Phosphatidylcholine analysis, separation

The ACE analysis of interactions between drugs and phospholipid bilayers of liposomes present as a pseudostationary phase was performed by Zhang et al. (32). The capillaries were treated to eliminate electroendosmosis. Freshly prepared and essentially neutral small unilamellar liposomes composed of egg phosphatidylcholine were sucked into the capillary. These liposomes increased both the retention of four negatively charged drugs and the separation between the substances (Fig. 6). The chromatographic retentions of these drugs on immobilized phosphatidylcholine liposomes, ex-... 

Fig. 21.6 Typical positive ion MALDI-TOF mass spectra of organic extracts of spermatozoa from a healthy volunteer. Before MS analysis, spermatozoa were separated into annexin V-negative (a) and annexin V-positive (b) spermatozoa. Peaks are labeled according to the corresponding m/z ratios. The most indicative peak groups are marked with grey bars. The asterisk indicates a typical DHB matrix peak (m/z = 551). Abbreviations LPC, lyso-phos-phatidylcholine PC, phosphatidylcholine. Reprinted from Glander et al.. Deterioration of spermatozoal plasma membrane is associated with an increase of sperm lyso-phosphatidyl-cholines, Andrologia, 2002 with permission from Blackwell...

Experiment I. In a time-course experiment, mucosal PGE production and phospholipid fatty acid profile were assessed at d 0,4,8,12, and 16 of dietary treatment in formula-fed and naturally reared piglets (n = 5 piglets per time per dietary treatment). Mucosal cells were scraped from proximal ends of the small intestine and frozen at -80°C for later lipid analysis. Lipids were extracted by a modified Folch procedure (15). Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were separated by thin-layer chromatography (16), and fatty acids in each phospholipid fraction were analyzed by gas chromatography. For eicosanoid measures, fresh mucosal tissue was incubated in Kreb s Ringer bicarbonate buffer as described previously (17). PGE2 was extracted from the incubation media with ethyl acetate and quantified using a competitive enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI). 

Hyphenated TLC techniques. TLC has been coupled with other instrumental techniques to aid in the detection, qualitative identification and, occasionally, quantitation of separated samples, and these include the coupling of TLC with high-pressure liquid chromatography (HPLC/TLC), with Fourier transform infra-red (TLC/FTIR), with mass spectrometry (TLC/ MS), with nuclear magnetic resonance (TLC/NMR) and with Raman spectroscopy (TLC/RS). These techniques have been extensively reviewed by Busch (1996) and by Somsen, Morden and Wilson (1995). The chemistry of oils and fats and their TLC separation has been so well established that they seldom necessitate the use of these coupling techniques for their identification, although these techniques have been used for phospholipid detection. Kushi and Handa (1985) have used TLC in combination with secondary ion mass spectrometry for the analysis of lipids. Fast atom bombardment (FAB) has been used to detect the molecular species of phosphatidylcholine on silica based on the molecular ion obtained by mass spectrometry (Busch et al, 1990). 

Figure 9.4 High-pressure liquid chromatography separation of 50 pg of a natural phosphatidylcholine mixture from egg yolk. The reconstructed ion chromatograms of diglyceride ions were selected from data acquired by full mass scanning from 120 amu to 820 amu. The relative intensity is shown based on the peak height. Column 3 pm Ultrasphere-ODS (4.6 mm x 7.5 cm). Mobile phase MeOH/hexane/0.1 m NH4OAC (71 5 7), 1 mlmin . Reprinted with permission from Kim, H. Y. and Salem, N. Jr, Phospholipid molecular species analysis by thermospray liquid chromatography/mass spectrometry. Anal. Chem., 58 (1), 9-14, 1986.

Fig. 1. A preparation of ARF, purified by a modification of the procedure of Kahn and Gilman (9), was chromatographed on Ultrogel AcA 54. Samples of fractions across a peak of protein corresponding in apparent size to ARF were taken for analysis by SDS-PAGE (left) and assay of their ability to stimulate choleragen-catalyzed ADP-ribosylation of Gsa. For the latter, samples were incubated with choleragen, Gsa (0.1 ig), 10 pM [32p]NAD, 250 pM GTP and 1 mM dimyristoyl phosphatidylcholine (15). Proteins were separated by SDS-PAGE. An autoradio am of the gel is on the right. Fractions that enhanced ADP-ribosylation of Gsa also increased auto-ADP-ribosylation of choleragen Ai. Both of these activities were correlated with the presence of a single protein of approximately 19 kDa. Data are from reference 15.

Figure 4. Analysis of the different polyphosphoinositides extracted from P-labelled cells. (A) Schematic representation of the expected separation of a mixture of P-labelled phospholipids by thin layer chromatography (TLC). Plates are silica gel 60 and the solvent for phosphoinositide separation is a mixture of CHCI3, CH3COCH3, CH3OH, CH3COOH and H O (80 30 26 24 14, v/v). MP, major phospholipids (phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine). (B) Typical high-performance liquid chromatography profile showing the separation of the various phosphoinositides from a mixture of P-labelled phosphoinositides. A specific gradient must be used to separate PtdIns(4)P and PtdIns(5)P (Rameh et ai, 1997 Niebhur et al., 2002).

Figure 4.9 Representative ESI-MS analysis of lipid classes resolved by intrasource separation. Lipid extracts from mouse liver samples were prepared by using a modified procedure of Bligh and Dyer [1]. MS analysis was performed with a TSQ Vantage triple-quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an automated nanospray apparatus (i.e., TriVersa, Advion Bioscience Ltd., Ithaca, NY) and Xcalibur system software. Mass spectra were acqnired directly from the diluted hpid extract in the negative-ion mode (a), after addition of 50 nmol LiOH/mg of protein in the diluted lipid extract and analyzed in the negative-ion mode (h), or the identical hpid solution to that in (b) in the positive-ion mode (c). IS denotes internal standard PC, PE, PG, PI, PS, TAG, NEFA, and CL stand for phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidyUnositoL phosphatidylserine, triacylglycerol, nonesterified fatty acid, and doubly charged cardioUpin, respectively.

Five As species were separated within 5 min from human blood serum by ion pair HPLC-ICP-MS. 20 pL sample was injected on a CIS column coated with phosphatidylcholine. The mobile phase was composed of 5 mM citrate buffer (pH 4.0)-5mM SDS-5mM TMAH-0.2mM 3-[(3-cholamidopropyl)-dime-thylammonio-1-propane sulfonate. The precision of the method, based upon analysis of 15 pg As, was better than... 

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