Rapid Quantitation of Digoxin in Human Plasma and Urine Using Isotope Dilution Liquid Chromatography‐Tandem Mass Spectrometry

Abstract

A rapid LC/MS/MS assay to quantitate digoxin in human plasma and urine has been developed and validated using a Packard liquid handling systems‐Multiprobe^® II AMP8E1. Following automatic solid phase extraction on Waters Oasis HLB 30 mg 96 plates, digoxin and its internal standard (Digoxin D3) were analyzed by reverse phase chromatography (LUNA C₁₈, 5 µm, 150×2 mm), and introduced into the mass spectrometer (Sciex ‐ API 3000) via the turboIonspray ion source operating in positive mode. The assay was validated for human plasma and human urine over a concentration range of 0.2–20 ng/mL and 1–100 ng/mL, respectively, using 0.5 mL of sample. The between day and within day coefficients of variation for all matrices were <17% at the concentrations of lower limit of quantification (LLOQ), and <13% at other quality control concentrations. The average recovery was 80.3% from plasma and 74.3% from urine. No matrix effect was observed. Freeze‐thaw stability, stability of digoxin in matrix, and stability of extracted samples were also evaluated. The mean concentration after three freeze‐thaw cycles was within±3.2% of the nominal value in plasma and within±11.4% of the nominal value in urine. After storage for 4 hours at room temperature, greater than 97.2% of digoxin remained in plasma and 96% remained in urine. Digoxin in extract was stable in the autosampler at+5°C for 77 hours in plasma and 80 hours in urine.

Keywords:

• Digoxin
• Mass spectrometry
• Human plasma
• Human urine
• High performance liquid chromatography

Acknowledgments

The authors thank M. Di Ioia for the review of the manuscript and Ms. K. Fredon and B. Dupraz for their technical assistance.