Dec 15, 2023

Public workspaceTargeted analysis of Short Chain Fatty Acids (SCFAs) in human serum using derivatization and LC-HRMS analysis

DOI
dx.doi.org/10.17504/protocols.io.e6nvwddj7lmk/v1
  • 1KU Leuven;
  • 2University of Edinburgh;
  • 3Centre for Cardiovascular Sciences, University of Edinburgh
Margaux Billen
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwddj7lmk/v1
Protocol Citation: Margaux Billen, Scott G Denham, Joanna Simpson, Natalie ZM Homer 2023. Targeted analysis of Short Chain Fatty Acids (SCFAs) in human serum using derivatization and LC-HRMS analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwddj7lmk/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: August 16, 2023
Last Modified: December 15, 2023
Protocol Integer ID: 86538
Keywords: Short Chain Fatty Acids, High Resolution Mass Spectrometry, SCFA profile, LC-MS method, SCFA derivatisation, ThermoFisher Exploris 240 Orbitrap, Human serum
Funders Acknowledgements:
Fonds Wetenschappelijk Onderzoek (FWO) Vlaanderen
Grant ID: V424223N
Biotechnology and Biological Sciences Research Council
Grant ID: BB/V019066/1
Abstract
This protocol describes the sample preparation and analysis of short chain fatty acids in human serum samples. Short chain fatty acids (SCFAs) - acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and 2-isobutoxyacetic acid (internal standard) were derivitised using 3-nitrophenylhydrazine (3-NPH). The derivatives were analysed by liquid chromatography high-resolution mass spectrometry (LC-HRMS).

A targeted LC-MS method was developed to measure six different SCFAs, by utilising approaches adapted from different published methods. These included Dei Cas et al, 2020 who profiled short and medium chain fatty acids following derivatisation (Dei Cas et al, 2020), Liao et al, 2021 who profiled kynurenine metabolites, short chain fatty acids and bile acids in samples following NPH derivatisation, and Nagatomo et al, 2022 who developed a method for application to plasma and tissue from a mouse model to profile SCFAs.

Short chain fatty acids were extracted from human serum using protein precipitation. Sample extracts were then derivatised alongside a calibration curve. Analysis of the derivatised samples was carried out by liquid chromatography high resolution mass spectrometry (LC-HRMS) in full scan negative mode on a ThermoScientific Exploris 240 Orbitrap. The amount of each analyte in each sample was calculated using linear regression of the peak area ratio of the analytes to the internal standard.

Our developed method used only 50 μL of serum with limits of detection of acetic acid at 1 ug/mL, 200 ng/mL for propionic acid and butyric, 20 ng/mL for isobutyric acid, isovaleric acid and valeric acid. Due to the low mass of the analytes in this method we used high resolution mass spectrometry in full scan mode as an alternative to triple quadrupole mass spectrometry which utilises fragmentation of analytes and multiple reaction monitoring. Sensitivity obtained using this HRMS method was found to be comparable to published literature.


Analytes

Short chain fatty acids (SCFAs) analysed using this protocol.
Internal standard

The internal standard used in this project is the SCFA-analogue 2-isobutoxyacetic acid.
Analyte information

NameAbbreviationChemical FormulaMonoisotopic mass (g/mol)NPH derivatised chemical formulaMonoisotopic Mass - NPH (g/mol)
Acetic acidAAC2H4O260.05C8H9N3O3194.06
Propionic acidPAC3H6O274.08C9H11N3O3208.07
Butyric acidBAC4H8O288.11C10H13N3O3222.09
Isobutyric acidisoBAC4H8O288.11C10H13N3O3222.09
Valeric acidVAC5H10O2102.13C11H15N3O3236.10
Isovaleric acidisoVAC5H10O2102.13C11H15N3O3236.10
2-isobutoxyacetic acid/C6H12O3132.08C12H17N3O4267.12


Derivatisation reaction

Hydrazide coupling used for SCFA derivatisation. 50mM of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) + 7% pyridine and 50mM of 3-nitrophenylhydrazine (3-NPH) in acetonitrile:H2O (1:1) are added to 50 µL of human serum.
Derivatisation protocol overview
Workflow for sample preparation (50 µL serum), derivatisation and analysis of short chain fatty acids in human serum samples.
Guidelines
Ensure all training is up-to-date for operating the necessary lab equipment.

