TITLE: Acetaldehyde Quantification in Microbial Fermentation Samples
AUTHORS: Isaiah D Richardson, Bishaldsharmath, Daniel G Olson

[DESCRIPTION]
This protocol details a broadly applicable approach for quantifying the concentration of acetaldehyde present in a microbial fermentation sample. To quantify acetaldehyde, a hydrazine derivatization reaction is performed following sampling using 2,4-dinitrophenylhydrazine (2,4-DNPH), which reacts with acetaldehyde in a 1:1 stoichiometric ratio to produce acetaldehyde-2,4-DNPH, a stable product at room temperature that can be quantified using high performance liquid chromatography (HPLC).

[STEPS]
SECTION: Preparation of the Derivatization Reagent
3. The primary objective of this section of the protocol is to prepare a 100 mL stock of derivatization reagent, which consists of 0.9 g/L 2,4-DNPH and 1.0% phosphoric acid (H3PO4) in acetonitrile.
SECTION: Preparation of the Derivatization Reagent
3.2. Carefully add the 0.09 g of 2,4-DNPH powder to a clean and dry 100 mL volumetric flask. The volumetric flask must be at room temperature for this step as 2,4-DNPH has explosive potential when exposed to high temperature environments.
SECTION: Preparation of the Derivatization Reagent
3.3. Carefully transfer the 100 mL volumetric flask containing the 2,4-DNPH powder to a fume hood.
SECTION: Preparation of the Derivatization Reagent
3.4. In the fume hood, use a repeat pipette to add 40.0 mL of acetonitrile (Sigma Aldrich CAS#: 75-05-8) to the 100 mL volumetric flask.
SECTION: Preparation of the Derivatization Reagent
3.5. Carefully swirl the flask to dissolve the 2,4-DNPH powder in the acetonitrile. The solution will appear as a vibrant, dark orange color following this step.
SECTION: Preparation of the Derivatization Reagent
3.6. Add 1.1765 mL of 85.0% phosphoric acid (H3PO4) to the 100 mL volumetric flask.
SECTION: Preparation of the Derivatization Reagent
3.7. Fill the volumetric flask to the 100 mL line with acetonitrile.
SECTION: Preparation of the Derivatization Reagent
3.8. Transfer the 100 mL of prepared derivatization reagent to a clean, dry, 200 mL glass bottle at room temperature in the fume hood.
SECTION: Preparation of the Derivatization Reagent
3.9. Wrap the bottle with aluminum foil to reduce exposure of the derivatization reagent to light.
SECTION: Preparation of the Derivatization Reagent
3.10. Use tape to label the bottle as "Derivatization Reagent". Include your initials and the date prepared.
SECTION: Preparation of the Derivatization Reagent
3.11. Store the derivatization reagent at -20.0ºC until use.
 

