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Purpose <p>Ticagrelor (TGR), a P2Y12 receptor inhibitor indicated for acute coronary syndromes, was chosen for establishing and validating a cost-effective RP-HPLC method. Employing a 3<sup>2</sup> full factorial design, the analytical conditions were optimized and validated for the quantification of the analyte in rat plasma, highlighting the method’s cost-effectiveness, robustness, and suitability as a practical alternative to LC–MS/MS for pharmacokinetic applications in resource-limited laboratories.</p> Methods <p>Chromatographic conditions were optimized on a C18 column employing an isocratic elution system comprising trifluoroacetic acid buffer, acetonitrile, and methanol (46:48:6, v/v/v) at a flow rate of 1.0 mL/min. UV detection was carried out at 278&#xa0;nm, with verapamil serving as the internal standard. Validation followed ICH M10 guidelines, and the method was applied to a rat pharmacokinetic study.</p> Results <p>The method yielded well-resolved, symmetrical peaks with high separation efficiency, with TGR eluting at 14.2&#xa0;min. A linear response was obtained in the range of 100–4000 ng/mL (R² = 0.9938 ± 0.0006). The sensitivity parameters showed a detection limit of 15.6 ng/mL and a quantification limit of 47.3 ng/mL. The validated LLOQ was 100 ng/mL. Accuracy ranged between + 0.47% and + 2.81%, while intra- and inter-day precision remained below 6.16% (%RSD 3.00 to 4.05%). Recovery was high (88.91 ± 2.92%), and matrix effects were minimal (&lt; 2.17%). Stability was confirmed under freeze–thaw, benchtop, autosampler, and long-term storage conditions. Pharmacokinetic evaluation in rats showed a C<sub>max</sub> of 428.56 ± 42.86 ng/mL, t<sub>max</sub> of 1.75 ± 0.27&#xa0;h, and AUC<sub>0–t</sub> of 2040.79 ± 204.08 ng·h/mL.</p> Conclusion <p>The developed factorial design–optimized RP-HPLC method demonstrated high sensitivity and precision, providing an economical tool for pharmacokinetic studies when LC–MS/MS is unavailable.</p> Graphical Abstract <p></p>

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Design of Experiments-Based RP-HPLC Bioanalytical Method Development and Validation for Ticagrelor Quantification in Rat Plasma with Pharmacokinetic Application

  • 期刊论文

  • 发表日期：

    2025年12月20日

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  • Mariyambibi A. Mandarawala,
  • Ashok N. Mahajan,
  • Shaileshkumar K. Koradia,
  • Moinuddin M. Soniwala,
  • Sohel A. Punasiya,
  • Dimal A. Shah,
  • Falgun A. Mehta

摘要

Purpose

Ticagrelor (TGR), a P2Y12 receptor inhibitor indicated for acute coronary syndromes, was chosen for establishing and validating a cost-effective RP-HPLC method. Employing a 32 full factorial design, the analytical conditions were optimized and validated for the quantification of the analyte in rat plasma, highlighting the method’s cost-effectiveness, robustness, and suitability as a practical alternative to LC–MS/MS for pharmacokinetic applications in resource-limited laboratories.

Methods

Chromatographic conditions were optimized on a C18 column employing an isocratic elution system comprising trifluoroacetic acid buffer, acetonitrile, and methanol (46:48:6, v/v/v) at a flow rate of 1.0 mL/min. UV detection was carried out at 278 nm, with verapamil serving as the internal standard. Validation followed ICH M10 guidelines, and the method was applied to a rat pharmacokinetic study.

Results

The method yielded well-resolved, symmetrical peaks with high separation efficiency, with TGR eluting at 14.2 min. A linear response was obtained in the range of 100–4000 ng/mL (R² = 0.9938 ± 0.0006). The sensitivity parameters showed a detection limit of 15.6 ng/mL and a quantification limit of 47.3 ng/mL. The validated LLOQ was 100 ng/mL. Accuracy ranged between + 0.47% and + 2.81%, while intra- and inter-day precision remained below 6.16% (%RSD 3.00 to 4.05%). Recovery was high (88.91 ± 2.92%), and matrix effects were minimal (< 2.17%). Stability was confirmed under freeze–thaw, benchtop, autosampler, and long-term storage conditions. Pharmacokinetic evaluation in rats showed a Cmax of 428.56 ± 42.86 ng/mL, tmax of 1.75 ± 0.27 h, and AUC0–t of 2040.79 ± 204.08 ng·h/mL.

Conclusion

The developed factorial design–optimized RP-HPLC method demonstrated high sensitivity and precision, providing an economical tool for pharmacokinetic studies when LC–MS/MS is unavailable.

Graphical Abstract