## 5 Analytical Techniques for Detection and Quantification of PFAS
### Extraction and Clean-Up  
As contaminants, in real matrices PFASs are usually present in very low concentration (trace levels) so it is necessary to extract them prior to the analysis. As a first step, the methods needed to be validated in water samples, and in 2019 the ISO 21675:2019 \"Water quality--Determination of perfluoroalkyl and polyfluoroalkyl substances (PFAS) in water--Method using solid phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS)\" was published. To validate the method an inter-laboratory trial (ILT) was conducted by [PERSON] et al. [135], involving 27 labs in total. The results suggested that the storage at 28 \({}^{\circ}\)C of water samples is preferred for those that are not analyzed within four weeks of collection, and it is also recommended to rinse the container wall with methanol prior to the elution step in order to improve recovery and achieve repeatable results for long-chain PFAS. More complex matrices have been already addressed, such as plant-based materials with the aim of developing and testing a straightforward extraction and clean-up approach for the measurement of five PFAS classes in various plant tissues [136]. The method demonstrated to be suitable for PFAS analysis, and adequate validation parameters were achieved for the majority of analytes. Addressing other matrices than water, [PERSON] et al. [137] studied the clean-up methods for fatty matrices, which is a critical step for PFAS analysis. Four different sorbents' lipid clean-up capabilities were assessed in samples of fatty fish following extraction using Quick Easy Cheap Effective Rugged Safe (QuEChERS) or the ethyl acetate technique. The sorbents tested were silica, Z-Sep, C\({}_{18}\), and EMR-lipid. The most efficient dispersive-Solid Phase Extraction (dSPE) sorbent was the EMR-lipid one performed after a QuEChERS extraction.  
The QuEChERSER (more than QuEChERS) method validated by [PERSON] et al. [138] increases the polarity range of QuEChERS. The technique is essentially performed by an ACN/H\({}_{2}\)O extraction followed by an ultra-centrifugation to obtain the fraction for LC. In addition, from the acetonitrile layer derived by the further addition of QuEChERS salts in the remaining initial extract it was possible to obtain, due to the salting-out effect into the Instrument Top Sample Preparation (ITSP) clean-up, a fraction for Gas Chromatography (GC). The method demonstrated advancement in clean-up efficiency, matrix effect, and recoveries compared to other PFAS analysis methods in food stuff previously used by US federal agencies. The ITSP proved to be a faster and automated approach to evaluate clean-up efficiency compared to the dSPE one.  
[PERSON] et al. [139] demonstrated the effectiveness of a QuEChERS extraction/clean-up method shortening the time required per analysis of environmental and food matrices, also increasing the efficiency and resulting in approximately 30 samples being ready for LC-MS/MS analysis in one working day. Based on these results, [PERSON] et al. [140] proposed a novel method based on a multistep sorption of PFASs for high volume direct injection of aqueous samples, which involves sorbent addition with biochar, centrifugation of samples, methanol addition, and a filtration with a 0.22 \(\upmu\)m syringe filter of cellulose prior to direct injection in a triple quadrupole LC-MS. The innovation of this method is the provision of high sample throughput due to the exclusion of instrument preparation and determination of equilibrium time.