Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Summary

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Abstract

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 µm, C8 100 Å; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Introduction

Cystic fibrosis (CF) is a common genetic disease involving the exocrine mucus glands of the lung, liver, pancreas, and intestines causing progressive multi-organ failure, such as a decline in lung function and pancreatic insufficiency^1,2,3. Ivacaftor (IVA) is the first Food and Drug Administration (FDA-US) and European Medicines Agency (EMA) approved cystic fibrosis trans-membrane conductance regulator (CFTR) potentiator drug, with evidenced clinical efficacy producing a significant improvement in the lung function over placebo in a small subset of CF patients bearing the G55....

Protocol

Approval for ethics was obtained from Monash University Human Research Ethics Committee (MUHREC).

1. Application of the Assay: Patient Sample Collection

1. Record the time when the patient takes their standard dose of either 150 mg ivacaftor or ivacaftor 125 mg /lumacaftor 200 mg.
2. Note down the exact time when the patient blood sample is collected.
   NOTE: We recommend collecting 4-5 samples over a 24 h time course. If the collection of only one sample is possible, the indicated time point is 2.5-4 h post ivacaftor or ivacaftor-lumacaftor combination dosing as C_max concentrations at steady state will be ac....

Results

We have recently reported a method, partially validated to FDA standards, on a triple-quadrupole LC-MS and an HPLC detector system, using a C8 column (5 µm, 3.9 mm x 50 mm i.d.) with the mobile phase consisting of 100% ACN and 0.1% formic acid in water (40:60, v/v) at a flow rate of 1 mL/min. A linear correlation of the peaks was observed over a concentration range from 0.01 to 10 µg/mL in human plasma for all metabolites, ivacaftor, Iva-M1, Iva-M6, and lumacaftor¹⁴. Here this method has been opti.......

Discussion

As previously reported, our group has for the first time developed and validated a HPLC and LC-MS method for rapid detection and quantification of ivacaftor and its major metabolites hydroxymethyl-IVA M1 (active) and IVA-carboxylate M6 (inactive); and lumacaftor in the plasma and sputum of CF patients¹⁴. The assay reported by our group previously was successfully used to quantify the concentration of LUMA, IVA, IVA-M1, and IVA-M6 in the plasma and sputum of CF patients undergoing steady state stan.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IVA (Cat#S114)	SelleckChem (USA).
LUMA (Cat#S1565)	SelleckChem (USA).
IVA-carboxylate (Cat# 510242247CS)	Clearsynth (Canada).
hydroxymethyl-IVA (Cat# 510240849CS)	Clearsynth (Canada).
Methanol (MeOH, LC-MS grade),	Sigma-Aldrich
acetonitrile (ACN, LC-MS grade)	Sigma-Aldrich
formic acid (FA)	Sigma-Aldrich
triple-quadrupole Shimadzu 8030 LC-MS
Phenomenex Kinetex (2.6 µm C8 100 Å; 50 × 2.1mm)
(KrudKatcher Ultrea HPLC In-Line Filter 0.5 m Depth Filter x 0.004inID).
1.5 mL polypropylene microcentrifuge tube (VWR).
Eppendorf Centrifuge 5430
13-mm syringe filter (0.45 µm nylon, GRACE, USA)
[Phenomenex VEREX, 9 mm, PP, 300 µL, PTFE/Silicone septa].