Definition:
Phospholipids are hydrolyzed to free their fatty acids from the sn-1 and sn-2 glycerol backbone. These fatty acids undergo methylation of the carboxylic acid function to form fatty acid methyl esters (FAME). This hydrolysis and methylation occur at room temperature when reacted with 1N sodium methoxide. The FAMEs are identified and quantitated using capillary gas chromatography with flame ionization detection (FID).

Apparatus:

• Hewlett Packard 5890 Gas Chromatograph
• J & W Fused Silica Capillary Column DB-225 30 M X 0.2 mm, 0.33 µ film
• IBM PC 486, 50 mHz
• HP-2D HPLC Chemstation Software
• Automated Liquid Sampler
• Test tubes
• Centrifuge

Reagents:

• Hexane (B&J)
• Reference Standard #68A (NU-CHEK-PREP, INC.)
• NIH-Reference standard F (Alltech)
• 1 N Sodium Methoxide

Procedure:

1. Prepare a reference standard. Transfer the contents of 1 vial of #68A STANDARD to a small bottle with a cap. Add 4 ml of hexane to the standard. Determine concentration by gravimetric analysis. Dilute to a final concentration of 20 mg/ml with additional hexane. Keep the reference standard refrigerated at 2-8^°C. Stable for 6 months.
   
2. Prepare the sample. Transfer 5 mg of sample into a test tube. If the sample is in powder form, dilute directly with 1 ml of hexane. If the sample is in chloroform, evaporate the solvent with nitrogen and add 1 ml of hexane. Add 50 µl of sodium methoxide to the sample in hexane. Vortex mix. Incubate the sample at room temperature for 5 minutes. Centrifuge the sample at 1600 rpm for 5 minutes.
   
3. Prepare the gas chromatograph.
   
4. Instrument conditions:
• Injector temp. 250^°C
• Detector temp. 220^°C
• Oven temp. 220^°C
• Column flow 1 ml/min
• Split flow 20:1
• Run time 25 min.
  
5. Run the standard. Program the sequence table to run the #68 FAME standard in first available position of the ALS, the NIH reference - F control in the second position, and a hexane solvent blank in the third position. Automatic calibration of retention times and response factors are automatically replaced with the calibrators data.
   
6. Run the sample. Program the samples into consecutive available positions and start the sequence. Reports for each injection will be reported.
   
7. Check GC Results. The lipid sample should be > 99% of the appropriate fatty acid for symmetric phospholipids (e.g. 18:1 PC) and approximately 50 ± 5% for each fatty acid component of an asymmetric phospholipid (e.g. 16:0-18:1 PC). For phospholipids of natural origin, the fatty acid profile should be consistent with historical data for that product. If the retention times match and there is <1% impurity, then the product is acceptable.
   
8. Limit of Detection. The limit of detection is 0.03% by weight of the original sample. The limit of quantitation for the method is 0.1% by weight of the original sample. No impurity or fraction should be reported that is is less than 0.1%.