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Evaluation and optimization of high‐performance liquid chromatography coupled with high‐resolution mass spectrometry for phospholipid quantitation
Author(s) -
Fu Mingkun,
Xu Wei,
Lu Qing,
Pan Guoyu,
Varga Csanad
Publication year - 2014
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.6835
Subject(s) - chemistry , chromatography , mass spectrometry , resolution (logic) , analytical chemistry (journal) , orbitrap , high performance liquid chromatography , artificial intelligence , computer science
RATIONALE 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 ±1/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 H 3 PO 4 , 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% ±1/MRP as MEW, 30 000 or 60 000 MRP, and profile algorithm, provide optimum results for quantitation of seven model phospholipids. Copyright © 2014 John Wiley & Sons, Ltd.