Vitamin E is an essential nutrient and measurement of alpha-tocopherol is critical in vitamin E research.
Method Article
alpha-Tocopherol Analysis by HPLC
https://doi.org/10.1038/protex.2011.244
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Vitamin E is an essential nutrient and measurement of alpha-tocopherol is critical in vitamin E research.
tocopherol
• 13x100 mm PTFE-faced rubber-lined cap (resistant to chloroform; Catalog No. 14-930-10A for tubes, Catalog No. 14-930-15D for PTFE caps; Fisher Scientific, Pittsburgh, PA USA, 1-800-766-7000, www.fishersci.com)
• 16x100 mm PTFE-faced rubber-lined cap (resistant to chloroform; Catalog No. 14-930-10B for tubes, Catalog No. 14-930-15E for PTFE caps; Fisher Scientific)
• Pestle tissue grinder with a Teflon pestle and a grinding vessel (10 mL chamber size, Catalog No. 3431D76; Thomas Scientific, Swedesboro, NJ USA)
• Grinding motor (Eberbach Corporation, Ann Arbor, Michigan)
• α-Tocopherol acetate (αTA, used as internal standard)
• α-Tocopherol (αTP, used for external standard curve)
• Wheaton serum vial (Amber, 5 mL, i.d.xo.d. 13x20 mm; Catalog No. 06-402A, Fisher Scientific)
• Wheaton aluminum seal (TFE lined on one side, rubber septa, 20 mm; Catalog No. 06-406-15A, Fisher Scientific)
• Wheaton hand crimper (20 mm; Catalog No. 10-319-490, Fisher Scientific)
• Wheaton hand decapper (20 mm; Catalog No. 06-451-132, Fisher Scientific)
• Acetone (HPLC grade) with BHT (with 151 µmol/L = 33.3 mg/L butylated hydroxy-toluene)
• Sodium sulfate (Na2SO4)
• Desktop Centrifuge
• 13 mm PTFE syringe filters (polytetrafluoroethylene, hydrophobic, high solvent resistance, high protein binding, 0.45 µm pore size; Catalog No. 605015; Alltech Associates, Inc., Deerfield, IL USA, 1-800-255-8324, www.alltechweb.com)
• Metal tip Micro-Mate interchangeable syringes (Catalog No. 14-825-1B for 2cc, Catalog No. 14-825-2B for 5cc; Fisher Scientific)
• N2 tank
• CM mixture 1:3 with BHT (chloroform:methanol, 1:3, v/v, with 151 µmol/L = 33.3 mg/L butylated hydroxy-toluene)
• PTFE glass-joint tape (1/2x600 inches; Catalog No. 14-831-300A; Fisher Scientific)
• Methanol (HPLC grade; Degased before being used as mobile phase)
• C-18 reverse-phase column (Alltima C18, 5 µm, 4.6 x 150 mm, Alltech Associates, Inc., Deerfield, IL USA)
• Autosampler vials (clear vial, Teflon cap, 2 mL, 8-425 cap, 100/pak; Part No. B7800-1; National Scientific Company, 1-800-332-3331)
• Beckman System Gold HPLC system: 507e Autosampler, 126 Solvent Module, 168 Detector, Gold Neuveau 1.6 Software (1996; Gold help line: 1-800-551-1150; Beckman Instruments, Inc., Fullerton, CA USA)
Stock solution (3 mg/mL): Weigh exactly 300 mg α-tocopherol acetate (αTA) to add into a 100 mL volumetric flask. Weigh 300 to 400 mg BHT to add into the flask. Tip to the marker with CM mixture (2:1).
TP:
Stock solution (3 mg/mL): Weigh exactly 300 mg -tocopherol (αTP) to add into a 100 mL volumetric flask. Weigh 300 to 400 mg BHT to add into the flask. Tip to the marker with CM mixture (2:1).
TA + TP:
Stock solution (TA: 30 ng/µL; αTP: 15 ng/µL): Use syringe to take exactly 1 mL of αTA stock solution and 500 µL of αTP stock solution into a 100 mL volumetric flask. Tip to the marker with CM mixture (2:1).
