Proteomics Strategies: Investigating Polysorbate Nanoencapsulation

Nanoencapsulation generally relies upon amphiphilic nanoparticles that form nanomicelles, or small globules that are impervious to aqueous matrices. The food industry uses these structures as a delivery system for small particles, including supplements, nutrients and preservatives. Outside the food sector, nanoencapsulation has applications for targeted drug therapy. An efficient method for the quantification of nanoparticles is imperative for accurate food labeling and to enable researchers to thoroughly assess the benefits and health risks associated with nanoencapsulation.

Krtkova et al. recently analyzed the specific nanocarriers polysorbate 20 and polysorbate 80 in filtered apple juice samples (10 mL).¹ The Scientific Committee on Food has set daily parameters for the intake of polysorbates at 10 mg kg⁻¹ bw/day, but the industry lacks an established protocol for the quantification of these analytes in micellar form. In this study, Krtkova et al. applied three proteomics approaches for this purpose: (1) ultra high-performance liquid chromatography with a size exclusion column and evaporative light scattering detector (UHPLC-SEC-ELSD), (2) direct analysis in real-time ion source Orbitrap-based mass spectrometry (DART-Orbitrap MS) and (3) ultra high-performance liquid chromatography with high-resolution time-of-flight mass spectrometry (UHPLC-HRTOF-MS).

For the UHPLC-SEC-ELSD, the team used one concentration (10 mg mL⁻¹) each for polysorbate micelles and polysorbate 20/80 and applied a 0.45-μm PVDF microfilter to avoid instrument clogging before LC separation. The instrument combined a chromatograph, a size exclusion column (150 × 4.6 mm; 1.7 μm, 0.3 mL min⁻¹ flow rate), and an optimized evaporative light-scattering detector.

For the DART-Orbitrap MS, the researchers used two concentrations (10 mg mL⁻¹, 5 mg mL⁻¹) each for polysorbate micelles and polysorbate 20/80, added caffeine (10 μg mL⁻¹) as an internal standard, and used a 0.45-μm PVDF microfilter before LC separation. They coupled a DART ion source with a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific) for a monitored range of m/z 100 to 1,100 and 100,000 resolving power (FWMH at m/z 200). With this method, the lowest calibrated levels for the micelles were 0.5 mg mL⁻¹ in water and 1 mg mL⁻¹ in apple juice. For this platform, the repeatability values were ~23% and ~46% for concentration values of 10 mg mL⁻¹ and 5 mg mL⁻¹, respectively.

As for the UHPLC-HRTOF-MS, the research team used three concentrations (100 μg mL⁻¹, 50 μg mL⁻¹, 10 μg mL⁻¹) each for polysorbate micelles and polysorbate 20/80, which they aliquoted (1 mL) and diluted in methanol (9 mL) before using a 0.45-μm PVDF microfilter and separating by LC. The platform combined a chromatograph, a reversed-phase column (100 × 2.1 mm and 1.8 μm particle, variable 0.3 to 0.6 mL min⁻¹ flow rate), and a TOF instrument set for full scan over a 100 to 2,000 Da range with 20,000 resolving power (FWMH at m/z 556.2766). The lowest calibrated level for in-source fragments was 0.5 μg mL⁻¹ in apple juice, and the calibration curve was linear over a range of 0.5 μg mL⁻¹ to 20 μg mL⁻¹. Repeatability values were ~2.5%, ~4% and ~20% for concentration values of 100 μg mL⁻¹, 50 μg mL⁻¹, and 10μg mL⁻¹.

Overall, the team found UHPLC-SEC-ELSD to be adequate for rapid screening for the presence of polysorbates in fruit juice. Micellar polysorbate, however, was indistinguishable from free polysorbate based on retention time, and free polysorbate coeluted with the matrix. On the other hand, both DART-Orbitrap MS and UHPLC-HRTOF-MS proved sensitive for the detection and quantification of polysorbates 20 and 80, including discerning between the two species in a fruit juice matrix. Neither approach distinguished micellar from free polysorbate. This study represents a step toward determining a universal quantification protocol with applications in food testing and consumer awareness programs.

Reference

1. Krtkova, V., et al. (2014, June) “Analytical strategies for controlling polysorbate-based nanomicelles in fruit juice,” Analytical and Bioanalytical Chemistry, 406(16) (pp. 3909–18), doi: 10.1007/s00216-014-7823-7.

Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.