RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2006; 20: 523–528 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2331 Simple, sensitive and rapid liquid chromatography/ atmospheric pressure chemical ionization mass spectrometric method for the quantitation of Ranolazine in rat plasma Yan Liang, Lin Xie*, Xiao-Dong Liu, Wei-Dong Chen, Tong Lu, Ye Xiong and Guang-Ji Wang Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, P. R. China Received 20 September 2005; Revised 2 December 2005; Accepted 2 December 2005 A sensitive and specific method using liquid chromatography with atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) has been developed and validated for the identifica- tion and quantification of Ranolazine in rat plasma. A simple liquid–liquid extraction procedure was followed by injection of the extracts onto a C 18 column with isocratic elution and detection using a single quadrupole mass spectrometer in selected ion monitoring (SIM) mode. The method was tested using six different batches of blank plasma. Linearity was established for the concen- tration range 0.02–10.0 mg/mL, with a coefficient of determination (r) of 0.9998 and good back- calculated accuracy and precision. The intra- and inter-day precision (relative standard deviation (RSD) %) was lower than 10%, and accuracy ranged from 85 to 115%. The lower limit of quantifica- tion was reproducible at 0.01 mg/mL with 20 mL plasma. The proposed method enables the unam- biguous identification and quantification of Ranolazine for pre-clinical and clinical studies. Copyright # 2006 John Wiley & Sons, Ltd. Ranolazine (RAN, RS-43285), (þ)-N-(2,6-dimethylphenyl)-4- [2-hydroxy-3-(2-methoxylphenoxy)propyl]-1-piperazine acetamide dihydrochloride, has been shown to modulate the metabolism of ischemia myocardial cells and improve the efficiency of oxygen use. It has been developed for treatment of angina and intermittent claudication. 1–3 In 1995, Herron et al. described a method using liquid chromatography/mass spectrometry (LC/MS) for estima- tion of RAN and metabolites, but this report did not provide a detailed description of the method for quantitating RAN in biological fluids. 4 To the best of our knowledge there are no other reports of a method for determination of RAN in vivo. Since RAN lacks strong characteristic UV absorption, detection at the more universal and thus non-selective wavelength of 200 nm required tedious isolation procedures and the use of large volumes (0.5–1.0 mL) of plasma. This might be suitable for human pharmacokinetic (PK) studies, but not for pre-clinical PK studies in rodents since the collection of multiple large-volume blood samples from each animal would be prohibited. In order to fully evaluate the pharmacokinetics of RAN in pre-clinical or clinical studies, it was necessary to develop and validate an assay with appropriate sensitivity, selectiv- ity, accuracy and precision. The potentially large numbers of samples in pre-clinical studies need a rapid and reliable assay. An ideal method should have simple sample prepara- tion, fast on-column separation, and sensitive and specific detection. LC/MS has become an analytical tool that meets most of the above needs. In many cases, highly specific mass spectrometric detection, especially using tandem mass spectrometry (MS/MS), requires only minimal chromato- graphic separation, but MS/MS instruments can be too expensive to justify for use in routine measurements in the pre-clinical laboratory. Solid-phase extraction (SPE) was successfully used to isolate RAN from plasma samples; 4 however, the recovery of RAN was found to depend strongly on the type of the hydrophobic adsorbent, and recoveries varying from 40–90% were observed. Furthermore, costs related to SPE automation as well as cartridge consumption during complete PK studies should be also considered (note that multiple use of a SPE cartridge is not recommended and that, for a complete study including validation, around 2000 samples are run). In the present work, isolation of RAN was achieved by a single-step liquid – liquid extraction (LLE) into diethyl ether, followed by solvent evaporation, re-dissolution of the residue, and injection onto the chromatographic column. An atmospheric pressure chemical ionization (APCI) inter- face, suitable for analysis of low-polarity compounds, was used because it provided more sensitivity and better reproducibility for RAN compared with an electrospray ionization (ESI) source. The analytical procedure was fully validated and successfully used to assess the pharmacoki- netics of RAN in rats. Copyright # 2006 John Wiley & Sons, Ltd. *Correspondence to: L. Xie, Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, P. R. China. E-mail: jsxielin@sina.com.cn