title: Evaluation and optimization of high-performance liquid chromatography coupled with high-resolution mass spectrometry for phospholipid quantitation.
abstract: BACKGROUND
  High-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC/HRMS) provides an attractive alternative to the traditional triple quadrupole mass spectrometry selected reaction monitoring (SRM) methodology in the field of quantitation.
  
  
  METHODS
  An LTQ Orbitrap Velos mass spectrometer coupled with a Shimadzu UHPLC system was used. A mass extraction window (MEW) was defined to mathematically correlate with mass resolving power (MRP). Five MRP measurement conditions at 7500, 15,000, 30,000, 60,000, and 100,000, and five MEW widths at 10%, 20%, 50%, and 100% of ±1000000/MRP, and were utilized as the experimental conditions. Comparison of profile and centroid algorithms was evaluated as well.
  
  
  RESULTS
  Selection of pH 8.5 buffer, C4 column, 20% IPA, and 10 ppm H3 PO4 , minimized hydrophobic and silanol interactions to enhance separation. Narrowing the MEW minimized background noise, while over-narrowing the MEW gave signal loss due to mass accuracy deviation. The mass accuracy deviation was larger for lower MRP measurements especially with centriod data, and hence a profile algorithm was recommended. The post-ionization signal suppression was observed with low MRP and was further confirmed with the analysis of multi-level linearity.
  
  
  CONCLUSIONS
  It was found that the HPLC/HRMS method with conditions of 20% ±1000000/MRP as MEW, 30,000 or 60,000 MRP, and profile algorithm, provide optimum results for quantitation of seven model phospholipids.
authors.affiliation: Analytical Development Laboratories, Takeda Pharmaceutical International Co., 35 Landsdowne Street, Cambridge, MA, 02139, USA.
authors.forename: Mingkun
authors.initials: M
authors.lastname: Fu
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
authors.forename: Qing
authors.initials: Q
authors.lastname: Lu
authors.forename: Guoyu
authors.initials: G
authors.lastname: Pan
authors.forename: Csanad
authors.initials: C
authors.lastname: Varga
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D016014:Linear Models; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D010743:Phospholipids / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D012680:Sensitivity and Specificity
chemical_list: D010743:Phospholipids