1. Cover hot plate with aluminum foil.
2. Place “raw” Pop-Tart on hot plate
3. Turn on hot plate and start timer.
4. Record Data:

· Heat Interval 1
· Heat Interval 2
· Flame Interval

Heat Interval 1 = 200o to Flame
Heat Interval 2 = 400o to Flame
Flame Interval = Flame start to finish

Lipids from Pop-Tart samples were extracted using organic solvents according to a modified procedure of Bligh and Dyer (1954). Fatty acids in the lipids were simultaneously saponified and methylated using boron trifluoride (BF3) in Methanol (Morrison, 1964). Fatty acid methyl esters are separated and identified by capillary column gas chromatography (Hoffman, 1995).

Extraction Procedures:

1. Add 3ml MeOH: CHCl3 :: 2:1 containing 0.02% butylated hydrxytoluene (BHT) to 16x100mm glass screw cap tubes labeled for homogenate sample.
2. Transfer 0.5 ml aliquots of homogenate into separate glass tubes, flush with N2, and seal with Teflon lined cap. Vortex for 10 min. (store in freezer for later extraction).

3. Add room temperature 23:0 fatty acid internal standard, 50ml to each extraction tube. Vortex 10 min., centrifuge at 3600 RPM x 10 min. and remove solvent (upper layer) to 16x100mm screw cap tube with dispo-pipet.
4. Add 0.5ml water to homogenate, vortex (10s). Then add 1.5ml MeOH:CHCl3 :: 2:1 + 0.02% BHT. Flush with N2 and cap.
5. Vortex for 5 min., centrifuge 10 min. and remove solvent (upper layer) with pervious solvent extract.
6. To solvent extract, add 2.0ml of 5mM CaCl2 and 1.5ml CHCl3, flush with N2, vortex 5 min. and centrifuge 5 min.
7. Prepare glass wool filters and wash with 2-3ml of 2:1 :: CHCl3:MeOH, discarding solvent. Using a 9” dispo-pipet transfer lower layer of solvent extract into 16x100mm tube.
8. Wash upper layer with 1.5ml CHCL3, flush with N2, vortex (5 min), centrifuge (5 min). Filter lower layer and pool solvent extract. Rinse filter with ~1ml of 2:1:: C:M.
9. Place sample tube in heating block (To = 37oC) and bring to dryness using N2. Azetrope water with ~1ml 2:1:: CHCl3:MeOH, N2 dry. Add 500ml 2:1:: CHCl3:MeOH to resuspend and vortex briefly.

Fatty Acid Methylation:

1. Turn on water bath (100oC), cool centrifuge (4oC), turn on drying block to 37oC, remove BF3 from freezer, and bring to room temperature.
2. Dry sample extract under N2.
3. Add 1.0ml BF3 to dried extract, flush with N2, cap tightly, and vortex gently.
4. Incubate in water bath for 20 min.
5. Immediately place in ice bath for 5 min, just enough to cool the tube.
6. Add 2.0ml hexane and 1ml distilled water, flush with N2 and cap.
7. Vortex, 5min. Centrifuge for 5 min.
8. Transfer upper hexane layer using 9” dispo-pipet into separate 4ml screw cap vials. Do not drip any aqueous layer into vial.
9. Add 2.0ml hexane to original tube, flush with N2 and cap. Vortex 5 min and centrifuge 5 min.
10. Transfer and pool upper phase as in step 8.
11. Dry tube under N2. Azetrope residual moisture with addition of ~1ml 2:1:: CHCl3:MeOH. Bring to dryness.
12. Resuspend residual in 400ml CH2Cl2, cap vial and vortex (5s). Transfer 50ml to conical GC vial, flush gently with N2 and cap.
13. The remainder of the sample in the 4ml vial should be flushed with N2 and stored in the freezer.

Gravimetric Lipid DTH:

1. Label and weigh empty Aluminum pan on Analytical Balance, handle with forceps only
2. Add 500ml sample, N2 dry at 40oC
3. Transfer to vacuum chamber, evacuate and leave for 12-24 hours
4. Weigh pan with sample
5. Difference equals the total lipid weight

General Phenol-Sulfuric Acid Assay for Carbohydrates:

1. Prepare the reagent by dissolving phenol in water (5%).
2. Mix samples, standards, and control solution (200mL containing up to 100mg carbohydrate) with 200mL phenol reagent
3. Add 1.0 mL of concentrated sulfuric acid rapidly and directly to the solution surface without allowing it to touch the sides of the tube.
4. Leave the solutions undisturbed for 10 minutes before shaking vigorously. Determine the absorbencies at 490nm after a further 30 min.

Previous Findings = PopTart Letter + Flaming PopTarts + Flaming Toaster

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