RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 2006; 20: 39–47 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2265 Rapid and direct measurement of free concentrations of highly protein-bound fluoxetine and its metabolite norfluoxetine in plasma Bin Guo 1{ , Chuan Li 1 * ,{ , Guangji Wang 2 and Laishun Chen 1 1 Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China 2 China Pharmaceutical University, Nanjing, Jiangsu 210009, China Received 3 June 2005; Revised 27 October 2005; Accepted 27 October 2005 Fluoxetine (F) and its active N-demethylated metabolite, norfluoxetine (NF), are selective serotonin re-uptake inhibitors that bind extensively to plasma proteins. Development and validation of a novel method for measuring free concentrations of F and NF in plasma are reported here. The plas- ma filtrate was prepared by a high-speed short-duration ultrafiltration (UF) and then submitted directly to a short-column liquid chromatography/tandem mass spectrometric (LC/MS/MS) assay. There was no significant matrix effect on the analysis, and non-specific binding of the analytes to the UF devices was negligible. For validation of the method, the recovery of the free analytes was compared to that from an optimized equilibrium dialysis method, and analyte stability was examined under conditions mimicking the sample storage, handling, and analysis procedures. The linearity range was 0.37–12 ng/mL for F and NF; the within-run and between-run relative stan- dard deviations were less than 11.9%, and accuracies across the assay range were 100  10.3%. This new method was then further validated in a pharmacokinetic (PK) study in beagle dogs receiving a single oral dose of fluoxetine hydrochloride. The integrity of the resulting PK data of free F and NF was absolute. The PK data indicate that the novel method is accurate and reliable. To our knowl- edge this is the first report describing a rapid and reliable method for direct measurement of free concentrations of F and NF in plasma, which will be useful for clinical pharmacokinetic/pharma- codynamic studies of F. Furthermore, the strategies described herein may be applied to the devel- opment and validation of methods for measuring the free concentrations of other drugs in plasma. Copyright # 2005 John Wiley & Sons, Ltd. As the magnitudes of both desired response and toxicity are functions of the drug concentration at the site of action, it is important to relate drug concentration measurements to their pharmacological responses and toxicities. However, scien- tists are rarely able to directly measure drug concentrations at the action sites. Such values are most often measured at alternative accessible sites, such as blood/plasma. Within blood, drugs can reversibly bind to plasma proteins such as albumin and alpha-1-acid glycoprotein, blood cells, and other components. Consequently, there can be large varia- tions in the drug concentrations measured from whole blood, plasma (or serum), and plasma water. Protein-bound drugs are too large to traverse the cell membrane, so in most cases only free unbound drugs are capable of entering cells and reaching the action site to generate a pharmacological response. Thus, the binding of drugs to plasma proteins is an important determinant of pharmacodynamic activities. Pharmacokinetic (PK) para- meters generated from free drug concentrations are often more clinically relevant than those derived from the total drug concentrations (the sum of free plus protein-bound concentrations). 1–3 The therapeutic ranges for most drugs have been established in terms of total drug concentration, but this may be sub-optimal for therapeutic monitoring of drugs that bind extensively to plasma proteins and may thus show inconsistent free fractions over the therapeutic range. 4–6 In the case of the anti-arrhythmic drug disopyramide, the bound fraction appears to be concentration-dependent at therapeutic plasma concentrations; 7 the peak response and the area under the response time curve for this drug were Copyright # 2005 John Wiley & Sons, Ltd. *Correspondence to: C. Li, Shanghai Institute of Materia Medica, SIBS, Chinese Academy of Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China. E-mail: chli@mail.shcnc.ac.cn {Also affiliated with the Graduate School of the Chinese Acad- emy of Sciences. Contract/grant sponsor: Science & Technology Commission of Shanghai Municipality; Contract/grant number: 04DZ19215. Contract/grant sponsor: Chinese Ministry of Science and Technology; Contract/grant number: 2003AA2Z347A, 2004CB720305, and 2005CB523403. Contract/grant sponsor: Chinese Academy of Sciences; Con- tract/grant number: KSCX1-SW-11 and KSCX2-XW-202.