This is the protocol for the sample preparation for the plasma assay. Please note, to ensure their integrity, all samples and standards should be kept at two to eight degrees Celsius during collection and processing. Once handling is completed, store all samples at minus 20 degrees.

For the standard preparation, prepare two independent stock solution of each anilide, ivacaftor, M1 metabolite, M6 metabolite, and lumacaftor in methanol at 100 microgram per milliliter and 10 microgram per milliliter. Please note, the compounds expire after 10 days days on bench top. For the standards, transfer 100 microliters of human plasma into the 1.5 milliliter polypropylene microcentrifuge tubes.

Continue to do so for all your other samples. Then add the internal standard to each tube. For example, we choose 0.01 microgram per milliliters out of the prepared standard.

Now this tube contains plasma spiked with the internal standard. Proceed like this with all other standards. Take your standards and spin it down for a few seconds to make sure that all liquid is at the bottom of the tube.

Continue to do this for all your 40 standards. For the patient samples, transfer 100 microliters out of the patient sample tube into the microcentrifuge tube. The real patient sample is not spiked with internal standard.

Spin the patient sample down for a few seconds to make sure that all liquid is at the bottom of the tube. The next steps are both designed for the samples and the standards. Add 200 microliters of 0.1%formic acid in acetonitrile into each sample tube to precipitate plasma proteins.

Continue to do so for all standards and patient samples. Vortex each sample vigorously for about 15 seconds. Continue to do so for all standards and incurred samples.

Allow to stand for 10 minutes in the fridge to facilitate precipitation. For demonstrating purposes, I will only centrifuge one sample and one standard. We are centrifuging at 10, 000 rpm for 10 minutes on an Eppendorf Centrifuge 5430 at four degrees.

We will now filter the supernatant into the Phenomenex vials. For this, we are using a 13 millimeter syringe filter with 0.45 micrometers nylon Grace filters purchased from USA, and we're filtering into the HPLC vials that can contain 1.5 milliliters. After filtration, we will transfer 100 microliters of supernatant into the LC/MS vials for LC/MS analysis.

This sample is now ready for analysis. Now simply put all your samples into the vial rack. We are using a Shimadzu 8030 LC/MS system, coupled with a triple-quadrupole mass spec.

Let the system equilibrate before analysis. Once equilibrated, which takes about 15 minutes, just press the Start button. For each metabolite, construct a calibration curve by plotting the peak area ratios of anilides versus the nominal concentration of calibration standards.

Use the regression equation for the calibration curve to calculate the concentrations of anilides in the incurred sample. A typical chromatogram is shown in this figure. Optimized chromatographic resolution was obtained using a mobile phase of 100%acetonitrile and 0.1%formic acid in water with a gradient elution on a Phenomenex Kinetex C8 column.

The LC retention times were as follows. For M6, one minute. For M1, 1.3 minutes.

For ivacaftor, 1.55 minutes. For lumacaftor, 2.3 minutes. In all blank plasma samples, no interference with the retention time of either of the anilides was observed, nor was interference detected from the combination of ivacaftor with lumacaftor.

Through the high-throughput analysis of a large amount of patient samples, we have optimized our reported method through the use of a smaller pore size reversed-phase chromatography column Phenomenex C8 column and a gradient solvent system instead of the current isocratic elution. This reduces the running time to six minutes per sample, including the washing phase. This reliable and novel method offers a simple, sensitive, and rapid approach for the therapeutic drug monitoring of ivacaftor and ivacaftor-lumacaftor combination and biological fluids.

Given the need to develop exposure-response relationships to maximize drug efficacy as well as contain healthcare costs, more sensitive tools need to be developed and validated to assist clinicians in using evidence-based therapy and high-throughput analytical techniques for patients suffering from CF.By applying the proposed analytical method to clinical pharmacokinetic and pharmacodynamic studies, the opportunity arises to further investigate the pharmacokinetic parameters of ivacaftor or ivacaftor-lumacaftor combination on a larger patient collective.