SECTION: Preparation of Acetaldehyde Standards
4. In a 4.0ºC cold room, carefully break the seal of the glass vial containing pure acetaldehyde ordered from Sigma Aldrich (Sigma Aldrich CAS#: 75-07-0). Carefully pour the 5.0 mL of pure acetaldehyde into a labeled 15 mL Falcon tube. Note: store acetaldehyde on ice at all times (whether working in a 4.0ºC cold room or not) and store the pure acetaldehyde stock at 4.0ºC when not in use to reduce evaporation.
SECTION: Preparation of Acetaldehyde Standards
5. In a 4.0ºC cold room, prepare 5.0 mL of a 1.0 M acetaldehyde stock solution following sub-steps 5.1-5.3:
SECTION: Preparation of Acetaldehyde Standards
5.1. Use a repeat pipette to add 4.72 mL of MilliQ water to a 15 mL Falcon tube.
SECTION: Preparation of Acetaldehyde Standards
5.2. Add 279.5 µL of pure acetaldehyde solution to the 4.72 mL of MilliQ water using a repeat pipette equipped with a 0.5 mL disposable plastic tip chilled to -20.0ºC for 20 minutes prior to use.
SECTION: Preparation of Acetaldehyde Standards
6. Prepare 100.0 mM, 10.0 mM, 1.0 mM, 0.1 mM, and 0.01 mM acetaldehyde standards in the 4.0ºC cold room following sub-steps 14.1-14.4:
SECTION: Preparation of Acetaldehyde Standards
6.1. Using a repeat pipette equipped with a 2.5 mL disposable plastic tip, add 900 uL of MilliQ water to 5 separate 2.0 mL Eppendorf tubes labeled "100 mM", "10 mM", "1.0 mM", "0.1 mM", and "0.01 mM".
SECTION: Preparation of Acetaldehyde Standards
6.2. Transfer 100 uL of the 1.0 M acetaldehyde stock solution to the 2.0 mL Eppendorf tube labeled "100 mM".
SECTION: Preparation of Acetaldehyde Standards
5.3. Following preparation, vortex the 1.0 M acetaldehyde solution for approximately 5-10 seconds to ensure complete mixing of the acetaldehyde with the MilliQ water.
SECTION: Preparation of Acetaldehyde Standards
6.3. Vortex the solution for 10 seconds to ensure thorough mixing.
SECTION: Preparation of Acetaldehyde Standards
6.4. Perform serial dilutions to prepare the remaining stock solutions by transferring 100 uL from the "100 mM" to the 10 mM tube and so on. Vortex each tube for 10 seconds prior to performing the next dilution to ensure thorough mixing of each solution.
SECTION: Preparation of Acetaldehyde Standards
7. Following preparation of the acetaldehyde stock solutions (100 mM, 10 mM, 1.0 mM, 0.1 mM, and 0.01 mM),  use a pipette to transfer 100 µL from each acetaldehyde stock solution into 500 µL of derivatization reagent in separate, labeled 2.0 mL Eppendorf tubes (final volume of each derivatized sample = 600 µL).
SECTION: Preparation of Acetaldehyde Standards
8. Vortex each derivatized acetaldehyde sample for 10 seconds.
SECTION: Preparation of Acetaldehyde Standards
9. Place and secure each of the derivatized samples on a nutating mixer in the 4.0ºC cold room.
SECTION: Preparation of Acetaldehyde Standards
10. Allow the derivatized acetaldehyde samples to mix overnight on the nutating mixer for approximately 12-16 hours to ensure complete derivatization of the acetaldehyde.
SECTION: Preparation of Acetaldehyde Standards
12. Following mixing, transfer 200 uL of supernatant from each derivatized sample to a labeled HPLC vial and run the samples on a Shimadzu Shim-Pack XR ODS column using the methods described below under the section titled: "HPLC System Setup and Method Parameters".
SECTION: Derivatization of Fermentation Samples
15. Acquire 1.0 mL of biological fermentation sample either via pipette (best for performing fermentations in Falcon tubes) or needle and syringe (best for performing fermentations in serum bottles) and transfer the 1.0 mL of sample to a labeled 2.0 mL Eppendorf tube stored on ice.
SECTION: Derivatization of Fermentation Samples
16. Immediately centrifuge the fermentation samples for 5.0 minutes at 21,300 rcf (x g). Note: It is best to use a centrifuge that is chilled to 4ºC to reduce the potential for evaporation of acetaldehyde from the sample, however using a centrifuge that is not chilled to 4.0ºC also works for this protocol.
SECTION: Derivatization of Fermentation Samples
17. Depending on the size of the cell pellet Carefully extract 700-900 µL of supernatant and transfer the supernatant to a labeled 2.0 mL Eppendorf tube. Store the sample on ice when not handling.
SECTION: Derivatization of Fermentation Samples
18. Transfer 100 uL of fermentation sample supernatant into 900 uL of MilliQ water (10x dilution). The purpose of this step is to ensure that there is sufficient 2,4-DNPH in the derivatization reagent to derivatize all of the acetaldehyde present in the fermentation sample. Store the sample on ice when not handling.
SECTION: Derivatization of Fermentation Samples
20. Transfer 100 uL of the 10x diluted fermentation sample into 500 uL of derivatization reagent in a clean, dry, labeled 2.0 mL Eppendorf tube.
SECTION: Derivatization of Fermentation Samples
21. Immediately vortex the derivatized sample for 10 seconds to ensure thorough mixing of the acetaldehyde with the 2,4-DNPH present in the derivatization reagent.
 

