TITLE: Case - Glycerol and Glucose Assays by GC-mass spectrometry
AUTHORS: Henri Brunengraber

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block"><span>Total glycerol is determined by first hydrolyzing glycerides (in plasma or tissues) in 1 N KOHethanol + heat and then the hydrolysate extracted following acidification ~pH 1.0. A two phase extract is created using chloroform. The aqueous phase contains the total glycerol and the organic phase contains total fatty acids. An aliquot of aqueous phase in dried down by nitrogen gas and the concentration and/or </span><span style = "vertical-align:super;">2</span><span>H-labelled glycerol is then determined by GC-MS following conversion of glycerol to its triacetate derivative.  </span></div><div class = "text-block"><span>Glucose assay involves extraction of glucose from blood, plasma or tissues and the concentration and/or </span><span style = "vertical-align:super;">2</span><span>H/</span><span style = "vertical-align:super;">13</span><span>C-labelled glucose is also determined by GC-MS following conversion to its pentaacetate derivative. Since the derivatizing methods are the same for glucose as glycerol, the can be assayed from a single sample preparation under the same GC-MS conditions and single run-time.</span></div><div class = "text-block"><span style = "font-weight:bold;">References:   </span></div><div class = "text-block">1. Triglyceride synthesis in epididymal adipose tissue: contribution of glucose and non-glucose carbon sources. Bederman IR, Foy S, Chandramouli V, Alexander JC, Previs SF. J Biol Chem.;284(10):6101-8 (2009)</div></div>

[STEPS]
?. Total / **Free Glycerol AssayProtocol:1. Place 20μl of plasma sample or 50 mg tissue into glass, screw-top tube 2. Add ~20μl of IS [2H5]glycerol, for concentration measurements 3. For total glycerol: add 200μl of KOH-EtOH (1M KOH in 70% ethanol) 4. Heat at 80ºC for 3 hours on a heating block to hydrolyze glyceride esters 5. Then add 50μl of 6N HCl 6. Add 300μl of chloroform, vortex for 2 min and then transfer to Eppendorf 7. Centrifuge for 2 min 8. Remove top aqueous layer and place in test tube and dry under nitrogen gas 9. React the dry residue with 50μl pyridine and 50μl acetic anhydride (100 µL of pyridine: acetic anhydride, 1:1) on heating block for until dry (don’t over dry) 10. Then add 70 µL ethyl-acetate, as running solvent for GC-MS 11. Transfer to GC insert, follow GC parameters as outlined below (1) 12. **For free glycerol: omit steps #3,#4 and ~20μl 6N HCL
?. Glucose AssayProtocol: 1. Place 10μl of blood sample in Eppendorf  2. Add 10μl of a 1mg/ml of IS U13C or 2H labelled glucose for concentration      a. may use ribose as an internal standard for 13C glucose labelling assays 3. Add 250μl of methanol to Eppendorf 4. Centrifuge for ~2 minutes 5. Place supernatant in test tube and dry under nitrogen gas6. React the dry residue with 50μl pyridine and 50μl acetic anhydride (100 µL of pyridine: acetic anhydride, 1:1)  7. Heat on heating block at 80ºC, until dry (do not over dry) 8. Add 70 µL ethyl-acetate, as running solvent for GC-MS 9. Transfer to GC insert, follow GC parameters as outlined below (1)
?. GC-MS Analysis: glycerol and/or/ glucose derivatives are analyzed using an Agilent 5973N-MSD equipped with an Agilent 6890 GC system, and a DB-17MS capillary column (30 m x 0.25 mm x 0.25 um). The mass spectrometer is operated under ammonia chemical ionization mode  ionization, and selective ion monitoring (SIM) of NH4 adducts are performed: For glycerol- triacetate, SIM m/z 236, 237, and 238 (M_0,M_1 and M_2) and for glucose-pentaacetate, SIM, m/z 408–414 (M_0 to M_6)