Applied solution (αTA: 6 ng/µL; αTP: 3 ng/µL): Use syringe to take exactly 100 µL of αTA + αTP stock solution into a HPLC autosampler vial. Then add 400 µL of CM mixture (2:1).
The ESTD solution needs to be made accurately. The ESTD can be stored in several sealed Wheaton vials for every batch of using.
Stock solution (about 3000 ng/L): Weigh 300 to 400 mg -tocopherol acetate to add into a 100 mL volumetric flask. Weigh 300 to 400 mg BHT to add into the flask. Tip to the marker with CM mixture (2:1).
Applied solution (30 ng/μL): Take about 1 mL of stock solution into a 100 mL volumetric flask. Tip to the marker with CM mixture (2:1).
The ISTD solution need approximate concentration when being made. The accurate concentration of ISTD needs to be checked by HPLC.
The ISTD can be stored in several sealed Wheaton vials for every batch of using.
HPLC priming:
• Check if mobile phase is enough.
• Check if the washing solvent bottle in 507E autosampler is enough.
It is recommended to use degassed methanol as washing solvent (Ref 3: pp 2-9).
• Wash the autosampler syringe to get rid of air bubbles. Click the button of “507E wash” 2-3 times.
• Check if the autosampler tubing is leaking.
• Change to the method for use. Click “single run” botton. Check if the mobile phase line is in the right solvent bottle.
• Prime the pumps (First prime lines, then prime pumps).
• Check if batch file options are right. Check the shutdown method to make sure that it uses the same mobile phase as the running method.
• Check the hard disk space in Windows File Manager.
• Check if the waste solvent bottle is full.
For Lymph/Bile/Serum:
• Prepare each set of 13x100 and 16x100 test tubes with Teflon caps. Check the mouth of tubes.
• Check the accuracy of automatic pipettes and syringes.
• Wash the HPLC syringe for sample injection. First wash it with concentrated HCl, then with D.I. water, last with methanol to remove water.
• Check windows to maintain the room temperature. This helps to keep the HPLC column condition constant.
Add 150 mg Na2SO4 into a 16x100 mm tube. Place the tube into a dessicator to avoid moisture.
Add 100 µL lymph/bile/serum with a 100 µL automatic pipette into the tube. Wash the pipette tip with 100 µL D.I. water. Critical step (be accurate)!
Add 2 mL acetone into the tube with a 5mL dispenser. Vortex vigorously 3 times to denature protein.
Add 500 µL internal standard solution into the tube with a 500 µL syringe. Vortex the tube vigorously 3 times. Stand 1-2 h for extraction.
Centrifuge the tube 20 minutes to precipitate Na2SO4.
Prepare another set of 13x100 mm test tubes. Check the tube mouth carefully. Wash with CM mixture.
Use a pasteur pipette to transfer the supernatant into a syringe connected to a 0.45 µm filter. Filter slowly. Wash the tube with 1.5 mL acetone twice. Transfer the washings into the syringe and filter again.
Evaporate the mixture under a gentle stream of N2 at 40 °C.
Cap the dry tube and put it in ice for 1-2 minutes. Place the CM mixture (1:3 with BHT) flask in ice also.
Use a 500 µL syringe to add 500 µL CM mixture (3:1) into the tube. Vortex the tube 3 times. Wrap the tube with PTFE tape. Place the tube into the freezer to stand for 1-2 hours. The sample should be injected at the day of preparation since aTP reduces at 8%/day. However, if the samples are placed in the freezer, it will be OK.
Inject 50 µL final solution into the HPLC system and run 6.5 minutes. Degased HPLC grade methanol was used as the mobile phase with a flow rate of 2 mL/min. Detection was monitored at 292 nm. Characteristic retention times were 4.1 min for α-tocopherol and 5.3 min for α-tocopherol acetate. The linear range of the αTP standard curve was from 50 to 500 ng (r = 0.999).
