Isomerization of octadecapentaenoic acid (18:5n‐3) in algal lipid samples under derivatization for GC and GC‐MS analysis

During gas chromatography (GC) analysis of fatty acid (FA) composition of the dinoflagellate Gymnodinium kowalevskii, we found unex‐pectedly low and irreproducible content of all‐cis‐3,6,9,12,15‐octadecapentaenoic acid (18:5n‐3), which is an important chemotaxonomic marker of several classes of microalgae. We compared chromatographic behavior of 18:5n‐3 methyl ester and other GC derivatives obtained using different conventional methods of derivatization. The use of methods based on saponification or base‐catalyzed transesterification resulted in a mixture of double‐bond positional isomers of 18:5. On a SUPELCOWAX 10 column, the equivalent chain length (ECL) value for authentic 18:5n‐3 methyl ester was 20.22, whereas the main component after base‐catalyzed methylation had ECL 20.88. Attempts to prepare N‐acyl pyrrolidides or 4,4‐dimethyloxazoline (DMOX) derivatives of 18:5n‐3 also gave inadequate results. These derivatives also showed a main peak corresponding to isomerized 18:5. Mass spectra for both DMOX and pyrrolidide derivatives of this compound showed the base peak at m/z 139, probably corresponding to 2,6,9,12,15‐18:5 acid. Of all methods tested for methylation, only derivatization with 5% HCl or 1% sulphuric acid in methanol gave satisfactory results. Therefore, GC or GC‐mass spectrometry analyses of algal lipids containing 18:5n‐3 may be inaccurate when base‐catalyzed methods of FA derivatization are applied. The best and simplest way to avoid incorrect GC results is to use standard acid‐catalyzed methylation.     

Utilization of (18‐Crown‐6)‐2,3,11,12‐tetracarboxylic Acid as a Chiral NMR Solvating Agent for Diamines and β‐Amino Acids

The compound (18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid was evaluated as a chiral nuclear magnetic resonance (NMR) solvating agent for a series of diamines and bicyclic β‐amino acids. The amine must be protonated for strong association with the crown ether. An advantage of (18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid over many other crown ethers is that it undergoes a neutralization reaction with neutral amines to form the protonated species needed for binding. Twelve primary diamines in neutral and protonated forms were evaluated. Diamines with aryl and aliphatic groups were examined. Some are atropisomers with equivalent amine groups. Others have two nonequivalent amine groups. Association equilibria for these systems are complex, given the potential formation of 2:1, 1:1, and 1:2 crown‐amine complexes and given the various charged species in solution for mixtures of the crown ether with the neutral amine. The crown ether produced enantiomeric differentiation in the ¹H NMR spectrum of one or more resonances for every diamine substrate. Also, a series of five bicyclic β‐amino acids were examined and (18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid caused enantiomeric differentiation in the ¹H NMR spectrum of three or more resonances of each compound. Chirality 27:708–715, 2015. © 2015 Wiley Periodicals, Inc.