Temperature optimization for improved determination of phosphatidylserine species by micro liquid chromatography with electrospray tandem mass spectrometric detection

Abstract

A sensitive method for determination of disaturated phosphatidylserine species in the presence of their monounsaturated analogs has been developed, using micro liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The hydrophobic nature of the phosphatidylserine species required a combination of low-eluting sample solvents and sub-ambient temperatures in order to focus large sample volumes up to 20 microL. The samples were dissolved in 2-propanol:hexane:water (20:10:4, v/v/v) prior to 1:9 dilution with ammonium formate buffer:2-propanol:tetrahydrofuran (30:55:15, v/v/v) and final 1:4 dilution with ammonium formate buffer (10 mM):2-propanol: tetrahydrofuran (55:37.5:7.5, v/v/v). The analytical column was a 0.5 x 150 mm stainless steel column packed with 5 microm C30 particles, while the mobile phase contained ammonium formate buffer (10 mM): 2-propanol:tetrahydrofuran (30:55:15, v/v/v). A temperature program from 5 degrees C (hold for 3 minutes) to 75 degrees C at 8 K/min provided separation of the disaturated phosphatidylserine species from their monounsaturated analogs, making available a sensitive determination of the isobaric species. The mass limit of detection for dipalmitoyl phosphatidylserine was 100 pg, corresponding to a concentration limit of detection of 5 pg/microL when using an injection volume of 20 microL. This is an improvement by a factor of 20 as compared to previously reported numbers obtained with conventional LC columns. The within-assay precision of dipalmitoyl phosphatidylserine was 11.9% RSD (n = 3), while the retention time precision was 4.1% RSD (n = 6).

Temperature-assisted on-column solute focusing: A general method to reduce pre-column dispersion in capillary high performance liquid chromatography

“…In their work temperature programming initiated at sub-ambient temperatures, ca. 5 °C, was used to focus samples of retinyl esters, polyolefin based Irganox antioxidants and ceramides made in 80-100% acetonitrile prior to separation using a neat acetonitrile mobile phase and C18 column [46-50]. High hydrophobicity and poor analyte solubility in water, necessary for on-column focusing was common to all of this work.…”

Detection Modes with High Temperature Liquid Chromatography—A Review

“…Today, as in classical LC-MS, APCI (150) and ESI (100,134,151) are the dominating ionization techniques used in HTLC-MS. The efficiency of these MS interfaces that are based on nebulisation, vaporization and desolvatation processes, can normally be expected to vary with mobile phase properties (surface tension, viscosity, and polarity).…”

Temperature-assisted solute focusing with sequential trap/release zones in isocratic and gradient capillary liquid chromatography: Simulation and experiment

“…Cooling cLC columns to sub-ambient temperatures is also advantageous. The Greibrokk group has done extensive work in this area with temperature programs initiated near 5 °C [48–52]. Recently, Schoenmakers et al immersed a capillary monolith in ice water to induce an in-column solvent switch [33].…”