Capillary electrophoretic enzyme immunoassay for digoxin in human serum

Area of Science:

• Analytical Chemistry
• Biochemistry
• Clinical Chemistry

Background:

• Conventional enzyme immunoassays (EMIT) face challenges analyzing drugs in complex biological matrices like hemolyzed, lipemic, or icteric serum.
• These sample interferences can lead to inaccurate drug level measurements, complicating patient treatment.

Purpose of the Study:

• To investigate the combined use of capillary electrophoresis (CE) and homogeneous enzyme immunoassay for reliable drug analysis in problematic serum samples.
• To develop and validate a method for quantifying digoxin in human serum, overcoming common interference issues.

Main Methods:

• Utilized an FDA-approved EMIT assay for digoxin, followed by CE separation of the enzymatic reaction products (NADH, NAD+) and an internal standard (p-nitrophenol, NP).
• Employed electrokinetic injection into a polyacrylamide-coated capillary and UV detection at 260 nm.
• Optimized CE conditions including electric field strength, capillary coating, and buffer composition (200 mM Tris-borate, pH 7.9, with 0.2% hydroxypropyl methylcellulose).

Main Results:

• Achieved separation of NADH, NAD+, and NP under optimized CE conditions (438 V/cm electric field strength).
• Demonstrated low detection variability (<2%) and high analytical recoveries (98-102%) for spiked serum samples.
• Confirmed no interference from common hemolyzed, lipemic, and icteric factors, as well as various pigmented blood components.

Conclusions:

• The combined CE-EMIT method provides a robust and accurate approach for digoxin analysis in complex serum matrices.
• This technique effectively addresses interferences that affect conventional EMIT assays, offering improved reliability for drug monitoring.
• The method shows potential applicability to other EMIT-based assays for therapeutic drug monitoring in challenging biological samples.