Lange Laboratory Protocol PS2
Phytosterol Extraction
Materials:
-
One 10mL (13mm x 100mm) glass culture tube with tight-fitting TFE cap for each sample
(SUN SRi; Rockwood, TN #13-415)
One 2mL glass GC vial for each sample (Agilent; Palo Alto, CA #5182-0714)
One 100uL glass insert with polymer feet for each sample (Agilent; Palo Alto, CA #5181-1270)
One blue screw cap for each sample (Agilent; Palo Alto, CA #5182-0717)
2 mL (5 ¾in) glass Pasteur pipettes
50 µL glass micropipettes
1 mL Hamilton syringe
Chemicals:
-
Chloroform (Omnisolv) (EMD Chemicals; Gibbstown, NJ)
Methanol (Omnisolv) (EMD Chemicals; Gibbstown, NJ)
Epicholesterol (Steraloids Inc.; Newport RI)
Liquid nitrogen
KOH (EMD Chemicals: Gibbstown, NJ)
Hexane (Omnsolv) (EMD Chemicals; Gibbstown, NJ)
Pressurized nitrogen tank (chromatographic grade)
MSTFA (Sigma; St. Louis, MO)
Distilled water
Solutions:
4mL CHCl3/MeOH (2:1 vol/vol) alloquoted into 10mL glass culture tubes and kept at -20oC
0.1mg/mL Epicholesterol standard in CHCl3/MeOH (2:1 vol/vol) kept at -20oC.
6% KOH (wt/vol) in MeOH
Equipment:
-
Single-tube vortexer (VWR Vortexer 2 G560 manufactured by Scientific Industries, Bohemia, NY #58815-234)
70oC water bath large enough to hold all your samples (PolyScience 2LS-M; Niles, IL)
Analog multi-tube vortexer large enough to hold all your samples (VWR VX-2500 manufactured by Henry
Troemner LLC; Thorofare, NJ #58816-115)
Speed vac large enough to hold all your samples (GeneVac; Valley Cottage, NY #EZ2Bio)
90oC water bath large enough to hold all your samples (Thermo NESLAB RTE7; Newington, NH)
Drying manifold large enough to hold all your samples (Custom fabricated – see attached drawing)
Fume hood
Gas chromatograph (GC) 6890N (Agilent Technologies)
Mass selective detector (MSD) 5973 inert (Agilent Technologies)
Software: MSD ChemStation G1701EA version E.02.00.493 (Agilent Technologies)
Lange Laboratory Protocol PS2 - Phytosterol Extraction
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Step-by-Step Instructions:
1.
All extractions and other sample manipulations must be performed in a fume hood.
2.
Aliquot 4 mL CHCl3/MeOH (2:1 v/v) into glass tubes. Take 0.1 mg/mL epicholesterol internal standard (IS)
solution out of freezer and allow it to reach room temperature before proceeding. Spike aliquot tubes with 100
µL of epicholesterol IS solution.
3.
For each sample, label one 10 mL glass tube containing 4 mL CHCl3/MeOH (2:1 v/v) with epicholesterol and one
2 mL-glass vial.
4.
Remove sample tubes from -80oC freezer and place immediately in liquid nitrogen.
5.
Make sure you have the right tube numbers for the experiment you are doing.
6.
Transfer sample into the CHCl3/MeOH/IS-containing glass culture tube.
7.
Cap tube and vortex on single tube vortexer until plant material is fully suspended.
8.
Incubate all tubes in 75°C water bath for 60 min.
9.
Shake tubes occasionally or vortex briefly several times while tubes are incubating. Check frequently to make
sure that the tubes are not boiling. If they are boiling, tighten or replace caps.
10.
Leave tubes on bench overnight (12 h).
11.
The next morning, uncap tubes (retain caps on bench.)
12.
At room temperature, evaporate solvents to dryness in the GeneVac EZ2-Bio Personal Evaporator for 2.5 h. Use
the program for “Mixture BP < 75oC”.
13.
To saponify dried residue, add 2000 µL 6 % (w/v) KOH in methanol to each tube. Cap very tightly!
15.
Place all tubes in 90°C water bath for 1 h to release the sterol moiety of sterol esters. Make sure the water bath
has gotten to 90oC before putting in tubes. Check frequently to ensure that samples are not boiling. If they are
boiling, tighten caps. If necessary, replace caps. Keep checking tubes frequently.
16.
Add 2 mL n-hexane and 2 mL H2O to each tube.
17.
Vortex all tubes together for 20 s in the multi-tube vortexer.
18.
Using a Pasteur pipette, remove the (upper) hexane phase from each tube and transfer to appropriately labeled
2 mL glass vial.
19.
At room temperature, evaporate solvent to dryness in the GeneVac EZ2-Bio Personal Evaporator for 2.5 h. Use
the program for “Mixture BP < 75oC”. Samples can be frozen at -80oC at this point.
21.
Using a glass micropipette, add 50 µL MSTFA to each sample and vortex on the single-tube vortexer for 20 s
(don’t use a Hamilton syringe, the MSTFA will plug it up.)
22.
Using a Hamilton syringe, add 150 µL n-hexane to each sample and vortex.
22.
Using a glass Pasteur pipette, transfer extract to a 100 µL glass insert, place insert containing the extract back
into the vial which originally contained the extract, cap vial, and incubate at room temperature for 5 min.
23.
Either run immediately on GC/MS or store at -80oC for up to 2 d.
Lange Laboratory Protocol PS2 - Phytosterol Extraction
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