| DOI: 10.29090/psa.2025.03.24.2970 | Pharm Sci Asia 2025; 52(3), 335-348 |
Development and validation of a highly specific for quantification of irbesartan in human plasma and its application to a bioequivalence studyPiyapat Pongnarin, Pinpilai Jutasompakorn, Weerawadee Chandranipapongse, Supornchai Kongpattanakul, Somruedee Chatsiricharoenkul*.
- Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
A simple LC-MS/MS method was developed for the determination of irbesartan in human plasma with high sensitivity and specificity. The extraction process utilized a liquid-liquid extraction technique with a mixture of ethyl acetate and hexane (90:10, v/v), achieving highly efficient recovery of irbesartan. Chromatographic separation was performed on a Luna? HST C18 column (50 mm x 3 mm, 2.5 ?m), using a mobile phase consisting of 0.1% aqueous formic acid and acetonitrile (33: 67, v/v) at flow rate of 0.2 mL/min, with a total runtime of 4.0 minutes. Irbesartan was detected by tandem mass spectrometer with positive ionization mode using the multiple reaction monitoring (MRM). The mass transition ion-pairs were m/z 428.95>206.96 and 428.95>195.01 amu for [Irbesartan+H]+ and m/z 435.98>234.97 and 435.98>291 amu for [Valsartan +H]+ (internal standard, IS), with, retention times of 1.44 and 2.24 minutes respectively. Key parameters of full validation were evaluated, and all consistently met the acceptance criteria. The limit of detection (LOD) and lower limit of quantification (LLOQ) for irbesartan were determined to be 60 pg/mL and 5.00 ng/mL, respectively. The method demonstrated linearity over concentration ranges of 5.00 - 6012.62 ng/mL with a correlation coefficient (r) consistently greater than 0.997(n = 3) using 1/X2 weighting. The within-run precision ranged from 2.43% to 7.61% and with accuracy from 92.42 to 106.20%. The between-run precision ranged from 4.73% to 8.66% with accuracy from 98.56%to 101.20%. Additionally, we investigated the effects of different plasma conditions, including hemolysed and hyperlipidemic plasma, on accuracy and precision. The results demonstrated that all measured values fell within acceptable tolerance limits. The relative recovery of irbesartan was determine to be 80.34%, 75.32%, and 74.26% for the LQC, MQC and HQC levels, respectively, while the IS demonstrated a relative recovery of 76.93%. The matrix effect exhibited no significant interference, as evidenced by the comparison of peak responses from six determinations at LQC and HQC levels, prepared in extracted drug-free human plasma obtained from six individual normal plasma sources, with those of neat standards at the corresponding concentrations. This ensures the reliability of the quantification. The calculated matrix factor values were 0.91 for LQC and 1.00 for HQC for irbesartan, while the matrix factor for valsartan was 0.92. Moreover, the IS-normalized matrix factor values were 0.99 for LQC and 1.09 for HQC for irbesartan. In addition, no significant matrix effect was observed when hemolysed and hyperlipidemic plasma samples were analyzed. Stability studies confirmed that irbesartan remained stable in human plasma under various conditions. It was stable for up to 29 days at -80?C for long term storage, up to 24 hours at 4?C and up to 6 hours at room temperature (25?C). The new LC-MS/MS method exhibit high sensitivity, specificity, and a broad wide linearity range, (5 to 6000 ng/mL) with a short run time of 4.0 minutes using valsartan as an internal standard. The method was developed and validated using a single-step liquid – liquid extraction requiring only 100 ?L of plasma. This positive ESI-LC-MS/MS method is simple, reproducible and robust, allowing high-throughput analysis with a large sample capacity per batch. It was successfully applied to the quantification of irbesartan in human plasma for bioequivalence studies of higher strength of irbesartan tablet (300 mg) in Thai volunteers.
Keyword:
irbesartan, LC-MS/MS, Method development and validation, bioequivalence, liquid-liquid extraction
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