Simplified fluorometric assay for diphenylhydantoin in plasma

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Pharmacology

Background:

• Accurate measurement of diphenylhydantoin (DPH) levels in plasma is crucial for therapeutic drug monitoring.
• Existing methods for DPH analysis often require complex extraction steps, increasing assay time and cost.

Purpose of the Study:

• To simplify and validate a direct fluorometric assay for diphenylhydantoin in human plasma.
• To assess the assay's sensitivity, reproducibility, and accuracy in the presence of common co-administered drugs.

Main Methods:

• A simplified fluorometric benzophenone procedure was developed, directly assaying 0.2 ml of plasma.
• Plasma was treated with alkaline permanganate to form benzophenone, followed by heptane extraction.
• Fluorescence was measured in the sulfuric acid layer after shaking with the heptane extract.

Main Results:

• The simplified assay is highly reproducible and sensitive, detecting 1 µg/ml of diphenylhydantoin.
• Results from this fluorometric method showed no significant difference compared to gas-liquid chromatography in 154 plasma samples.
• Assay accuracy was unaffected by the presence of phenobarbital, other anticonvulsants, barbiturates, tranquilizers, or other common drugs.

Conclusions:

• This direct fluorometric assay offers a simplified, rapid, and cost-effective method for diphenylhydantoin quantification in plasma.
• The assay's robustness and accuracy make it suitable for routine therapeutic drug monitoring, even in patients on multiple medications.