruchte1,
- Yuzhe Weng1,
- Mylene Huebecker1,
- Kerri-Lee Wallom1,
- Frances M Platt1,2,3,4
- 1Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, United Kingdom;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
- 3Royal Society Wolfson Research Merit Award Holder;
- 4Wellcome Trust Investigator in Science
Protocol Citation: Danielle te Vruchte, Yuzhe Weng, Mylene Huebecker, Kerri-Lee Wallom, Frances M Platt 2025. Analysis of sphingosine, sphinganine and glucosylsphingosine from cells, animal tissues and plasma. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6zkz1gqe/v1
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: July 02, 2025
Last Modified: July 21, 2025
Protocol Integer ID: 221572
Keywords: sphingolipid metabolism, glucosylsphingosine from cell, analysis of sphingosine, glucosylsphingosine in cell line, extracted sphingosine, complex sphingolipid, glucosylsphingosine comprise, glucosylsphingosine, labelled c20 sphingosine, complex sphingolipids like ceramide, plasma sphingosine, sphingosine, c20 sphingosine, labelled c20 glucosylsphingosine standard, c20 glucosylsphingosine standard, glucosylceramide, sphinganine, glucose, sphingoid base, phthaldialdehyde, crucial molecule, cell line, cell, molar quantities of these molecule, molecule, fluorescent derivative
Funders Acknowledgements:
The Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Aligning Science Across Parkinson’s (ASAP) Initiative
Grant ID: ASAP-000478
Welcome Investigator Award
Grant ID: 202834/Z/16/Z
Abstract
Sphingosine (SphO), sphinganine (SphA) and glucosylsphingosine (GlcSph) are crucial molecules involved in sphingolipid metabolism. Sphingosine and sphinganine are long-chain bases that serve as building blocks for more complex sphingolipids like ceramides and glucosylceramides (GlcCer). Glucosylsphingosine comprises of a sphingoid base with glucose attached.
Here we describe a method which can measure sphingosine, sphinganine and glucosylsphingosine in cell lines, animal tissue and plasma. The extracted sphingosine, sphinganine and glucosylsphingosine
are reacted with Phthaldialdehyde (OPA) reagent, in the presence of the thiol compound mercaptoethanol, to form a fluorescent derivative which can be measured by reverse phase HPLC. With the inclusion of OPA-labelled C20 sphingosine or OPA-labelled C20 glucosylsphingosine standards, it is possible to obtain molar quantities of these molecules.
Guidelines
This protocol requires the use of some hazardous materials. As such, users must be appropriately trained and hazardous materials stored, used and disposed of in accordance with your institution's health and safety policies, and approved laboratory policies, risk assessments and codes of practice.
Materials
Acetone for HPLC, ≥99.8%
34850-M Sigma-Aldrich
Acetonitrile for HPLC, gradient grade, ≥99.9%
34851 Sigma-Aldrich
Bicinchonic Acid solution
B9643-1L Sigma-Aldrich
Boric acid
194810 MP Biomedicals, LLC
Chloroform Stabilized with Amylene, for HPLC, ≥99.8%
C/4966/17 Fisher Scientific
Chromolith Performance RP-18e 100-4.6 HPLC column (Merck, Darmstadt, Germany)
1.02129.0001 VWR International Ltd.
Copper(II) sulfate solution
C2284 Sigma-Aldrich
Ethanol, ≥99.8%
32221-M Sigma-Aldrich
C18-glucosylsphingosine standard (1 mM in EtOH, store at -80°C)
860535 Glucosyl(ß) Sphingosine (d18:1), Avanti Polar Lipids
C20-glucosylsphingosine standard (1 mM in EtOH, store at -80°C)
860438 Glucosyl (ß) Sphingosine (d20:1), Avanti Polar Lipids
2-Mercaptoethanol, ≥99.0%
63689 Sigma-Aldrich
Methanol for HPLC grade, ≥99.9%
34860 Sigma-Aldrich
OPA (Phthaldialdehyde), ≥99.0% (HPLC)
79760 Sigma-Aldrich
Sodium chloride, AR
S/3160/60 Fisher Scientific
Sodium hydroxide
06203 Sigma-Aldrich
C18-sphinganine standard (1 mM in EtOH, store at -80°C)
860498 Sphinganine (d18:0), Avanti Polar Lipids
C18-sphingosine standard (1 mM in EtOH, store at -80°C)
860490 Sphingosine (d18:1), Avanti Polar Lipids
C20-sphingosine standard (1 mM in EtOH, store at -80°C)
860660 Sphingosine (d20:1), Avanti Polar Lipids
Tris base
sc-3715A Santa Cruz
ISOLUTE® NH2 100 mg/1 mL
#470-0010-A Biotage
Equipment:
Pipettes
Homogeniser
Vortex mixer
Ultrasonic water bath
Centrifuge
Sample concentrator (Heatblock with a nitrogen supply)
RP-HPLC
Safety warnings
This protocol requires the use of some hazardous solvents, reagents and chemicals. Refer to the Safety Data Sheets (SDS) provided by supplier and applicable Control of Substances Harmful to Health (COSHH). The correct personal protective equipment must be worn, and incidents reported in line with your institution's policy and procedures.
