PROTOCOL 122 | VOL.5 NO.1 | 2010 | NATURE PROTOCOLS p u o r G g n i h s i l b u P e r u t a N 0 1 0 2 © natureprotocols / m o c . e r u t a n . w w w / / : p t t h INTRODUCTION In recent years, there have been major breakthroughs in the devel- opment of improved instrumentation, especially toward the inte- gration of different steps into one system. Today’s sophisticated instruments, such as the gas chromatograph–mass spectrometer (GC–MS) and liquid chromatograph–MS (LC–MS), can perform extremely challenging operations by separating and quantifying target compounds from complex samples as well as applying chemo- metric methods to evaluate results statistically 1 . On the other hand, the combination of sampling and sample preparation steps is not easily accomplished, primarily because conventional sample prepa- ration techniques use multistep labor-intensive procedures and/or require organic solvents. For this reason, the development of an automated method that integrates sampling and sample prepa- ration with separation methods has been greatly hindered and, consequently, more than 80% of the analysis time is usually spent on conventional sampling and sample preparation procedures 1,2 . As a result, this approach becomes highly unsuitable in routine analyses, as it drastically decreases the throughput of analysis and moreover is incompatible with the promising developments in chromatographic and mass spectrometric instrumentation 1,2 . Thus, the selection of an appropriate sample preparation method is the most crucial step in the entire analytical process and this is one of the major reasons behind the development of solid-phase microextraction (SPME) 3 . SPME was developed by Pawliszyn et al. 1,2 in an attempt to address the need to facilitate rapid sample preparation both in the laboratory and on-site where the investigated system is located. Since its invention, the technique has evolved in numerous ways including the development of new SPME formats, advances in the automation of SPME processes and contribution to high-through- put analyses 1–5 . Although the initial and current development and evolution trends in SPME technology have shown the great capa- bility of this technique in all fields of modern environmental, food and biological analytical chemistry 1,3–5 , the current protocol is focused on the initially developed ‘fiber’-SPME format, which still remains the most widely used form of the technique 1 . The principles and theory of SPME operation will be addressed in one of the sections to follow. Once the familiarity with SPME theory and principles is achieved, the current protocol can serve as a guide for the development of analytical methods in both targeted and untargeted applications involving the quantitative and qualitative determinations of organic molecules (such as pesti- cides, pharmaceuticals, drugs of abuse, dioxins, polycyclic aromatic hydrocarbons (PAHs), personal care product residues) in various samples such as food products (plant-based food commodities, alcoholic/nonalcoholic beverages, meat products, etc.), biological samples (urine, blood, plasma, etc.) and environmental samples (air, drinking water, waste water, soil, etc.) available in solid, liquid and gaseous forms. The objective of the current protocol is to introduce the basic principles of fiber-SPME and provide a comprehensive general strategy for developing SPME methods without emphasis on a single target application. In addition, specific implications unique to GC or LC SPME-based methods will be outlined. The compati- bility of SPME for the development of high-throughput automated methods with an emphasis on particular SPME-GC applications of interest will be addressed elsewhere 6 . SPME device The fiber-SPME device consists of a fiber holder (Supplementary Fig. 1a) (available in two different formats to support manual or automated SPME processes) and the fiber assembly (Supplementary Fig. 1b) with a built-in fiber/solid support inside a needle, which resembles a modified syringe 7 . The needle protects the fiber from damage, which might occur during penetration of the septa of sample vials or the chromatographic injectors 8 . The fused-silica fiber is coated with a thin layer of a suitable polymeric station- ary phase (described later), which enables extraction and enrich- ment of the analytes by concentrating them during absorption or adsorption processes from the sample matrix 3 . Theory of SPME Fiber-SPME sampling consists of exposing a thin polymeric coat- ing to the sample matrix for a predetermined amount of time (Supplementary Fig. 2) 1,2 . The transport of analytes from the sample to the SPME fiber coating is initiated immediately when Protocol for solid-phase microextraction method development Sanja Risticevic, Heather Lord, Tadeusz Górecki, Catherine L Arthur & Janusz Pawliszyn Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada. Correspondence should be addressed to J.P. (janusz@sciborg.uwaterloo.ca). Published online 7 January 2010; doi:10.1038/nprot.2009.179 Solid-phase microextraction (SPME) is a sample preparation method developed to solve some of the analytical challenges of sample preparation as well as sample introduction and integration of different analytical steps into one system. Since its development, the utilization of SPME has addressed the need to facilitate rapid sample preparation and integrate sampling, extraction, concentration and sample introduction to an analytical instrument into one solvent-free step. This achievement resulted in fast adoption of the technique in many fields of analytical chemistry and successful hyphenation to continuously developing sophisticated separation and detection systems. However, the facilitation of high-quality analytical methods in combination with SPME requires optimization of the parameters that affect the extraction efficiency of this sample preparation method. Therefore, the objective of the current protocol is to provide a detailed sequence of SPME optimization steps that can be applied toward development of SPME methods for a wide range of analytical applications.