For tissues (except adipose tissues):
• Wash grinding vessels and pestles with detergent, D.I. water, and CM mixture.
• Prepare 2 sets of 16x100 test tubes with Teflon caps. Check the mouth of tubes.
• Check the accuracy of syringes.
• Wash the HPLC syringe for sample injection. First wash it with concentrated HCl, D.I. water, then methanol to remove water.
• Check windows to maintain the room temperature. This helps to keep the HPLC column condition constant.
It is very important to get accurate weighing data. Place the grinding vessel in the right place and balance it to avoid drifting when reading. When using a small volume grinding vessel, such as a 5 mL one, no drifting occurs.
Add proper amount of internal standard solution into the grinding vessel before homogenization.
Attached a Teflon pestle to a grinding motor and move the pestle up and down in the grinding vessel for 1.5 min in ice.
Use a Pasteur pipette to transfer the first extract into a 16x100 mm test tube. Place the tube in ice.
Wash the grinding vessel with 2 mL acetone w/BHT. Move the pestle up and down in the grinding vessel for 1.5 min. Use the Pasteur pipette to transfer the second extract into the tube.
Repeat step 5 twice. Collect the washings in the tube. Centrifuge the tube for 20 minutes.
Use a Pasteur pipette to transfer the supernatant into another 16x100 mm test tube through a syringe connected to a 0.45 µm filter. Filter slowly. Wash the original tube with 1.5 mL acetone twice. Transfer the washings into the syringe and filter again. Don’t wash the tube if HPLC is clogged by the sample injection. Sometimes the filter does not work well enough.
Evaporate the mixture under a gentle stream of N2 at 40 °C.
Cap the dry tube and put it in ice for 1-2 minutes. Place the CM mixture (3:1 with BHT) flask in ice also.
Use a 500 µL syringe to add 300 µL CM mixture (1:3) into the tube. Vortex the tube 3 times. Wrap the tube with PTFE tape. Place the tube into the freezer to stand for 1-2 hours. Sometimes CM (2:1) is needed to dissolve all the lipids stuff.
The sample should be injected at the day of preparation since aTP reduces at 8%/day. However, if the samples are placed in the freezer, it will be OK.
First run should be 60 minutes to check if there is any unknown peak at around 30 minutes. This peak will affect the next injection if it is not eluted.
For adipose tissues:
Use a syringe to add certain amount of total lipid extracts into a 13x100 mm test tube.
Use a syringe to add proper amount of internal standard solution into the tube.
Evaporate the mixture under a gentle stream of N2 at 40 °C.
Cap the dry tube and put it in ice for 1-2 minutes. Place the CM mixture (2:1 with BHT) flask in ice also.
Use a syringe to add proper amount of CM mixture (2:1) into the tube. Vortex the tube 3 times. Wrap the tube with PTFE tape. Place the tube into the freezer to stand for 1-2 hours. The sample should be injected at the day of preparation since aTP reduces at 8%/day. However, if the samples are placed in the freezer, it will be OK.
Inject 50 µL final solution into the HPLC system and run 6.5 minutes. Degased HPLC grade methanol was used as the mobile phase with a flow rate of 2 mL/min. Detection was monitored at 292 nm. Characteristic retention times were 4.1 min for α-tocopherol and 5.3 min for α-tocopherol acetate. The linear range of the αTP standard curve was from 50 to 500 ng (r = 0.999).
Extraction: about 4 hours for 20 samples
HPLC: about 7 minutes/sample
Zou, W., Noh, S.K., Owen, K.O., & Koo, S.I. (2005) Dietary carnitine enhances the lymphatic absorption of fat and a-tocopherol in ovariectomized rats. J. Nutr. 135: 753-756.
"http://jn.nutrition.org/content/135/4/753.full":http://jn.nutrition.org/content/135/4/753.full
Zaspel, B.J. & Csallany, A.S. (1983) Determination of alpha-tocopherol in tissues and plasma by high-performance liquid chromatography. Anal. Biochem. 130: 146-150.
Thanks to Koo, S.I. for mentoring and financial supporting efforts.
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