IntroductionMercury speciation has long been a field of concern. Such interest is mainly due to toxicological impact, ecological problems and biogeochemical cycling of mercury involving distribution, accumulation, transformations and transport pathways in the natural environment.1Mercury is a very toxic element. However, the toxicity of mercury is highly dependent on its chemical form. Methylmercury is one of the most toxic mercury species. To understand the toxicological impact and pathways of mercury species in the environment, the determination of total mercury is frequently not sufficient. Therefore, the assessment of inorganic mercury and methylmercury concentrations, specifically in sediments and soils, is very important to the interpretation of biogeochemical cycles of mercury in aquatic environments.2Determination of different mercury species from various complex matrices,e.g., soils and sediments, is still considered a difficult task due to the frequently very low concentration of methylmercury in soils and sediments (less than 1.5% of the total mercury).3The quality of the results mainly depends on the sample pre-treatment stages (sampling, storage and sample preparation), in spite of significant improvements in the instrumentation techniques. The most widely used methods for the extraction and separation of inorganic and methylmercury are the Westöö technique4–7(acidic leaching method), alkaline digestion,8–10steam distillation,9–11solvent extraction,12–14a modified Westöö methodology15(alkaline based technique), and supercritical fluid extraction,16followed by one or two separation steps. The separation and detection techniques associated with these methods include gas chromatography (GC), HPLC coupled with element-selective detection techniques, such as ICP-MS, atomic emission spectrometry (AES), atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), or cold vapor atomic absorption spectrometry (CV-AAS). As all of the aforementioned sample preparation methods use either acid or base with organic solvents, and, after extraction, most of them implement sample pre-concentration steps (e.g., ethylation or reduction with SnCl2or hydride generation with NaBH4), there is a possibility of interconversion or unidirectional transformation of inorganic mercury to organic mercury13,16–29orvice versa29–32during sample storage, shipment, extraction, pre-concentration or analysis steps. Therefore, the results obtained using these procedures frequently introduce positive or negative biases for either inorganic mercury or methylmercury, or both. Besides such drawbacks, these methods require much solvent, labor and time.The efficiency of the less solvent- and time-consuming microwave-assisted extraction (MAE) technique for sample preparation in environmental applications has been evaluated elsewhere in different matrices (soils, sediments, and biological tissues) in different applications (total digestion for elemental analysis, extraction of selected organic compounds), and in speciation analysis (organotin). Vazquezet al.33,34used the focused microwave-assisted extraction (FMAE) technique to extract methylmercury with HCl and toluene, a modified method of Westöö,4,5from sediment and biological tissue samples. Tsenget al.35–37also implemented FMAE for the extraction of methylmercury, also from sediment and tissue samples. There are several drawbacks to FMAE: samples must be extracted at atmospheric pressure and below the boiling point of the solvent; simultaneous extraction of multiple samples is not possible; it is difficult to preset a constant temperature profile as this technique only allows control of the applied power which, in turn, is directly dependent on the number of samples or the total mass, and, there is a high possibility of losing the volatile organomercury compounds during extraction. However, no one has yet tried the closed-vessel microwave-assisted extraction technique (which is free from the aforementioned drawbacks) for mercury speciation in soils or sediments.Therefore, the purpose of this study was to develop a microwave-assisted extraction procedure capable of quantitative extraction with little or no transformation of inorganic mercury and methylmercury from soils and sediments in a closed-vessel microwave system, and incorporate it into EPA draft Method 3200 as an alternative extraction procedure for mercury species. Careful optimization of the conditions for the microwave extraction procedure is required to stabilize the mercury species in the microwave field, prior to speciation analysis. Essential parameters, such as concentration of the extraction medium, amount of sample, temperature and time of exposure must be optimized. The literature35suggests that nitric acid (HNO3) is a better solvent for microwave-assisted extraction because it introduces little or no interferences to the ICP-MS. Therefore, nitric acid has been evaluated as an extraction solvent. The irradiation power, one of the most useful parameters for microwave extraction, was not optimized during this study due to its dependency on the number of samples or the total mass of the extraction medium.This paper describes a fast and easy method for the quantitation of inorganic mercury and methylmercury using closed-vessel microwave-assisted extraction, followed by separation with HPLC and detection with ICP-MS. The stability of the mercury species in a microwave field and the optimization of different parameters are also described in detail. The developed method was then validated by using different standard reference materials and reference soils obtained from Environmental Resource Associates®. The developed method was also validated using EPA Method 6800 [Elemental and Speciated Isotope Dilution Mass Spectrometry, (IDMS and SIDMS, respectively)].38EPA Method 6800 was used as a diagnostic tool to check whether any interconversion between inorganic mercury and methylmercury is taking place during or after extraction. One of the unique applications of SIDMS is to trap errors related to specific portions of a protocol. This is accomplished by using multiple spikings with multiple isotope-labeled species at specific method protocol points. The error of the specific steps can be discovered, and their contribution to the overall transformation of a species known. To perform these types of applications, inorganic mercury and methylmercury labeled with multiple isotopes are required. Although methylmercury labeled with different isotopes has recently become commercially available,39–41the methylmercury labeled with a minor mercury isotope was synthesized in the laboratory.