Materials
Consumables

ItemSupplierPart no.Quantity
1.75 mL glass vials with lids Scientific Laboratory Supplies Ltd TUB1200 10
7 mL glass vials with lids Scientific Laboratory Supplies Ltd TUB1220 5
28 mL tall form glass vials with lids VWR T008/04 2
TruView Total Recovery 2mL glass vials with screw capWaters 186005663CV 54
1.5 mL Microcentrifuge SafeLock TubesEppendorfSTK-TUBE-035 54
Kinetex 2.6 um C18 50 x 2.1mm Phenomenex 00B-4462-AN 1
Chemicals
ItemSupplierArticle no.
Water (HPLC grade) Fisher ScientificC-10449380-X
Acetonitrile (LC-MS grade) VWR 83640.320
Methanol (LC-MS grade) VWR 83638.320
Water (LC-MS grade) VWR 83645.320
Formic acid (LC-MS grade) Fisher Scientific10596814
2-Propanol (LC-MS grade) VWR 84881.320
Acetic acid (5 mL)Sigma Aldrich71251-5ML-F
Propionic acid (1 mL)Sigma Aldrich94425-1ML-F
Butyric acid (5 mL)Sigma Aldrich19215-5ML
Isobutyric acid (500 mg)Sigma Aldrich46935-U
Valeric acid (1 mL)Sigma Aldrich75054-1ML
Isovaleric acid (1 mL)Sigma Aldrich78654-1ML
2-isobutoxyacetic acidSigma AldrichCDS014100-250MG
Pyridine (LC-MS grade)Fisher Scientific3951366
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimideSigma Aldrich341006
3-Nitrophenylhydrazine hydrochloride (5 g) Sigma AldrichN21804-5G
Equipment
ItemModelSupplier
Dri-block DB.3A Techne
Microtube centrifuge 1-15 Sigma
Liquid Chromatography Pump Vanquish uHPLC Thermo
Autosampler Vanquish uHPLC Thermo
Column oven Vanquish uHPLC Thermo
Mass spectrometer Exploris Orbitrap 240 Sciex
Balance PS-100 Fisher Scientific
Safety warnings
Adhere to local lab rules.
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is a highly toxic reagent, handle with care and follow all necessary safety rules.
Ethics statement
When handling human clinical samples, ensure you are following local guidelines including adherence to Good Clinical Practice. In particular, ensure that samples are analysed without identifiable patient data.

