Onion Flavor Compound Analysis
Flavor Compound Extraction:  

1.  A wedge is cut from each onion in the sample.

2.  The wedges are weighed.

3.  The onion wedges are placed in a jar.  5ml of 80% methanol by volume (4 L methanol to 1 L deionized water) per 1 gm of onion tissue is poured into the jar.

4.  The jar is placed into a -20° C freezer overnight.

5.  The next morning, the extract is removed from the freezer and the onion tissue is strained.   The liquid is placed into a second jar and returned to the freezer.  The onion tissue is placed back into the jar and extracted a second time, using the same volume of the methanol/water mixture as before.  The jar is placed back into the -20° C freezer for 4 hours.

6.  The onion tissue is strained and the liquid is combined with the first extractant and returned to the freezer. The tissue is returned to the jar and an 80% ethanol by volume (4 L ethanol to 1 L water) mixture is poured into the jar, using the same volume as the methanol/water mixture. The jar is returned to the freezer for 2 hours.

7.  After 2 hours, the onion tissue is strained and discarded.  The ethanol/water extractant is mixed with the first two extractants.   This mixture is returned to the -20° C freezer until ready to use.

*Note - Since 15 ml of methanol/ethanol/water was added per 1 gm fresh weight (gfw) of tissue, 15 ml of extract is equivalent to 1 gfw of tissue.
Flavor Compound Sample Preparation:
Three internal standards are added to the samples for HPLC quantification:    L-glutamyl-L-glutamic acid (g-glu glu), S-methyl glutathione (S-MeG), and Butyl-L-cysteine sulfoxide (BCSO).  The g-glu glu and S-MeG can be purchased, but the BCSO must be synthesized.  These concentrations are added to each sample:  0.2 mg of g-glu glu per gfw of tissue, 0.5 mg of S-MeG per gfw, and 1.0 mg BCSO per  mg gfw.
 

Preparation of internal stock solutions:
1.  The internal standard stock solutions are prepared:  (these are made to allow 1 ml of each standard stock solution to be added per sample to get the concentrations stated above)
     g-glu glu            5.0 mg in 25 ml of dd (double deionized) water to get 0.2 mg per ml
     S-MeG              12.5 mg in 25 ml of dd water to get 0.5 mg per ml
     BCSO               25.0 mg in 25 ml of dd water to get 1.0 mg per ml
 

Concentrating MEW onion extracts and addition of internal standards:
2.  15.0 ml of sample extract is placed into 40 ml test tubes.  1.0 ml of each standard stock solution is added to each sample and vortexed. 

3.  The test tubes are placed in a drying rack in the fume hood and blown to dryness.  This takes 8-10 hours. Upon dryness, the samples are capped and placed into the -20° C freezer.

4.  Samples are rehydrated in 1.0 ml of dd water, vortexed, and tranferred to a 1.5 ml capped centrifuge tube. 

 

Ion Exchange of Samples:
5.   Prepare the four acetic acid elutions: 
     0.1 M           6.0 ml glacial acetic acid (HOAc) per 1 L dd water
     0.2 M         12.0 ml HOAc per 1 L dd water
     2.0 M       120.0 ml HOAc per 1 L dd water                                        
     5.0 M       300.3 ml HOAc per 1 L dd water                                     
6.  The ion exchange columns (15 ml) are prepared as follows:  place anion exchange resin (we use Bio-Rad AG 1-X8 , 200-400 mesh, acetate form) into beaker and add dd water until viscous enough to pull into a 5 ml pipet.  Pipet resin to the 3 ml mark and top with upper bed support.  Flush column with 20-30 mls of water.

7.  0.5 ml of the sample is loaded onto the column.  10 ml of  0.1 M acetic acid  is loaded onto the column.  This is collected in a 20 ml scintillation vial and labeled Fraction A.  It contains the S-alk(en)yl-L-cysteine sulfoxides, or flavor precursors.   14.0 ml of 0.2 M acetic acid  is added to the column and allowed to drip through. This flushes the column.  After that is done, 15.0 ml of 2.0 M acetic acid is loaded and collected as Fraction B.  This contains the g-glutamyl peptides.    10.0 ml of  5.0 M acetic acid is loaded onto the column to remove any compounds still bound to it.  After that has dripped through, flush the column with 30-40 ml of dd water to regenerate the column.  It is now ready for the next sample.  The A and B fractions are placed in the drying rack (see step 8) or into the  -20° C freezer for storage. 

8.  The A and B fractions are blown down in a drying rack.  Upon dryness, the vials are capped and placed in the -20° C freezer.

 

Derivitization of Samples:

9.  The samples are removed from the freezer, rehydrated with 1.0 ml of dd water, and vortexed.
10.  100 µl of the sample is pipetted into a 1.5 ml plastic centrifuge tube with the cap cut off.  The centrifuge tubes are loaded into the centrivap (we use a Labconco centrivap concentrator and cold trap with a Fisher Scientific Maxima C Plus vaccuum pump). 
11.  A 1:1:1 solution of ethanol:water:triethylamine is made up.  This is to counter the acidity of the samples.  250 ml of the 1:1:1 solution is added to each sample.  Samples are again dried in the centrivap.  This should take 2-3 hrs.

12.  A 7:1:1:1 solution of ethanol:water:triethylamine:phenylisothiocyanate is made up.  100 ml of the solution is added to each sample to derivatize the sample.  Each sample is then flushed with nitrogen gas, capped immediatly, and allowed to sit for 20 minutes.  After 20 minutes, the samples are uncapped and placed back in the centrivap.  This should take 1 to 1 1/2 hrs.   Upon dryness, the samples are capped and placed into the  -20° C freezer.   They can be stored in the freezer for up to 30 days.

 

Running the samples on the HPLC:
13.  The samples are removed from the freezer, rehydrated with 1.0 ml of a 2:7 acetonitrile:water solution and vortexed.  The sample is pipetted into the HPLC sample vials.
14.  We use a Waters 2690 HPLC separations module with a 996 Photodiode array detector and an autosampler for analysis.   Our column is a  RP-C18, 5 micron, 250 x 4.6-mm from Applied Biosystems.  The column temperature is maintained at 30°C.   40 mls of sample is injected onto the column and compounds are detected at 254 nm.

15. Buffers used in HPLC analysis are 0.14 M sodium acetate with 0.05% TEA buffered to pH 6.35 with acetic acid and 60% acetonitrile.  Buffers are filtered using a 0.45µm filter before use.

16. The HPLC running conditions are as follows: the   gradient begins with 85% of the sodium acetate buffer, 15% of the acetonitrile buffer and linearly decreases over 21 minutes to a concentration of 45% sodium acetate, 55% acetonitrile. It continues downward to 0% sodium acetate, 100% acetonitrile over the next minute and remains there for 14 minutes before re-equilibrating to the original conditions.  A sample chromatogram of an A fraction  is shown below.

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