May 06, 2026
LC-MS quantitation
- Ryan Sheldon1,
- Christine Isaguirre1,
- Lindsay Meyerdirk1,
- Naman Vatsa1
- 1Van Andel Institute
Protocol Citation: Ryan Sheldon, Christine Isaguirre, Lindsay Meyerdirk, Naman Vatsa 2026. LC-MS quantitation. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3mjpel25/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: May 04, 2026
Last Modified: May 06, 2026
Protocol Integer ID: 316320
Keywords: van andel institute mass spectrometry core, gne 2861 l, ms quantification, ms quantitation, gne, lc
Funders Acknowledgements:
ASAP
Grant ID: 025191
Abstract
This is the protocol used for GNE 2861 LS-MS quantification at the Van Andel Institute Mass Spectrometry Core.
Materials
- GNE2861, HY-12632, MedChemExpress
- 80% methanol (A456, Fisher Scientific)
- D3 octanoyl-carnitine (HY-139392S, MedChemExpress)
- Acetonitrile (A955, Fisher Scientific)
- Water (W6, Fisher Scientific)
- D8 Tryptophan (DLM-6903, Cambridge Isotope Laboratories)
- Agilent 6470 triple quadrupole mass spectrometer
- Agilent ultra-high performance liquid chromatography 1290 Infinity II
- Acquity Premier HSS T3 Column (1.8 μm, 2.1mm × 150mm, 186009472, Waters)
- VanGuard cartridge (1.8 μm, 2.1 mm × 5 mm, 186009473, Waters)
- Autosampler vial with insert (03452247, Thermo Fisher)
- Skyline Software (v 25.1.0.237; RRID:SCR_014080)
Safety warnings
Be careful to avoid thawing.
Ethics statement
This protocol requires prior approval upon usage of mice tissue by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
The Henderson Lab attained approval from our Institutional Animal Care and Use Committee (IACUC), #23-10-023.
LC-MS quantitation
Prepare an external calibration curve from a high concentration of the drug (GNE2861, HY-12632, MedChemExpress) at 10µg/mL followed by eight additional points via a half-log serial dilution.
Place a hippocampus from each mouse into a bead mill homogenizer tube (19-627, Revvity) and weigh. Be careful to avoid thawing.
Extract brain samples and 10µl of each curve point by homogenization in 1 mL of an 80% methanol (A456, Fisher Scientific) (v/v) solution.
The extraction solution is spiked with D3 octanoyl-carnitine (HY-139392S, MedChemExpress) as an internal standard such that the final concentration in solution is 5ng/mL.
After solvent addition, vortex each sample for 10s.
Homogenize samples for 30s at 6m/s at 6°C ±2°C (cooled via liquid nitrogen).
Sonicate samples for 5min in a water bath sonicator.
Incubate samples on wet ice for 1 hour.
Centrifuge for 10 minutes at 4°C at 17,000g.
Transfer 850µl of supernatant to a new 1.5mL Eppendorf Tube.
Repeat the centrifuge step to fully clear the precipitate.
After the second spin, collect 800µl of supernatant to a fresh 1.5mL Eppendorf tube and dry in a rotary vacuum.
Resuspend dried extracts by adding 40µl of acetonitrile (A955, Fisher Scientific).
Vortex samples for 10 sec.
Sonicate samples for 5 min in a water bath sonicator.
Add 40µl of water solution added to each sample such that the final resuspension solvent composition is 50/50 acetonitrile/water (v/v).
Repeat the 10 sec vortex and 5 min water bath sonication.
Centrifuge samples for 10 minutes at 4°C at 17,000g.
Transfer 70µl of supernatant to an autosampler vial with insert (03452247, Thermo Fisher).
Samples and standards were analyzed with an Agilent 6470 triple quadrupole mass spectrometer coupled with an Agilent ultra-high performance liquid chromatography 1290 Infinity II.
2µL of each sample was injected and separated using a 12-minute gradient on an Acquity Premier HSS T3 Column (1.8 µm, 2.1mm × 150mm, 186009472, Waters, Eschborn, Germany) equipped with a VanGuard cartridge (1.8 µm, 2.1 mm × 5 mm, 186009473, Waters).
Mobile phase A consisted of LC/MS grade water (W6, Fisher) with 0.1% formic acid (A117, Fisher Scientific).
Mobile phase B consisted of 99% Acetonitrile (A955-4, Fisher), 1% LC/MS grade water (W6, Fisher) and 0.1% formic acid.
Column temperature was kept at 50 °C, flow rate was held at 0.4 mL/min, and the chromatography gradient was as follows:
-0-2 min held at 0% B, 2-7.
-1 min ramp from 0 to 99% B,
-7.1-9 min, then held at 99%B.
-At 9.1 min flow was switched to 100% A at 0.6 mL/min and held until 11 min.
-By 11.1 min flow rate is reduced to 0.4ml/min and allowed to stabilize at the flowrate until 12 min.
Gas flow at 13 L/min at 200°C, sheath gas flow at 12 L/min at 325 °C and the nebulizer was set to 45 psi.
Capillary voltage was +3000 and nozzle voltage was +500.
Data was acquired using dynamic multiple reaction monitoring (dMRM) including at least two transitions per compound. The transition list was developed and optimized using neat analytical standards; parameters are provided in Table 1.
Peak picking and integration were performed using Skyline Software (v 25.1.0.237; RRID:SCR_014080). D3 octanoyl-carnitine was used as an internal standard to assess variability induced by extraction. D8 Tryptophan was used as a measure of run-to-run variability during the course of the LC-MS analysis. The relative standard deviation of each internal standard across all samples was 9.2% and 3.2% respectively, indicating low extraction induced variability and highly stable LC-MS performance.
Acknowledgements
Method development and analysis was conducted the Van Andel Institute Mass Spectrometry Core (RRID:SCR_024903)