BioProtocols--Met5

Lactate GC/MS Analysis

Lactate is isolated from the tissue culture medium and analysis of lactate can provide information on pentose

cycle activity relative to glycolysis.

Lactate clean up:

Use 1 ml tissue culture medium. Add 2 drops HCl and extract with 1 ml ethyl acetate. Transfer to a tube with a pinch of sodium sulfate to keep dry. Dry carefully under a nitrogen stream flow. This is ready to derivatize.

Lactate derivative: Lactic acid n-propylamide-heptafluorobutyric ester.

Reagents:

1. 2,2 Dimethoxypropane (Supelco 3-3053)

2. 0.5N HCl in methanol (Supelco 3-3095)

3. N-propylamine (Sigma P-5893)

4. Heptafluorobutyric anhydride (HFB, Aldrich 39,491-2)

5. Methylene chloride (Dichloromethane, Fisher D151-4)

Standards: unlabeled lactic acid.

Procedure:

1. To a dried lactate sample, add 200 ml of 2,2 Dimethoxypropane and 50 ml of methanolic HCl. Incubate in low heating block (~75^oC) for at least an hour.

2. Add 60 ml of n-propylamine. The mixture will smoke. Incubate on high heating block (~100^oC) for at least an hour. This allows the methyl ester to be displaced by n-propylamine.

3. Dry under a stream on nitrogen and there will be a formation of a white precipitate.

4. Add 400 ml of dichloromethane and 15 ml of HFB. Incubate at room temperature for about 10 minutes. Then dry under stream of nitrogen.

5. To prepare for the gas chromatography, add 50 to 2000 ml of methylene chloride.

GC/MS:

GC column: HP5, length 30m, 250mm, programmed at constant He flow of 1ml/min.

Inlet temp. 200^oC, split ratio 1:100 adjusted depending on sample concentration.

Oven temperature programmed as follows: 100^oC for 3min, then a 20^oC/min ramp to 160^oC and hold for 1min. Retention time: 3.96 min

Relevant ion clusters for SIM analysis:

CI mode (20% methane): m/z 328 cluster for C1-C3. (monitor 327 to 332)

EI mode: m/z 327 cluster for C1-C3 fragment, and m/z 241 cluster for C2-C3 fragment.

Comments:

Ref. 1 is the general method for mass spec analysis of lactate. Ref. 2 is an example of application of lactate analysis to estimate the pentose cycle activity.