Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

1. Initial Considerations, Preparation of Instruments and Standards

1. Always use glass for storing (glass culture tubes, glass autosampler vials) or transferring (glass pipettes) lipids and organic solvents.
2. Minimize exposure of all lipid samples and standards to air. Seal polytetrafluoroethylene (PFTE) caps tightly to avoid air exposure and evaporation. Immediately resuspend dried lipids in the next solvent or keep them in a steady stream of nitrogen.
3. Use 100 µl of sample. In instances where more (or less) sample is available, adjust volumes accordingly. However during the lipid fractionation on the NH₂ SPE column, stated volumes must remain as indicated.
4. Turn on centrifuge and set to 0 °C prior to start of sample preparation.
5. This technique uses many volatile solvents so keep all solvents capped during sample preparation.
6. Prepare two types of standards per recommendation.
7. Prepare a negative control comprised of spiked-in standards all at constant concentration. This is spiked into all samples as well as a pooled sample which is used as a batch QC (spiked pooled sample used to monitor sample preparation reproducibility on separate days) and instrument QC (spiked pooled samples previously prepared, sub-aliquoted, and used to monitor instrument conditions/fluctuations during analysis and on separate days).
   1. Prepare positive controls at 1x, 2x, and 4x standard spikes. The positive controls are added to all samples as well as a pooled plasma sample and are used as quality control compounds for both the sample preparation steps and instrumental analysis steps to analyze and identify the fold change differences during data analysis to ensure that all aspects of the preparation and instrumental analysis were performed correctly.
8. Store standards at -20 °C and store samples at -80 °C. Certain internal standards which are prone to degradation following long term storage should be stored at -80 °C.
9. This procedure requires the use of hazardous, flammable or volatile solvents such as MTBE. Perform all steps in a fume hood.

2. Internal Standards

1. Choose internal standards (ISTD) based on individual projects and their specific experimental design. For biofluids such as plasma, BALF, or urine, isotopically labeled ISTDs which are similar in nature to those found biologically in these samples would be ideal. These would include but are not limited to amino acids, hormones or lipids. For plant metabolomic samples, labeled standards such as flavonoids, or carotenoids could be used. The same applies to other metabolomic studies whereby the investigator should choose an internal standard which is representative of the sample type being analyzed.
2. Ensure that the chosen ISTD covers a wide range of the chromatogram; for example if the acquisition time is 20 min, standards that elute every 5 min could be used.
3. Create hydrophilic stock solutions at 2 mg/ml using a variety of isotopically labeled standards and/or other polar compounds which are exogenous to the sample being analyzed. From each stock solution, create one solution in 1:1 methanol:water with all standards to a final concentration of 25 µg/ml creatinine-D₃, 100 µg/ml lysine-D₄, and 200 µg/ml valine-D₈.
4. Create hydrophobic stock solutions using a variety of isotopically labeled standards and/or other non-polar compounds which are exogenous to the sample being analyzed; stock concentrations of 17:0 fatty acid (4 mg/ml), 19:1 fatty acid (4 mg/ml), 17:0 ceramide (2 mg/ml), 17:0 PE (1.75 mg/ml), 15:0 PC (2 mg/ml), and testosterone-D₂ (1 mg/ml). From each stock, create one solution in 1:1 chloroform:methanol with all standards to a final concentration of 50 µg/ml 17:0 ceramide, 100 µg/ml 15:0 PC, 100 µg/ml testosterone-D₂, 200 µg/ml 17:0 fatty acid, 200 µg/ml 19:1 fatty acid, and 200 µg/ml 17:0 PE in the standard mix.
5. Test the degree of ionization of each standard ahead of time using at least five different concentrations for each standard to determine the limit of detection and linearity of the instrument for these compounds. The concentration of the stock solution for each individual standard will vary depending on experimental design, and the concentration of each standard in the combined spiked standard may range from 20 μg/ml to 2 mg/ml depending on how well they ionize.
6. Create a spike mix of positive controls and add them to the samples at 1x, 2x, or 4x concentration levels to monitor quantitatively the strength of the sample preparation and the accuracy of the instrumental data. Final concentration of positive controls can be: 2 mg/ml D-glucose, 100 µg/ml alanine-D₃, 200 µg/ml methylmalonic acid-D₃, 20 µg/ml triglyceride-D₅, and/or other hydrophobic and hydrophilic standards. Adjust the standard concentrations based on the sensitivity of the MS and HPLC instrumentation used for analysis.

