Automated 96-well solid phase extraction and hydrophilic interaction liquid chromatography–tandem mass spectrometric method for the analysis of cetirizine (ZYRTEC ®) in human plasma—with emphasis on method ruggedness

Abstract

A high-throughput bioanalytical method based on automated sample transfer, automated solid phase extraction, and hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) analysis, has been developed for the determination of cetirizine, a selective H 1-receptor antagonist. Deuterated cetirizine (cetirizine-d 8) was synthesized as described and was used as the internal standard. Samples were transferred into 96-well plates using an automated sample handling system. Automated solid phase extraction was carried out using a 96-channel programmable liquid-handling workstation. Solid phase extraction 96-well plate on polymer sorbent (Strata X) was used to extract the analyte. The extracted samples were injected onto a Betasil silica column (50 × 3, 5 μm) using a mobile phase of acetonitrile–water–acetic acid–trifluroacetic acid (93:7:1:0.025, v/v/v/v) at a flow rate of 0.5 ml/min. The chromatographic run time is 2.0 min per injection, with retention time of cetirizine and cetirizine-d 8 both at 1.1 min. The system consisted of a Shimadzu HPLC system and a PE Sciex API 3000 or API 4000 tandem mass spectrometer with (+) ESI. The method has been validated over the concentration range of 1.00–1000 ng/ml cetirizine in human plasma, based on a 0.10-ml sample size. The inter-day precision and accuracy of the quality control (QC) samples demonstrated <3.0% relative standard deviation (R.S.D.) and <6.0% relative error (RE). Stability of cetirizine in stock solution, in plasma, and in reconstitution solution was established. The absolute extraction recovery was 85.8%, 84.5%, and 88.0% at 3, 40, and 800 ng/ml, respectively. The recovery for the internal standard was 84.1%. No adverse matrix effects were noticed for this assay. The automation of the sample preparation steps not only increased the analysis throughput, but also increased method ruggedness. The use of a stable isotope-labeled internal standard further improved the method ruggedness. Practical issues of analyzing incurred samples were discussed. This HILIC–MS/MS method for analysis of citirizine in human plasma was successfully used to support clinical studies.