Carrageenan analysis. Part 3: Quantification in swine plasma

Abstract

Development and validation of this method was conducted to support a 28-day piglet feeding study of swine-adapted infant formulations stabilised with carrageenan. The validation was performed in accordance with USFDA Good Laboratory Practice (GLP) Regulations and associated current bioanalytical guidelines. Separation of carrageenan from plasma protein was unsuccessful using saturated sodium chloride due to the extremely strong cross-linking interactions between carrageenan and protein. Poligeenan is the deliberately acid-hydrolysed low molecular weight polygalactan non-food product produced from carrageenan. Poligeenan molecules are nearly identical to carrageenan molecules with respect to molecular structure, the primary difference being molecular weight. These poligeenan molecules have similar molecular weight when compared with the lowest molecular weight fraction of carrageenan called the low molecular-weight tail (LMT). Poligeenan was separated from plasma protein using the salting procedure, this being due to the significantly weaker interaction with protein caused by its shorter molecular chain length. Thus, poligeenan was applied as a chemical analyte surrogate for the LMT of carrageenan solely for the development and validation of the method. This method was used to try to detect the LMT of the carrageenan test material during the 28-day piglet feeding study, and if such was absorbed into the bloodstream. Successful development and validation of the method was achieved using LC-MS/MS coupled with ESI in negative-ion mode. A standard curve of instrument response versus poligeenan concentration was developed using swine plasma spiked with a range of poligeenan concentrations. The lower level of quantification (LLOQ) of poligeenan was 10.0 µg ml^–1, and the quantification range was 10.0–100.0 µg ml^–1. No animals were fed poligeenan.

Keywords:

• carrageenan
• poligeenan
• swine plasma
• LC-MS/MS
• ESI

Acknowledgements

The authors thank: Bjorn A. Thorsrud, Study Director (MPI), S. Karina Kwok (MPI), Lori L. Cochrane (MPI), Holly Bridges (MPI), Roger N. Hayes (MPI), David J. Humphries (MPI), Mario Pellerin (MPI), Myra L. Weiner (TOXpertise LLC), Brinda Mahadevan (Abbott Nutrition) and Christopher J. Sewall (FMC) for technical discussions; Eunice M. Cuirle (FMC), Theresa M. Hedrick (IFC) and Brenda Frantz (MPI Research) for project management and coordination. William Blakemore is owner and principal of Celtic Colloids Inc., a consulting company providing advice on hydrocolloids technology and including leadership on analytical methods development to private companies. Ashley Brant was a senior project manager with MPI Research, a contract research organisation, and Jonathan Bissland is a bioanalytical method developer with MPI Research. Natalie Bissland was formerly a bioanalytical validation specialist with MPI Research and has since joined Eurofins Laboratories. The research work detailed in this paper was carried out for FMC Corporation (FMC) and the International Formula Council (IFC) under cost reimbursable contracts. FMC is a manufacturer of carrageenan and products containing carrageenan. The IFC is an association of manufacturers and marketers of formulated nutrition products, e.g. infant formulas and adult nutritionals, whose members are based predominantly in North America. The method development and validation reported in this paper is the professional work product of the authors. FMC and IFC were given the opportunity to review this paper and to offer comments on the content. Those comments did not alter the professional opinions of the authors. The authors have not appeared in any legal proceedings related to the findings reported in this paper. The conclusions drawn are not necessarily those of FMC or IFC.