Bioanalytical Assay for Poloxamer Detection
Why is it crucial to develop bioanalytical detection assays for poloxamers?
Amphiphilic copolymers, such as poloxamers, have been gaining significant attention due to their ability to function as both hydrophilic and hydrophobic polymers. The hydrophilic moieties promote water solubility and compatibility with biological environments, while the hydrophobic moieties allow interaction with hydrophobic targets [1]. This “dual nature” characteristic enables these polymers to be used in a wide range of clinical and pharmaceutical applications, including gene therapy, DNA delivery, and as therapeutic excipients [2].
Poloxamers are non-ionic, poly(ethylene oxide)–poly(propylene oxide) (PEO–PPO) triblock copolymers that have been widely used in the pharmaceutical and biomedical industries. As shown in the figure below, poloxamers contain three polymer subunits covalently linked together. While different types of poloxamers share a similar backbone, their molecular weights differ based on the number of hydrophilic (PEO) and hydrophobic (PPO) blocks [3]. For example, Poloxamer 188 (P-188) contains 80 PEO and 27 PPO subunits, while Poloxamer 338 (P-338) contains 141 PEO and 44 PPO subunits.
Recently, the bioanalytical team at Emery Pharma has undertaken multiple projects involving poloxamer detection in emerging therapeutics. Poloxamers have shown promise as a therapy for Duchenne Muscular Dystrophy (DMD), a degenerative disease marked by muscle membrane instability. In this application, poloxamers have been shown to stabilize dystrophic cardiac muscle membranes in animal models (see figure below) [4].
Despite the wide range of applications, there is limited information in the literature regarding bioanalytical techniques for trace-level quantification of poloxamers. To achieve the sensitivity, accuracy, and precision required by FDA bioanalytical method validation guidelines, it is critical to develop a robust and sensitive bioanalytical assay for poloxamer detection.
Traditional size-exclusion chromatography (SEC) and liquid chromatography with evaporative light scattering detection (HPLC-ELSD) methods have been used to quantify poloxamers in biological samples. However, these techniques often suffer from low sensitivity, poor reproducibility, and non-linear responses. Similarly, electrospray ionization mass spectrometry (ESI-MS) techniques have shown limitations due to poor ionization efficiency and suboptimal sensitivity, particularly with complex poloxamer structures [5].
To overcome these limitations, our bioanalytical scientists at Emery Pharma have developed a highly sensitive, robust LC-MS/MS method for poloxamer quantification in various biological matrices. This method includes a protein precipitation extraction protocol to isolate poloxamers prior to LC-MS/MS analysis, as shown in the figure below.
The LC-MS/MS method, operating in Multiple Reaction Monitoring (MRM) mode, significantly improves detection specificity, quantification limits, and dynamic range for poloxamers in both biological and pharmaceutical samples.
The method uses a standard curve in the target matrix (free of endogenous poloxamers) to quantify concentrations. It offers a linear range from 2.0 µg/mL (0.0002% w/v) to 50.0 µg/mL (0.005% w/v), with the possibility of extending dynamic range via pre-concentration or sample dilution.
Precision was assessed using independent biological replicates, with a %CV under 15–25%, depending on the matrix. Accuracy, evaluated through quality control (QC) samples within the linear range, showed a %error also under 15–25%, consistent with FDA bioanalytical guidelines.
Operating in MRM mode enhances both sensitivity and selectivity by removing matrix interference, making the method ideal for complex biological fluids such as plasma, serum, and cellular product matrices. The method’s limit of detection (LOD) is approximately 1.5 µg/mL (0.00015% w/v), and the limit of quantitation (LOQ) is 2.0 µg/mL (0.0002% w/v) in therapeutic cell product matrices (with values subject to change depending on matrix).
Robust LC-MS/MS analysis of poloxamers requires meticulous optimization of parameters like chromatographic column selection, mobile phase composition, operating temperature, injection volume, and sample cleanup procedures. Even factors like the choice of microcentrifuge tubes can impact detection at trace concentration levels.
The team at Emery Pharma brings extensive experience in the development and validation of bioanalytical assays for detecting poloxamers at trace levels. We welcome the opportunity to hear more about your analytical method development or biopharmaceutical testing needs. For additional information, please contact us online or call us at +1 (510) 899-8814!
About the Author
Originally authored by Ali Najafi. This article was reviewed and updated on July 11, 2025 by Dr. Prajita Pandey, current Associate Director of Chemistry.
References
- Hitesh R. Patel et al /Int.J. PharmTech Res. 2009,1(2)
- D. Ramya Devi et al /J. Pharm. Sci. & Res. Vol.5(8), 2013, 159 – 165
- Anaïs Pitto-Barry et al/Polym. Chem., 2014, 5, 3291-3297
- Houang et al. Skeletal Muscle (2018) 8:31
- M. Nair et al. / Journal of Pharmaceutical and Biomedical Analysis 41 (2006) 725–730
