Phospholipids in liquid chromatography/mass spectrometry bioanalysis: comparison of three tandem mass spectrometric techniques for monitoring plasma phospholipids, the effect of mobile phase composition on phospholipids elution and the association of phospholipids with matrix effects.
Because plasma phospholipids may cause matrix effects in bioanalytical liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods, it is important to establish optimal mass spectrometric techniques to monitor the fate of phospholipids during method development and application. We evaluated three MS/MS techniques to monitor phospholipids using positive and negative electrospray ionization (ESI). The first technique is based on using positive precursor ion scan of m/z 184, positive neutral loss scan of 141 Da and negative precursor ion scan of m/z 153. The second technique is based on using class-specific positive and negative selected reaction monitoring (SRM) transitions to monitor class-representative phospholipids. The third technique, previously reported, utilizes in-source collision-induced dissociation (CID)-based positive SRM of m/z 184 --> 184. We recommend the all-inclusive technique 1 for use in qualitative assessment of all classes of phospholipids and technique 2 for use in quantitative assessment of class-representative phospholipids. Secondly, we evaluated the elution behaviors of the plasma phospholipids under different reversed-phase mobile phase conditions. The phospholipid-eluting strength of a mobile phase was mainly dependent on the type and amount (%) of the organic eluent and the strength increased in the order of methanol, acetonitrile and isopropyl alcohol. Under the commonly used gradient and isocratic elution schemes in LC/MS/MS bioanalysis, not all the phospholipids are eluted off the column. Thirdly, we investigated the association between phospholipids and matrix effects in positive and negative ESI using basic, acidic and neutral analytes. While the phospholipids caused matrix effects in both positive and negative ESI, the extent of ionization suppression was analyte-dependent and was inversely related to the retention factor and broadness of the phospholipids peaks. The lysophospholipids which normally elute earlier in reversed-phase chromatography are more likely to cause matrix effects compared to the later-eluting phospholipids in spite of the larger concentrations of the latter in plasma.
Xia
Yuan-Qing
YQ
Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.
Jemal
Mohammed
M
D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D010743:Phospholipids / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
D010743:Phospholipids