Solid Phase Extraction (SPE) of Patulin

 

 

 

Equipment and Solutions

 

Waters Oasis Extraction Cartridges (Part # WAT094226)

Plastic Syringes (Luer-lok) – 10.0 ml (Becton Dickinson 10 cc # 904)

Plastic Syringes (Luer-lok) – 5.0 ml (Becton Dickinson 5 cc # 309603)

Sodium Bicarbonate (NaHCO3), 1.0% solution (1 g/100 ml water)

Acetic Acid, 1.0% solution (1.0 ml conc. Acetic Acid/100 ml water)

Acetic Acid, 0.1% solution (0.1 ml conc. Acetic Acid/100 ml water)

10% ethyl acetate in ethyl ether (5 ml ethyl acetate and 45 ml ethyl ether)

Methanol

 

 

Read through the procedure and understand it before attempting to complete.  The procedure must be completed in a timely manner with no prolong time stops.  Patulin will degrade in alkaline conditions (sodium bicarbonate solution) and on a dry support cartridge.  Therefore, time is of the utmost importance.  Complete procedure as fast as possible.

 

 

Method

1.     Test samples at single strength, record the Brix.  Dilute all apple concentrates to 11.5 Brix.

2.     Using a 5 ml plastic syringe, draw up about 2 ml air and then exactly 2.0 ml  of 1.0% sodium bicarbonate solution.  Set this syringe down for latter use.

3.     Using another 5 ml plastic syringe, draw up about 2 ml air and then exactly 2.0 ml of 1.0% acetic acid solution.  Set this syringe down for latter use.

4.     Using a 10 ml syringe, draw up about 4 ml of air followed by 2-3 ml water and attached syringe to top of Water Oasis Cartridge.  Force the water down into the cartridge and out the bottom.  Use the air in the syringe to force all water through the cartridge.  Do this slowly with a drip rate of 2-3 drips per second.  Detach syringe from cartridge.

5.     Draw up about 4 ml air followed by about 2 ml methanol into the same 10 ml syringe.  Again, attach syringe to cartridge and force methanol through at the same drip rate.  Detach syringe from cartridge.

6.     Repeat step 4.  The cartridge is considered “activated” at this step.

7.     Using a different 5 ml syringe, draw up about 2 ml air and exactly 2.5 ml of sample (apple juice).  Attach syringe to top of “activated” cartridge and slowly force juice through cartridge at the rate of approximately 1 drip per second, no faster.  The liquid flowing from bottom of cartridge should be clear at all times.  If it becomes colored, you could be forcing the juice through the cartridge too fast and causing channeling or the cartridge was not properly activated.  If the liquid becomes colored before all the juice is expressed out of the cartridge, start over with another cartridge and sample.  Use the excess air in the syringe to blow the juice through the cartridge.

8.     Immediately without hesitation, attach the 5 ml syringe containing the 1.0% sodium bicarbonate to the top of the cartridge and force the solution through at the rate of approximately 1 drop per second, no faster.  The liquid from the cartridge will be colored.  Blow out the excess solution using the air in the syringe.  Detach syringe.

9.     Immediately without hesitation, attach the 5 ml syringe containing the 1.0 % acetic acid to the top of the cartridge and force the solution through at the rate of approximately 1 drop per second, no faster.  The liquid from the cartridge will be colorless.  Blow out the excess solution using the air in the syringe.  At this point, detach the syringe and draw in about 3 to 5 ml air.  Attach to cartridge and blow out any excess liquid.

10.  Attach cartridge to vacuum apparatus to draw air through cartridge to dry.  This should take about 3 to 5 min.  While drying, label a small vial for sample.

11.  When cartridge is dry, move to hood area and add approximately 1.0 ml of 10% ethyl acetate in ethyl ether solution to cartridge using a disposable glass pipette.  Make sure the cartridge is inserted into the labeled vial prior to adding ethyl ether solution.  Attach a empty 5 ml syringe containing 3 to 4 ml air to top of cartridge.  Very carefully force the solvent through the cartridge into the vial.  Do this at a drip rate less than 1 per second.

12.  Evaporate off solvent under stream of helium or nitrogen until vial is dry.  May need to hold vial in “luke warm” water bath.  Be very careful here.  The boiling point of ethyl ether is 35 C, it is also very flammable.  If there is liquid at the bottom of the vial after 5 to 6 minutes of evaporation, this indicates that the cartridge was not completely dry prior to adding the solvent.  If too much liquid, the sample will need to be extracted again from the start.

13.  Immediately after drying the vial, add 0.5 ml of 0.1% acetic acid solution to vial, cap, and mix vigorously (vortex).  Run HPLC method to determine patulin content.  If cannot test at this time, put vial into a freezer for future testing.  Do not store at room temperature or in refrigerator.

14.  Calculations:  2.5 mls of sample has been concentrated to 0.5 ml final volume.  The value obtained from the chromatogram should be corrected by multiplying by 2, and then dividing by the recovery percentage for a 100 ppb patulin standard in apple juice.  Typical recoveries for the 100 ppb apple juice standard should range from 92 to 98%.  Report results as ppb patulin on a single strength basis or at 11.5 Brix (for concentrates).