Experimental - Polar Metabolite Analysis
Typical polar metabolite analysis report as of September 2026.
Sample Preparation
Lipid samples extracted using 2-phase or a Wessel-Flügge extraction. Extracted analysts are dried and reconstituted in ……………… internal standard (ISTD). Following vortex and centrifugation at ~12k RPM/~18k RCF, 35 µL of each sample is transferred to HPLC vials (p/n 6ERV11-03PPCT / 6ARC11NRT1RT, Thermo) and the remaining 5 uL is used to generate a pool sample. Sample injection order is randomized prior to analysis by LC-MS/MS. All solvents are LCMS grade (Optima, Thermo).
Data acquisition
Typically, 2 µL sample, maintained at 4°C, is injected by a UHPLC system (Vanquish Duo, Thermo) and separated by Hydrophilic Interaction Liquid Chromatography (iHILIC®-(P) Classic, PEEK-SS, 100×2.1mm, 5µm, 200Å, P/N 180.052.0520, HILICON). The column is heated to 40 °C. The gradient, generated at 300uL/min) starts at 90% B /10% A and decreases linearly to 40% B / 60% A over 15 min and held at 40% B / 60% A for 2 min. hereafter the gradient is returned to 90% B /10% A and conditioned for 4 min. Buffer A: 20 mM ammonium carbonate in water adjusted to pH 9.6, Buffer B: acetonitrile. All solvents are LCMS grade (Optima, Thermo). For targeted profiling, MS data are acquired using polarity switching with alternating full-scan mass ranges of m/z 55–202 and 200–1000. For Data Dependent Acquisition (DDA) positive and negative data are acquired in separate runs. Source conditions: spray voltages in positive and negative mode are set to +3.5 kV and −3.0 kV, respectively, Capillary temperature: 350 °C, Sheath gas: 60, Auxiliary gas: 20, Sweep gas = 1, and S-lens RF level: 60
Typically, 2 µL sample, maintained at 4°C, is injected by a UHPLC system (Vanquish Duo, Thermo) and separated by Hydrophilic Interaction Liquid Chromatography (iHILIC®-(P) Classic, PEEK-SS, 100×2.1mm, 5µm, 200Å, P/N 180.052.0520, HILICON). The column is heated to 40 °C. The gradient, generated at 300uL/min) starts at 90% B /10% A and decreases linearly to 40% B / 60% A over 15 min and held at 40% B / 60% A for 2 min. hereafter the gradient is returned to 90% B /10% A and conditioned for 4 min. Buffer A: 20 mM ammonium carbonate in water adjusted to pH 9.6, Buffer B: acetonitrile. All solvents are LCMS grade (Optima, Thermo). For targeted profiling, MS data are acquired using polarity switching with alternating full-scan mass ranges of m/z 55–202 and 200–1000. For Data Dependent Acquisition (DDA) positive and negative data are acquired in separate runs. Source conditions: spray voltages in positive and negative mode are set to +3.5 kV and −3.0 kV, respectively, Capillary temperature: 350 °C, Sheath gas: 60, Auxiliary gas: 20, Sweep gas = 1, and S-lens RF level: 60
Standard HILIC method
Data Query
Targeted profiling data are processed using SkyLine while DDA data typically are queried using Compound Discoverer. Precursor and fragment ion mass search accuracies are set to 5 ppm and 10 ppm, respectively. ….
Targeted profiling data are processed using SkyLine while DDA data typically are queried using Compound Discoverer. Precursor and fragment ion mass search accuracies are set to 5 ppm and 10 ppm, respectively. ….
Data Analysis
Targeted data are processed…. DDA data are processed….
Targeted data are processed…. DDA data are processed….
Results
Results are shared via email. Larger files are shared via DropBox. The results are typically presented in an Excel report with multiple tabs. Important tabs includes: …….. Explanation of typical Excel Report format and an example Excel Report are available: How to read an untargeted lipid report & Example Report.
Results are shared via email. Larger files are shared via DropBox. The results are typically presented in an Excel report with multiple tabs. Important tabs includes: …….. Explanation of typical Excel Report format and an example Excel Report are available: How to read an untargeted lipid report & Example Report.
How to read a untargeted lipid analysis report.
Expectations and Limitations
A typical untargeted….
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*) For larger sample sets: A library is generated by analyzing pool sample(s) using DDA, separate injections using positive and negative polarity. Samples are analyzed using polarity-switch MS1-only and measured MS1 masses are compared to the library generated from the pool(s).