Feb 27, 2024
Version 1
Extraction, derivatisation and GC-MS/MS of d2-glucose and d5-glycerol from human plasma V.1
This protocol is a draft, published without a DOI.
- Joanna Simpson1,2,
- Scott G Denham1,2,
- Natalie ZM Homer1,2
- 1University of Edinburgh;
- 2Mass Spectrometry Core, Edinburgh Clinical Research Facility, University of Edinburgh
- Metabolomics Protocols & Workflows
- Clinical Mass Spectrometry
Protocol Citation: Joanna Simpson, Scott G Denham, Natalie ZM Homer 2024. Extraction, derivatisation and GC-MS/MS of d2-glucose and d5-glycerol from human plasma. protocols.io https://protocols.io/view/extraction-derivatisation-and-gc-ms-ms-of-d2-gluco-c9r8z59w
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: February 27, 2024
Last Modified: February 27, 2024
Protocol Integer ID: 95776
Keywords: tandem mass spectrometry, GC-MS/MS, tracer glucose, tracer glycerol, stable isotope tracers
Funders Acknowledgements:
NRS Research Scotland
Grant ID: EDCRF
Abstract
Tracer glucose and tracer glycerol is used to assess glucose kinetics and turnover.
Here we have improved upon existing gas chromatography mass spectrometry (GC-MS) methods for the quantitation of glucose, D2-glucose, glycerol and D5-glycerol in plasma.
Plasma samples (200 μL) were extracted by automated 96-well protein precipitation (PPT+) using acetonitrile and as an organic solvent, carried out on an Extrahera automated sample handler.
Extracts were derivatised using acetic anhydride to form glucose pentacetate and glycerol triacetate, with internal standard butanetriol and 13C6-glucose pentacetate.
Derivatives were injected using a TriPlus autosampler and separated on a Trace GC Ultra on an Innowax MS column (30 m x 0.25 mm; 0.25 μm) and a carrier gas mobile phase of helium. The run time was 30 minutes, followed by mass spectral analysis on a TSQ Quantum tandem quadrupole mass spectrometer (ThermoFisher) operated in chemical ionisation mode.
This method has been used to analyse glucose, D2-glucose, glycerol, D5-glycerol. Validation demonstrates that this method is sensitive, specific, and suitable for simultaneous measurement of glucose and glycerol in plasma (200 µL) and glucose turnover
Attachments
Guidelines
Ensure all training is up-to-date for operating the necessary laboratory instrumentation and equipment.
Materials
Consumables Table
| A | B | C | D | |
| 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 | 10 | |
| 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+ 96 well plate | Biotage | 120-2040-P01 | 1 | |
| 96 Extrahera 1000 L pipette tips | Biotage | 414141 | 2 | |
| 2 mL deep well 96 well collection plate | Waters | 186002482 | 1 | |
| 96 well plate sealing film | Merck | Z369659-100EA | 1 | |
| 300 uL fixed insert glass autosampler vials (100 pk) | Agilent | 9301-1388 | 1 | |
| Crimp caps aluminium 11 mm (100 pk) | Agilent | 5181-1211 | 1 | |
| Innowax 30 m x 0.25 mm, 0.25 µm | 1 |
Consumables for extraction and derivatisation of glucose and glycerol
Chemicals and Analytical Standards Table
| A | B | C | |
| Item | Supplier | Article no. | |
| Water (HPLC grade) | Fisher Scientific UK Ltd (QMRI Stores) | STK-SOLV-175 | |
| Acetonitrile (HPLC grade) | Fisher Scientific UK Ltd (QMRI Stores) | STK-SOLV-025 | |
| Pyridine, anhydrous 99.8% | Merck | 270970-100ML | |
| Acetic Anhydride (Reagent Plus) | Merck | 320102-100ML | |
| n-Heptane | Fisher Scientific UK | 10598800 | |
| Glucose | Sigma-Aldrich | 1.08337.1000 | |
| d2-Glucose (6,6-d2) | Cambride Isotope Laboratories | DLM-349-SP-PK | |
| Glycerol (liquid) | Sigma-Aldrich | G9012 | |
| d5-Glycerol (1,1,2,3,3-d5) | Cambridge Isotope Laboratories | DLM-1229-1 | |
| 13C6-glucose | Sigma-Aldrich | 389374-1G | |
| Butanetriol (liquid) | Sigma-Aldrich | 309710 |
Chemicals and analytical standards
Solutions Required
- 1:1 Acetic anhydride:Pyridine (20 mL
o Add 10 mL acetic anhydride to 10 mL pyridine. Mix thoroughly. This will give enough solution for
derivatisation of 100 samples. Prepare freshly for each batch.
