Dec 15, 2023
Targeted analysis of 5-Fluorouracil (5-FU) and Fluoroacetate (FAC) in human plasma by automated PPT+ extraction and LC-HRMS analysis
- Margaux Billen1,2,
- Scott Denham2,
- Joanna P Simpson2,
- Natalie Homer3
- 1KU Leuven;
- 2University of Edinburgh;
- 3Mass Spectrometry Core, Centre for Cardiovascular Sciences, University of Edinburgh
- Metabolomics Protocols & Workflows
- Clinical Mass Spectrometry
Protocol Citation: Margaux Billen, Scott Denham, Joanna P Simpson, Natalie Homer 2023. Targeted analysis of 5-Fluorouracil (5-FU) and Fluoroacetate (FAC) in human plasma by automated PPT+ extraction and LC-HRMS analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3p2kg8j/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: July 14, 2023
Last Modified: December 15, 2023
Protocol Integer ID: 85008
Keywords: 5-fluorouracil, fluoroacetate, capecitabine metabolism, High Resolution Mass Spectrometry, LC-MS method, ThermoFisher Exploris 240 Orbitrap, Human Plasma, catabolite fluoroacetate, fluorouracil, fluoroacetate, labelled lactate, liquid chromatography high resolution mass spectrometry, metabolite, resolution mass spectrometry analysis, liquid chromatography, high resolution mass spectrometry, using automated protein precipitation, human plasma sample
Funders Acknowledgements:
Fonds Wetenschappelijk Onderzoek (FWO) Vlaanderen
Grant ID: V424223N
Biotechnology and Biological Sciences Research Council
Grant ID: BB/V019066/1
Abstract
This protocol describes the extraction and targeted high-resolution mass spectrometry analysis of two compounds - the anti-cancer drug 5-fluorouracil (5FU) and its catabolite fluoroacetate (FAC), in human plasma samples.
A targeted LC-MS method was developed to measure FAC and 5-FU, by adapting methods from Deenen et al, 2012 and from Leong et al, 2022. Samples were enriched with isotopically labelled lactate and the drug and its metabolite were extracted using automated protein precipitation on an Extrahera liquid handling robot (Biotage) alongside calibration standards.
Analysis of the extract was carried out by liquid chromatography high resolution mass spectrometry (LC-HRMS) in full scan negative mode on an Exploris 240 Orbitrap (ThermoScientific). The amount of each compound (5FU and FAC) in each sample was calculated using linear regression of the peak area ratio of the analytes to the isotopically labelled internal standard.
Analytes
Internal standard
Analyte information
| Name | Abbreviation | Chemical Formula | Monoisotopic mass | |
| Fluoroacetate | FAC | C2H3FO2 | 78.0117 | |
| 5-Fluorouracil | 5-FU | C4H3FN2O2 | 130.0179 | |
| 13C3-lactate | 13C3-Lac | [13C]3H6O3 | 93.042 |
Guidelines
Ensure all training is up-to-date for operating the laboratory equipment included in this protocol.
