TITLE: Extraction, derivatisation and GC-MS/MS of d2-glucose and d5-glycerol from human plasma
AUTHORS: Joanna P Simpson, Scott G Denham, Natalie ZM Homer

[DESCRIPTION]
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

[BEFORE_START]
Ensure all consumables are in stock and all compounds and reagents are freshly prepared

[GUIDELINES]
Ensure all training is up-to-date for operating the necessary laboratory instrumentation and equipment.

[STEPS]
SECTION: Glucose and Glycerol and tracer acetate derivative analysis by GC-MS/MS
13. 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.
15. 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

 

2. 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.
SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4. Perform protein precipitation of samples and standards in a 96-well plate with 200 µLe and 600 μL acetonitrile


5. 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. 

 

 
3. 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
10. Set up the Gas Chromatography system with autosampler, injector and oven temperature settings as below

 
μ 

 



SECTION: Set up of mass spectrometry settings following GC separation
11. TSQ GC-MS/MS mass spectrometry settings
 
 
 
7. 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 µLand then a 200 µL aliquot is taken for extraction. No internal standard is required for the enrichment curve.
 

 

 
14. 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: 
 
 

 

SECTION: Set up of GC-MS/MS instrumentation into Chemical Ionisation mode
9. Ensure the GC-MS/MS INSTRUMENT is in CI mode using Methane as reagent gas and Argon as collision gas
 

SECTION: Set up of mass spectrometry settings following GC separation
12. Mass transitions for each acetate derived compound monitored:

 

SECTION: Preparation of solutions, calibration standards and enrichment curves
1. 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 mLn water - Make fresh every 3-4 weeks
        (This should be sufficient for 5 plates of samples)
SECTION: Derivatisation of glucose and glycerol and tracers with acetic anhydride
8. 
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.	
SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4.1. 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): 

 

SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4.2. Defrost plasma samples, vortex, and centrifuge (100 x g, 5 min)

SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4.3. 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.
SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4.4. Place a labelled PPT+ plate in deck position 3. Check plate orientation is correct.
SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4.5. Place a labelled Waters 2 mL 96-well collection plate is in carousel position A
SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4.6. 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. 
SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4.12. 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.
SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4.7. 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. 
SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4.8. Add 200 uL of each plasma sample from the sample tubes to the appropriate well plate positions
SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4.9. 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. 
SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4.10. 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.
SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4.11. Remove the plate seal and place the sample plate on the deck of the Extrahera in position 4.
SECTION: Extraction  of glucose, glycerol, D2-glucose and D5-glycerol from human plasma
4.13. 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.
SECTION: Transfer of extracts to glass vials for derivatisation
6. 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.