- Heptane + 5% Acetic anhydride (10 mL):
o Add 10 mL acetic anhydride to 10 mL pyridine. Mix thoroughly. This will give enough solution for
resuspension of 100 samples. Prepare freshly for each batch.
Equipment
| A | B | C | |
| Item | Model | Supplier | |
| Liquid Handling Robot | Extrahera | Biotage | |
| Evaporator | TurboVap 96 Dual | Biotage | |
| Manual adjustable volume pipettes, various | ErgoOne | Starlab | |
| Repeater pipette | Repetman | Gilson | |
| Microtube centrifuge | 1-15 | Thermo Scientific | |
| Deepwell Plate shaker | - | Starlab | |
| Gas Chromatography System | Trace GC Ultra | Thermo Scientific | |
| Autosampler | TriPlus (liquid injection) | Thermo Scientific | |
| Mass spectrometer | TSQ Quantum | Thermo Scientific |
Equipment required for extraction and mass spectrometry analysis
Safety warnings
Ensure risk assessments are up to date and that all local laboratory guidelines are followed for handling chemicals and biological samples
Ethics statement
All human studies were approved by the University of Edinburgh NHS Lothain ACCORD Ethical Review Board and samples analysed in a Good Clinical Practice laboratory.
Before start
Ensure all consumables are in stock and all compounds and reagents are freshly prepared
Preparation of solutions, calibration standards and enrichment curves
Preparation of solutions, calibration standards and enrichment curves
Preparation of Internal Standard Solution
Label vials as follows:
• 1 x 28 mL glass vials labelled INT STD mix
• Internal standard mix (13C6 Glucose 5 mg/mL + Butanetriol 0.25 mg/mL)
o Weight out 50 mg of 13C6 Glucose in a 28 mL glass vial. Add 2.5 µL butanetriol and make up to 10 mL n water - Make fresh every 3-4 weeks
(This should be sufficient for 5 plates of samples)
Preparation of Calibration Standard Solutions
Pipette volume of each solution into glass vials as defined below.
Note: Amount of glucose is 50 x that of d2 glucose and 200x that of Glycerol and d5-glycerol
• GLUCOSE A - 50 mg/mL Glucose + 1 mg/mL d2-glucose (need 100 µL max) so – Prepare 2 mg/mL d2-glucose in water. Weigh out 50 mg glucose in a 7 mL glass vial. Add 500 µL x 2 mg/mL d2-glucose + 500 µL water to the 50 mg glucose. This dilutes the 2 mg/mL d2-glucose 1:1 and results in 1 mL of a 50 mg/mL Glucose/ 1 mg/mL d2 glucose solution. Vortex thoroughly.
• GLUCOSE B - 5 mg/mL Glucose + 0.1 mg/mL d2-glucose: Add 100 µL of Glucose A solution to 900 µL water in a 1.75 mL glass vial. Vortex thoroughly.
• GLUCOSE C - 0.5 mg/mL Glucose + 0.01 mg/mL d2-glucose: Add 100 µL of Glucose B solution to 900 µL water in a 1.75 mL glass vial. Vortex thoroughly.
• GLYCEROL A – 250 µg/mL (Glycerol + d5-glycerol): Prepare by weighing out 2 mg glycerol and 2 mg d5-glycerol in separate 7 mL glass vials. Add 4 mL water to each to produce 500 µg/mL solutions of each. Vortex thoroughly. Combine 500 µL of the 500 µg/mL glycerol solution and 500 µL of the 500 µg/mL d5-glycerol solution in a 1.75 mL glass vial giving a combined 250 µg/mL solution. Vortex thoroughly.
• GLYCEROL B – 25 µg/mL (Glycerol + d5-glycerol): Add 100 µL of Glycerol A solution to 900 µL water in a 1.75 mL glass vial. Vortex thoroughly.
• GLYCEROL C – 2.5 µg/mL (Glycerol + d5-glycerol): Add 100 µL of Glycerol B solution to 900 µL water in a 1.75 mL glass vial. Vortex thoroughly.
