MULTI-PREP® GRAVITY SERIES GVSA-1OO METHOD FOR THE ANALYSIS OF HOMOCYSTEINE IN SERUM OR PLASMA USING GC-MS
This method is a preliminary procedure for investigational use only. Although it has performed well in our laboratory it must be validated by your laboratory before it is used to report patient values. We would appreciate your comments on it's performance and welcome your suggestions for improvements or enhancements.
Revised:January, 2001
EXTERNAL STANDARDS - (Please see Notes and Supplemental Information before proceeding)
Homocysteine, and Homocysteine D4 will gradually oxidize respectively to Homocystine and Homocystine D8 when stored in solution. Standards which are dried down without extraction must be protected from oxidation. This method incorporates the use of Stabilizer "A", a proprietary reducing agent, to maintain Homocysteine in a reduced state.
EXTRACTED STANDARDS
When preparing standards for extraction or internal standardization, it is preferable to start with Homocystine and Homocystine D8 which will be reduced respectively to Homocysteine and Homocysteine D4 during the extraction procedure.
Values for Total Homocysteine in serum or plasma vary somewhat from lab to lab but 5-15 µ MoL/L is usually considered normal.
SAMPLE COLLECTION:
Blood should be collected and stored on ice. Serum or plasma should be separated within 1 hour of collection and refrigerated (up to 5 days) or frozen until assayed.
SAMPLE PREPARATION:
1. 100 µL of serum or plasma containing 200 ng Homocystine D8 in
20 µL Methanol:H20 (85:15) solution as internal standard.
2. Add 100 µL Stabilizer "A" concentrate and mix gently. Incubate
at room temperature for at least 2 min.
3. Add 100 µL 0.5N Sodium Hydroxide, mix and incubate at 400C for
30 min.
4. Add 3 mL of 0.1M Tris Buffer, pH 9.0.
5. Centrifuge 3000 RPM for 3 min.
COLUMN PREPARATION:
Wash each GVSA-100 column sequentially with the following solutions,
allowing each wash to drain through the
column by gravity flow.
1. 3 mL 0.2M Acetic Acid in Acetone
2. 3 mL 10% HCL
3. 3 mL H2O
SAMPLE EXTRACTION AND PURIFICATION:
1. Pour prepared samples onto the columns and allow them to drain
by gravity flow.
2. Wash each column as follows:
a. 2 mL Acetone:H20 (1:1).
b. 5mL H2O
SAMPLE ELUTION
1. Elute with 1.5 mL 0.5M Acetic Acid in Methanol.
2. Dry at 600C under Nitrogen or Argon. Remove from heat as soon
as eluates have dried.
Elution Solvent Preparation: Add 2.8 mL of Glacial Acetic Acid to
95 mL of Methanol.
DERIVATIZATION
1. Add 50 µL of n-Butylchloride and 50 µL MTBSTFA to each dried
eluate and standard. Mix well.
2. Cap tubes and incubate at 750C for 20 min.
