TITLE: Targeted analysis of Short Chain Fatty Acids (SCFAs) in human serum using derivatization and LC-HRMS analysis
AUTHORS: Margaux Billen, Scott G Denham, Joanna P Simpson, Natalie ZM Homer

[DESCRIPTION]
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

 Internal standard

 Analyte information

 


Derivatisation reaction

  Derivatisation protocol overview  

[GUIDELINES]
Ensure all training is up-to-date for operating the necessary lab equipment.


[STEPS]
SECTION: Solvent preparation
1. Prepare Mobile Phase A: water + 0.1% Formic Acid
Add 1 L of LC-MS grade water to a 1L glass bottle.
Add 1 mL of LC-MS grade Formic Acid to the water.
Mix thoroughly.
SECTION: Solvent preparation
2. Prepare Mobile Phase B: Acetonitrile
Add 1 L of LC-MS grade Acetonitrile to a 1L glass bottle.
SECTION: Solvent preparation
3. Prepare Autosampler Seal Wash: 10% Acetonitrile
Add 100 mL LC-MS grade Acetonitrile to 900 mL LC-MS grade water in a 1L glass bottle. 
Mix thoroughly.
SECTION: Internal standard preparation
10. 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.
SECTION: Internal standard preparation
11. 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''.

 

SECTION: Calibration standards mix preparation
12. Prepare calibration standard stock solutions.

SECTION: Calibration standards mix preparation
12.2. 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 name       MW      chemical
  formula      chemical
  formula-NPH      Density      mg of std
  in 5 uL based on density      vol of
  methanol added (uL)        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        Propionic acid      74.08      C3H6O2      C9H11N3O3      0.99      4.95      4950        Acetic acid      60.05      C2H4O2      C8H9N3O3      1.05      5.25      5250   

SECTION: Calibration standards mix preparation
12.3.  Prepare Calibration Standard mix dilutions according to the table:
   
SECTION: Calibration standards mix preparation
12.1. 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.
SECTION: Derivatisation procedure with NPH
13. 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.
SECTION: Derivatisation procedure with NPH
15. Defrost calibration standard solutions, internal standard solutions and human serum samples.
SECTION: Derivatisation procedure with NPH
16. Label 54 Eppendorf Microcentrifuge SafeLock Tubes (1.5 mL) according to the plate map.
SECTION: Derivatisation procedure with NPH
17. Add water (LC-MS grade) to the Eppendorfs according to this guide:

 
SECTION: Derivatisation procedure with NPH
18. 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.
 
SECTION: Derivatisation procedure with NPH
19. Add 50 µL of each serum sample into the appropriately labelled Eppendorfs.
SECTION: Derivatisation procedure with NPH
20. Add 100 µL of ice-cold isopropanol and 50 µL of ice-cold methanol to all Eppendorfs, except for the solvent blank (A1). Vortex each Eppendorf tube.

SECTION: Derivatisation procedure with NPH
21. Centrifuge the samples for 20 minutes at 14 000 RPM and 4°C.
SECTION: Derivatisation procedure with NPH
22. CAREFULLY transfer 150 µL of the supernatant, without disturbing the pellet, of all Eppendorf tubes to TruView Total Recovery 2mL glass vials.

SECTION: Derivatisation procedure with NPH
23. Add 10 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).

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

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

SECTION: Derivatisation procedure with NPH
27. Screw solid caps on all samples and vortex the solutions.
SECTION: Derivatisation procedure with NPH
28. Place all vials in a heating block (Techne dri-block) at 40°C for 20 min.

SECTION: Derivatisation procedure with NPH
29. After 20 minutes, put all vials back on ice.
SECTION: Derivatisation procedure with NPH
30. Add 250 µL of the quenching solution (water + 5% Formic Acid) to each vial, except for the solvent blank (A1).

SECTION: Derivatisation procedure with NPH
31. Vortex all samples, exchange the solid caps for caps with a septum.
SECTION: Derivatisation procedure with NPH
14. 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.

 
SECTION: Derivatisation procedure with NPH
32. Add500 µL of LC-MS grade water to the solvent blank. Samples are ready for LC-MS analysis.


SECTION: Derivatisation solution preparation
5. 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.
SECTION: Derivatisation solution preparation
6. 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.
SECTION: Derivatisation solution preparation
7. 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. 
SECTION: Derivatisation solution preparation
8. 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. 
SECTION: Derivatisation solution preparation
9. Prepare quenching solution: water + 5% Formic Acid
Add 475 L of LC-MS grade water to a 500 mL glass bottle.
Add 25 mL of LC-MS grade Formic Acid to the water.
Mix thoroughly.
SECTION: Set up of SCFA LC-HRMS method and analysis
33. Put the freshly prepared mobile phases onto the uHPLC system. Purge lines with mobile phase A and mobile phase B.
SECTION: Set up of SCFA LC-HRMS method and analysis
34. 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.
SECTION: Set up of SCFA LC-HRMS method and analysis
35. 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
  (%)      B
  (%)      Curve        Initial      0.300      100      0      Initial        1.00      0.300      100      0      5        2.80      0.300      5      95      5        3.50      0.300      5      95      5        3.60      0.300      100      0      5        7.00      0.300      100      0      5   

SECTION: Set up of SCFA LC-HRMS method and analysis
36. Add the following mass spectrometry method parameters to the acquisition method:
 
     Instrument      Thermo Exploris 240 Orbitrap        Source, Ionisation Mode      Thermo Scientific™ OptaMax™ NG ion source (H-ESI)        Scan Mode, Polarity      MRM, Negative        Mass range      Full Scan        Acquisition time      7.0 min        Delay time      0 sec        Sheath Gas       50        Aux Gas      15        Sweep Gas      1        IonSpray Voltage (IS) (Negative)      -2500 V        Ion Transfer Tube Temperature
  (TEM)      350°C        Vaporizer Temperature      450°C        Probe position (x – axis)      2        Probe position (y – axis)      2   

SECTION: Set up of SCFA LC-HRMS method and analysis
41. 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).

 
SECTION: Set up of SCFA LC-HRMS method and analysis
37. Place the Thermovial 54 rack with the samples into the autosampler of the chromatography system.
SECTION: Set up of SCFA LC-HRMS method and analysis
38. 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. 
SECTION: Data analysis using TraceFinder software
42. 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
SECTION: Set up of SCFA LC-HRMS method and analysis
39. 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.
SECTION: Derivatisation solution preparation
4. 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.
SECTION: Set up of SCFA LC-HRMS method and analysis
40. Use the deprotonated molecular mass m/z [M-H]- for each compound in the table below to interrogate the data.