Quantification of nortriptyline in plasma by HPLC and fluorescence detection

Area of Science:

• Analytical Chemistry
• Pharmacokinetics
• Biomedical Analysis

Background:

• Accurate quantification of nortriptyline (NT) in biological matrices is crucial for therapeutic drug monitoring and pharmacokinetic studies.
• Existing methods may require larger sample volumes or more complex procedures.
• Development of a simple, sensitive, and specific assay is needed.

Purpose of the Study:

• To develop and validate a simple, sensitive, and specific high-performance liquid chromatography (HPLC) method for determining nortriptyline (NT) in plasma samples.
• To establish a reliable method for pharmacokinetic analysis of NT.

Main Methods:

• HPLC with isocratic reversed-phase (C18) chromatography and fluorescence detection.
• Derivatization of NT using 9H-fluoren-9-ylmethyl chloroformate (Fmoc-Cl).
• Analysis of 100 microl plasma samples with a one-step deproteinization and derivatization process.

Main Results:

• Linear calibration curves were obtained over the concentration range of 5-5000 ng/ml.
• The derivatization reaction was rapid (20 min at room temperature) with a stable derivative (≥48 h).
• High sensitivity was achieved with a limit of detection (LOD) of 2 ng/ml and a lower limit of quantification (LLOQ) of 10 ng/ml, with relative standard deviation below 8%.

Conclusions:

• A simple, sensitive, and specific HPLC method for nortriptyline determination in plasma has been successfully developed.
• The method is suitable for pharmacokinetic studies, demonstrated by its application to rat plasma samples after NT administration.
• This assay offers a reliable tool for quantifying nortriptyline in biological samples with minimal sample volume.