Mar 05, 2026
Protocol for the LC-MS/MS-based metabolomics analysis of samples generated using the MPLEx method
- Priscila M Lalli1,
- Christopher Anderton2,
- Geremy Clair1,
- Chaevien S Clendinen2
- 1Pacific Northwest National Laboratory;
- 2Pacific Northwest National Lab
- Human BioMolecular Atlas Program (HuBMAP) Method Development Community
- Metabolomics Protocols & Workflows
Protocol Citation: Priscila M Lalli, Christopher Anderton, Geremy Clair, Chaevien S Clendinen 2026. Protocol for the LC-MS/MS-based metabolomics analysis of samples generated using the MPLEx method. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj1zmpvk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 30, 2026
Last Modified: March 05, 2026
Protocol Integer ID: 242363
Keywords: Metabolomics, LC-MS/MS, HubMAP, metabolomics analysis of sample, based metabolomics analysis, metabolomic, metabolite extract, tandem mass spectrometry, mass spectrometry, liquid chromatography, reverse phase chromatography, mplex method, mplex protocol, mplex method this protocol, m, hubmap sample
Funders Acknowledgements:
NIH - Biorepository for Investigation of Neonatal Diseases of Lung-Normal (BRINDL-NL)
Grant ID: U01HL122700
NIH - Biorepository for INvestigation of Diseases of the Lung (BRINDL) - Phase II
Grant ID: U01HL148861
NIH - The Human Lung BioMolecular Multi-Scale Atlas Program (HuBMAP-Lung)
Grant ID: U54HL165443
NIH - Research Center for Spatiotemporal Lung Imaging and Omics
Grant ID: U01HL148860
Abstract
This protocol describes how Metabolite Extracts (ME) obtained from the MPLEx protocol are analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in HILIC and Reverse Phase chromatography. The details provided here indicate analyses performed at the Pacific Northwest National Laboratory (PNNL) on HubMAP samples.
Materials
80:20 MeOH:H2O
Waters glass vials
Thermo Scientific Vanquish Flex UHPLC system
Thermo Scientific Q-Exactive-HF Orbitrap mass spectrometer
Thermo Hypersil GOLD column (2.1 × 150 mm, 3 μm particle size)
Reverse phase mobile phase A (RP-MBA): Water with 0.1% formic acid
Reverse phase mobile phase B (RP-MBB): ACN with 0.1% formic acid
ACQUITY UPLC BEH HILIC column (2.1 × 100 mm, 1.7 μm particle size)
HILIC mobile phase B (HILIC-MBB): 5% ACN: 95% 10 mM NH4OAc in H2O with 0.05% NH4OH
HILIC mobile phase B (HILIC-MBB): 100% ACN with 0.05% NH4OH
ACQUITY UPLC BEH Amide VanGuard Pre-column (130Å, 1.7 µm, 2.1 mm X 5 mm)
Thermo HPLC Filter Holder with a ID filter cartridge (0.2 µm, 2.1mm)
Pierce LTQ Velos ESI Positive Ion Calibration kit
Pierce Negative Ion Calibration kit
Troubleshooting
Sample preparation
Metabolites are extracted using a modified Folch extraction (Folch 1957; Nakayasu, et al., 2016) termed MPLEx. The protocol utilized is provided on the protocols.io titled:
To each of the original vials, 150 µL of 80:20 MeOH:H2O was added and then sonicated for 10 minutes.
Samples were then vortexed well and centrifuged at 4500 rpm for 3 minutes.
Samples were then transferred to labeled Waters glass vials and stored at -20 ˚C in 80:20 MeOH:H2O (4/1, v/v) until metabolomics LC-MS/MS analysis.
LC-MS/MS system
Metabolite extract (ME) are analyzed using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) using a Thermo Scientific Vanquish Flex UHPLC system interfaced with a Thermo Scientific Q-Exactive-HF Orbitrap mass spectrometer. Samples were analyzed in reverse phase and HILIC chromatography both in positive and negative ionisation mode (4 runs per sample).
