1. Analytical Techniques for Detection and Quantification of PFAS
The established methods for performing PFAS analysis are based on Liquid Chromatography-Mass Spectrometry (LC-MS). Both the sample preparation and the development of the chromatographic set-up are crucial steps for reliable, precise, and accurate measurements. According to the literature, the conventional reverse phase separation stationary phase column is the most widely utilized 
approach. To improve the chromatographic performance, columns equipped with polar functionalized C18
alkyl chains were introduced 
. The adoption of columns with polar groups on the surface of the stationary phase permits them to better retain the more polar compounds, in particular the shortest one at the first minutes of elution. In addition, the implementation of an isocratic elution method 
enabled the thorough investigation of C2
, and alternative PFAS by using a special hybrid HILIC/ion-exchange column. In this way, it was possible to quickly and easily analyze legacy, alternative, and ultrashort-chain PFAS in water samples.
The development of novel analytical methods is a crucial step because the legal limits are changing rapidly so that the actual techniques can become rapidly obsolete. Decreasing the limit of quantification is the biggest challenge for the ultra-trace search for PFAS in environmental and food matrices.
An overview of the analytical technique strategy is depicted in Table 1.
Table 1. Scheme of analytical techniques strategy.
2. Sampling and Storage
The first step to improve the level of detection and quantification in complex matrices, in particular from the environment (air, water, soil), is the sampling. For analyzing 26 PFASs in drinking water treatment plant (DWTP) Gobelius et al. 
used two approaches, based on hydrophilic-lipophilic balance (HLB) and weak anion exchange (WAX) of polar organic chemical integrative samplers (POCIS) which were calibrated and used in the investigation. The average PFAS concentration was measured to be rather stable over the course of the days, demonstrating the suitability of the calibration method used for the passive samplers POCIS-WAX and POCIS-HLB. In general, there were no appreciable differences in the detection of individual PFAS contents in drinking water between POCIS-WAX, POCIS-HLB, and composite water samples (collected in aliquots during periods). It is noteworthy that POCIS-HLB had low absorption rates, which may account for the fact that perfluorobutanoic acid (PFBA) was only found using POCIS-WAX and composite water sampling. New microporous polyethylene (PE) diffusion passive sampler was also tested for PFAS groundwater monitoring 
. Using this design, the linear absorption phase of PFASs was successfully extended and a mean t1/2
of 240 days was estimated.
For testing the best condition for storing the samples, Woudneh et al. 
measured the stability of 29 PFAS in different types of water stored at different temperatures. PFAS were spiked in different water samples that were then kept in HDPE containers for up to 180 days at +20, +4, and −20 °C. The study showed that keeping the samples below 0 °C is the only way to ensure the stability of PFAS over time because the effects of the analyte interconversions were noticeable within 7 days of storage at 4 °C, which is the temperature that is normally used to stock aqueous samples for analysis of PFAS.
In case of air samples, to overcome the issue of sampling air including the particulate matter and the gaseous phase for the discovery of unknown novel PFASs, the study of Yu et al. 
showed the use of a cryogenic air sampler (CAS) to simultaneously collect atmospheric gaseous phase and particulate matter comprehensively. Five groups of chlorinated perfluoropolyethers were reported for the first time in this study.
Simon et al. 
studied a fast and simple extraction method for PFAS contaminated soil. The samples of soil were freeze-dried or air-dried before analysis and were also collected from different locations. Johnson 
analyzed soils samples collected at different depth levels. It was the first known study of this type and the results showed that this sort of sampling provides insightful information about the type, source and the consequence of the contamination.
3. 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 Taniyasu et al. 
, involving 27 labs in total. The results suggested that the storage at 28 °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 
. 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, Drábová et al. 
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, C18
, 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 Taylor et al. 
increases the polarity range of QuEChERS. The technique is essentially performed by an ACN/H2
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.
Gallocchio et al. 
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, Askeland et al. 
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 μm 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.
4. Analytical Detection Techniques
Mass spectrometry in all of its forms is the elite technique for the identification and quantification of PFAS, and there are numerous examples 
Wu et al. 
, aiming to speed up the analysis of PFAS, developed and applied a hyphenated nano-electrospray ionization to HRMS (Nano-ESI-HRMS) to analyze wastewater and aqueous film-forming foams (AFFFs) samples collected from three local wastewater treatment plants (WWTPs). Nano-ESI-HRMS enables detection of various PFAS with the inclusion of the ultra-short chain and other very polar molecules which are often underestimated in LC. This approach has the advantages of saving time (lower than two minutes per sample) without compromising accuracy and sensitivity. Taking a step forward in the rapid identification of PFAS, Dodds et al. 
devised a quick and efficient method for separating isobar molecules such as 6:2 FTS and Hydro-EVE that differs only for 0.023 m/z. Even with HRMS instruments, mass separation of these precursors is extremely difficult. Collision cross section (CCS) is the phenomenon that underpins ion mobility separation; whereas other LC techniques separate molecules based on their interaction with the stationary phase of the column (polarity) over time, the additional step of this analysis allows for separation based on atom spatial distribution (molecular size).