3. Protein Precipitation

1. Thaw samples to RT and spike 10 µl of ISTD to each sample as described below.
2. Spike 10 µl of both hydrophilic and hydrophobic standard solutions (created from the stock in steps 2.3 and 2.4) to each sample. Adjust the standard concentrations as necessary based on the sensitivity of the MS and HPLC instrumentation used for analysis
   1. Spike 10 µl of either 1x, 2x, or 4x positive control solution (created from the stock in step 2.6) to each sample. Adjust the standard concentrations as necessary based on the sensitivity of the MS and HPLC instrumentation used for analysis
   2. Vortex each sample for 10 sec prior to the protein precipitation step
3. Add 400 µl of ice cold methanol (stored at -20 °C) to each sample.
4. Vortex for 10 sec per tube.
5. Centrifuge at 0 °C for 15 min at 18,000 x g.
6. Transfer all the supernatant to a new glass culture tube, and then dry under N₂.
7. If analyzing the protein pellet fraction, proceed to steps 3.8-3.11. If not analyzing the protein pellet, skip to Section 4.
   Note: The protein pellet may contain compounds with high hydrophobicity which would be useful when performing drug studies¹⁸, food analyses involving hydrophobic flavonoids¹⁹ or metabolomic studies related to diseases where very hydrophobic compounds accumulate, such as neuronal ceroid-lipofuscinoses²⁰ and lysosomal lipid storage diseases²¹.
8. Add 1 ml of MTBE to the white (or off-white) protein pellet, vortex for 30 sec per tube, then centrifuge at 0 °C for 15 min at 18,000 x g. Decant the MTBE layer to a new glass culture tube.
   This is a critical step as errors here will drastically affect results (see Figure 5 in representative results).
   1. Consistently aspirate the same amount of MTBE for ALL samples since the size of the pellet will vary among samples. Therefore, if only 900 µl can be decanted for the sample with the least amount of supernatant, then decant 900 µl for all samples.
9. Repeat step 3.9, and combine the organic layer to the same glass culture tube prepared in step 3.7.
10. Dry samples by N₂ flow and resuspend in 200 µl of 1:1 chloroform:methanol. Vortex briefly.
11. Transfer to a centrifuge tube. Centrifuge at 0 °C for 15 min at 18,000 x g, then transfer supernatant to autosampler screw cap vials using glass pipettes.

4. Liquid-liquid Extraction

1. Using a glass pipette, add 3 ml MTBE to the dried methanol residual (from section 3, step 6), vortex 30 sec, add 750 µl of water, then vortex 10 sec per tube.
2. Spin ~200 x g for 10 min in a centrifuge at RT.
3. Aspirate 2.5 ml of the MTBE layer (without getting water) and transfer to a clean glass culture tube.
   Note: This is a critical step as differences here will affect results. 2.5 ml of MTBE should be carefully decanted from the top layer because it allows a fixed volume to be decanted without pipetting the aqueous layer below. If the volume of sample at the start of the experiment was less than the indicated 100 μl for this method, scale the volume of MTBE to proportionally reflect this starting sample volume.
4. Add 3 ml MTBE to the remaining water part of sample, and vortex 10 sec per tube.
5. Spin ~200 x g for 10 min in the centrifuge at RT.
6. Aspirate 3 ml of MTBE (without getting water) and combine with MTBE tube.
7. Concentrate the remaining aqueous layer by drying under N₂.
8. Re-suspend residue in 100 µl of water.
9. Add 400 µl of ice cold MeOH to the glass culture tube, vortex briefly, and then transfer to microcentrifuge tube.
10. Leave at -80 °C for 20-30 min. Spin at 0 °C for 15 min at 18,000 x g.
    Note: It is recommended to place the entire sample rack in a -80 °C freezer as this will allow any remaining protein to precipitate out in the methanol. If a -80 °C freezer is not available, other options are: storing at -20 °C, placing the samples on dry ice, or keeping them in an ice bucket. It is important to consistently store the samples in a cold environment and at the same temperature to allow precipitation.
11. Aspirate 450 µl of supernatant and transfer to a clean microcentrifuge tube. Dry completely in a vacuum centrifugal concentrator at no more than 45 °C. (Takes about 1-2 hr).
12. Resuspend dried supernatant in 200 µl of 5% acetonitrile/water. Vortex briefly. Freeze at -80 °C.

