Gastric

Perspiration

Composition of fluid

Reagent

mL/L

Reagent

g/L

Hydrochloric acid (30%)

7.41

Sodium chloride

Urea

Lactic acid (90%)

Concentrated ammonia until a stable pH of 6.5 ± 0.1 was reached.

5.0

1.0

1.06

pH

1.5 ± 0.1

6.5 ± 0.1

Temp (˚C)

37 ± 1

30 ± 1

Loading (g/L)

0.2

2

Time (hours)

2

24, 72, 120 and 168*

Agitation rate (rpm)

171

No agitation

Protocol overview

One borosilicate Erlenmeyer flask of 250 ml was used for the blank control. In the test vessels, 10 mg of test item was weighed in triplicate into three separate 250 ml Erlenmeyer flasks. 50 mL of extraction fluid was added to each test item and blank control vessel. After swirling the flasks to mix the test item and the medium, and adjusting the pH, the flasks were covered with a screw cap and placed in a temperature controlled orbital shaker at an agitation rate of 171 rpm (revolutions per minute) for one hour. Flasks were allowed to sit without agitation for one additional hour before sampling.

One hundred mg of test item was weighed into twelve separate 250mL Erlenmeyer flasks, three replicate flasks for each sampling time (i.e. at 24, 72, 120 and 168 hours). 50mL of extraction fluid was added to each test item and blank control vessel. After swirling the flasks to mix the test item and the medium, and adjusting for pH, the flasks were covered with a screw cap and placed in a temperature controlled orbital shaker without agitation for 24, 72, 120 and 168 hours.

A syringe was used to remove a 15 ml sample from each test vessel at a depth of two third of the supernatant. The samples were filtered through a 0.1 µm or 0.2 µm syringe filter** and transferred to tubes for storage of less than one month.