Stabilization and quantification of carnosic acid and metabolites in plasma using antioxidant-assisted salting-out extraction coupled with LC-MS/MS.

Carnosic acid (CA), a diterpene abundant in rosemary, is well known for its antioxidant activity but is chemically unstable in plasma. During sample handling it rapidly undergoes autoxidation, which often makes its quantification unreliable. We developed an LC-MS/MS method that combines a simple acetonitrile-NaCl salting-out extraction with the use of antioxidant additives. Extraction conditions-solvent composition, salt ratio, and aqueous pH-were optimized using design of experiments and response surface methodology. In parallel, several antioxidants were tested for their ability to stabilize CA during preparation. The final protocol, using 100% acetonitrile, 100% NaCl, and acidic aqueous phase (pH < 1.5), together with pyrogallol as an additive, provided consistent recoveries above 80% and matrix effects within 15%. Linearity was excellent (r > 0.99) across the calibration range, and the method showed low quantification limits (5 nmol/L for CA; 1 nmol/L for its metabolites). Accuracy and precision met regulatory criteria, and reproducibility was achieved using podocarpic acid as an internal standard. In a small mouse study, the approach was able to follow CA in plasma after oral administration, with a peak observed at 0.25 h. By combining stabilization with a straightforward extraction, this work offers a practical way to quantify CA and related compounds in plasma. The strategy may also be useful for other polyphenols that share similar instability issues and could support future pharmacokinetic or bioavailability studies.