Physiologically based extraction test
(PBET)
Chemicals:
Gastric fluid:
• Pepsin
• Citric acid
• L(-)-Sodium malate
• Acetic acid
• Lactic acid
• Concentrated HCl
Small intestinal fluid:
• Pancreatin
• Bile salts
• Saturated solution of NaHCO3
Equipment:
• Incubator (rotator in oven with temperature 37 °C)
• Water bath 37 °C
• Centrifuge
• Vials
• Filters
Procedure:
Day before test
• Weight reagents and samples, store in the fridge
• Turn on incubator
Procedure
• Preparation of gastric fluid solution (40 ml for sample, the amount is for 0.5 dm3
of fluid)
• Mix in the beaker on magnetic stirrer these reagents:
o 450 ml MQ water
o 0.625 g pepsin
o 0.25 g citric acid
o 0.25 g L(-)-Sodium malate
o 0.25 ml acetic acid
o 0.21 ml lactic acid
• Add concentrated HCl for pH adjusting on 2.5 (add 0.6 ml and then slowly step by step add
0.05 ml).
• Transfer to volumetric flask and add water to volume 0.5 dm3
.
• Transfer gastric fluid to the vial and put to the water bath (37 °C) for 30 minutes.
Preparation of gastric phase extract
• 0.4 g dust + 40 ml gastric fluid
• Put into the incubator for 60 minutes
• Check and adjust pH in 15 and 45 minutes
• Take 8 ml of extract to conical vials, centrifugate for 10 min (3 000 rpm), filtrate to the vials
and store in the fridge until solvent exchange
Preparation of small intestinal phase extract (the rest extract 32 ml)
• Adjust pH on 7 with saturated solution of NaHCO3 (normally 1.2 ml, but in some cases of our
dust samples it should be much more – 4 ml)
• Add reagents:
o 20 mg pancreatin
o 70 mg bile salts
• Put into the incubator for 4 hours (no pH adjusting is needed)
• Centrifugate for 10 min (3 000 rpm), filtrate to the vials and store in the fridge until solvent
exchange
Solvent exchange
Chemicals:
• NaCl
• Acetic acid
• Dichloromethane
• Na2SO4
Equipment:
• Vials
• Pasteur pipettes
Procedure:
8 ml gastric phase extract
• Add:
o 0.3 g NaCl
o 0.03 ml acetic acid
o 2 ml DCM
• Shake 3 min
• Collect DCM fraction
• Add 2 ml DCM and shake 3 min
• Collect DCM fraction
• Dehydration by Na2SO4, filtrate to the vials and set the volume to 10 ml and store in the
fridge until clean-up
32 ml gastric and intestinal phase extract
• Add:
o 1 g NaCl
o 1 ml acetic acid
o 4 ml DCM
• Shake 3 min
• Collect DCM fraction
• Add 4 ml DCM and shake 3 min
• Collect DCM fraction
• Dehydration by Na2SO4, filtrate to the vials and set the volume to 10 ml and store in the
fridge until clean-up
Clean-up for flame retardants
Chemicals
• cleaned activated silica gel (activation for 12 hours at 150 ° C)
• cleaned non-activated silica gel
• sulfuric acid modified silica gel (22 ml concentrated H2SO4 + 50 g activated silica)
• hexane, DCM, nonane
• internal standards
Equipment
• glass column, internal diameter 1 cm
• 20 ml vial
• Pasteur pipette, cotton
Procedure
• prepare the separation column:
o put a cotton on the bottom of the column
o add about 1 cm high layer of cleaned activated silica gel, then 5 g of activated sulfuric
acid-modified silica gel
o gently tap the column with the stick
o load 1-2 cm layer of non-activated silica gel at the top of the column, and then tap
again
• transfer the sample to the column, wash the original vial at least 2x with 1 ml
dichloromethane, add to the sample in the column
• elute with 30 ml of 50% dichloromethane in hexane into a 20 ml vial
• concentrate the sample under a gentle stream of nitrogen to 500 μl
• transfer the sample to a minivial, add 40 μl of nonane and evaporate to the final volume
of 40 μl
• add internal standards
• carefully close the minivial and store it in the refrigerator
Determination of analytes by GC-MS
Analyte determination will be done by GC-MS.