Protocol for In-vitro Permeation Testing Using Strat-M^® Membranes

Most pharmaceuticals and cosmetic actives currently on the market are delivered transdermally or topically via a passive diffusion route. Strat-M^® membranes were developed to mimic this passive diffusion process. This synthetic membrane was designed to provide a non-animal model for transdermal diffusion and safety testing of cosmetic actives, formulations, personal care products, active pharmaceutical ingredients (API), and pesticides and chemicals. We developed a protocol for diffusion testing of caffeine formulations using Strat-M^® membranes. This protocol can be used as a basis for diffusion testing of other compounds using Strat-M^® membranes.

Transdermal diffusion testing of caffeine formulations

Procedure

1. Initial Setup

1. Turn on the circulating water baths.
2. Vacuum filter 500 mL PBS buffer by vacuum filtration and keep under vacuum for at least 30 min.
3. Allow 30 min. for the water baths to reach temperature (37 °C).
4. Ensure that stirring is on for both zones on the Logan instrument by turning the stirring switch on in front of the Logan instrument.
5. Place 25 mm Strat-M^® membrane between the donor and receiver compartment and clamp together.
6. Ensure that the shiny side (skin) of the membrane is facing the donor compartment (top).
7. Fill the receptor compartment with PBS, overflowing through the side tubes to ensure no air bubbles. The receptor compartment platform can be tilted to ensure that there are no bubbles in the compartment.
8. Attach 5 mL syringes filled with PBS to sampling ports. For a particular receptor chamber, the same 5 mL syringe is used during entire experiment.
9. Wait at least 30 min. for PBS in receptor compartment to reach 37 °C.
10. Ensure that no bubbles are generated while waiting for the temperature to reach 37°C. Add buffer as needed if there are air bubbles.
11. Add 500 µL of formulation to donor compartment using a pipettor or appropriate device. Note time.

2. Preparation of Formulations

Caffeine Solution in Propylene Glycol + Oleic Acid

1. First prepare saturated solution of caffeine in propylene glycol by weighing 3 ± 0.1 g of caffeine in a 200 mL glass bottle. Add 150 ± 1 mL propylene glycol (PG). Cap the bottle and place on roller mill for at least 48 hrs at room temperature (19 – 25 °C).
2. Filter solution of caffeine in propylene glycol using Whatman^® Filter Paper # 1 and vacuum filtration assembly.
3. To prepare solution of caffeine in propylene glycol + oleic acid (OA), mix 95 ± 0.4 mL of caffeine solution in propylene glycol with 5 ± 0.1 mL oleic acid in a 200 mL glass bottle and cap.
4. Label the caffeine solution bottle with date prepared, operator name, and date.
5. Storage of donor solution: 90 days from date of preparation at room temperature (19-25 °C).

Receptor Solution (Phosphate Buffered Saline, pH 7.4)

1. Dissolve 1 packet of PBS salt to 1000 mL using Milli-Q^® water. 1000 mL certified Class A volumetric flask is used for preparation of this solution.
2. Label the buffer solution with date prepared, operator name, and date.
3. Storage of buffer solution: 7 days from date of preparation at room temperature (19-25 °C)
4. Volume of receptor solution: 5.0 mL
5. Temperature of receptor solution: 37 °C ± 0.5 °C
6. Stirring: Constant (300 RPM as factory set)
7. Sampling Time: 0, 1, 2, 3, 4, 5, 6, 8, 10, 24, 26 hrs

3. Sampling Protocol

1. Prior to collecting each sample, check for formation of air bubbles.
2. At each sampling time point, remove 5 mL syringe attached to individual chamber and replace it with 3 mL syringe.
3. Withdraw 1.0 ± 0.1 mL of sample from the receptor chamber and discard.
4. Reattach the 3 mL syringes to sampling port and withdraw 500 ± 100 µL of sample for analysis. The same 3 mL syringe is used for sampling from a single cell throughout the experiment.
5. Add sample to deep well plate using a pre-defined layout.
6. Once sample is withdrawn, the original 5 mL syringes are reattached to their respective ports and receptor solution is added back to receptor chamber making sure that no air bubbles are left behind. This operation can be done by tilting the Franz cell set up to ensure that bubbles can be removed completely.

