Rapid characterization of commercial polysorbate 80 by ultra-high performance supercritical fluid chromatography combined with quadrupole time-of-flight mass spectrometry.

Polysorbate 80, as a nonionic surfactant, is widely used in the food, personal care, and pharmaceutical industries due to the advantages of high surface activity, low toxicity, etc. In fact, the products of polysorbate 80 are complex mixtures of oligomers. In this work, a novel and fast method was developed to characterize the commercial polysorbate 80 by ultra-high performance supercritical fluid chromatography (UHPSFC) combined with quadrupole time-of-flight mass spectrometry (QTOF-MS). Some crucial parameters, such as temperature, back pressure and flow rate were optimized. UHPSFC could distinguish n-mer from (n-1)-mer and (n+1)-mer in the same series, which provided the high separation resolution needed for quantitative determination of each oligomer in same series. It was not achieved in previous studies. Furthermore, the characteristic ion fragments were found in MS/MS experiment and used to identify different series. The results revealed that main components of this nonionic surfactant comprise polyethylene oxide (PEO), PEO-monooleate, PEO-isosorbide, PEO-isosorbide monooleate, PEO-isosorbide dioleate, PEO-sorbitan, PEO-sorbitan monooleate, PEO-sorbitan dioleate and PEO-sorbitan trioleate, etc. The separation was performed using BEH stationary phase, so the relationship between molecular structure of these oligomers and chromatographic retention behavior in supercritical fluid chromatography were also investigated for first time. The whole analytical process only takes 8min for one sample. Therefore, UHPSFC-QTOF-MS is a simple, novel and efficient tool to analyze polysorbate 80.

Identification of the Allergenic Ingredients in Reduning Injection by Ultrafiltration and High-Performance Liquid Chromatography.

Reduning injection is a traditional Chinese medicine injection which has multiple functions such as clearing heat, dispelling wind, and detoxification. Although Reduning injection was widely utilized, reports of its allergenicity emerged one after another. However, there is little research on its allergenic substances. The aim of this study is to evaluate the sensitization of Reduning injection and explore the underlying cause of the anaphylactic reaction. The main ingredients in Reduning injection were analyzed before and after ultrafiltration. Ultrafiltrate Reduning injection, unfiltered Reduning injection, egg albumin, Tween-80, and nine effective components in Reduning injection were utilized to sensitize guinea pigs. The serum 5-hydroxytryptamine level was used to assess the sensitization effect of Reduning injection. We found a significant decrease in Tween-80 content comparing to other components in the injection after ultrafiltration. Unfiltered Reduning injection, Tween-80, chlorogenic acid, and cryptochlorogenin acid caused remarkable anaphylactoid reaction on guinea pigs while ultrafiltration Reduning resulted in a significantly lower degree of sensitization. Our results suggest that ultrafiltration could significantly reduce the sensitization of Reduning injection, which is likely due to the decrease of Tween-80. We also conjectured that the form of chlorogenic acid and cryptochlorogenin acid within the complex solution mixture may also affect the sensitizing effect.

Removal of Polysorbate 80 by complexation prior to LC-MS analysis.

The presence of Polysorbate 80 in samples can challenge liquid chromatography-electrospray ionisation mass spectrometry (LC-ESI-MS) analysis as it is easily ionised and detected. In this study, we demonstrate that interference from Polysorbate 80 can be reduced by complexation with a metal ion followed by precipitation by thiocyanate. The precipitation procedure was tested on a mixture of low molecular weight compounds (e.g. amino acids and non-amino organic acids) and it was shown that none of the tested compounds were precipitated.

Quantitation of low concentrations of polysorbates in high protein concentration formulations by solid phase extraction and cobalt-thiocyanate derivatization.

A spectrophotometric method was developed to quantify low polysorbate (PS) levels in biopharmaceutical formulations containing high protein concentrations. In the method, Oasis HLB solid phase extraction (SPE) cartridge was used to extract PS from high protein concentration formulations. After loading a sample, the cartridge was washed with 4M guanidine HCl and 10% (v/v) methanol, and the retained PS was eluted by acetonitrile. Following the evaporation of acetonitrile, aqueous cobalt-thiocyanate reagent was added to react with the polyoxyethylene oxide chain of polysorbates to form a blue colored PS-cobaltothiocyante complex. This colored complex was then extracted into methylene chloride and measured spectrophotometrically at 620 nm. The method performance was evaluated on three products containing 30-40 mg L(-1) PS-20 and PS-80 in ≤70 g L(-1) protein formulations. The method was specific (no matrix interference identified in three types of protein formulations), sensitive (quantitation limit of 10 mg L(-1) PS) and robust with good precision (relative standard deviation ≤6.4%) and accuracy (spike recoveries from 95% to 101%). The linear range of the method for both PS-20 and PS-80 was 10 to 80 mg L(-1) PS. By diluting samples with 6M guanidine HCl and/or using different methylene chloride volumes to extract the colored complexes of standards and samples, the method could accurately and precisely quantify 40 mg L(-1) PS in up to 300 g L(-1) protein formulations.

Quantification of unadsorbed protein and surfactant emulsifiers in oil-in-water emulsions.

Unadsorbed emulsifiers affect the physical and chemical behaviour of oil-in-water (O/W) emulsions. A simple methodology to quantify unadsorbed emulsifiers in the aqueous phase of O/W emulsions has been developed. Emulsions were centrifuged and filtered to separate the aqueous phase from the oil droplets and the concentration of unadsorbed emulsifiers in the aqueous phase determined. The quantification of unadsorbed surfactants based on the direct transesterification of their fatty acids was validated for Tween 20, Tween 80, citric acid ester (Citrem), Span 20 and monolauroyl glycerol. To determine unadsorbed proteins, results obtained with Folin-Ciocalteu reagent or UV-spectrophotometry were compared on emulsions stabilized by ß-lactoglobulin (BLG), ß-casein (BCN) or bovine serum albumin (BSA). The first method gave more accurate results especially during aging of emulsions in oxidative conditions. The whole methodology was applied to emulsions stabilized with single or mixed emulsifiers. This approach enables optimization of emulsion formulations and could be useful to follow changes in the levels of unadsorbed emulsifiers during physical or chemical aging processes.

Comparable virus inactivation by bovine or vegetable derived Tween 80 during solvent/detergent treatment.

A mixture of Tri-n-butyl phosphate (TNBP) and Polysorbate 80 (Tween 80) is often used for virus inactivation during the manufacture of medicinal products derived from human plasma. This procedure, known as solvent/detergent treatment, is of high effectiveness for inactivation of enveloped viruses. Tween 80 can be manufactured from bovine tallow or from vegetable material. As the bovine-derived Tween 80 is normally used for the solvent/detergent treatment, the question has been raised whether vegetable-derived Tween 80 can be applied as an alternative substance for the solvent/detergent treatment. Comparable inactivation studies were therefore performed using Vesicular Stomatitis Virus (VSV), Pseudorabiesvirus (PRV), Semliki Forest Virus (SFV) and Bovine Diarrhoea Virus (BVDV). In principle, no differences were observed in the effectiveness of the solvent/detergent treatment when bovine or vegetable-derived Tween 80 was used. The comparability in the efficiency of both detergents for virus inactivation was shown to be independent of solvent/detergent concentration, of temperature (16 degrees C and 6 degrees C vs. 27 degrees C and 25 degrees C) and protein concentration (10% and 5% human albumin). In summary, vegetable-derived Tween 80 is of the same effectiveness as bovine-derived Tween 80, when used for virus inactivation by the solvent/detergent treatment.