article · Biomedical Chromatography
A sensitive liquid chromatography-tandem mass spectrometry method has been developed and fully validated to measure sofosbuvir and daclatasvir simultaneously in human blood plasma. These antiviral drugs form a dual therapy used to treat hepatitis C genotype 3 infections over a sixteen-week period. The procedure isolates both medications alongside an internal standard using liquid-liquid extraction with methyl tert-butyl ether, followed by separation on a specialised column and detection through a triple quadrupole mass spectrometer. Validated in line with regulatory bioanalytical standards, the assay demonstrated strong linearity, precision, accuracy, and stability across clinically relevant concentration ranges. The protocol has already been tested in human pharmacokinetic analyses, demonstrating high reproducibility and ruggedness for monitoring drug levels in patient samples.
Combination therapy using sofosbuvir and daclatasvir provides an effective treatment for hepatitis C genotype 3. Accurately tracking both medications simultaneously in patient blood is essential to verify proper drug exposure, ensure patient safety, and evaluate treatment efficacy. A reliable combined testing protocol simplifies clinical laboratory analysis, supporting therapeutic monitoring and the assessment of bioequivalence in drug development.
This bioanalytical method is an applied and tested laboratory tool ready for immediate use by contract research organisations, diagnostic laboratories, and pharmaceutical companies running clinical trials. It enables efficient quantification of two frontline antivirals to support therapeutic drug monitoring and bioequivalence studies. Because the protocol has already been validated against formal regulatory standards and applied in pharmacokinetic testing, adoption requires routine laboratory transfer rather than basic research development.
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A simple and highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical method was developed and fully validated for the first time for the simultaneous determination of newly discovered antiviral drugs, namely sofosbuvir (SOF) and daclatasvir (DAC) in human plasma. Tadalafil (TAD) was used as internal standard (IS). SOF, DAC and TAD (IS) were extracted from plasma using liquid-liquid extraction technique with methyl tert-butyl ether. The chromatographic separation was carried out using ZorbaxSB-C<sub>18</sub> column (4.6 × 50 mm,5 μm) and 5 mm ammonium formate buffer (pH 3.5)-acetonitrile (50:50, v/v) as mobile phase in an isocratic elution mode pumped at a flow rate 0.7 mL min<sup>-1</sup> . The quantitation was performed on API4500 triple quadrupole tandem mass spectrometer with positive electrospray ionization interface in multiple reaction monitoring mode. Validation was applied according to US Food and Drug Administration guidelines for bio-analytical methodswith respect to linearity, precision, accuracy, selectivity, carry-over, stability and dilution integrity. Linearity was obtained over concentration ranges of 0.3-3000 and 3-3000 ng mL<sup>-1</sup> for SOF and DAC, respectively, by applying a weighted least-squares linear regression method (1/x<sup>2</sup> ). The proposed method could be applied successfully in bioequivalence and/or clinical studies for therapeutic drug monitoring of patients undergoing dual combination therapy as the latter combination proved more efficacious and powerful tool for the complete treatment of hepatitis C genotype 3 within 16 weeks. The suggested method has been applied successfully to pharmacokinetic studies with excellent assay ruggedness and reproducibility.
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DOI: 10.1002/bmc.4186
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