Materials
Consumables
| Item | Supplier | Part no. | Quantity | |
| 1.75 mL glass vials with lids | Scientific Laboratory Supplies Ltd | TUB1200 | 10 | |
| 7 mL glass vials with lids | Scientific Laboratory Supplies Ltd | TUB1220 | 5 | |
| 28 mL tall form glass vials with lids | VWR | T008/04 | 2 | |
| 2 mL deep well 96 well collection plate | Biotage | 121-5203 | 1 | |
| Biotage PPT+ plate | Biotage | 120-2040-P01 | 1 | |
| 96 Extrahera 1000 mL pipette tips | Biotage | 414141 | 2 | |
| 2 mL deep well 96 well collection plate | Waters | 186002482 | 1 | |
| 96 well plate sealing film | Merck | Z369659 | 1 | |
| Adhesive Plate Seal | Waters | 186006336 | 1 | |
| ACQUITY Premier HSS T3 Column 1.8 µm, 2.1 x 150mm | Waters | 186009469 | 1 |
Chemicals
| Item | Supplier | Article no. | |
| Water (HPLC grade) | Fisher Scientific UK Ltd (QMRI Stores) | C-10449380-X | |
| Acetonitrile (HPLC grade) | VWR | C-20060320-X | |
| Acetonitrile (LC-MS grade) | VWR | 83640.320 | |
| Water (LC-MS grade) | VWR | 83645.320 | |
| Formic acid (LC-MS grade) | Fisher | 10596814 | |
| 2-Propanol (LC-MS grade) | VWR | 84881.320 | |
| 5-fluorouracil - 500MG | Merck Life Sciences | PHR1227-500MG | |
| Fluoroacetate - 250MG | Merck Life Sciences | 796875-250MG | |
| L-Lactic acid-13C3 (100 mg) | Merck Life Sciences | 746258-100MG |
Equipment
| Item | Model | Supplier | |
| Liquid Handling Robot | Extrahera | Biotage | |
| Evaporator | TurboVap 96 Dual sample concentrator | Biotage | |
| Microtube centrifuge | 1-15 | Sigma | |
| 96 well plate centrifuge | Heraeus Megafuge 16R | Thermo | |
| Deepwell Plate Thermoshaker | TS-DW | Grant Scientific | |
| Liquid Chromatography Pump | Vanquish | Thermo | |
| Autosampler | Vanquish | Thermo | |
| Column oven | Vanquish | Thermo | |
| Mass spectrometer | Exploris 240 Orbitrap+ | Thermo | |
| Balance | PS-100 | Fisher Scientific |
Safety warnings
Adhere to local laboratory rules.
Ethics statement
When handling human clinical samples, ensure you are following local guidelines including adherence to Good Clinical Practice. In particular, ensure that data files of samples analysed do not contain identifiable patient information.
Solvent preparation
15m
Mobile Phase A: H2O + 0.1% Formic Acid
- Add 1 L of LC-MS grade H2O to a 1L glass bottle.
- Add 1 mL of LC-MS grade Formic Acid to the H2O.
- Mix thoroughly.
Mobile Phase B: Acetonitrile
- Add 1 L of LC-MS grade Acetonitrile to a 1L glass bottle.
Autosampler Seal Wash: 10% Acetonitrile
- Add 100 mL LC-MS grade Acetonitrile to 900 mL LC-MS grade H2O in a 1L glass bottle.
- Mix thoroughly.
Internal standard preparation
20m
All solutions (internal standards and calibration standards) are kept at -20°C after preparation until use.
Prepare Internal standard stock solution.
13C3-Lactate stock solution (1 mg/mL)
Using the PS-100 balance, weigh out approximately 2 mg of 13C3-Lactate into a 7 mL glass vial, add appropriate volume of water (HPLC grade) and vortex thoroughly to give a 1 mg/mL 13C3-Lactate stock solution e.g. 2 mL for 2 mg.
Prepare Internal Standard dilutions according to the table below in:
- 1 x 1.75 mL glass vials labelled '50 µg/mL 13C3-Lactate in water'.
- 1 x 3.5 mL glass vials labelled '5 µg/mL 13C3-Lactate in water'.
| Stock conc | Amount of stock | Vol water (µL) | Final vol (µL) | |
| 50 mg/mL | 50 µL x 1 mg/mL 13C3-Lactate | 950 | 1000 | |
| 5 µg/mL | 200 µL x 50 mg/mL 13C3-Lactate | 1800 | 2000 |
Calibration standards mix preparation
30m
Prepare calibration standard stock solutions.
FAC stock solution (1 mg/mL)
Using a PS-100 balance, weigh out approximately 2 mg of FAC into a 7 mL glass vial, add appropriate volume of water (HPLC grade) and vortex thoroughly to give a 1 mg/mL FAC stock solution e.g. 2 mL for 2 mg.
Store at -20 °C.
5-FU stock solution (1 mg/mL)
Using the PS-100 balance, weigh out approximately 2 mg of 5-FU into a 7 mL glass vial, add appropriate volume of water (HPLC grade) and vortex thoroughly to give a 1 mg/mL 5-FU stock solution e.g. 2 mL for 2 mg.
Store at -20 °C.
Prepare Calibration Standard mix dilutions according to the table below, in:
- 4 x 1.75 mL glass vials labelled: 50 µg/mL, 5 µg/mL, 500 ng/mL, 50 ng/mL.