5. Solid-phase Extraction

1. Dry the MTBE fraction under nitrogen at 35 °C with a good flow of nitrogen (takes about 10-15 min).
2. When completely dry, stop the flow of nitrogen and quickly resuspend in 1 ml chloroform (CHCl₃) using a glass pipette. Vortex briefly.
   Note: Solvents such as CHCl₃ have low viscosity. During pipetting, low surface tension causes solvent loss from the pipette. It is recommended that the pipette tip be prewet at least twice to allow equilibration between the solvent being pipetted and the space in the pipette. A gas-tight syringe can also be used if available.
3. Set up an SPE vacuum manifold and NH₂ SPE columns for fractionation.
4. Warm samples to RT and always keep suspended under a steady flow of N₂. Warming to RT will allow lipid resuspension and the nitrogen will prevent oxidation and polymerization of lipids.
5. Wash and condition SPE cartridge 2x with 400 µl hexane. Discard waste and replace with new glass collection tube.
6. Add sample to SPE column, collect flow through in glass tubes.
7. With glass pipette add 1 ml of 2:1 CHCl₃:IPA, collect flow through in same glass tubes (this is the Neutral Fraction).
8. Dry the neutral fraction under N₂ to minimize oxidation (takes about 10-15 min).
9. With glass pipette add 1 ml of 5% acetic acid in diethyl ether, collect flow through in new glass tubes (this is the Fatty Acid Fraction).
10. Dry the fatty acid fraction under N₂ to minimize oxidation (takes about 10-15 min).
11. Use plastic tips to add 800 µl of methanol to SPE cartridge, and collect flow-through in 15 ml plastic conical tubes (this is the Phospholipid Fraction).
12. Transfer phospholipid fraction to 1.5 ml centrifuge tubes. Dry the samples with a vacuum centrifugal concentrator at 45 °C (takes about 1-1.5 hr).
13. Resuspend each of the samples from the three fractions in 200 µl of 100% methanol, vortex, and transfer to autosampler vials screw-cap for storage.

6. Sample Storage Conditions

1. Store all samples at -80 °C until ready for instrument analysis.
   Note: Extracted samples, reconstituted in organic solvent may be stored at -20 °C. However this is not recommended as potential metabolites of interest will degrade at this temperature. Liquid nitrogen storage is also not recommended as investigators have reported contamination issues, special storage vials are needed, and there is lack of homogeneity in the chamber causing wide temperature fluctuations.
2. If sample volumes are small (<100 µl), use inserts in the vials to prevent solvent evaporation in the headspace during storage.
3. Avoid freeze-thaw of the samples. Thaw the samples only once, right before instrument analysis. Repeated freeze-thaws result in sample degradation.

7. Liquid Chromatography Conditions

1. Use a C-18 2.1 mm x 50 mm (1.8 μm) analytical column with a C-18 2.1 mm x 12.5 mm (5 μm) guard column to analyze the hydrophobic fraction.
2. Set the autosampler temperature to 4 °C, the column temperature to 60 °C, the injection volume to 2 µl and the flow rate to 0.25 ml/min.
3. Use 0.1% formic acid in water for mobile phase A, and 0.1% formic acid in isopropanol:acetonitrile:water (60:36:4) for mobile phase B.
4. Run the following gradient elution profile: Start at 30% B, and increase to 70% B from 0 to 1 min, then increase to 100% B from 1 to 15 min and hold for 5 min, followed by 5 min 10% B washing and 5 min post run.
5. Use a HILIC 2.1 mm x 50 mm (2.6 μm) analytical column with a guard column to analyze the hydrophilic fraction.
6. Set the autosampler temperature to 4 °C, the column temperature to 20 °C, the injection volume to 2 µl and the flow rate to 0.5 ml/min.
7. Use 50% acetonitrile in 10 mM ammonium acetate, pH 5.8 for mobile phase A, and 90% acetonitrile in 10 mM ammonium acetate pH 5.8 for mobile phase B.
8. Run the following gradient elution profile: Start at 100% B from 0 to 2 min, then decrease to 50% B from 2 to 15 min, followed by 5 min 0% B washing and 10 min post run.