High-performance liquid chromatography determination of clonazepam in plasma using solid-phase extraction

Area of Science:

• Analytical Chemistry
• Pharmacology
• Clinical Chemistry

Background:

• Therapeutic drug monitoring (TDM) of clonazepam via high-performance liquid chromatography (HPLC) traditionally faces challenges with low sensitivity and extensive sample preparation.
• Existing methods often involve labor-intensive liquid-liquid extractions, limiting efficiency and throughput.

Purpose of the Study:

• To develop a rapid and sensitive HPLC method for therapeutic drug monitoring of clonazepam in plasma.
• To overcome the limitations of previous methods by employing a streamlined sample workup procedure.

Main Methods:

• A rapid solid-phase extraction (SPE) technique was utilized, processing 1 ml of plasma.
• Clonazepam and methyl-clonazepam were extracted using C18 SPE columns, eluted with methanol, dried, and reconstituted.
• Chromatographic separation was performed on a RP18 column with UV detection at 306 nm.

Main Results:

• The developed method achieved analytical sensitivity down to 2 micrograms/L.
• Linearity was established over a concentration range of 5-200 micrograms/L with low intra-assay coefficients of variation (1.2% at 200 µg/L, 4.8% at 5 µg/L).
• Results from patient samples showed no statistical difference compared to an established gas chromatographic method, and external quality assessment data were consistently accurate.

Conclusions:

• The new HPLC method with rapid SPE offers improved sensitivity and reduced sample workup time for clonazepam TDM.
• This method provides a reliable and efficient alternative for monitoring clonazepam levels in clinical settings.
• No interference was observed from other benzodiazepines or antiepileptic drugs, ensuring specificity.