 

* Blank sample: Phosphate buffered saline, pH 7.4 (PBS)

Plate Map
		1	2	3	4	5	6	7	8	9	10	11	12
	Sample time (hrs)	0	1	2	3	4	5	6	8	10	24	26	Blank*
	Cell #
A	1	S0	S1	S2	S3	S4	S5	S6	S8	S10	S24	S26	BL
B	2	S0	S1	S2	S3	S4	S5	S6	S8	S10	S24	S26	BL
C	3	S0	S1	S2	S3	S4	S5	S6	S8	S10	S24	S26	BL
D	4	S0	S1	S2	S3	S4	S5	S6	S8	S10	S24	S26	BL
E	5	S0	S1	S2	S3	S4	S5	S6	S8	S10	S24	S26	BL
F	6	S0	S1	S2	S3	S4	S5	S6	S8	S10	S24	S26	BL
G	Standard, µg / mL	0.5	2.5	5.0	25.0	50.0	250						BL
H	Blank	BL	BL	BL	BL	BL	BL	BL	BL	BL	BL	BL	BL

4. Preparation of Caffeine Solution for HPLC (calibration curve)

1. Weigh 50 ± 1 mg of caffeine using analytical balance and transfer into glass vial.
2. Note down actual weight of caffeine in the batch record.
3. Add 10 mL methanol to make a stock solution of caffeine.
4. Actual concentration of caffeine stock solution [µg/mL = Actual weight of caffeine (µg)
5. Actual volume of methanol (mL)]
6. Label the bottle with date prepared, name of operator, and date.
7. This stock solution can be stored at 4 °C in a refrigerator for 7 days.
8. Dilute the stock solution as per following protocol using PBS buffer to prepare various standards of caffeine solution.
9. Based on the actual weight of caffeine weighed, actual concentration of standards is calculated and recorded in the batch record. Once the standards are prepared, the following standards are used for generating calibration curve.

*Actual caffeine concentration will depend on the weight of caffeine dissolved in methanol
Concentration of caffeine in solution 1 (µg/mL) = Concentration of caffeine stock solution / 2
Concentration of caffeine in solution 2 (µg/mL) = Concentration of caffeine stock solution / 10
Concentration of caffeine in solution 3 (µg/mL) = Concentration of caffeine solution 1 / 10
Concentration of caffeine in solution 4 (µg/mL) = Concentration of caffeine solution 2 / 10
Concentration of caffeine in solution 5 (µg/mL) = Concentration of caffeine solution 3 / 10
Concentration of caffeine in solution 6 (µg/mL) = Concentration of caffeine solution 4 / 10
Concentration of caffeine in solution 7 (µg/mL) = Concentration of caffeine solution 5 / 10
Concentration of caffeine in solution 8 (µg/mL) = Concentration of caffeine solution 6 / 10

Final caffeine concentration, µg/mL		Solution number	Caffeine solution (Volume)	PBS buffer	Used for calibration
Specified	Actual
2500 µg/mL	*	1	0.5 mL (Stock Solution)	0.5 mL	No
500 µg/mL	*	2	0.1 mL (Stock Solution)	0.9 mL	No
250 µg/mL	*	3	0.1 mL (Solution 1)	0.9 mL	Yes
50 µg/mL	*	4	0.1 mL (Solution 2)	0.9 mL	Yes
25 µg/mL	*	5	0.1 mL (Solution 3)	0.9 mL	Yes
5 µg/mL	*	6	0.1 mL (Solution 4)	0.9 mL	Yes
2.5 µg/mL	*	7	0.1 mL (Solution 5)	0.9 mL	Yes
0.5 µg/mL	*	8	0.1 mL (Solution 6)	0.9 mL	Yes

5. HPLC Analysis of Samples

*ACN = Acetonitrile

Chemical	Column Used	Mobile Phase	Flow Rate	Detection Wavelength	Injection Volume
Caffeine	Agilent Eclipse XDB, 4.6 mm X 150 mm, 5 µm (# 993967-902)	Water with 0.1% formic acid + ACN* with 0.1% formic acid (80:20 v/v)	1 mL/min	272 nm	10 µL

Equipment and Supplies

Product	Vendor	Part number
Franz cell instrument (Logan)	Logan	Model FDC-6T
Temperature controller	Logan	VTC-200
Assorted beakers for chemical transfer	Fisher Scientific or equivalent	Class A
Assorted graduated cylinders for chemical transfer	Fisher Scientific or equivalent	Class A
Assorted Volumetric Flasks for preparation of buffers / solutions	Fisher Scientific or equivalent	Class A
Lab balance	Mettler Toledo®	Model PG5002-S or equivalent
Plastic disposable transfer pipettes	Fisher Scientific or equivalent	Class A
Digital Pipettor (100 – 1000 µl)	Gilson or equivalent	H8315780 or equivalent
Digital Pipettor (20 – 200 µl)	Eppendorf® or equivalent	3530085 or equivalent
Filter Paper (# 1)	Whatman®	1001-042 or equivalent
Syringes - 3 mL	Fisher Scientific	14-823-435 or equivalent
Syringes - 5 mL	Fisher Scientific	S30467-6 or equivalent
Stericup® sterile filtration unit with 0.2 µm PVDF membrane	Millipore®	SCGVU05RE
Deep Well 96 well plate	Fisher Scientific or equivalent	12565606 or equivalent