Solvent preparation
Solvent preparation
15m
15m
Prepare Mobile Phase A: water + 0.1% Formic Acid
  • Add Amount1 L of LC-MS grade water to a 1L glass bottle.
  • Add Amount1 mL of LC-MS grade Formic Acid to the water.
  • Mix thoroughly.
Prepare Mobile Phase B: Acetonitrile
  • Add Amount1 L of LC-MS grade Acetonitrile to a 1L glass bottle.
Prepare Autosampler Seal Wash: 10% Acetonitrile
  • Add Amount100 mL LC-MS grade Acetonitrile to Amount900 mL LC-MS grade water in a 1L glass bottle.
  • Mix thoroughly.
Derivatisation solution preparation
Derivatisation solution preparation
20m
20m
Prepare all solution fresh each time the protocol is carried out.
All solutions (for derivatisation, internal standards and calibration standards) are stored at -20°C after preparation until use.
Prepare derivatisation solution A: Acetonitrile:Water (50:50)
  • Add 10 mL of LC-MS grade water to a 28 mL glass vial labelled Acetonitrile:Water (50:50).
  • Add 10 mL of LC-MS grade Acetonitrile.
  • Mix thoroughly.
Prepare derivatisation solution B: Acetonitrile:Water (50:50) + 7% pyridine
  • Add 8.6 mL of LC-MS grade water to a 28 mL glass vial labelled Acetonitrile:Water (50:50) + 7% pyidine.
  • Add 1.4 mL of LC-MS grade pyridine to give a 14% pyridine in water solution.
  • Dilute with 10 mL of LC-MS grade Acetonitrile to give a Acetonitrile:Water (50:50) + 7% pyridine solution.
  • Mix thoroughly.
Prepare derivatisation solution C: 50 mM 1-Ethyl-3(3dimethyl-aminopropyl)carbodiimide (EDC) + 7% pyridine.
  • Using the PS-100 balance, an appropriate amount of EDC was weighed into a 7 mL glass vial. EDC is toxic and highly corrosive and so this was added to the 7 mL vial in the fume hood and transferred to the balance with the lid on the vial.
  • To this the correct amount of acetonitrile:water (50:50) + 7% pyridine was added to achieve a 50 mM EDC solution in 50:50 acetonitrile:water + 7% pyridine. EDC has a molecular weight (MW) of 155.24 g/mol and so 7.72 mg is used per mL.
Prepare derivatisation solution D: 50 mM 3-NPH (3-nitrophenylhydrazine)
  • Using the PS-100 balance, an appropriate amount of 3-NPH was weighed into a 7 mL glass vial.
  • To this the correct amount of acetonitrile:water (50:50) was added to achieve a 50 mM 3-NPH solution in 50:50 acetonitrile:water. 3-NPH has a MW of 189.60 g/mol and so 9.45 mg is used per mL.
Prepare quenching solution: water + 5% Formic Acid
  • Add Amount475 L of LC-MS grade water to a 500 mL glass bottle.
  • Add Amount25 mL of LC-MS grade Formic Acid to the water.
  • Mix thoroughly.
Internal standard preparation
Internal standard preparation
20m
20m
Prepare Internal Standard stock solution:

2-isobutoxyacetic acid stock solution (1 mg/mL)
Add 500 µL of water (HPLC grade) to a manufacturer's vial of 0.5 mg 2-isobutoxyacetic acid and vortex thoroughly to give a 1 mg/mL 2-isobutoxyacetic acid stock solution.
Prepare Internal Standard mix dilution according to the table below:
  • 1 x 28 mL glass vials labelled ''2.5 µg/mL 2-isobutoxyacetic acid (IS) in water''.

Stock concentrationAmount of stockVolume of water (µL)Final volume (µL)
2.5 µg/mL 25 µL x 1 mg/mL of 2-isobutoxyacetic acid stock solution 997510000

Calibration standards mix preparation
Calibration standards mix preparation
30m
30m
Prepare calibration standard stock solutions.

Label vials as follows:
  • 6 x 7 mL glass vials labelled 1 mg/mL acetic acid (AA), propionic acid (PA), butyric acid (BA), isobutyric acid (isoBA), valeric acid (VA), isovaleric acid (isoVA)
  • 5 x 1.75mL glass vials labelled: 100 µg/mL, 50 µg/mL, 5 µg/mL, 500 ng/mL and 50 ng/mL.
SCFA stock solutions (1 mg/mL)

The following standards were prepared as 1 mg/mL solutions in methanol. 5 µL of each standard was added to a glass 7 mL vial. As all of the standards are liquids, the density was used to account for the volume of methanol added to each standard and is shown below.

Standard nameMWChemical FormulaChemical formula-NPHDensity (g/ml)Mass of std (mg) in 5 µLVol MeOH (µL)
Acetic acid 60.05 C2H4O2 C8H9N3O3 1.05 5.25 5250
Propionic acid 74.08 C3H6O2 C9H11N3O3 0.99 4.95 4950
Butyric acid 88.11 C4H8O2 C10H13N3O3 0.96 4.80 4800
Iso-butyric acid 88.11 C4H8O2 C10H13N3O3 0.95 4.75 4750
Valeric acid 102.13 C5H10O2 C11H15N3O3 0.94 4.70 4700
Iso-valeric acid 102.13 C5H10O2 C11H15N3O3 0.93 4.65 4650

Prepare Calibration Standard mix dilutions according to the table:
Derivatisation procedure with NPH
Derivatisation procedure with NPH
2h
2h
Prepare an electronic list of the samples to be analysed using this method. The sample list needs to include a unique ID, as well as recording all relevant experimental details.
Complete a 54-vial (Thermo Vial Rack) map for standards and samples (make sure to place them column-wise) using the design as shown. The number of samples that can be analysed per batch is 36, alongside a 15-point calibration curve.

