High-performance thin-layer chromatography was directly combined with electrospray mass spectrometry (ESI-MS) for structural identification issues below the level of lipid classes in complex samples through a portable, automated, elution-based interface. For samples as diverse as biodiesel and human plasma, separation conditions using Automated Multiple Development were selected in each case to provide lipid classes as zones narrow enough to ensure a direct transfer of them to ESI-MS. The respective zone of interest can be selected at will. ESI spectra of neutral lipids and sphingolipids showed sodium adducts when recorded from the plate. By using the described technique and ion-trap technology, the respective sodium adducts were fragmented. Sodium remained as the charge of the fragment ions and, thus, was useful for their structural identification through MS. In this way, composition profiles of each class by ESI-MS, and further identification of individual lipids and the molecular species belonging to each of them, were obtained by MS/MS and/or high-resolution MS. Thus, mono and diacylglycerides in ESI and fatty acids (in ESI-) were identified as low-concentration impurities in a fatty acid methyl ester-based biodiesel sample. Likewise, molecular species of sphingomyelins and globotriaosylceramides were unequivocally identified in human plasma samples.

High-performance thin-layer chromatography (HPTLC)-densitometry was directly combined with electrospray (ESI) tandem mass spectrometry for obtaining rapid and relevant structural identification of phospholipids (PL) species associated to membrane proteins (MP), in non-sulfur, purple bacteria having photosynthetic activity. Thus, species belonging to phosphatidylcholines (PC), phosphatidylethanolamines (PE), cardiolipins (CL) and phosphatidylglycerols (PG) associated to MP were investigated in bacterial membrane extracts from Rhodobacter (Rb.) blasticus, Rhodospirillum (R.) rubrum and Rhodobaca (Rbc.) bogoriensis, as well as those which are bound to a purified MPphotosynthetic complex from Rbc. bogoriensis. PL-classes were separated using a 7-step gradient-solvent sequence with a previous acid plate preconditioning, using Automated Multiple Development. Band zones of the plate corresponding to PL classes were selected to ensure their direct transfer to ion-trap MS equipment through an elution-based interface. Under the studied conditions, ESI þ-MS spectra of PC and CL mostly showed sodium adducts ([M þ Na] þ) and [M-2H þ 3Na] þ , respectively, when recorded from the plate. The respective sodium adducts were fragmented in the ion-trap, and sodium remained as the charge of the fragment ions, thus being useful for their structural identification through MS/MS. ESI-MS and MS/MS spectra of CL were also obtained as [M-2H] 2À , as well as those of PE and PG species as [M-H]and [M] À , respectively. In this way, relative composition profiles of each studied PL-class by ESI-MS, and further identification of individual PL and the molecular species belonging to each of them by MS/MS were obtained.

In this paper we present a rapid method for determining phosphorylcholine (ChoP). ChoP was first hydrolyzed by the enzyme alkaline phosphatase (AP); the choline formed was then submitted to a reaction with Choline Oxidase (ChOx). Both reactions were carried out simultaneously, in the same test without previous steps of incubation. The analytical signals used were the intrinsic fluorescence of ChOx due to FAD and that corresponding to a fluorescein derivative bonded to ChOx (ChOx-FS); both can be related with the concentration of ChoP. Once the conditions were optimized, the response range for ChoP was 5.2•10-7-1.0•10-5 M using the peak area as the analytical parameter with a precision of about 4% (RSD) at both ChOx and ChOx-FS wavelengths. In the experimental conditions found, it was also possible to determine free choline (Ch) with figures of merit similar to those obtained for ChoP. These results have made possible the sequential determination of Ch and ChoP in milk powder, using only one aliquot of the mixture, with good results. The recoveries obtained for both analytes were close to 100 %. The method is rapid because an incubation step is not necessary. Moreover, the enzymatic reaction is autoindicating and thus the additional detection step required by other published enzymatic methods is avoided.