different retention time between standards and EDTA plasma pool

Hello everyone,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.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).The best UPLC–MS/MS conditions that we get as follow: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.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.But there is difference in retention time for phosphocholine between standards and physiological sample (human EDTA plasma), 3.50 min in standards vs 1.88 min in plasma.Table. Instrument parameters for LC–MS/MS   analysis of choline, phosphocholine, and glycerophosphocholine, and internal   standard choline. AnalyteCone voltage (V)Collision energy (eV)Precursor ion (m/z)Product ion (m/z)Retention time (min)Choline3416104.0860.051.79Phosphocholine3921184.11124.863.50 Standards1.88 PlasmaGl...
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