Defrost calibration standard solutions, internal standard solutions and human serum samples.
Label 54 Eppendorf Microcentrifuge SafeLock Tubes (1.5 mL) according to the plate map.
Add water (LC-MS grade) to the Eppendorfs according to this guide:

Add the required amount of standards to the Eppendorfs according to the table below. Due to small volumes being pipetted ensure that the standard is pipetted into the water.
Add Amount50 µL of each serum sample into the appropriately labelled Eppendorfs.
Add Amount100 µL of ice-cold isopropanol and Amount50 µL of ice-cold methanol to all Eppendorfs, except for the solvent blank (A1). Vortex each Eppendorf tube.

Centrifuge the samples for 20 minutes at 14 000 RPM and 4°C.
CAREFULLY transfer Amount150 µL of the supernatant, without disturbing the pellet, of all Eppendorf tubes to TruView Total Recovery 2mL glass vials.

Add Amount10 µL of 2.5 µg/mL 2-isobutoxyacetic acid (IS) to each vial, except for the solvent blank (A1) and the reagent blanks (B1 and F3).

Place all vials on ice.
Add Amount50 µL of derivatisation solution C (50 mM EDC + 7% pyridine in 50:50 ACN:H2O) to each vial, except for the solvent blank (A1).

Add Amount50 µL of derivatisation solution D (50 mM 3-NPH in 50:50 ACN:H2O) to each vial, except for the solvent blank (A1).

Screw solid caps on all samples and vortex the solutions.
Place all vials in a heating block (Techne dri-block) at 40°C for Duration00:20:00 .

After 20 minutes, put all vials back on ice.
Add Amount250 µL of the quenching solution (water + 5% Formic Acid) to each vial, except for the solvent blank (A1).

Vortex all samples, exchange the solid caps for caps with a septum.
AddAmount500 µL of LC-MS grade water to the solvent blank. Samples are ready for LC-MS analysis.


Set up of SCFA LC-HRMS method and analysis
Set up of SCFA LC-HRMS method and analysis
30m
30m
Put the freshly prepared mobile phases onto the uHPLC system. Purge lines with mobile phase A and mobile phase B.
Install a Kinetex 2.6 µm C18 (50 x 2.1 mm) column into the column oven and set the column temperature to 50 °C. Equilibrate at 90% mobile phase A, 0.4 mL/min for at least 15 minutes. Ensure that the pressure is stable and there are no leaks detectable on the system.
Create an acquisition method in Xcalibur for chromatography and mass spectrometry settings. For chromatography include the following chromatographic gradient conditions in table below.
Add the detail of the column and mobile phases in the method. Make sure the right column position is selected for the valves and the column oven temperature and column pre-heater are set to 50 °C.

Time (min)Flow (mL/min)%A%BCurve
Initial 0.400 90 10 Initial
2.50 0.400 90 10 5
10.0 0.400 70 30 5
10.5 0.400 0 100 5
12.5 0.400 0 100 5
13.0 0.400 90 10 5
15.0 0.400 90 10 5
Chromatographic gradient for separation of derivatized SCFAs in serum samples on a Kinetex 2.6 µm C18 50 x 2.1mm using a system of water + 0.1% Formic Acid (Mobile Phase A) and Acetonitrile (Mobile Phase B).

