Quantitative determination of phospholipid compositions by ESI-MS: effectsof acyl chain length, unsaturation, and lipid concentration on instrument response

Electrospray ionization-mass spectrometry (ESI-MS) is a very promising tool for the analysis of phospholipid compositions, but is hampered by the fact that not all molecular species are detected with equal efficiency, We stud ied this and other issues that need to be taken into account to obtain trul y quantitative compositional data, The key findings were as follows: First, the instrument response for both saturated and unsaturated phospholipid sp ecies decreased with increasing acyl chain length. This effect became incre asingly prominent with increasing overall lipid concentration, Second, the degree of acyl chain unsaturation also had a significant effect on instrume nt response, At the highest concentration studied (10 pmol/mul), polyunsatu rated species gave 40% higher intensity than the fully saturated ones. The effect of unsaturation diminished and nearly disappeared with progressive d ilution, Third, the instrument response for the different head group classe s varied markedly depending on the infusion solvent used, Notably, inclusio n of ammonia in the infusion solvent eliminated sodium adduct formation in the positive ion mode, thus greatly simplifying the interpretation of the s pectra, The fact that instrument response is dependent on many structural f eatures, overall lipid concentration, solvent composition, and instrument s ettings makes it necessary to include several internal standards for each p hospholipid class to obtain accurate data, Preferably, both unsaturated and saturated standards should be used, Finally, we quantified the major phosp holipid classes of BHK cells using ESI-MS, The data agreed closely with tho se obtained with thin-layer chromatography and phosphorus analysis. This st udy indicates that quantitative compositional data can be obtained with ESI -MS, provided that proper attention is paid to experimental details, partic ularly the choice of internal standards.