Before start
Check that you have the required reagents, solvents, chemicals, equipment and PPE.
Cells homogenisation
Lyse and homogenise cell pellet (≥ 1 x 106 cells) in 200 µL dddH2O through three cycles of freeze-thawing with vortexing after each cycle.
Measure protein concentration using the Bicinchonic acid assay (BCA) method in triplicate (repeat if triplicate values are not consistent).
Using determined protein concentrations, prepare samples as 200 µg protein in 100 µL ddH2O in a 2 ml screw-cap tube on ice.
Animal tissue homogenisation
Homogenise tissue in ddH2O yielding tissue concentration of 25 mg tissue (wet weight) per 1 mL ddH2O.
Measure protein concentration using the Bicinchonic acid assay (BCA) method in triplicate (repeat if triplicate values are not in consistent).
Using determined protein concentrations, prepare samples as 200 µg -500 µg protein in 100 µL ddH2O in a 2 ml screw-cap tube on ice.
Plasma
Use 50 µL plasma and add 50 µL ddH2O in a 2 ml screw-cap tube on ice.
Extraction of Sph and GlcSph from cells, tissue and plasma
35m
Example of the experiment layout:
| Sample/Standard name | ddH2O | Sample | Standard Master Mix | Standard | |
| Sample 1 | 50 μl | 50 μl | 500 μl C20 MM | - | |
| Sample 2 | 50 μl | 50 μl | 500 μl C20 MM | - | |
| Sample 3 | 50 μl | 50 μl | 500 μl C20 MM | - | |
| Sample 4 | 50 μl | 50 μl | 500 μl C20 MM | - | |
| Sample 5, etc. | 50 μl | 50 μl | 500 μl C20 MM | - | |
| Blank | 100 μl | - | 500 μl CHCl3:MeOH (1:2) | - | |
| C18 SphO | 100 μl | - | 500 μl C20 SphO mix | 3 μl 0.1 mM C18 SphO | |
| C18 SphA | 100 μl | - | 500 μl C20 SphO mix | 3 μl 0.1 mM C18 SphA | |
| C20 SphO | 100 μl | - | 500 μl C20 SphO mix | - | |
| C18 GlcSph | 100 μl | - | 500 μl C20 GlcSph mix | 3 μl 0.1 mM C18 GlcSph | |
| C20 GlcSph | 100 μl | - | 500 μl C20 GlcSph mix | - |
See steps 9 and 10 below on how to make up the standards and standard master mixes.
5 standards and a blank are needed:
- 1 mM C18 sphingosine (C18 SphO),
- 1 mM C18 sphinganine (C18 SphA),
- 1 mM C20 sphingosine (C20 Sph),
- 1 mM C18 glucosylsphingosine (C18 GlcSph),
- 1 mM C20 glucosphingosine (C20 GlcSph),
- Blank (with only CHCl3:MeOH (1:2)).
Make 0.1 mM C18 SphO, 0.1 mM C18 SphA and 0.1 mM C18 GlcSph standard solutions:
50 µL stock (1 mM) + 0.5 mL EtOH.
Make the different C20 (master) mixes as described below in Steps 10.1 to 10.3:
For the samples make a C20 master mix (C20 MM) in a 15 ml tube:
6 µL 1 mM C20 Sph and 6 µL 1 mM C20 GlcSph in 10 mL CH3Cl:MeOH (1:2) (need 500 µL MM C20 per sample, scale up if necessary).
For the Sph standards make a C20 SphO mix (C20 SphO mix) in a 15 ml tube:
3 µL 1 mM C20 Sph in 5 mL CHCl3:MeOH (1:2) (need 500 µL MM C20 SphO per Sph standard).
For the GlcSph standards make a C20 GlcSph mix (C20 GlcSph mix) in a 15 ml tube:
3 µL 1 mM C20 GlcSph in 5 mL CHCl3:MeOH (1:2) (need 500 µL MM C20 GlcSph per GlcSph standard).
For the blank and the standards put 100 µL ddH2O in a 2 ml screw-cap tube.
Add 500 µL C20 MM to the sample tubes,
500 µL C20 SphO mix to the C18 SphO, C18 SphA and C20 SphO standard tubes,
500 µL C20 GlcSph mix to the C18 GlcSph and C20 GlcSph standard tubes and just
500 µL pure CHCl3: MeOH (1:2) into the Blank tube.
Add 3 µL of 0.1 mM C18 standards according to the standard tubes labels (see experiment layout).
Sonicate all tubes for 00:10:00 at Room temperature .
10m
Add 500 µL NaCl (1 M).
