Journal of Chromatography A, 1216 (2009) 7664–7669 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Simplified kinetic calibration of solid-phase microextraction for in vivo pharmacokinetics Xu Zhang a,b , Ali Es-haghi c , Jibao Cai a,b,∗ , Janusz Pawliszyn b,∗∗ a Department of Chemistry, University of Science and Technology of China, Hefei 230026, China b Department of Chemistry, University of Waterloo, Waterloo, Ontario, N2L 3G1 Canada c Department of Physico Chemistry, Razi Vaccine & Serum Research Institute, P.O. Box 31975/148, Karaj, Iran article info Article history: Received 16 April 2009 Received in revised form 29 August 2009 Accepted 9 September 2009 Available online 12 September 2009 Keywords: In vivo SPME Single-point kinetic calibration Pharmacokinetics abstract Solid-phase microextraction (SPME) has been demonstrated to be useful for in vivo sampling in phar- macokinetic studies. In this study, a single time-point kinetic calibration for in vivo dynamic monitoring was developed by simplification of the laborious multiple time-point kinetic calibration, based on the independent desorption kinetics of the preloaded standards from SPME fibers with the changing ana- lyte concentrations. The theoretical foundation and practical application conditions, such as the replicate numbers, the optimal time-point for desorption, and the sampling time, were systematically investigated. Furthermore, the feasibility of using regular standards rather than deuterated ones for the kinetic cali- bration was justified by comparing to the data obtained using the deuterated standards. All the methods were verified by in vitro and in vivo experiments. The results from in vivo SPME were validated by the blood drawing and chemical assay. These simplified calibration methods improved the quantitative appli- cations of SPME for dynamic monitoring and in vivo sampling, enhance the multiplexing capability and automatic potentials for high throughput analysis, and decrease expenses on reagents and instruments. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Solid-phase microextraction (SPME) has been demonstrated to be a promising approach to in vivo sampling for pharmacokinetic (PK) studies, due to its simple configuration and implementation, its time-effectiveness and cost-effectiveness, its miniaturized for- mat, and the little disturbance it causes to the system under study [1–4]. However, improving the quantitative capability (accuracy and precision) of SPME remains the main task for further develop- ment. For example, the matrix effects such as the fouling and the diffusion-layer effect was found to affect the sorption parameters and thus the quantitative analysis of complicated environmental and biological samples [5–9]. Except choosing appropriate fiber coatings, these issues can be addressed significantly by better- ing the calibration method, since the accuracy of SPME approach depends on whether the appropriate calibration procedure is adopted; meanwhile, the operational procedure of SPME is mainly determined by the calibration approach. The simpler the operation, the easier the automation of the procedure would be. Moreover, ∗ Corresponding author at: Department of Chemistry, University of Science and Technology of China, Hefei 230026, China. Tel.: +86 551 360 6642; fax: +86 551 360 6642. ∗∗ Corresponding author. Tel.: +1 519 888 4641; fax: +1 519 746 0435. E-mail addresses: jbcai@ustc.edu.cn (J. Cai), janusz@uwaterloo.ca (J. Pawliszyn). temporal resolution is with importance deserving consideration for a dynamic monitoring in a living system, as in vivo PK stud- ies [10–11]. To achieve high temporal resolution, fast sampling based on pre-equilibrium SPME would be more suitable than the time-consuming equilibrium SPME [1,3,4]. However, the external calibration method is not suitable for pre-equilibrium in vivo sam- pling, because this calibration is neither able to correct for the unknown blood flow, nor even reproduce the blood hydrodynamics in the in vitro laboratory experiment when performing the calibra- tion. However, the agitation of the sample matrix (the blood flow) can seriously affect the extraction kinetics of SPME, thus affecting the quantitation under pre-equilibrium conditions. Additionally, for in vivo or on-site analysis, it is often challenging to have the exact blank sample matrix to prepare the calibration standards, for exam- ple, in the PK study, to obtain the blank blood from the same animal, especially for the small animals such as mice that have limited amount of blood. In this case, the blank blood must be from differ- ent animals, which could result in significant interanimal variation, as previous study showed [4]. Accordingly, quantitative analysis was conducted by adopting the kinetic calibration method, or standard-on-fiber calibration, by which analyte extraction is calibrated by determining desorp- tion of the preloaded standards from the SPME fiber to the sample matrix [12–13]. This method compensates for the effects of agi- tation, temperature, and matrix for the simultaneous processes of standard desorption and analyte extraction in the very sampling 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.09.021