SECTION: Derivatization of Fermentation Samples
19. Immediately vortex the 10x diluted sample for 8-10 seconds to ensure thorough mixing.
SECTION: Derivatization of Fermentation Samples
22. Transfer the derivatized samples on ice to a 4.0ºC cold room. Place and secure each derivatized sample on a nutating mixer in a 4.0ºC cold room as shown below.
 

SECTION: Derivatization of Fermentation Samples
23. Allow the derivatized sample to mix undisturbed on the nutating mixer for approximately 12-16 hours to ensure complete derivatization.
SECTION: Derivatization of Fermentation Samples
25. Transfer 200 uL of supernatant from the derivatized sample to a labeled HPLC vial.
SECTION: Derivatization of Fermentation Samples
26. Run the derivatized sample on a Shimadzu HPLC system using the setup parameters described below along with a set of derivatized acetaldehyde standards (10 mM, 1.0 mM, 0.1 mM, and 0.01 mM). 
SECTION: Preparation of Acetaldehyde Standards
13. Use the height values acquired on the HPLC system (in units of mAu) for each derivatized acetaldehyde to create a standard curve of height (mAu) (x-axis) vs. concentration (mM) (y-axis). Add a trendline and the trendline equation on Excel. Use the trendline equation (in the linear form of y = mx + b) to convert height values (in units of mAu) to concentrations (in mM) by plugging the height value in for "x" to solve for "y" (concentration of acetaldehyde in mM).

 

SECTION: Derivatization of Fermentation Samples
27. Refer to step 13 of this protocol (under the "Preparation of Acetaldehyde Standards" section) for a description of how to convert height values (in mAu) on the HPLC system to concentration values (in mM) to quantify the amount of acetaldehyde present in each biological fermentation sample.
SECTION: Introduction and Protocol Structure
1. This protocol details a standardized approach for quantifying acetaldehyde in microbial fermentation samples. Accurately quantifying the concentration of acetaldehyde in biological samples poses challenges, as acetaldehyde is a volatile chemical with a boiling point of 20.2ºC (68.4ºF), and thus readily evaporates at room temperature (20.0-22.0ºC or 68.0-72.0ºF). To quantify acetaldehyde, a hydrazine derivatization reaction is performed immediately upon sampling using 2,4-dinitrophenylhydrazine (2,4-DNPH), which reacts with acetaldehyde in a 1:1 stoichiometric ratio to produce acetaldehyde-2,4-DNPH, a stable product at room temperature that can be quantified using high performance liquid chromatography (HPLC) at an absorbance of 254 nm.
2. This protocol is structured into the following sections:
1. Preparation of the Derivatization Reagent (Step 3 [3.1-3.11])
2. Preparation of Acetaldehyde Standards (Steps 4-12)
3. Derivatization of Fermentation Samples (Steps 13-24)
4. HPLC System Setup and Method Parameters (Step 25 [25.1 - 25.10])
SECTION: Preparation of the Derivatization Reagent
3.1. Weigh out 0.09 g of 2,4-dinitrophenylhydrazine (2,4-DNPH) (red, crystalline powder; Sigma Aldrich CAS#: 119-26-6) on a scale using a small weigh boat. Note that 2,4-DNPH is highly reactive and volatile, use extreme caution and wear proper PPE at all times when handling this chemical. Record the exact mass of 2,4-DNPH weighed out in a laboratory notebook.
SECTION: HPLC System Setup and Method Parameters
28. Detailed below are the high-performance liquid chromatography (HPLC) system setup parameters for running the derivatized acetaldehyde samples on a Shimadzu HPLC system:
SECTION: HPLC System Setup and Method Parameters
28.1. HPLC System: Shimadzu Prominence-i LC-2030C 3D Liquid Chromatograph
SECTION: HPLC System Setup and Method Parameters
28.2. Refractive Index Detector (RID) Unit: Shimadzu RID-20A Refractive Index Detector
SECTION: HPLC System Setup and Method Parameters
28.3. HPLC Column: Shim-Pack XR ODS column (75 mm L. x 4.6 mm I.D>, 2.2 µm)
SECTION: HPLC System Setup and Method Parameters
28.4. Column Temperature: 40ºC (left and right)
SECTION: HPLC System Setup and Method Parameters
28.5. Tray Temperature: 4.0ºC
SECTION: HPLC System Setup and Method Parameters
28.6. Mobile Phases (2 separate solutions):
Eluent A: Acetonitrile (100% acetonitrile)
Eluent B: Water/Tetrahydrofuran (THF) in a 8:2 ratio (ex: 800 mL milliQ water, 200 mL THF = 1.0 L solvent)
SECTION: HPLC System Setup and Method Parameters
28.7. Mobile Phase Flow Rate: 1.0 mL/minute
SECTION: HPLC System Setup and Method Parameters
28.8. Injection Volume: 10.0 µL
SECTION: HPLC System Setup and Method Parameters
28.9. Time Program (Gradient Run): Total run time per sample = 17.0 minutes
Tables 1-2 and Figure 9 below detail the gradient run setup for Eluent A (acetonitrile) and Eluent B (Water/THF) for running derivatized acetaldehyde samples on the Shimadzu Shim-Pack XR ODS Column.