Extraction Procedure
2h
Acquire or build a sample list for the samples in Microsoft Excel including experimental details and unique sample identification codes.
Complete plate map for standards and samples (make sure to place them column-wise) using the design as shown. The number of samples that can be analysed per batch is 83, alongside a 10-point calibration curve.
Defrost control pooled human male plasma for calibration standard and blanks preparation.
Defrost calibration standard mixes, internal standards and samples.
Take a 2mL deep well 96 well collection plate (Biotage) and add 100 µL control pooled to the calibration standard and blank wells (A1-D2).
Add required amount of standards to the plasma according to the table shown here. Due to the small volumes of water being pipetted ensure that the standard is pipetted into the plasma.
Add 100 µL of each plasma sample to the appropriate wells.
Add 20 µL of the 5 µg/mL internal standard solution to all wells, except for Double Blank and Solvent Blank wells.
Seal the plate using a 96 well plate sealing film (Merck) and shake the plate on a plate shaker for 00:05:00 to ensure that the standards and internal standards are sufficiently mixed.
Set up Extrahera liquid handling robot for PPT+ extraction.
Turn on Air Compressor. Make sure a pressure of ~9 bar is achieved and that the compressor goes into Standby (indicated by green flashing light).
Make sure the fume cupboard is switched on and the duct hose is in place in a fume cupboard to ensure proper ventilation.
Turn on Extrahera and wait for it to boot up.
From the Maintenance menu select 'Flush Solvent Inlets' and purge the line which contains LC-MS grade Acetonitrile. Throw away the purged liquid and fill up the container with fresh LC-MS grade Acetonitrile.
Ensure that sufficient number of standard bore solvent tips (deck position 1 and 2) are on the deck.
Place a PPT+ plate in deck position 3. Make sure that it is in the correct orientation and is properly clicked in place.
Take a Waters 2mL 96 well collection plate, and mark it with the project title, investigator name, plate number, extraction date and the initials of the person doing the extraction. Place the plate in carousel position A and make sure that well A1 is on the outside of the carousel next to the A1 label!
Remove the plate seal and place the sample plate on the deck of the Extrahera in position 4.
Select 'Run Single Method' from the Extrahera menu and select an appropriate PPT+ extraction method, then press 'Prepare Run'. Select the columns of the PPT+ plate for processing and update the tip numbers/locations if necessary.
Press Run. The Extrahera loads 300 µL of Acetonitrile into each well. It then transfers the sample plate contents onto the acetonitrile in the PPT+ plate. The Extrahera applies positive pressure to pass the samples through the PPT+ plate and collects the eluent in the Waters 2mL deep well 96 well plate.
Once complete, check the volumes of elution solvent in the collection plate are approximately equal indicating good performance of the positive pressure head. Check that the samples and standards were correctly aspirated from the sample plate.
Place the colection plate on the TurboVap Dual 96 sample concentrator with the gas temperature set to 40 °C and the gas flow to 30 mL/min.
Once dry, resuspend the dry samples in 80 µL of H2O and seal the plate with a Waters Adhesive plate sealing film.
Shake the plate for00:10:00 at 600 rpm to ensure the samples are resolubilised.
Set up of FAC and 5FU LC-HRMS method and analysis
30m
Put the freshly prepared mobile phases onto the UPLC system. Purge lines with mobile phase A and mobile phase B.
Install an ACQUITY Premier HSS T3 Column 1.8 µm, 2.1 x 150mm column into the column oven. Set the column temperature to 40°C and equilibrate at 100% mobile phase A, 0.3 mL/min for at least 15 minutes. Ensure that the pressure is stable and there are no leaks detectable on the system.
Create an acquisition method in Xcalibur for chromatography and mass spectrometry settings. For chromatography include the following chromatographic gradient conditions in the table below.
Add the detail of the column and mobile phases in the method. Make sure the right column position is selected for the valves and the column oven temperature and column pre-heater are set to 40°C.
| Time (min) | Flow (mL/min) | %A | %B | Curve | |
| Initial | 0.300 | 100 | 0 | Initial | |
| 1.00 | 0.300 | 100 | 0 | 5 | |
| 2.80 | 0.300 | 5 | 95 | 5 | |
| 3.50 | 0.300 | 5 | 95 | 5 | |
| 3.60 | 0.300 | 100 | 0 | 5 | |
| 7.00 | 0.300 | 100 | 0 | 5 |
Chromatographic gradient for separation of FAC and 5-FU in extracted plasma samples on an ACQUITY Premier HSS T3 Column 1.8 µm, 2.1 x 150mm using H2O + 0.1% Formic Acid (Mobile Phase A) and Acetonitrile (Mobile Phase B).
