2.5mL of the stock was dissolved in HLM working solution to appropriate
concentrations. All working solutions were on ice before TECAN transfer and
dilution. Serial dilutions were conducted by TECAN for all samples. Seven
concentrations for a standard curve and four concentrations (3 LLQ, low,
median, and high) for QC were prepared. Eight concentrations of each positive
inhibitor or test compound were used for determination of IC 50.
The assay was designed to run six compounds (five test compounds and one
positive control) for each CYP enzyme. Two plates were used to determine
IC 50 values for five test compounds. A standard, a positive control and two test
compounds at the highest concentration were manually prepared and spiked
into the last row (H) of each column (1, 3, 5, and 7) in a 2-mL 96-well
preparation plate for serial dilution by TECAN. The HLM solution was
transferred to column 1, 3, 5, and 7 except the row H. The individual test
compounds were diluted serially with HLM to eight concentrations. DMSO
was adjusted to0.16% (v/v) as a final concentration for each diluted sample.
The tip used for dilution was rotated after each transfer of the sample,
aspirated and dispensed three times before use to ensure the sample was well
mixed in each dilution.
After serial dilution by TECAN, 180mL of the mixture in column 1, 3, 5,
and 7 was transferred to an incubation plate in triplicate. After preincubation
at 37C in a 96-well temperature-controlled heater block for 5 min, 20mL
NADPH (10 mM in 100 mM phosphate buffer) was added to the reaction plate
to a final volume of 200mL to initiate the reaction. During the course of
reaction, 240mL of acetonitrile containing internal standard was prepared and
added into a filter plate. After the incubation, 120mL reaction mixture in the
well was transferred into the filter plate for termination of the reaction. One
hundred and eight microliters mixture in the well containing standard sample
and 12mL of NADPH were also transferred into a separate filter plate as a
need for standard calibration and QC. Thus, the filter plates containing
terminated incubation mixtures, standard and QC samples, respectively, was
stacked on a 2 mL 96-well receiver plate, vortexed for 30 s, and the mixtures
were filtered (0.45mm, hydrophobic or hydrophilic PTFE membranes) by a
centrifuge for 5 min at 2000 g, into the receiver plate. The receiver plate was
vortexed and sealed with a polypropylene film, and 10–25mL of each sample
was injected to LC/MS/MS for analysis. Filtration technology was used to
separate soluble metabolites from matrix. A newly 96-well filtration plate with
chemically resistant 0.45mm polytetrafluoroethylene (PTFE) membrane
(Millipore Inc.) is recommended, which processes up to 1.8 mL of aqueous
organic phase and allows for good separation of the metabolites from precipitated
protein. Samples in the 96-well filtration plate can be quantitatively transferred
into a common 96-well collection plate by either vacuum or centrifuge (5 min)
equipped with a plate carrier. The filtered samples are clean and directly injected
into LC/MS from the collection plate with no clog in tubing and needle sprayer.
Thus, the sample variation is low.
522 ANALYSIS OFIN VITROCYTOCHROME P450 INHIBITION