Add 500 µL CHCl3.
Add 100 µL NaOH (3 M).
Vortex and leave for 00:15:00 at Room temperature (vortex every 5 min).
15m
Centrifuge at 16.000 x g for 00:10:00 at Room temperature .
10m
Transfer the lower, organic phase very carefully to a new 1.5 ml Eppendorf tube (keep!), while leaving the upper phase and protein interphase (which can be discarded).
Purification of Sph and GlcSph
Condition the right number of ISOLUTE ®NH2 100 mg/1 mL columns with 2 x 1 mL CHCl3.
Apply the extracted organic phase to the columns, let drip through gravity flow, discard flow-through.
Elute first sphingosine and sphinganine, then glucosylsphingosine from the same column:
To elute sphingosine and sphinganine: Apply 900 µL acetone to the column and collect the eluent in a new 1.5 ml screw-cap tube. After elution, use syringe to push to empty the column.
To elute glucosylsphingosine: Apply 900 µL acetone:EtOH (6:1) to the column and collect the eluent in a new 1.5 ml screw-cap tube. After elution, use syringe to push to empty the column.
Dry down the solvents under a slow stream of N2 gas.
The samples are now ready for OPA-labelling (or they can be stored at -20°C for OPA-labelling next day).
OPA-labelling of Sph and GlcSph
22m
Solutions:
3% boric acid, pH 10.5:
dissolve 6 g boric acid in 180 mL H2O
adjust pH with 10 M KOH to 10.5
fill up to 200 mL (Filter sterilize).
OPA-labelling solution: weigh 1 mg – 5 mg OPA in 15 ml tube covered with aluminium foil.
Dissolve OPA in EtOH (20 µl/mg OPA) (e.g. 1 mg OPA: add 20 µl EtOH),
add 2-mercaptoethanol (1 µl/mg OPA) (e.g. 1 mg OPA: add 1 µl 2-mercaptoethanol),
dilute 1:2000 with 3% boric acid (e.g. 1 mg OPA: add 2 ml 3% boric acid).
Prepare the OPA-labelling solution just before labelling (light-sensitive, instable).
Resuspend the lipids in 50 µL pre-warmed EtOH (37 °C water bath/heat block), vortex gently.
Add 50 µL fresh OPA-labelling solution to each sample and vortex.
Incubate at Room temperature in the dark for 00:20:00 . Vortex again after 10 min.
20m
Add 100 µL MeOH:5 mM Tris pH 7 (9:1) and vortex.
Centrifuge at 3000 rpm for 00:02:00 at Room temperature .
2m
Transfer 150 µL of the supernatant (carefully pipette from the top) to a HPLC vial with plastic insert.
For RP-HPLC inject 20 µL -50 µL .
RP-HPLC
30m
The RP-HPLC system consists of a VWR Hitachi Elite LaChrom HPLC system with a L-2485 fluorescence detector set at Ex λ340nm and Em λ455nm.
The solid phase used is a Chromolith Performance RP-18e 100-4.6 HPLC column (Merck, Darmstadt, Germany).
The chromatographic flow rate is 2.0 mL/min, and total run time per sample is 00:30:00 .
| Time | 100% MeOH | 100% ddH2O | 100% acetonitrile | 80% ddH2O/20% acetonitrile | |
| 0.0 | 0.0 | 0.0 | 64.0 | 36.0 | |
| 2.0 | 0.0 | 0.0 | 64.0 | 36.0 | |
| 7.0 | 0.0 | 0.0 | 88.0 | 12.0 | |
| 8.0 | 5.0 | 0.0 | 89.0 | 6.0 | |
| 22.0 | 100.0 | 0.0 | 0.0 | 0.0 | |
| 24.0 | 100.0 | 0.0 | 0.0 | 0.0 | |
| 26.0 | 0.0 | 0.0 | 64.0 | 36.0 | |
| 30.0 | 0.0 | 0.0 | 64.0 | 36.0 |
Gradient conditions for reverse phase HPLC. All chromatography was controlled and data were collected and processed using Waters Empower software.
30m
The mobile phases are MeOH, acetonitrile and 80% ddH2O/20% acetonitrile mix.
The gradient conditions for the RP-HPLC are as described in the table above.
Identification of Sph-species: Individual sphingosine species are identified by their retention time in comparison to standards.
Approximate Retention Times:
C18 GlcSph ∼ 3.80 min
C20 GlcSph ∼ 6.06 min
C18 SphO ∼ 6.78 min
C18 SphA ∼ 8.00 min
C20 SphO ∼ 9.18 min
Quantification: Sph species are quantified by comparison of integrated peak areas with a known amount of spiked OPA-labelled C20 sphingosine standard (Avanti Polar Lipids) or OPA-labelled C20 glucosylsphingosine standard (Avanti Polar Lipids) in each sample.
Results should be normalized to protein content or per ml of plasma.