# Chemicals from Brominated Flame Retardants: Analytical Methods, Occurrence, Transport and Risks
## 2 Analytical Methods for the Detection of BFRs in Environmental and Biological Samples
### Sample Collection and Extraction Methods
#### 2.1.1 Biotic Samples  
Humans and Animal Tissues  
Due to their lipophilic nature, BFRs tend to accumulate in various animal tissues such as adipose tissue, liver tissue, blood plasma, breast milk, muscle tissue, brain tissue, reproductive organs (ovaries and testes), and bone tissue. The analysis of BFRs in animal tissues can vary depending on factors such as species, habitat, diet, and exposure levels. Solvent extraction by liquid-liquid extraction (LLE) and Soxhlet extraction were the main methods used for the extraction of BFRs in tissue [21]. Alternative methods for the extraction of BFRs from tissues include Ultrasound-Assisted Extraction (UAE) [22] and Microwave-Assisted Extraction (MAE) [23]. Further methods include accelerated Solvent Extraction (ASE), Solid-Phase Extraction (SPE) [24], and Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) [25]. QuEChERS is a robust and versatile sample preparation technique designed to efficiently isolate analytes from complex matrices. This multi-step process, encompassing extraction, partitioning, and cleanup, is optimized to yield a final extract that is highly suitable for chromatographic analysis. Shortly after PBDE restrictions were implemented in Europe, some researchers in Denmark measured for the presence of novel brominated flame retardants (NBFRs), hexabromocyclododecanes (HBCDDs), PBDEs, and dechlorane in samples of human breast milk [26]. The samples were collected manually using a breast milk pump, dried with diatomaceous earth, and extracted using Soxhlet with hexane:acetone (4:1). The clean-up of the samples was accomplished in a multilayer column containing activated aluminum oxide, sulfuric acid-impregnated silica, and some Na\({}_{2}\)SO\({}_{4}\), eluted with hexane and analyzed by GC-ECNI-MS. Although MAE has been used mainly for the extraction of solid samples like sediments and soil, [PERSON] et al. [23] described extraction of PBDEs from human milk samples using MAE. Using a Mars X microwave extraction system, they optimized a MAE extraction method [which involved 5 g of freeze-dried milk + 10 mL water + HCOOH + 2-propanol + 40 mL n-hexane:acetone(1:1, \(v/v\)); and extraction with MAE at 105 \({}^{\circ}\)C for 20 min], cleaned up the extract using 1 multilayer (2 + 6) g SPE column with 20 mL n-hexane:dichloromethane (4:1, \(v/v\)) elution solvent, and used GC-\(\mu\)ECD or GC-MS/MS for detection. Employing ultrasound extraction (UAE) coupled with dispersive solid-phase extraction (d-SPE) as a cleanup procedure, [PERSON] et al. [22] compared UAE extraction to conventional SE techniques, revealing significant improvements. Analytical parameters were thoroughly assessed, and the innovative method demonstrated notable advantages, reducing extraction and purification times by approximately 74-80% and solvent consumption by around 94-97%. While the Soxhlet method has been the traditional method for years, the excessive use of solvent and the long extraction time have always been its limitations. The advantages of MAE over other extraction methods include lower solvent consumption and analyte loss, shorter processing time, and higher sample throughput. The QuEChERS method, which was originally developed for pesticide analysis in food, involves a combination of solvent extraction and dispersive solid-phase extraction (dSPE). [PERSON] et al. [25] employed QuEChERS techniques for the simultaneous determination of PBDEs and hexabromocyclododecanes (HBCDs) in fish, shellfish, and the muscle of terrestrial animals. The method involved the use of QuEChERS-like extraction and a two-step clean-up, followed by a dual instrumental detection, GC-MS/MS for PBDEs and LC-MS/MS for HBCDs [25]. The simultaneous application of ASE and SPE for the extraction and clean-up analysis of BFRs in the subcutaneous adipose tissue of Guiana Dolphins sampled across the Brazilian coast have also been reported by [PERSON] et al. [24].