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Publisher Summary This chapter describes a gas chromatography-mass spectrometry (GC-MS) method that can directly measure the actual mole amounts of folate in a sample. In the GC-MS method, a known quantity of a mixture of a bacterially synthesized stable isotope-labeled ([ 13 C 6 ]) folate internal standard is added to the blood or tissue sample, and the folate coenzymes are specifically purified using a large excess of folate-binding protein to bind all folates and remove any free p -aminobenzoyl (PABA), p -aminobenzoyl glutamate (PABA-Glu), or p -aminobenzoyl polyglutamate (PABA-Glu n ). The C-9-N-10 linkage of PABA to the pterin moiety is chemically cleaved to pterin and PABA-Glu n . Folates are released from tissue-binding sites by heating in detergent and freshly prepared ascorbate. Prior to raising the pH for binding of the folates to a strong anion-exchange resin, the sample is cooled and 2-mercaptoethanol is added because ascorbate is rapidly inactivated as a reducing agent at pH 9.5. After the application of the crude sample, the strong anion-exchange resin is washed and eluted with 2 M sodium chloride and folate-binding protein. Recovery is maximized by warming the resin to 37° for 30 min prior to elution. This step serves to purify the folates in a concentrated solution of 0.5 ml. |
