# Recent Advance in the Sample Pretreatments for Drug Analysis in Zebrafish

## TL;DR

This paper reviews methods for preparing zebrafish samples for drug analysis, highlighting trends and challenges in improving efficiency and standardization.

## Contribution

A systematic review of zebrafish sample pretreatment methods and their evolution toward more efficient and automated techniques.

## Key findings

- Traditional methods like LLE and protein precipitation are still widely used but limited by low efficiency and matrix effects.
- Advanced SPE techniques, especially SPME, are gaining popularity due to higher selectivity and compatibility with advanced analytical tools.
- Current challenges include lack of standardized protocols and the need for rapid, on-site pretreatment methods.

## Abstract

Zebrafish, as an emerging model organism, are widely used in in vivo pharmacodynamic and pharmacokinetic studies. Unlike direct chemical analyses that require no sample preparation, most biological samples must undergo preprocessing steps—procedures that profoundly affect analytical outcomes. This paper systematically summarizes the main methods and types of zebrafish sample pretreatment currently in use, aiming to provide a reference for future research in zebrafish sample analysis and preparation. A systematic search was conducted across PubMed, Web of Science, and CNKI for studies published between 2014 and 2024 focusing on liquid–liquid extraction (LLE), solid phase extraction (SPE), and related techniques for zebrafish drug analysis. The results indicate that traditional methods, including LLE and protein precipitation, remain prevalent due to their operational simplicity, but are limited by low enrichment efficiency and pronounced matrix effects (MEs). In contrast, advanced SPE techniques, particularly solid phase microextraction (SPME), are increasingly favored for complex biological sample processing, with key trends including technique hyphenation (e.g., SPME–high-performance liquid chromatography (SPME-HPLC), and micro-SPE–mass spectrometry (µSPE-MS)) and the development of novel sorbents. Despite these advances, current challenges persist, such as immature rapid on-site pretreatment protocols, the difficulty of balancing analytical efficiency with operational simplicity, and the lack of standardized procedures across studies. Overall, zebrafish sample pretreatment techniques are evolving toward higher efficiency, selectivity, and automation. Future research efforts should prioritize the development of intelligent, eco-friendly pretreatment methods and the establishment of unified standards to enhance the reproducibility and comparability of zebrafish-based pharmacological studies.