FAME analysis of pollen fatty acids

Dry down the pollen sample in a freeze dryer.

Add 1mm zirconia beads to a 2 mL homogenizer tube and fill tube 1/4 full for each pollen sample. Keep the amount the same among samples and standards.

Add 10.0 mg pollen (usually about 1-2 mg lipid equivalent) to the homogenizer tube.

Add a FAME internal standard (ISTD) in stock solution directly to the vial (for our preparations, 56 µg cis-10-heptadecenoic acid, 56 µL of a 1 µg/uL stock solution in 2:1 chloroform-methanol). Use a synthetic fatty acid that does not occur naturally in the pollen or obscure chromatographic peaks of pollen fatty acids through co-elution. Allow the ISTD carrier solvent to evaporate off.

Critical note: add the ISTD here only if the sample is being analyzed only for FAME. If the sample is to be analyzed separately for FAME and total lipids (chromic acid), the ISTD should only be added to the sample after Folch extraction solution subsample removal. Total lipid assays such as chromic acid cannot distinguish between native (sample) and synthetic (ISTD) lipids and ISTD should not be added to chromic acid subsamples.

Add 1000 µL 2:1 chloroform: methanol (Folch solution) to the homogenizer tube.

Pulverize the pollen in the homogenizer tube with the BeadBeater homogenizer for 30 seconds. Tough materials may require additional 30 second rounds of homogenization. Avoid homogenizing for more than 30 seconds at a time to avoid sample overheating. Check the homogenate under a microscope to confirm that the pollen exine wall was fractured.

Turn off the power to the BeadBeater when loading or unloading tubes. Secure the tube caps tightly to avoid sample loss.

Add 210 µL 0.25% KCl to the homogenizer tube and briefly vortex.

Centrifuge the tube contents for 10 minutes at maximum speed in a microcentrifuge.

Two layers will form - an upper aqueous/methanolic layer and a lower chloroform/methanolic layer, with a possible boundary layer between the two phases. The amount of solvent in these two phases will be different than the two reagents' volumes since some methanol goes into each layer. Estimate the volume of both layers (often 310 µL upper layer and 690 µL lower layer) and record. Pipette 160 uL of the lower chloroform/methanolic layer into a crimp cap vial being careful not to transfer any solid materials.

Save a second subsample of the lower chloroform layer as a backup in a deep freezer in case the reaction fails.

Stopping point - The Folch extract can either be dried down immediately for FAME acid methanolysis or stored capped with a red rubber crimp cap in the -80°C freezer until later.

Turn on the SpeedVac vacuum centrifuge 30 min before dry down to allow the solvent vapor trap refrigerator to cool.

Reduce the sample and external standards in the vacuum centrifuge to complete dryness (about 1 to 2 hrs).

Make a fresh solution of methanolic HCl reagent (8% HCl in methanol) every day. For 100 samples, add 3.5 mL concentrated hydrochloric acid to 14.9 mL methanol in a 20 mL scintillation vial for a total volume of 18.4 mL. For 300 samples, add 9.7 mL concentrated hydrochloric acid to 41.5 mL methanol for a total volume of 50.0 mL.

Always add solvent slowly to strong acids to avoid violent boiling and spattering.

Perform an acid methylation to convert fatty acids to their FAME equivalents. First dissolve the dried vial FAME extract in 100 µL toluene.

Add 750 µL methanol to the crimp cap vial followed by 150 µL methanolic HCl. Seal with a red rubber crimp cap and vortex well. Incubate the vials at 45°C in heating blocks on a thermally controlled hot plate for 16 hours. Cover the heating blocks loosely with aluminum foil to reduce convective heat loss.

Double check that each crimp cap seal is tight before incubating the reaction vials.

Critical note: once the fatty acids are converted into their methyl ester equivalents (FAMEs), the compounds are slightly volatile and will be lost by drying down in a vacuum centrifuge or by prolonged uncapping.

Stopping point – The methylated FAMEs can be stored capped in the fridge or freezer immediately after the reaction but should be processed and analyzed by GC-MS within a few weeks maximum.

After the incubation, allow the vial to cool to room temperature. Briefly centrifuge the sealed vials for 5 seconds in the SpeedVac vacuum centrifuge without vacuum to draw down any condensation.

Unseal (decrimp) the crimp cap and transfer the entire vial contents (~ 1 mL) to a new 2 mL Eppendorf tube.

Partition the methanolysis products between two solvents. Add 300 µL DI water and 600 µL hexane to each tube. To limit hexane solvent losses, keep the vials tightly capped except when in active use. Vortex for 5 seconds.

Centrifuge the tube contents for 10 minutes at full speed in a microcentrifuge. The contents should form two layers, an upper hexane layer and a lower methanol-water layer. The upper hexane layer has the FAME compounds of interest. These layers will be different volumes than the amounts of water and hexane originally added as methanol will go into both layers.

Perform aqueous washes of the hexane layer to remove residual acids from the organic solvent. Transfer as much of the top hexane layer (200 top hexane layer (200 µ L) to a new 2 mL Eppendorf tube as possible, but do not pull in any water from the lower layer. Estimate the volume transfered and the total volume of the top layer, and remove about the same volume for all samples and ESTD standards.

Phase transfers do not have to be exact or include all of the upper hexane layer, as the fraction transferred will be ultimately be calculated by the amount of ISTD recovered.

Wash the transferred hexane twice with water to remove acid residues. Add 300 µL DI water to the tube, vortex 5 seconds, and centrifuge for 10 minutes at full speed. Remove about 200 µ L of the aqueous layer (bottom layer) after the first wash and dispose of it, leaving the top hexane layer in the tube. bottom layer ) after the first wash and dispose of it, leaving the top hexane layer in the tube. Add a second wash 300 µL DI water to the tube, vortex, and centrifuge for 10 minutes. Leave the water in after the second wash - the hexane layer will be removed shortly.

Remove 100 µL of the upper hexane layer into a glass insert in a screw top vial. Seal the sample with a screw top vial cap. Store the vial in the -80°C freezer until GC-MS analysis. Make sure that the screw top vial cap is on securely as hexane can escape through leaky threads.

Stopping point – for longer term storage (more than a few days), transfer the hexane layer to a crimp cap vial, seal the vial with a red rubber crimp cap, and store in the deep freezer. FAME compounds degrade with a month or two in the deep freezer.

FAME compounds in the hexane layer can be analyzed by EI GC-MS on a gas chromatograph coupled to a mass spectrometer detector (for the author's group, an Agilent 7890A gas chromatograph coupled to an Agilent 5975D mass spectrometer run by Agilent Mass Hunter software. 1 µL sample is injected at 220°C onto a HP-5MS column (30 m x 0.25 mm x 0.25 µm film; Agilent, Inc.) with helium as a carrier gas at 1.2 mL/min. Compounds are separated by oven temperatures programmed at 10°C/min from 35°C with an initial 1 min hold to 230°C with an 8 min hold, then to 320°C with a 0.5 min final hold).

FAME compounds are elucidated by comparison of mass spectra and retention times with esterified standards. FAME compounds are quantified by comparison of characteristic mass fragments (m/z) with known amounts of authentic standards. The fraction of the total sample injected is calculated from the amount of internal standard (for the author's group, cis-10-heptadecenoic acid) recovered in each injected sample.

Obtain FAME compound retention times and characteristic mass fragments (m/z) from authentic standards. The m/z fragments are used in SIM mode to generate single ion chromatograms with compound peaks. Select m/z fragments for peak quantification that are prominent and free of background interference from contaminants.