Calibration standards
| A | B | C | D | E | F | G | H | I | |
| Standard name | Glucose (µg) | d2-Glucose (µg) | Glycerol (µg) | d5-Glycerol (µg) | Vol Glucose and d2-Glucose (µL) | Vol Glycerol and d5-Glycerol (µL) | Vol Water (µL) | Vol Int Std (uL) 13C6-Glucose/ Butanetriol | |
| Blank | 0 | 0 | 0 | 0 | 0 | 0 | 200 | 0 | |
| Std 0 | 0 | 0 | 0 | 0 | 0 | 0 | 200 | 20 | |
| Std 1 (10 µg Glucose) | 10 | 0.2 | 0.05 | 0.05 | 20 x Glucose C | 20 x Glycerol C | 160 | 20 | |
| Std 2 (20 ug) | 20 | 0.4 | 0.1 | 0.1 | 40 x Glucose C | 40 x Glycerol C | 120 | 20 | |
| Std 3 (50 ug) | 50 | 1 | 0.25 | 0.25 | 10 x Glucose B | 10 x Glycerol B | 180 | 20 | |
| Std 4 (100 ug) | 100 | 2 | 0.5 | 0.5 | 20 x Glucose B | 20 x Glycerol B | 160 | 20 | |
| Std 5 (150 ug) | 150 | 3 | 0.75 | 0.75 | 3 x Glucose A | 3 x Glycerol A | 194 | 20 | |
| Std 6 (200 ug) | 200 | 4 | 1 | 1 | 4 x Glucose A | 4 x Glycerol A | 192 | 20 | |
| Std 7 (300 ug) | 300 | 6 | 1.5 | 1.5 | 6 x Glucose A | 6 x Glycerol A | 188 | 20 | |
| Std 8 (400 ug) | 400 | 8 | 2 | 2 | 8 x Glucose A | 8 x Glycerol A | 184 | 20 | |
| Std 9 (500 ug) | 500 | 10 | 2.5 | 2.5 | 10 x Glucose A | 10 x Glycerol A | 180 | 20 |
Table Y - Preparation of Calibration standards
Note that Std 1 (10 ug) is 10 ug glucose, 0.2 ug D2-glucose, 0.05 ug glycerol and 0.05 ug D5-glycerol. Std 9 (500 ug) is 500 ug glucose, 10 ug D2-glucose, 2.5 ug glycerol and 2.5 ug D5-glycerol
Extraction of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
Extraction of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
Perform protein precipitation of samples and standards in a 96-well plate with 200 µL e and 600 μL acetonitrile
Prepare a map of calibration standards and samples in a 96-well format as below, using Column-wise plate layout for automated extraction on an Extrahera liquid handling robot (Biotage, Sweden):
Plate Map design for automated 96-well extraction by protein precipitation (PPT+)
Defrost plasma samples, vortex, and centrifuge (100 x g, 00:05:00 )
5m
Set up Extrahera 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). Switch on fumehood. Turn on Extrahera. Purge line S3 for acetonitrilensure sufficient number of standard bore solvent tips (Deck position 1) and standard bore sample tips (Deck position 2) are on the deck.
Place a labelled PPT+ plate in deck position 3. Check plate orientation is correct.
Place a labelled Waters 2 mL 96-well collection plate is in carousel position A
Take a Biotage 2 mL deep well collection plate and label with batch details. Add 200 uL water to blank wells according to plate map designed.
Add required amount of water and Glucose and Glycerol standards to the calibration standard wells according to the table in the PREPARATION OF CALIBRATION STANDARDS section. Due to the small volumes of standard being pipetted, ensure that the standard is pipetted INTO the water.
Add 200 uL of each plasma sample from the sample tubes to the appropriate well plate positions
Using the Gilson Repetman and a 1 mL tip, add 20 uL of the internal standard solution to wells except for Double Blank and Solvent Blank wells.
Seal the plate using a Merck well plate sealing film and shake the plate on a plate shaker for 2 mins to ensure that the standards and internal standards are sufficiently mixed.
Remove the plate seal and place the sample plate on the deck of the Extrahera in position 4.
Select and run the PPT+ method. It will load 600 uL acetonitrile onto the PPT+ plate. Then it will transfer the contents of the sample plate column by column onto the acetonitrile loaded PPT+ plate.
