In Situ Thiolysis Procedure – Outline with videos

Safety
Normal lab safety procedures should be followed at all times and in addition any steps that involve Benzyl Mercaptan (BM) must be carried out in a fume cupboard and disposable gloves must be worn. This compound is very pungent and unpleasant; every effort must be made not to contaminate any surfaces or to handle open samples in the lab. Keep all samples in sealed containers, use dilute bleach to destroy BM residues.

Reaction Preparation
1 - Turn on the waterbath

 

2 - Take the reagents out of the fridge and take the magnetic stirrer bars ('fleas') out of the acetone and let them dry.

 

3 - Weigh the samples, you should weigh 180-200 mg into each tube, all samples should be run in duplicate or triplicate. Include a positive control sample and don’t forget to record the weights!

 

4 – Make up the reagent, in this example we mix
20 ml of methanol
10 ml of 3.3% HCl in Methanol*
1 ml of Benzyl Mercaptan

This is enough for 10 reactions (i.e. 5 samples in duplicate)

* 3.3% HCl in Methanol is prepared by taking approx 90 mls of methanol, adding 3.3 mls of concentrated HCl and then making up to 100 ml with more methanol.

 

Start the Reaction
5 - Add a flea (magnetic stirrer bar) and 3 ml of reagent to each tube. Screw the lid down tightly and transfer the tubes to the water bath. Check the sample is being mixed.
Set the timer and leave for 60 minutes.

 

Stop the Reaction
6 - Take the tubes out of the waterbath and add 9 ml of 1% Formic acid (in water) to each tube. Whirlymix and centrifuge (5 minutes at 4,400 rpm).

Finally
7 – Remove approximately 1 ml of the centrifuged sample to a labelled HPLC vial and cap the vial. Load the vials onto the HPLC autosampler and analyse.

 

8 – The LC/MS instructions (with screenshots) are here

Cleaning
The unused reagent and reaction contents are poured into a waste solvent bottle using a sieve to catch the magnetic stirrer bars. The tubes and caps are rinsed with dilute bleach before leaving to soak overnight in more dilute bleach. Finally the tubes are scrubbed with a test tube brush in warm soapy water and then put through the dishwasher.

Thiolysis of extracts and partially purified fractions
The procedure for extracts and fractions is very similar, it is just that much smaller sample sizes are required and the reagent is made up slightly differently. Also different reaction and diluent volumes are used. Full details are available the “current protocols” file.

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THIOLYSIS ON PURIFIED FRACTIONS

Step 1: Weigh 4 mg into a 10 ml tube (make a note of the exact weight!). It is best to run these samples in duplicate (4mg and 8mg) or triplicate (4mg, 6mg and 8mg).

Step 2: Add 1.5 mL of MeOH and 0.5 mL of 3.3% HCl/ in MeOH.

Step 3: Add 50 µL of Benzyl Mercaptan and a magnetic stirrer.

Step 4: Cap and heat tubes (with stirring) at 40 ^oC for 1 hour.

Step 5: Add 2.5 mL of 1% formic acid in water plus 0.5 mL of internal standard (àTaxifolin 0.05 mg/mL).

*Prepare 1mg/ml Taxifolin standard in MeOH and dilute to 0.05 mg/mL)

Alternative Procedure – Premix all the reagents.

5 samples – 7.5 mls MeOH, 2.5 mls 3.3% HCl/ in MeOH, 250 µL of Benzyl Mercaptan, 2.05mls per tube.

10 samples – 15 mls MeOH, 5 mls 3.3% HCl/ in MeOH, 500 µL of Benzyl Mercaptan, 2.05mls per tube.

15 samples – 22.5 mls MeOH, 7.5 mls 3.3% HCl/ in MeOH, 750 µL of Benzyl Mercaptan, 2.05mls per tube.

20 samples – 30 mls MeOH, 10 mls 3.3% HCl/ in MeOH, 1000 µL of Benzyl Mercaptan, 2.05mls per tube.

25 samples – 37.5 mls MeOH, 12.5 mls 3.3% HCl/ in MeOH, 1250 µL of Benzyl Mercaptan, 2.05mls per tube.

If not using an internal standard use 3mls of 1% formic acid in water at the end of the reaction, and use an external standard calibration against taxifolin (a number of taxifolin vials have been prepared and stored in the top shelf of Freezer 4).

Chris Drake September 2014

 

Extraction of free flavanols

Procedure

Sample = finely ground plant material (ball-milled or 1mm screen).

1. Approx 200mg sample weighed accurately into a screw capped tube
2. Add 1.5 mls  MeOH-WFA (70mls methanol, 29mls water, 1mls formic acid)
3. Add a magnetic stirrer bar
4. Incubate in a water bath (at max stirrer speed) at 40C for 1 hour.
5. diluted with 6mls of 1% formic acid (Total volume = 7.5mls)
6. Incubate in a water bath (stirred) at 40C for another 1 hour.
7. Centrifuge and remove 1ml for HPLC analysis.
8. Load HPLC vials on to the autosampler and include a taxifolin standard (vials prepared in freezer).
9. Run the samples using the "CHRIS FLAVANOLS 60C" method with a 5ul injection volume.

Use this template for the results…../../Chris_D/Thiolysis/Protocols%20and%20templates/Thiolysis%20calc%20-%20subtract%20free%20flavanols.xlsx