Quantitative Analysis of Dairy Phospholipids by ³¹P NMR

Abstract

31 P NMR analysis of samples prepared in a sodium cholate detergent system was assessed as a method for the quantitative measurement of dairy phospholipids. Major phospholipid (PL) classes measured included: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin (SM) and dihydrosphingomyelin (DHSM). The 31 P NMR method was validated by comparison with a quantitative two-dimensional thin-layer chromatography (2D-TLC) technique. The 2D-TLC system was more sensitive, able to detect some minor compounds not observed by 31 P NMR. However, 31 P NMR is more suited to routine analysis, with sample analysis taking 36 min. The method was also more versatile and sample analysis was possible on high phospholipid containing materials without prior lipid extraction (e.g. buttermilk protein concentrate, beta serum liquid).

Phospholipid fingerprints of milk from different mammalians determined by 31P NMR: Towards specific interest in human health

“…Previous 31 P NMR characterisation of PLs conducted mainly on cow milk (Andreotti et al, 2006;MacKenzie et al, 2009;Murgia et al, 2003) can be directly compared with our data (Table 4). The use of the monophasic solvent system trimethylamine/dimethyl-formamide/guanidinium hydrochloride resulted in an underestimation of PE quantification due to the formation of different adducts (Andreotti et al, 2006;Murgia et al, 2003) when compared to a protocol using a detergent system at pH 7.1 (MacKenzie et al, 2009), or to our biphasic system at pH 7.7, which showed the best resolution of the PE peak; in addition, the PS peak was not well-resolved and was underestimated that was solved also by the two latter protocols.…”

“…The use of the monophasic solvent system trimethylamine/dimethyl-formamide/guanidinium hydrochloride resulted in an underestimation of PE quantification due to the formation of different adducts (Andreotti et al, 2006;Murgia et al, 2003) when compared to a protocol using a detergent system at pH 7.1 (MacKenzie et al, 2009), or to our biphasic system at pH 7.7, which showed the best resolution of the PE peak; in addition, the PS peak was not well-resolved and was underestimated that was solved also by the two latter protocols. Ether lipids (ethanolamine plasmalogen and alkyl-acyl-phosphatidylcholine) were not detected in two published works (Andreotti et al, 2006;MacKenzie et al, 2009), but an EPLAS level close to the one we found was reported by Murgia et al in spite of their very different protocol and of their weak resolution of this peak (Murgia et al, 2003). By comparison to these three published protocols, our current approach provides better resolution (well separated and narrower peaks, especially for PI), probably due to the setting of an optimal temperature (25°C for cow, camel and human milk, and 5°C for mare milk); this might also explain why our cow milk PI level is lower than the published values.…”

“…Milk PL composition is currently analysed by thin layer chromatography (Bitman, Wood, Mehta, Hamosh, & Hamosh, 1984;van Beusekom, Martini, Rutgers, Boersma, & Muskiet, 1990), HPLC (Rombaut & Dewettinck, 2006), or more recently by tandem mass spectrometry (Gallier, Gragson, Jimenez-Flores, & Everett, 2010), with a prior separation of PLs and neutral lipids. An alternative method is 31 P NMR spectroscopy that was used to determine PL classes of cow, buffalo and ewe milk (Andreotti, Trivellone, & Motta, 2006;MacKenzie, Vyssotski, & Nekrasov, 2009;Murgia, Mele, & Monduzzi, 2003), but showed some limitations that could be solved by adapting protocols previously used for characterising the PLs of tissues (Larijani, Poccia, & Dickinson, 2000;Lutz & Cozzone, 2010). We aimed to establish an improved sensitive 31 P NMR quantification procedure with minimal sample preparation for analysing the exact composition of PL classes at levels varying from 0.2% to 2% of total lipids in different milk sources (human, cow, mare and camel) (El-Agamy, 2009;Jensen, 1999;Morrison, 1968;Rombaut & Dewettinck, 2006).…”

Chlamydia trachomatis Relies on Autonomous Phospholipid Synthesis for Membrane Biogenesis

“…The lipids were extracted using the Bligh and Dyer method (39). The NMR analysis was performed as described (40). Briefly, the lipid extracts were suspended into 500 l of the cholate detergent system through vortexing and dispersion in a sonicating bath at 60°C for 30 min.…”

Determination of phosphatidylserine in milk-based nutritional products using online derivatization high-performance liquid chromatography

“…Reported techniques to determine PS in dairy products following a lipid extraction include thin layer chromatography (TLC) [12], 31 P-Nuclear Magnetic Resonance (NMR) spectroscopy [13] and high-performance liquid chromatography coupled with an evaporative light scattering detector (HPLC-ELSD) [14]. While TLC is more suitable for qualitative and semi-quantitative purposes [15], 31 P-NMR is unsuitable for routine analysis due to its high cost.…”