Polysorbate 80 is a surfactant and emulsifier frequently found in foods, drug products, as well as in biochemical solutions and preparations. Although its presence in foods is required to be documented by law, the exact concentration or proportion in which it is added is not. Determining such concentration is a relatively straightforward matter by quantitative NMR and/or quantitative LCMS if an authentic sample of the material can be obtained.
Polysorbate 80 is commercially available from many sources, but its specifications can vary widely. As such, we begin with quantitative NMR to determine the exact concentration of polysorbate 80 in the “pure” polysorbate 80 sample. Quantitative NMR requires the careful weighing of the sample as well as an internal standard, in this case, sodium 1,1,2,2,3,3-hexadeutero-3-(trimethylsilyl)-1-propanesulfonate (d6-DSS). The ratio of the integrals gives the molar ratio of the polysorbate 80 to d6-DSS. It’s important to set the relaxation delay (d1) long enough so that the sample is fully relaxed between pulses.
Figure 1: Quantitative NMR on a “pure” sample of polysorbate 80 shows it to be only 92%.
In principle, quantification can be performed using any of the resulting peaks, but in practice, only a few selected peaks are fully separated from other signals in the sample. In this case, the terminal methyl of the oleate group provides a clean signal, and is used for quantitation in the present example analysis vs the TMS signal in the standard. A molar ratio of 0.61:1 polysorbate:d6-DSS corresponds to an approximate purity of 92% for the polysorbate 80 standard based upon our weighed amounts in the prepared sample.
The concentrations of polysorbate 80 are too low for the NMR technique to be effective in the common foods, drugs and other formulations where it is used. For these analyses, we turn to LCMS which is more sensitive to levels of polysorbate 80 in these samples. Even though polysorbate 80 has a very low molar absorptivity, we are able to detect the compound readily using an Evaporative Light Scattering Detector (ELSD). ELSD responses are non-linear with concentration, but a concentration-response curve is readily built with serial dilutions of a 920 ppm (0.1% w/v of 92% polysorbate 80) solution.
Finally, with the quantitative method in place, the unknown sample is run employing the same method. Several ELSD peaks are detected, and it’s possible to generate multiple concentration-response curves for multiple species and fully quantify the mixture. But in the present case, the polysorbate 80 peak is sufficiently resolved to demonstrate the power and utility of quantitative NMR and quantitative LCMS when used together.
Figure 2: ELSD trace of the unknown sample. Each peak represents a different chemical compound. The scale is exaggerated to show the peak corresponding to polysorbate 80.
Figure 3: Quantitation of polysorbate 80 in the unknown sample. Based on the concentration-response curve, there is 800 ppm (0.08%) polysorbate 80 in the product.
If you’re interested in NMR and LCMS and how it can help you with quantitation, contact Emeryville Pharmaceutical Services.