The scientific community is also working to develop new approaches to fight PFAS contamination through various techniques that may be more rapid or “in situ”.
Jackson et al. 
studied albumin as the main protein involved as a carrier for some PFAS compounds in human serum, aiming to use this affinity to rapidly characterize the contaminants. Albumin has numerous nonspecific sites that selectively bind hormones, fatty acids, drugs, and several xenobiotics, particularly PFASs. Prior to the development of the current technology, the methods for determining this affinity, such as surface plasmon resonance or titration chemistry, required too much time because of the large number of distinct PFAS that needed to be examined. Differential Scanning Fluorimetry (DSF) is a quick, high-throughput technique for determining ligand-binding interactions, and it is most frequently used to assess the stability of proteins under various circumstances. The findings showed that DSF can specify protein binding affinities and pinpoint the physicochemical factors that contribute to protein binding for a significant number of PFAS. A different approach to perform a rapid and in situ analysis was found by Park et al. 
who discovered the aptamer binding to PFOA (KD
= 5.5 μM) phenomenon for the first time, owing to the alkyl length, which is important in the physicochemical event. To investigate this behavior, a fluorescence-based aptasensor was created, which allows for easy and rapid monitoring of PFAS and other emerging pollutants in situ. The results obtained utilizing a multimode plate reader were compared to traditional LC-MS detection methods. NMR spectroscopy and circular dichroism analysis were performed to compare the binding strengths of the aptamer with and without the addition of PFOA, and the results show that the aptamer can bind long alkyl chains with specificity.
In parallel to routine quantification of legacy PFAS, in order to monitor the global contamination levels, it is also necessary to detect all possible fluorinated substances, which may be toxic as well. The untargeted approach is the only way to discover all the organic fluorinated compounds, and it is usually performed with high-resolution mass spectrometry 
Xiao et al. 
developed for the first time a non-targeted method using fluorine extraction using fluoro-cotton fibers put in SPE cartridges. The interaction F-F aids in the extraction and, as a result, enhancement of organic fluorine compounds. In this study, rice samples were grown in artificially contaminated soils with perfluoroalkyl substance soils to validate the method. This method improved the use of extraction methods to aid in untargeted characterization, but it is also possible to improve chromatographic conditions for the purpose. A novel analytical method for the non-targeted detection of total PFAS was developed by Renai et al. 
for the analysis of fire-fighting AFFFs. The researchers performed the untargeted identification due to a LCxLC chromatography system coupled to a high-resolution mass spectrometer. The peak capacity of chromatographic systems was increased by using the 2D-LC method and this was essential to map the chemical space of complicated mixtures such as AFFFs.
For screening all possible PFAS on AFFFs, Young et al. 
tested a method based on the Fourier-transform (FT) ion cyclotron resonance (ICR) MS. One of the purposes of this study was to show the effectiveness of FT-ICR MS direct infusion for suspect and non-target detection of PFAS in a complex mixture of AFFFs. Molecular structure cannot be determined definitively by direct infusion MS (or any mass analyzer) by detecting the molecular ion alone, but the study shows that the list of known PFASs can be expanded during subsequent analyses using accepted structural identification standards. Another alternative approach for untargeted analysis of perfluorinated compounds is the inductively-coupled plasma (ICP) MS. This method allows Jamari et al. 
to identify all fluorinated compounds whether or not they are ionized under electro spray conditions, and a fluorinated standard is available. This non-targeted approach is well adapted to assist in identifying a large proportion of organofluorines not detected.
Analytical techniques for the detection of PFAS have come a long way and continue to develop. Currently, modern PFAS analysis can use various methods, including the combination of various hyphenated chromatography techniques such as liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). These techniques allow for accurate and sensitive detection of individual PFAS and sometimes even identifying specific PFAS precursors. So far, the main innovations rely on in situ and high throughput techniques which are necessary to answer the monitoring requests. In parallel, the implementation of non-targeted screening methods will allow the understanding of the novel classes of PFAS introduced in the industry products and, also, the characterization of their presence in environment and food. The development of even more accurate and sensitive in situ and screening techniques will constitute the main strategy to fight PFAS contamination and to provide reliable inputs to regulatory bodies and lawmakers.