 


 

 

SECTION: HPLC System Setup and Method Parameters
28.10. Detection Method: Photodiode array (PDA) [Ultraviolet (UV)] at 254 nm
SECTION: HPLC System Setup and Method Parameters
28.11. Provided below is a photograph of the touchscreen interface on a Shimadzu Prominence-i LC-2030C 3D HPLC system while running a derivatized sample using the setup parameters described above:
 

SECTION: Preparation of Acetaldehyde Standards
11. Centrifuge the derivatized samples for 5.0 minutes at 2,000 rcf (x g) to pellet any residual debris.
SECTION: Derivatization of Fermentation Samples
24. Centrifuge the derivatized sample for 5 minutes at 2,000 rcf to pellet any residual debris from the derivatization reaction. This step is not essential but is best to perform this step to reduce the potential of any debris from the derivatized sample clogging the HPLC needle, tubing, or column.
SECTION: Preparation of Acetaldehyde Standards
12.1. Note: Although it can be run on the Shimadzu HPLC system, the 100 mM acetaldehyde standard should be omitted in the creation of a standard curve for the quantification of acetaldehyde. The reason for this is that there is not a sufficient concentration of 2,4-DNPH present in the derivatization reagent to derivatize all of the acetaldehyde present in the 100 mM standard regardless of how long the sample is mixed on the nutating mixer.
SECTION: Derivatization of Fermentation Samples
26.1. Note: Due to an insufficient concentration of 2,4-DNPH in the derivatization reagent, the acetaldehyde present in a 100 mM standard cannot be fully derivatized and thus should not be used to create a standard curve to quantify acetaldehyde. Only the 10 mM, 1.0 mM, 0.1 mM, and 0.01 mM acetaldehyde standards should be used in the creation of a standard curve to quantify acetaldehyde.
SECTION: Preparation of Acetaldehyde Standards
14. Provided below are images of the HPLC spectra from running 10 mM, 1.0 mM, 0.1 mM, and 0.01 mM derivatized acetaldehyde standards on a Shimadzu HPLC system using the setup and method parameters detailed later in this protocol under the section titled, "HPLC System Setup and Method Parameters". These derivatized acetaldehyde standards were used to produce the standard curve provided above (step 13) to quantify the concentration of acetaldehyde in microbial fermentation samples. Acetaldehyde elutes off the Shim-Pack XR ODS HPLC column at approximately 6.45 minutes as a double peak comprised of a leading short peak and a subsequent tall peak. As shown below, the short and tall acetaldehyde peaks should be split and only the tall peak should be used for the quantification of acetaldehyde. It is also important to note that 0.01 mM acetaldehyde is the limit of detection for the Shimadzu HPLC system.