Quantitation of Amiodarone in Explanted Human Heart Tissue using MALDI-TOF MS

Andrea Garcia, Catherine M. Bentzley

Department of Chemistry & Biochemistry, University of the Sciences in Philadelphia, 600 South 43^rd Street, Philadelphia, PA 19104

INTRODUCTION

Amiodarone, an iodine-rich, benzofuran derivative, functions therapeutically as a class III anti-arrhythmic drug.¹ (Figure 1). It is used to treat life threatening ventricular arrhythmias including tachycardia and fibrillation by functioning as a beta-blocker. By acting on Na⁺ and K⁺ channels of the myocardial cell, amiodarone prolongs the action potential and refractory period of a heartbeat, thereby reducing the overall heart rate.²

Therefore the development of a method to quantitate amiodarone from a heart transplant patient would prove not only valuable, but also life saving. The goal of this project is to develop a suitable assay using MALDI-TOF to quantitate amiodarone concentrations from a human heart tissue biopsy slide. Upon receipt of a new heart, transplant patients are required to undergo monthly biopsies of the new heart tissue. During this procedure 10-20 milligrams of the new heart are extracted and prepared as a slide for testing. We intend to quantitate amiodarone concentrations from the biopsy slide and eventually determine correlations between amiodarone concentrations and clinical cardiac effects. Only Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) offers the convenience and speed necessary for the detection of amiodarone directly from heart tissue.^4-6

EXPERIMENTAL

Standard solutions of amiodarone (Sigma Chemical Co., St. Louis, MO) ranging in

concentrations from 50 to 1mM were prepared in methanol.

The 5.0 mg/ml solution of the matrix 6-aza-2-thiothymine (Sigma Chemical Co.) was

prepared daily in MeOH.

MALDI solutions were prepared by vortexing a 10gram piece of unmedicated heart tissue with 500 mL of the amiodarone standard and 500 ml of the matrix solution for 15 seconds. The medicated heart tissue was prepared in a similar manner with the exclusion of the amiodarone standard.

MALDI spots were prepared by depositing 8mL of the MALDI solution from an electrospray probe at a flow rate of 2 ml/minute. All samples were prepared and analyzed in triplicate.

A PerSeptives Biosystem Voyager-DE Biospectrometry Workstation (Framingham, MA)

was used to acquire the MALDI spectra.

RESULTS

Analysis of the spectra obtained from the standard solutions showed well-resolved peaks for both amiodarone and ATT at various concentrations. As illustrated in corresponding Figures, the most intense peak from the spectra occurred at 288 m/z, which was characteristic of (2ATT+H) ⁺¹. Amiodarone was assigned a value of 645 m/z. The matrix peak was used as the internal standard and allowed for the production of a calibration curve of amiodarone: the ratio of the relative peak area of amiodarone to matrix was plotted versus the known concentrations of amiodarone in solution. A MALDI spectrum was acquired for the detection of amiodarone from

an explanted, clinical heart. Based upon our standard curve the concentration of amiodarone in the explanted heart was determined as 6.01 mM These results compare favorably to early LC/MS results obtained on the same sample of 7.45mM.

Figure 1 MALDI-TOF Spectrum of 20mM amiodarone solution

Figure 2. MALDI-Tof spectra of 10 mM amiodarone