Per- and polyfluoroalkyl substances （PFASs） are a large group of synthetic chemicals that have been widely used in various industrial and commercial products owing to their unique physicochemical properties. However， accumulating evidence suggests that PFASs are persistent， transmissive over long distances， bioaccumulative， and toxic； consequently， their adverse effects on ecosystems and humans is of widespread concern. Serum is the most commonly used human matrix for assessing internal exposure to environmental pollutants， and several analytical methods have been developed to measure PFASs in sera. Current methods are generally fast， convenient， and robust； however， their pretreatment steps require large amounts of organic solvents and materials， such as solid-phase extraction cartridges and/or sorbents. In this study， a novel and low-cost analytical method based on cold-induced phase separation （CIPS） strategy was developed for the simultaneous determination of 31 legacy and emerging PFASs in serum. The core mechanism and distinctive feature of CIPS involves cooling an acetonitrile-water （ACN-water） mixture at a low temperature to produce two clear-cut layers： one with a high ACN proportion （the ACN layer） and an aqueous layer （water layer）. Certain chemicals are significantly enriched in the ACN layer during cooling； at the same time， impurities， especially water-soluble impurities， remain in the aqueous layer. CIPS only requires the temperature to be varied， and no external impurities are introduced during pretreatment， which dramatically reduces material costs and avoids new impurities from intervening. Our method involves the following procedure： serum was drawn accurately （0.2 mL） into a 1.5 mL Eppendorf （EP） tube， 2 ng of each isotopically labeled internal standard was added， the mixture is vortexed， and 350 µL of ACN was added， followed by vortexing and ultrasonic extraction. Subsequently， 450 µL of water is added to adjust the volume proportion of ACN to 35% （the volume percentage of ACN in the total solution）. The protein at the bottom of the tube was collected following centrifugation at 15 000 r/min for 10 min， and the supernatant was transferred to a 1 mL syringe. The syringe was frozen in a -20 ℃ refrigerator for 1 h to obtain the two layers， after which the upper layer （approximately 80-100 μL） containing ACN and the target compounds was finally transferred to a glass vial for instrumental analysis. Liquid chromatography coupled with triple quadrupole mass spectrometry augmented with electrospray ionization （LC-ESI-MS/MS） was used to quantify the PFASs. The analytes were separated using a C18 column， with methanol and 2 mmol/L of ammonium formate-HO used as mobile phases. Linearities， limits of detection （LODs） and， limits of quantification （LOQs）， recoveries， precisions， and matrix effects were determined under the optimal conditions. The LODs and LOQs of PFASs in serum were 0.01-25 and 0.03-83 pg/mL， respectively. Under two spiked levels， namely 5 ng/mL and 25 ng/mL， average recoveries ranged between 60.5% and 129.6%， with relative standard deviations （RSDs） of less than 22.8%. Under 5 pg/mL as LOD spiked level， average recoveries ranged between 61.6% and 199.1%，with RSDs<29.4%. While matrix-effect testing revealed slightly enhanced signals， the use of isotopically labeled internal standards compensated for these effects. Real samples were subsequently analyzed， with 50 human serum samples collected in first trimester of pregnancy women living in the Shunyi District， Beijing. Nine PFASs exhibited high detection frequencies （>80%）， which suggests that PFASs are ubiquitous in the population. The median and mean levels of ΣPFASs （sum of 31 PFASs） in serum were 21.8 and 22.9 ng/mL， respectively， and the range was 0.456-73.9 ng/mL. Both legacy and emerging PFASs were detected at high frequencies and contamination levels， which suggests that they are widely used. In summary， the method developed in this study is fast， sensitive， and solvent- and material-efficient； it is also very linear and highly accurate， and exhibits satisfactory extraction recovery and enrichment factors； hence， it is suitable for surveying large populations as well as for use in environmental epidemiology. 2 31