Add the following mass spectrometry method parameters to the acquisition method:
| A | B | |
| Instrument | Thermo Exploris 240 Orbitrap | |
| Source, Ionisation Mode | Thermo Scientific™ OptaMax™ NG ion source (H-ESI) | |
| Scan Mode, Polarity | Full Scan, Negative | |
| Mass range | 50 - 200 m/z | |
| Resolution | 120 000 | |
| Acquisition time | 7.0 min | |
| Sheath Gas | 50 | |
| Aux Gas | 15 | |
| Sweep Gas | 1 | |
| IonSpray Voltage (IS) (Negative) | -2500 V | |
| Ion Transfer Tube Temperature | 350°C | |
| Vaporizer Temperature | 450°C | |
| Probe position (x – axis) | 2 | |
| Probe position (y – axis) | 2 |
Place the sealed 96-well plate into the autosampler of the chromatography system.
Create a batch in Xcalibur - use the correct position for the 96-well plate, the correct position of the column, the correct lines for the mobile phases and the correct LC-MS/MS method. Name and save the Batch acquisition file. Use the same naming convention to name the resulting data file.
Set volume of injection to 20 µL and submit batch to analyse.
Test the system with a mid-standard curve point injection and then complete the batch in order from A1 to H12.
Use the m/z [M-H]- for each compound in the table below to interrogate the data:
| FAC | 5FU | 13C3-Lactate | ||
| m/z [M-H]- | 77.0044 | 129.0106 | 92.0345 | |
| Retention time (min) | 1.50 | 2.32 | 1.97 |
Example chromatography of FAC, 5-FU and 13C3-Lactate separation is shown below. Separation performed on an ACQUITY Premier HSS T3 Column 1.8 µm, 2.1 x 150mm using a system of H2O + 0.1% Formic Acid (Mobile Phase A) and Acetonitrile (Mobile Phase B).
Chromatographic Separation of FAC (1.50 mins), 5FU (2.32 mins) and the internal standard 13C3-Lactate (1.97 mins) on an ACQUITY Premier HSS T3 (Waters) Column 1.8 µm, 2.1 x 150mm using a system of H2O + 0.1% Formic Acid (Mobile Phase A) and Acetonitrile (Mobile Phase B). Flow rate 0.3 mL/min, 40°C and a gradient elution over 7 minutes.
Data Analysis using TraceFinder
2h
Use the protocol below and the compound specific table details (mass and retention time) in Step 32 to evaluate the data and obtain the FAC and 5FU concentrations in the samples analysed:
Margaux Billen, Scott G Denham, Joanna P Simpson, Natalie ZM Homer 2023. Using TraceFinder and Excel software to evaluate and report multi-analyte targeted LC-MS data acquired on an ThermoScientific Exploris 240 Orbitrap. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldm8z7l5b/v1
Protocol references
Leong, B. J.; Folz, J. S.; Bathe, U.; Clark, D. G.; Fiehn, O.; Hanson, A. D., Fluoroacetate distribution, response to fluoridation, and synthesis in juvenile Gastrolobium bilobum plants. Phytochemistry 2022, 202, 113356. https://doi.org/10.1016/j.phytochem.2022.113356.
Deenen, M. J.; Rosing, H.; Hillebrand, M. J.; Schellens, J. H. M.; Beijnen, J. H., Quantitative determination of capecitabine and its six metabolites in human plasma using liquid chromatography coupled to electrospray tandem mass spectrometry. Journal of Chromatography B 2013, 913-914, 30-40. https://doi.org/10.1016/j.jchromb.2012.11.033
Margaux Billen, Scott G Denham, Joanna P Simpson, Natalie ZM Homer 2023. Using TraceFinder and Excel software to evaluate and report multi-analyte targeted LC-MS data acquired on an ThermoScientific Exploris 240 Orbitrap. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldm8z7l5b/v1