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Development and Validation of Bioanalytical Method for Efavirenz in Dried Blood Spot Using High Performance Liquid

Dysaprita Nabila Putri1, Yahdiana Harahap1,2, Febrina Amelia Saputri1

  • 1Faculty of Pharmacy, Universitas Indonesia, Depok, Indonesia.

Journal of Experimental Pharmacology
|April 2, 2026
PubMed
Summary

Developing an improved High-Performance Liquid Chromatography-Photodiode Array (HPLC-PDA) method for quantifying efavirenz in dried blood spots (DBS) ensures accurate therapeutic drug monitoring (TDM). This validated method enhances treatment safety and effectiveness by providing reliable efavirenz concentration measurements.

Keywords:
HIVHPLCdried blood spotefavirenzhigh performance liquid chromatographytherapeutic drug monitoring

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Area of Science:

  • Analytical Chemistry
  • Pharmacokinetics
  • Clinical Diagnostics

Background:

  • Efavirenz requires specific serum concentrations (1-4 µg/mL) for efficacy and to prevent CNS toxicity.
  • Interindividual pharmacokinetic variability necessitates therapeutic drug monitoring (TDM) for efavirenz.
  • Existing dried blood spot (DBS) methods for efavirenz quantification have shown limitations in performance and guideline compliance.

Purpose of the Study:

  • To develop and validate an improved High-Performance Liquid Chromatography-Photodiode Array (HPLC-PDA) method for efavirenz quantification in DBS.
  • To establish a reliable bioanalytical method suitable for routine TDM of efavirenz.
  • To ensure the method meets current bioanalytical validation guidelines.

Main Methods:

  • Optimization of sample preparation including protein precipitation and methanol extraction.
  • Development of chromatographic separation using isocratic conditions and UV detection at 245 nm.
  • Validation of the method according to US FDA (2018) and EMA (2022) guidelines, assessing linearity, accuracy, precision, recovery, carryover, and matrix effect.

Main Results:

  • The method demonstrated excellent linearity (r² = 0.9995) over the 0.1-30 µg/mL range.
  • High accuracy (±9.3%) and precision (CV < 4.6%) were achieved.
  • The method showed acceptable recovery (91.4-95.7%) with no significant carryover or matrix effects, meeting all validation criteria.

Conclusions:

  • The developed HPLC-PDA method provides a sensitive, accurate, and precise approach for efavirenz quantification in DBS.
  • This validated method is suitable for routine therapeutic drug monitoring of efavirenz.
  • The improved method supports enhanced patient safety and treatment effectiveness through reliable drug concentration monitoring.