Anti-tumor drugs have high toxicity and multiple adverse reactions, while liposomal formulations can reduce drug toxicity and decrease drug activity degradation, making them a promising targeted carrier. Currently, most pharmacokinetic studies of liposomal anti-tumor drugs mainly focus on total concentration. However, free drugs are the part that truly exerts pharmacological effects in vivo, so the separation and determination of free drugs are crucial. Among them, sample pretreatment is a key step in the separation and extraction of free drugs after liposomal administration.

Why Test Liposomal Antitumor Drugs
In recent years, the development of anti-tumor drugs has been rapid. However, currently used anti-tumor drugs all have varying degrees of adverse reactions, limiting their usage. To reduce side effects, improve bioavailability, and prolong the duration of action in the body, the development of liposomal formulations has become a hot topic in pharmaceutical research. Liposomes (composed of phospholipids or cholesterol , etc) encapsulate drugs within the phospholipid layer, which not only protects the drug's activity and reduces the occurrence of drug resistance but also shields normal tissues from drug toxicity. Therefore, liposomes offer a promising and safe drug delivery method due to their ability to accumulate in tumor tissues with higher permeability. Studies indicate that the concentration of free drugs in blood or tissues post-liposomal administration correlates with efficacy and side effects. However, most pharmacokinetic studies focus on total drug concentration, potentially providing unreliable data for liposomal pharmacology and toxicology. Thus, establishing methods to analyze free drug concentration in liposomal formulations is essential.

Common Assays for Liposomes of Antitumor Drugs
Currently, the in vivo analysis methods of liposomal anti-tumor drugs include conventional chromatography-mass spectrometry analysis, capillary electrophoresis, metal complex-based imaging techniques, and fluorescence imaging analysis.

Conventional Chromatography-Mass Spectrometry Analysis
High-performance liquid chromatography (HPLC) is commonly used to determine the total concentration of liposomal anti-tumor drugs, with relatively fewer reports on determining the free concentration. HPLC analysis of liposomes typically utilizes C18, pentafluorophenyl (PFP), or cyanopropyl (XDB-CN) columns, among which, C18 columns are the most commonly used in determining the concentration of liposomal anti-tumor drugs. The mobile phase often consists of acetonitrile-water or methanol-water. When detecting weakly basic drugs such as doxorubicin and irinotecan (CPT-11), adding acid-base modifiers to the mobile phase can adjust retention time, optimize chromatographic peak shape. Formic acid, acetic acid, and ammonium acetate are commonly used acid-base modifiers. Currently, gradient elution is commonly used, which can shorten the analysis cycle, improve separation efficiency, enhance peak shape, reduce tailing, and increase sensitivity.

Capillary Electrophoresis Analysis
Capillary electrophoresis (CE) has the advantage of high separation efficiency and requires small sample volumes. However, comparing to chromatography methods, CE has higher detection limits and lower sensitivity.

Metal Complex-based Imaging Techniques
Metal complex-based imaging techniques include single-photon emission computed tomography (SPECT) and positron emission computed tomography (PET). SPECT utilizes gamma rays emitted by single-photon radioactive isotopes for imaging. Commonly used gamma emitters include technetium-99m(99mTc) and indium-111 (111In). PET is a technique that collects images by detecting the concentration of positron-emitting isotopes in various parts of the body. Commonly used metal isotopes include copper-64 (64Cu), zirconium-89 (89Zr), and manganese-52 (52Mn).

Fluorescence Imaging Analysis
Fluorescence imaging analysis involves labeling liposomes with fluorescent tracers to detect their changes in vivo. Commonly used fluorescent tracers include 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiD), self-quenching carboxyfluorescein, and polydiacetylene.

In addition, enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry (LC-MS) can also be used to determine the blood drug concentration of anticancer drug liposomes. After liposome administration, over 98% of the drugs in the plasma exist in encapsulated form, with low concentrations of free drugs. Liquid chromatography-mass spectrometry, due to its high sensitivity, is widely used for the analysis of free drug concentrations in anticancer drug liposomes.