Reverse phase chromatography (RP)
5-10 µl of ME are injected and compounds are separated using a Thermo Hypersil GOLD column(2.1 × 150 mm, 3 μm particle size) with a column temperature of 40 °C and a flow rate of 400 μL min–1. Reverse phase mobile phase A (RP-MBA : Water with 0.1% formic acid) and B (RP-MBB : ACN with 0.1% formic acid) were initially 90:10, respectively. The gradient method continued as follows: 0–2 min held at 90% A; 2–11 min 10% A; 11–12 min 10% A; 12–12.5 min held at 10% A with an increased flow rate to 500 μL min–1; 12.5-13.5 min at 90% A flow rate at 500 μL min–1; 13.5-14 min at 90% A flow rate at 500 μL min–1 ; 14-14.5 min at 90% A flow rate decrease flow rate to 400 μL min–1; 14.5-16 min held at 90% A. The elution gradient is provided in the table below.
| A | B | C | D | |
| Time(min) | % MPA | % MPB | Flow rate (mL/min) | |
| 0 | 90 | 10 | 0.4 | |
| 2 | 90 | 10 | 0.4 | |
| 11 | 10 | 90 | 0.4 | |
| 12 | 10 | 90 | 0.4 | |
| 12.5 | 10 | 90 | 0.5 | |
| 13.5 | 90 | 10 | 0.5 | |
| 14 | 90 | 10 | 0.5 | |
| 14.5 | 90 | 10 | 0.4 | |
| 16 | 90 | 10 | 0.4 |
Hydrophilic interaction liquid chromatography (HILIC)
3-5 µl of ME are injected and compounds are separated using a using a ACQUITY UPLC BEH HILIC column (2.1 × 100 mm, 1.7 μm particle size) with a column temperature of 50 °C and a flow rate of 300 μL min–1. Chromatography was adapted from Clendinen et. al 2019. Mobile phase A (HILIC-MBA : 5% ACN: 95% 10 mM NH4OAc in H2O with 0.05% NH4OH) and B (HILIC-MBB : 100% ACN with 0.05% NH4OH) were initially 5% and 95%, respectively at a flow rate of 300 μL min–1. The gradient method continued as follows: 0–6.0 min. 63% A; 6.0-7.0 min 63% A; 7.0–7.1 min 5% A. 7.1-7.2 min. 5% A with an increased flow rate of 500 μL min–1; 7.2–9.5 min held at 5% A. 9.5-9.7 min. 5% A with a decreased flow rate of 300 μL min–1; and 9.7–12.0 min held at 5% A. The elution gradient is provided in Table below.
| A | B | C | D | |
| Time(min) | % MPA | % MPB | Flow rate (mL/min) | |
| 0 | 5 | 95 | 0.3 | |
| 6 | 63 | 37 | 0.3 | |
| 7 | 63 | 37 | 0.3 | |
| 7.1 | 5 | 95 | 0.3 | |
| 7.2 | 5 | 95 | 0.5 | |
| 9.5 | 5 | 95 | 0.5 | |
| 9.7 | 5 | 95 | 0.3 | |
| 12 | 5 | 95 | 0.3 |
ACQUITY UPLC BEH Amide VanGuard Pre-column (130Å, 1.7 µm, 2.1 mm X 5 mm) and Thermo HPLC Filter Holder with a ID filter cartridge (0.2 µm, 2.1mm) was added to the HILIC and RP analytical columns respectively to guard against sample particulate and are swapped out every 500-600 injections. Analytical columns are monitored and replaced when performance drops (increased backpressure, poor resolution and peak shape) or after 1,200 injections.
Mass spectrometry parameters
Samples are analyzed in both positive and negative ionization modes using HCD (higher-energy collision dissociation).
The HESI source parameters are set as follows: spray voltage 3.7 or 3.0 kV for positive and negative modes respectively; capillary temperature 300 °C; S lens RF level 50 arbitrary units, and aux gas heater temperature 350 °C.
Full MS scan data are acquired at a resolving power of 120,000 FWHM at m/z 200 with a scanning range of m/z 80–800. The automatic gain control (AGC) target is set at 3E6 ions, with a maximum injection time of 20 ms. The data dependent acquisition (dd-MS2) parameters used to obtain product ion spectra are as follows: resolving power 15,000 FWHM at m/z 200, AGC target 1E5 ions with maximum injection time of 100 ms, loop count , and normalized collision energy (NCE): 20, 30, 40 eV.
Metabolites identifications/relative quantification preprocessing
For data generated, confident metabolite identifications are made using Thermo Compound Discoverer 3.3.
