different retention time between standards and EDTA plasma pool

Hussain

<p>Hello everyone,</p><p></p><p></p><p style="line-height:150%;mso-layout-grid-align:none; text-autospace:none">We try to develop a stable-isotope dilution ultra performance liquid chromatography–mass spectrometry (UPLC–MS/MS) method for the simultaneous quantification of choline, phosphocholine, and glycerophosphocholine.</p><p style="line-height:150%;mso-layout-grid-align:none; text-autospace:none">We still working on this method, we are out using an Acquity Ultra Performance LC system coupled to a MicroMass Quattro Premier XE tandem quadrupole mass spectrometer (Waters Corporation, Milford, MA, USA).</p><p></p><p style="line-height:150%;mso-layout-grid-align:none; text-autospace:none">The best UPLC–MS/MS conditions that we get as follow:</p><p style="line-height:150%;mso-layout-grid-align:none; text-autospace:none">The samples were separated on an Acquity UPLC BEH HILIC column (100mm×2.1mm (i.d.); 1.7µm particle size) with an Acquity HILIC VanGuard pre-column (5mm×2.1mm (i.d.); 1.7µm particle size) and a 0.2µm in-line filter (Waters Corporation). The column temperature was set to 30 ?C.</p><p style="line-height:150%;mso-layout-grid-align:none; text-autospace:none">The flow rate was 0.6 mL/min. The solvents were 15 mmol/L ammoniumformate (solvent A, pH 3.5) and acetonitrile (solvent B) with a gradient over the 6.5 min run time as follow: 0.0–3.5 min (12.5% A), 3.5–3.6 min (30% A), 4.5–4.6 min (20% A), and 4.6–6.5 min (12.5% A). All gradient steps were linear. The sample injection volume was 10µL.</p><p style="line-height:150%;mso-layout-grid-align:none; text-autospace:none">But there is difference in retention time for phosphocholine between standards and physiological sample (human EDTA plasma),<span style="color: #ff0000;"><strong> 3.50 min in standards vs 1.88 min in plasma.</strong></span></p><table border="1" cellpadding="0" cellspacing="0" style="width: 648px;"><tbody><tr><td colspan="6" width="648"><p style="line-height:150%"><strong>Table. Instrument parameters for LC–MS/MS   analysis of choline, phosphocholine, and glycerophosphocholine, and internal   standard choline.</strong></p></td></tr><tr><td width="182"><p style="line-height:150%">Analyte</p></td><td width="93"><p align="center" style="text-align:center;line-height:150%">Cone voltage (V)</p></td><td width="92"><p align="center" style="text-align:center;line-height:150%">Collision energy (eV)</p></td><td width="76"><p align="center" style="text-align:center;line-height:150%">Precursor ion (m/z)</p></td><td width="84"><p align="center" style="text-align:center;line-height:150%">Product ion (m/z)</p></td><td width="122"><p align="center" style="text-align:center;line-height:150%">Retention time (min)</p></td></tr><tr><td width="182"><p style="line-height:150%">Choline</p></td><td width="93"><p align="center" style="text-align:center;line-height:150%">34</p></td><td width="92"><p align="center" style="text-align:center;line-height:150%">16</p></td><td width="76"><p align="center" style="text-align:center;line-height:150%">104.08</p></td><td width="84"><p align="center" style="text-align:center;line-height:150%">60.05</p></td><td width="122"><p align="center" style="text-align:center;line-height:150%">1.79</p></td></tr><tr><td width="182"><p style="line-height:150%">Phosphocholine</p></td><td width="93"><p align="center" style="text-align:center;line-height:150%">39</p></td><td width="92"><p align="center" style="text-align:center;line-height:150%">21</p></td><td width="76"><p align="center" style="text-align:center;line-height:150%">184.11</p></td><td width="84"><p align="center" style="text-align:center;line-height:150%">124.86</p></td><td width="122"><p align="center" style="text-align: center; line-height: 150%;"><span style="color: #ff0000;">3.50 Standards</span></p><p align="center" style="text-align: center; line-height: 150%;"><span style="color: #ff0000;">1.88 Plasma</span></p></td></tr><tr><td width="182"><p style="line-height:150%">Gl...