Parallel Artificial Membrane Permeability Assay (PAMPA)

Product List

Equipment

• 96-well Teflon block acceptor plate (Millipore MSSACCEPT0R)
• 96-well 0.45um Teflon membrane donor plates (Millipore MAIPNTR10 or MAIPN4550)
• 96-well plates for HPLC-MS sampling (optional)
• 96-well plate seals (heat or sticky aluminum foil) (optional)
• Deep-well (~2mL) 96-well plate (optional)
• Development chamber (e.g. desiccator chamber without desiccant)
• 12-channel pipette (50-300 uL) (optional)
• Many extra tips, pre-racked
• Multichannel pipette solvent wells

Chemicals

• Analytes, 200uM in DMSO
• Lecithin (reasonably high purity is important; 90% is affordable and satisfactory)
• Dodecane (99+% purity)
• 1X PBS pH7.4 buffer
• Methanol
• DMSO
• Propranolol, 200uM in DMSO (optional)

Process

Overview

1. Prepare solutions
2. Prepare membrane plate
3. Load sample plates
4. Run assay
5. Prepare analytical samples
6. Analyze / Calculate

Notes

The PAMPA assay is standardized to use 5% DMSO in all solutions, increasing ease and efficiency for DMSO stocks.
Target Concentration: 1uM (Orbitrap) or 10uM (LTQ)
Sample Limit: (quadruplicate): 24 compounds per 96-well plate
Multichannel recommended (but not necessary)
Permeability Standards:

Compound	Pe (x10^-6)	Std Dev	Comments
Propanolol	?	?	Polar, might not elute in certain chromatography methods
Progesterone	7.0	2.4
Carbamazepine	9.0	0.5	also useful as hydrocarbon partition coefficient standard
1NMe3	3.0	1.0
CSA	0.5	0.2

There are two periods of time where the assay is time sensitive—reserve some undistracted time:

• Everything between preparing the donor plate membrane, and starting the assay.
• Transferring solutions from the finished assay to sealed plates/vials for analysis.

It can be very helpful to create a plate layout spreadsheet with exact masses annotated.

Prepare Solutions

Prepare target analyte (typically from a stock concentration in DMSO) to a dilution of 1-10 uM in a buffer of 1X PBS pH 7.4 and 5% DMSO.

• If analyte is 200uM in DMSO; add 50uL to 950uL of 1X PBS.
• Preparing these in deep-well plates makes later transfer (with a multichannel pipette) easier
• Mix well with pipette
• Be sure to calculate enough volume for preparing the recovery plate from this stock!

Prepare a 1% lecithin in dodecane solution in an Eppendorf tube, and sonicate until fully mixed (~5 min).

• 10mg of lecithin in 1mL of dodecane
• Always prepare fresh

Prepare Membrane Plate

Inspect membranes for tears
Using a pipette (single or multichannel), gently dispense 5uL of 1% lecithin / dodecane solution onto donor plate membrane

• Turn plate “upside-down” for ease of tip access
• Gently apply single 5uL drops of solution onto the membrane
• Hang drop from tip right above membrane, then expel air to release drop
• Do not touch the membrane with tip!
• Multichannel pipettes can be used, but time efficiency is not urgent here
• Ignore any observational disparities between membranes; as long as it’s not a tear, the results work out consistently
• Let the plate sit for at least 10 minutes (while preparing other samples) for the lipids to adsorb fully

Load Sample Plates

Using a multichannel pipette and solvent reservoir, dispense 300 uL of 1X PBS 5% DMSO buffer into the acceptor plate wells

• Use good pipette technique; volume disparities will influence results
• Cover the plate with a loose cover to minimize evaporation

Using a multichannel pipette, transfer 150 uL of the 10uM analyte in 1X PBS 5% DMSO into the donor plate wells

• Gently dispense solutions into the donor plate, running the solution down the side of the well to minimize membrane disturbance
• Use good pipette technique; volume disparities will influence results
• Important: save (and seal) source analyte stock for recovery analysis!

(Optional) Reverse technique

• The analyte can be applied to the larger Teflon-block plate instead (requiring a larger volume of analyte!) in order to double the signal from the membrane plate.
• Useful for especially-dilute analytes, or slowly-permeating analytes
• Adjust volumes appropriately in the calculations

Run Assay

Gently lower the donor plate wells into the acceptor plate wells.

• Confirm the absence of any bubbles; these will impair PAMPA performance
• Gently lift and re-lower the donor plate if observed
• Place a plastic cover over the whole assembly (not airtight, just protective)
• Record current time / start a timer

Gently transfer the assembly to a moist separate chamber (not necessarily airtight)

• Wet some paper towels, to lie over and under the assembly
• Temperature control has been shown to be important for results, but we do not bother

Allow assembly to sit for 10-20 hr

• 14-16 hr is standard; adjust for faster- or slower-permeating analytes
• The PAMPA Pe calculations account for assay duration

Gently separate the plates

• Slowly, to minimize any contamination in the acceptor plate (very full wells)
• Record total time of immersion
• Immediately begin sample transfer

Prepare Analytical Samples

Repeat for the acceptor plate, donor plate, and source stock 10 uM deep-well plate (“recovery plate”):
Transfer 100 uL from each well to a labeled 96-well plate for HPLC-MS injection.

• Use good pipette technique
• Mix well with pipette sparging!
• Use good pipette technique
• Switch tips between transfers
• Seal the plate immediately upon finishing the transfer (Plate sealer, or sticky aluminum foil)

Analyze / Calculate

Analyze

Analyze all simples within 24-48 hr after running the assay

• There is anecdotal evidence that compounds decompose or crash out, or that solvent levels change

Create an appropriate HPLC-MS method; suggestions:

• 3-10 min total time for efficiency and generality
• 1-1.5min high-polarity wash (to waste; not to MS) to remove PBS buffer
• Ramp up to (and hold at) 100% acetonitrile, ensuring good elution of analyte (and injection standard)
• Consider using single ion monitoring to increase sensitivity, and ease analysis.

Consider frequent rinse methods (methanol or DMSO)

• Minimize analyte carry-over
• Reduce analyte precipitation in the injection loop (a frequent problem with the polar PBS wash)
• Ensure that wash solvent is appropriate to solubilize analyte and PBS buffer

Avoid sequential injection of compounds with the same mass in sequence to avoid carry-over interference
Quantify TIC intensity; not UV absorbance

Calculate

Parameters

Active surface area of membrane (mm2)	Msa = 240
Volume of acceptor well (uL)	Va = 300
Volume of donor well (uL)	Vd = 150
Assay run time (s)	Ts
Donor intensity	Id
Acceptor intensity	Ia
Recovery intensity	Ir

Calculations

• Analyte equilibrium

(1)

\begin{align} AnalyteEquil =\frac{(Ia*Va)+(Id*Vd)}{Va + Vd} \end{align}

• %Transmittance; discard data if >95%

(2)

\begin{align} T = \frac{Ia}{AnalyteEquil} \end{align}

• %Recovery; low values represent material loss (aggregation or adherence)

(3)

\begin{align} R =\frac{(Ia*Va)+(Id*Vd)}{Ir*Vd} \end{align}

• constant

(4)

\begin{align} C =\frac{Vd*Va}{(Vd+Va)*Msa*Ts} \end{align}

• Pe (x10^-6), often displayed in log scale

(5)

\begin{align} P_e = (-C * \ln(1-T)) * 10^{-6} \end{align}