The robot will leave the sample to interact with the acetonitrile for 20 minutes, before applying positive pressure onto the PPT+ plate and collecting the eluent of the sample in the Waters 2 mL deep well 96-well plate. Check the full volume has been eluted into the plate.
Preparation of Enrichment curve for glucose and D2-Glucose
- Label 7 mL glass vials for Glucose enrichment curve (i.e Blank, 0%.....8%) and a ‘tracer only’ sample (n=9).
- Pipette volume of each stock solution into 7 mL glass vials as defined in table below.
- All solutions made fresh every 3-4 weeks:
- 2 mg/mL d2-glucose prepared by 10 mg d2-glucose in 5 mL water
- 2 mg/mL Glucose prepared by 100 mg in 50 mL water
These are made up to 5000 µL or 500 µL and then a 200 µL aliquot is taken for extraction. No internal standard is required for the enrichment curve.
| A | B | C | D | |
| Standard | d2-glucose (mL) | Glucose (mL) | Water (mL) | |
| Blank | 0 | 0 | 500 | |
| 0% | 0 | 500 | 0 | |
| 0.26% | 12.5 | 4987 | 0 | |
| 0.5% | 25 | 4975 | 0 | |
| 1% | 50 | 4950 | 0 | |
| 2% | 100 | 4900 | 0 | |
| 4% | 200 | 4800 | 0 | |
| 8% | 400 | 4600 | 0 | |
| d2 TRACER ONLY | 40 | 0 | 160 |
Table X - Enrichment curve for D2-glucose / Glucose
Transfer of extracts to glass vials for derivatisation
Transfer of extracts to glass vials for derivatisation
Transfer eluent to individually labelled glass vials and dry down on a Dry Block under nitrogen gas at 40C, gas flow at 25 L/min. Typical dry down is 35-40 mins. Do not overdry. Store at -20C at this stage if needed.
Preparation of Glycerol Enrichment curve for glycerol and d5-glycerol
- Label 7 mL glass vials for Glycerol enrichment curve (i.e Blank, 0%.....8%) and a ‘tracer only’ sample (n=9)
- Pipette volume of each stock solution into 7 mL glass vials as defined in table below.
- All solutions made fresh every 3-4 weeks:
- 2 mg/mL d5-glycerol prepared by adding 10 mg d2-glucose in 5 mL water
- 2 mg/mL Glycerol prepared by adding 100 mg into 50 mL water
These are made up to 500 µL and then a 200 µL aliquot is taken for extraction. No internal standard is required for the enrichment curve.
| A | B | C | D | |
| Standard | d5-glycerol (µL) | Glycerol (µL) | Water (µL) | |
| Blank | 0 | 0 | 500 | |
| 0% | 0 | 500 | 0 | |
| 0.26% | 12.5 | 4987 | 0 | |
| 0.5% | 25 | 4975 | 0 | |
| 1% | 50 | 4950 | 0 | |
| 2% | 100 | 4900 | 0 | |
| 4% | 200 | 4800 | 0 | |
| 8% | 400 | 4600 | 0 | |
| d5 TRACER ONLY | 40 | 0 | 160 |
Table X - Enrichment curve for D5-glycerol
Derivatisation of glucose and glycerol and tracers with acetic anhydride
Derivatisation of glucose and glycerol and tracers with acetic anhydride
1) Using the Gilson Repetman and a 5 mL tip, add 200 µL of Pyridine:Acetic Anhydride (1:1) to the dried extracts. Cap vial and vortex thoroughly.
2) Leave on the bench at room temperature for 15 minutes.
3) Dry down the vial contents using the TurboVap 96 Dual system with Gas Flow set to 25L/min, Gas Temperature set to 40°C and the Plate Temperature set to 40°C. Typical dry down time is 30-45 mins. Do not over dry.
4) Using the Gilson Repetman and a 5 mL tip, resuspend the dry sample in 100 µL of Heptane + 5% Acetic Anhydride. Cap and vortex thoroughly.
5) Transfer the contents of each 1.75 mL sample vial to individual 300 µL fixed insert glass autosampler vials. Crimp caps and store at -20°C until analysis.
