OPTIMIZATION AND VALIDATION OF ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY COUPLED WITH TANDEM MASS SPECTROMETRY FOR QUANTIFICATION OF EFAVIRENZ IN HUMAN PLASMA

GAYATRI S.; SURYA M.; KARTHIK R.; VASUNDRA L.

doi:10.22159/ijap.2025v17i6.55345

Authors

GAYATRI S. Sri Ramachandra Faculty of Pharmacy, SRIHER (DU), Chennai-600116, Tamil Nadu, India https://orcid.org/0000-0002-8995-7882
SURYA M. Sri Ramachandra Faculty of Pharmacy, SRIHER (DU), Chennai-600116, Tamil Nadu, India https://orcid.org/0009-0004-9344-6462
KARTHIK R. Bioanalytical and Analytical, Scitus Pharma Services Private Ltd, Thirumazhisai-600124, Tamil Nadu, India https://orcid.org/0000-0002-4963-1204
VASUNDRA L. Sri Ramachandra Faculty of Pharmacy, SRIHER (DU), Chennai-600116, Tamil Nadu, India https://orcid.org/0009-0004-5690-6239

DOI:

https://doi.org/10.22159/ijap.2025v17i6.55345

Keywords:

FDA guidelines, Internal standard, Non-nucleoside reverse transcriptase inhibitor, QC samples

Abstract

Objective: This study presents the optimization and validation of a sensitive, rapid, and reliable ultra-performance liquid chromatography method for the quantification of efavirenz in human plasma, using efavirenz ¹³C₆ as the internal standard.

Method: The liquid-phase extraction technique was used to isolate the internal standard and the analyte from human plasma. The extracted samples were analyzed using chromatography on an Acentis® Express 90 Å C₁₈ column (5 cm x 4.6 mm, 2.7µm), elution carried out using a mixture of ammonium acetate buffer (5 mmol) and acetonitrile as the mobile phase (70:30). The flow rate was maintained at 0.6 ml/min with an injection volume of 5 µl. The ionization mode was negative ion electrospray, with a source energy of 150 °C.

Results: The linearity of the method was confirmed by a correlation coefficient of 0.9979, and the calibration range for efavirenz was found to be 50.00-5000 ng/ml. The precision and accuracy for both intra-day and inter-day measurements showed a %CV of less than 2.3% which is within the acceptable limit. The total run time was 3 min. The mean percentage recovery was 83.9 %.

Conclusion: The developed method offers a significantly reduced run time, enhancing analytical throughput and efficiency compared to previously reported methods with longer durations. This makes it particularly suitable for high-throughput analysis in routine quality control or pharmacokinetic studies.

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