Common Methods of Liposomal Drug Concentration Assay
In the analysis of lipid-based anticancer drug formulations, the predominant method involves measuring the total drug concentration in biological samples. Although only about 2% of the drug exists in its free form after entering the body, the free drug fraction is essential for exerting pharmacological effects. Hence, the separation and determination of free drugs from lipid-based anticancer drug formulations are pivotal in drug analysis. After lipid-based drug administration, biological samples such as plasma contain not only drugs and their metabolites but also salts, acids, bases, proteins, and other endogenous or exogenous small molecules. The primary objective of sample pretreatment is to employ appropriate techniques to eliminate, to the greatest extent possible, the influence of these factors on drug analysis in plasma. The efficacy of sample pretreatment methods significantly impacts the accuracy and reliability of drug and metabolite concentration determination. Therefore, sample pretreatment stands as a decisive step in the analysis of drugs in biological samples.

Total Liposomal Drug Concentration Assay
Protein Precipitation Method (PPT)
The protein precipitation method is the most commonly used pretreatment method for determining the total concentration of lipid-based anticancer drugs. It effectively disrupts liposomes and releases drugs. When using the PPT method, it is necessary to determine the type of precipitant, volume, and system pH. Common protein precipitants include methanol, acetonitrile, and their mixtures. The PPT method is simple and fast, but it may not completely remove soluble proteins and can lead to the co-precipitation of some analytes, reducing extraction recovery and making it unsuitable for the analysis of trace substances.

Liquid-Liquid Extraction (LLE)
Liquid-liquid extraction is also used for the analysis of total drug concentration in lipid-based anticancer drug formulations. It primarily removes phospholipids to achieve separation and purification while reducing matrix effects during instrument detection. Commonly used extraction agents include ethyl acetate, chloroform, and propanol. For paclitaxel liposomes, methyl tert-butyl ether is often used for extraction. Adding a buffer solution to the extraction solvent can adjust the pH to improve solubility and increase recovery. LLE is highly selective and suitable for lipophilic drugs but requires a longer extraction time and may result in emulsification if not handled properly, reducing drug extraction recovery.

Free Liposomal Drug Concentration Assay
Several methods have been reported for separating free drugs from liposomes, including ion exchange chromatography, size exclusion chromatography (gel chromatography), ultrafiltration, and solid-phase extraction (SPE). The first three methods have limitations: ion exchange chromatography has poor repeatability and low recovery rates, gel chromatography requires high sample dilution and has slow separation speed, and ultrafiltration, while effective, may lead to drug adsorption on the equipment and significantly reduce recovery rates due to the low concentration of free drugs in plasma. Till now, SPE has been widely used for separating free drugs from lipid-based anticancer drug formulations. This method can effectively remove endogenous substances from plasma, has high recovery rates, and is easy to enrich, making it suitable for separating free drugs and metabolites in plasma.

Selection and Pretreatment of Solid Phase Extraction (SPE) Columns
Solid-phase extraction columns include C18, C8, strong cation exchange, mixed-mode anion exchange, and hydrophilic-lipophilic balance (HLB) columns. Currently, the most commonly used type of solid-phase extraction column in lipid-based anticancer drug SPE is the OasisHLB SPE column. The OasisHLB SPE column is a hydrophilic-lipophilic balanced solid-phase extraction column with a larger sample loading capacity than C18 columns, high recovery rates, and wide pH adaptability. It can separate and purify samples and concentrate and enrich them, making it widely used for separating free drugs in plasma after liposome administration. The choice of solid-phase extraction column should be based on the properties of the drug and the separation requirements.

Impact of Eluent pH
The pH of the eluent affects not only the elution capacity of the solid-phase extraction column for free drugs but also the stability of the drug. Therefore, appropriate eluents should be selected based on the nature of the drug and actual conditions to accurately determine the free drug concentration in lipid-based anticancer drug formulations.

Online Solid-Phase Extraction
Online solid-phase extraction offers advantages such as simplicity, stability, low detection limits, and high sensitivity when combined with HPLC, making it a comprehensive method for sample pretreatment and analysis detection.