Add the following mass spectrometry method parameters to the acquisition method:
AB
Instrument Thermo Exploris 240 Orbitrap
Source, Ionisation Mode Thermo Scientific™ OptaMax™ NG ion source (H-ESI)
Scan Mode, Polarity Full Scan, Negative
Mass range 50-500 m/z
Resolution 120 000
Acquisition time 15.0 min
Sheath Gas 30
Aux Gas 5
Sweep Gas 1
IonSpray Voltage (IS) (Negative) -2500 V
Ion Transfer Tube Temperature 300°C
Vaporizer Temperature 450°C
Probe position (x – axis) 2
Probe position (y – axis) 2

Place the Thermovial 54 rack with the samples into the autosampler of the chromatography system.
Create a batch in Xcalibur using the electronic plate map - use the correct position for the Thermovial 54 rack, the correct position of the column, the correct lines for the mobile phases and the correct LC-MS/MS method. Name and save the Batch acquisition file. Use the same naming convention to name the resulting data file.
Set volume of injection to 5 µL and submit batch to analyse.
Test the system with a mid-standard curve point injection and then complete the batch in order from A1 to F9.
Use the deprotonated molecular mass m/z [M-H]- for each compound in the table below to interrogate the data.

AAcetate NPHPropionate-NPHButyrate-NPHIsobutyrate-NPHValerate-NPHIsovalerate-NPH
m/z [M-H]-194.0571208.0728222.0883222.0883236.1039236.1039
Retention time (min)1.182.305.074.577.927.45



Typical chromatography of NPH-derivatised acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and 2-isobutoxyacetic acid separation is shown below. Separation performed on a Kinetex 2.6 µm C18 50 x 2.1mm using a system of water + 0.1% Formic Acid (Mobile Phase A) and Acetonitrile (Mobile Phase B).

Chromatographic Separation of Acetic acid-NPH (1.18 mins), Propionic acid-NPH (2.30 mins), Butyric acid-NPH (5.07 mins), Isobutyric acid-NPH (4.57 mins), Valeric acid-NPH (7.92 mins), Isovaleric acid-NPH (7.45 mins) and 2-isobutoxyacetic acid-NPH (10.07 mins) on a Kinetex 2.6 µm C18 50 x 2.1mm using a system of water + 0.1% Formic Acid (Mobile Phase A) and Acetonitrile (Mobile Phase B). Flow rate 0.4 mL/min, 50 °C and a gradient elution over 15 minutes.
Data analysis using TraceFinder software
Data analysis using TraceFinder software
2h
2h
Use this protocol to evaluate the data and obtain the SCFA profile of the samples analysed:
Margaux Billen, Scott G Denham, Joanna P Simpson, Natalie ZM Homer 2023. Using TraceFinder and Excel software to evaluate and report multi-analyte targeted LC-MS data acquired on an ThermoScientific Exploris 240 Orbitrap. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldm8z7l5b/v1
Protocol references
Liao, H.-Y.; Wang, C.-Y.; Lee, C.-H.; Kao, H.-L.; Wu, W.-K.; Kuo, C.-H., Development of an Efficient and Sensitive Chemical Derivatization-Based LC–MS/MS Method for Quantifying Gut Microbiota-Derived Metabolites in Human Plasma and Its Application in Studying Cardiovascular Disease. Journal of Proteome Research 2021, 20 (7), 3508-3518. https://doi.org/10.1021/acs.jproteome.1c00147

Nagatomo, R.; Kaneko, H.; Kamatsuki, S.; Ichimura-Shimizu, M.; Ishimaru, N.; Tsuneyama, K.; Inoue, K., Short-chain fatty acids profiling in biological samples from a mouse model of Sjögren’s syndrome based on derivatized LC-MS/MS assay. Journal of Chromatography B 2022, 1210, 123432. https://doi.org/10.1016/j.jchromb.2022.123432

Dei Cas, M.; Paroni, R.; Saccardo, A.; Casagni, E.; Arnoldi, S.; Gambaro, V.; Saresella, M.; Mario, C.; La Rosa, F.; Marventano, I.; Piancone, F.; Roda, G., A straightforward LC-MS/MS analysis to study serum profile of short and medium chain fatty acids. Journal of Chromatography B 2020, 1154, 121982. https://doi.org/10.1016/j.jchromb.2020.121982

Margaux Billen, Scott G Denham, Joanna P Simpson, Natalie ZM Homer 2023. Using TraceFinder and Excel software to evaluate and report multi-analyte targeted LC-MS data acquired on an ThermoScientific Exploris 240 Orbitrap. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldm8z7l5b/v1