For RP and HILIC positive and negative mode, spectra are aligned using an adaptive curve with a maximum of 0.3 or 0.6 RT shift respectively and a 3 ppm mass tolerance. In most instances, peaks are selected based on a minimum intensity of 1e6 and a chromatographic S/N of 3. Detected features are grouped based on a mass tolerance of 3ppm and a RT tolerance of 0.25. Features are then filtered out depending on the number of samples. Compounds are assigned based on Isotopic pattern, RT, MS1, and/or MS2. All identifications and integrated peaks are manually validated and exported. We provide, on average, 4 levels of identification starting from the most to least confidence: 1. Match with MS1, MS2, AND RT; 2. Match with MS1 and RT; 3. Match with MS2 only; 4. Match primarily with MS1 and partial MS2 match.
Quality Assurance/Quality Control (QA/QC)
Checks and calibrations are done on the Q-Exactive-HF instrument using Thermo Fisher calibration solutions: Pierce LTQ Velos ESI Positive Ion Calibration or Pierce Negative Ion Calibration. Using a HESI source, the calibrant solution is infused at a flow rate of 5-10 µL/minute and the ionization source settings are adjusted to get a stable spray (total ion current variation of 5% or less).
Mass calibration is performed at least once a week or if mass accuracy drops above 5 ppm. Other calibrations (trapping gas control, electronics, and basics positive and negative ions) are performed after the instrument has been vented for deep cleaning.
Prior to the analysis of the samples, at least two solvent blanks (MeOH:H2O 80:20) are injected to check for contamination. If the system is free of contamination and there is no variation of peak intensities between the second and first blanks, an instrument standard QC (synthetic mixture of metabolites) is injected to check the instrument performance.
Sensitivity decreases are checked by monitoring the intensity of metabolites in the instrument QC. Mass error is calculated for a few metabolites across the mass range in the instrument QC and should be below 5 ppm.
The instrument performance is verified throughout the sequence by injecting a solvent blank and instrument QC after every 20 samples and at the end of the sequence. They also serve to check for carryover and retention time drifts.
Prior to MS analysis, 5-10 µL of total metabolite extract from each reconstituted sample is combined to form a Pooled QC. The pooled QC samples are analyzed before the first and after the last sample of each sequence and approximately after every 20th (or middle) sample within a sequence. This results in at minimum 3 or more pooled QC samples being analyzed in each sequence. The pooled QC injections are used to monitor variations in peak intensity and retention time across the sequence.
At least three process blanks are created with each sample set to check for contaminants that were introduced during the preparation and/or storage process.
In most cases, samples are injected using a 5 µL injection for RP and 3 µL for HILIC. The first pooled QC sample is manually inspected for overall ion intensity. If samples appear to be very dilute, injection volume is increased to 10 µL (RP) or 5 µL (HILIC) injection.
If a decrease in sensitivity is observed, the ion transfer tube is cleaned. If that doesn’t improve sensitivity, the mass spectrometer is vented and cleaned from the S-lens to the octapole. The instrument is then restored under vacuum and is baked out for 8 hours. After the vacuum is restored, the transfer lens, electron multiplier gain, pAGC scaling, mass calibration, and advanced signal processing are calibrated in both modes in addition to isolation waveforms and activation waveforms being calibrated in just positive mode.
Acknowledgements
This work was supported by the National Heart, Lung, and Blood Institute (NHLBI) Molecular Atlas of Lung Development Program Human Tissue Core (LungMAP HTC) and LungMAP BioRepository for INvestigation of Diseases of the Lung (BRINDL) through grants U01HL122700 and U01HL148861 (to GS Pryhuber), and U01HL148860 (G Clair) and by the NIH Common Fund grant U54HL165443 (to GS Pryhuber with Co-Investigators G Clair, and C Anderton). Part of this work was performed in the Environmental Molecular Science Laboratory, a U.S. Department of Energy (DOE) national scientific user facility at Pacific Northwest National Laboratory (PNNL). Battelle operates PNNL for the DOE under contract DE-AC05-76RLO01830. The opinions expressed in this article are the authors’ own and do not reflect the view of the NIH, the Department of Health and Human Services, or the U.S. government. We are very grateful for the generosity of the donor families and honor their loss.