Set up of GC-MS/MS instrumentation into Chemical Ionisation mode
Set up of GC-MS/MS instrumentation into Chemical Ionisation mode
Ensure the GC-MS/MS INSTRUMENT is in CI mode using Methane as reagent gas and Argon as collision gas
Note
Ensure argon and methane cylinders are regularly checked for supply level
Set up the Gas Chromatography system with autosampler, injector and oven temperature settings as below
| A | B | |
| Injection | Helium | |
| Carrier gas | Helium | |
| Carrier gas flow (mL/min) | 2.0 | |
| Liner | PTV Siltek Metal Liner 120 mm x 2.75 mm OD x 2mm | |
| Septum | BTO 50 mm septum | |
| Temperature | 750 °C | |
| Operating mode | Splitless | |
| Split flow control | On | |
| Split flow | 15.0 | |
| Splitless time | 1.00 min | |
| Purge flow control | On | |
| Purge flow | 5.00 mL/min | |
| Constant septum purge | On | |
| Vacuum compensation | On | |
| Enable gas saver mode | Off | |
| Enable evaporation phase | On | |
| Enable clean phase | On | |
| Enable pressure ramps | Off | |
| Post cycle temperature | CoolDown |
Gas Chromatography Injector Settings
μ
| A | B | C | D | |
| Time (min) | Rate (°C/min) | Target value (Rate (°C) | Hold time (min) | |
| 0.00 | 0.0 | 60.0 | 1 | |
| 1 | 30 | 150 | 0.00 | |
| 10 | 10 | 260 | 3 | |
| 20.00 | Stop run |
Gas Chromatography temperature gradient settings, fitted with TGWAX-MS (30 m x 0.25 mm; 0.25 m)
Set up of mass spectrometry settings following GC separation
Set up of mass spectrometry settings following GC separation
TSQ GC-MS/MS mass spectrometry settings
| A | B | |
| Instrument | Thermo TSQ9000 | |
| Ion volume, Ionisation Mode | Chemical Ionisation | |
| Reagent gas | Methane | |
| Reagent gas flow | 1 mL/min | |
| Scan Mode, Polarity | SIM & SRM, Positive & Negative | |
| Resolution (Q1/Q3) | unit/unit | |
| MS transfer line tempature | 240 °C | |
| Ion source temperature | 175 °C |
TSQ GC-MS/MS mass spectrometry settings for glucose and glycerol analysis
Mass transitions for each acetate derived compound monitored:
| A | B | C | D | E | |
| Compound name | Mode | Ionisation | Q1 (m/z) | Retention time (mins) | |
| Glucose | SIM | CI negative | 287 | ||
| D2-glucose | SIM | CI negative | 289 | ||
| Glycerol | SIM | CI negative | 217 | ||
| D5-glycerol | SIM | CI negative | 222 | ||
| 13C6-glucose | SIM | CI negative | 293 | ||
| butanetriol | SIM | CI negative | 231 |
Compound specific mass spectrometry parameters for acetate derivatives on TSQ Quantum, CI - chemical ionisation, SIM - single ion monitoring
Glucose and Glycerol and tracer acetate derivative analysis by GC-MS/MS
Glucose and Glycerol and tracer acetate derivative analysis by GC-MS/MS
16m
16m
Set up an acquisition batch in Xcalibur software using the electronic Microsoft Excel file of the calibration standards and sample list. Set to inject 2 µL per sample and use a method of chromatographic separation as described in step 10 and mass spectrometer settings as outlined in step 11.
Example Chromatogram of Glucose, Glycerol and tracers of Glucose, Glycerol and tracers with associated retention times
Check the retention times of glucose, glycerol, tracers and internal standards are as expected, as shown in the chromatogram below:
Expected result
Retention times; glycerol at 6 mins, D5-glycerol at 6 mins, glucose at 12.6 mins and D2-glucose at 12.6 mins and internal standards 13C6-glucose at 12.6 mins and butanetriol at 6.8 mins.
Extracted ion chromatograms of glucose, D2-glucose, glycerol, D5-glycerol, 13C6-glucose and butan
Once the chromatography of a test solution has been checked and the retention times are consistent, set the batch of samples to analyse. Use Tracefinder software and excel to evaluate the GC-MS/MS data to calculate the concentration of glucose and glycerol and tracers in each sample
Protocol
NAME
Using TraceFinder and Excel software to evaluate and report multi-analyte targeted LC-MS data acquired on an ThermoScientific Exploris 240 OrbitrapCREATED BY
Margaux Billen