Apr 15, 2026

Analysis of vitamin A and vitamin E in serum Using HPLC with UV and Fluorescence Detection 

DOI
https://dx.doi.org/10.17504/protocols.io.261gerk5ol47/v1
  • 1Kansas State University;
  • 2U.S. FDA
  • Vet LIRN
Geraldine Magnin
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DOI: https://dx.doi.org/10.17504/protocols.io.261gerk5ol47/v1
Protocol Citation: Geraldine Magnin, andriy tkachenko 2026. Analysis of vitamin A and vitamin E in serum Using HPLC with UV and Fluorescence Detection . protocols.io https://dx.doi.org/10.17504/protocols.io.261gerk5ol47/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 11, 2025
Last Modified: April 15, 2026
Protocol  Integer ID: 119978
Keywords: hplc with uv, using hplc, retinol, hplc, serum, tocopheryl acetate, uv, tocopherol, fluorescence detection, retinyl acetate, fluorescence
Funders Acknowledgements:
HHS - US Department of Health and Human Services
Grant ID: 5U18FD005147-05/UFD005147B
Abstract
This procedure described the quantification ofvitamin A (all-Trans-retinol) and E (a-tocopherol) in serum. This analysis is performed by HPLC with UV and fluorescence detection. Recoveries are calculated using internal standard calibration with a-tocopheryl acetate (for a-tocopherol) and retinyl acetate (for retinol).
Guidelines
Keep serum samples in the refrigerator if analyzed the same day or freeze at -20 °C until analysis.
Materials
Materials
  1. HPLC column Phenomenex Luna C18(2) 150 x 4.6, 5 m equipped with a guard column cartridge (P/N AJ0-4287) inserted into a guard column holder (P/N KJ0-4282)
  2. Centrifuge tubes, polypropylene, 15 mL (Genesee Scientific, P/N 21-101).
  3. Microcentrifuge tubes, 0.5mL Safe-Lock Tubes, Clear, Eppendorf (Genesee Scientific, P/N 86-942).
  4. Disposable Borosilicate Glass Tubes with Plain End (Fisher Scientific, P/N 14-961-25).
  5. Pipette, single channel, variable, 0.5-5 mL (Eppendorf North America, 492000084).
  6. Pipette, single channel, variable, 100-1,000 µL (Eppendorf North America, 492000060).
  7. Pipette, single channel, variable, 10-100 µL (Eppendorf North America, P/N 492000106).
  8. Pipette, single channel, variable, 2-20 µL (Eppendorf North America, P/N 492000033).
  9. Quartz cuvette, micro, 1 mL

Reagents
  1. Ethanol with 0.04% BHT: To 250 mL of ethanol, add 100 mg of BHT. Mix gently until dissolved.
  2. Cyclohexane with 0.04% BHT: To 1 liter of cyclohexane, add 400 mg of BHT. Mix gently until dissolved.

Chemicals
  1. Methanol, HPLC grade (Fisher Scientific P/N A4524) or equivalent.
  2. Acetonitrile, HPLC grade (Fisher Scientific, P/N A9984) or equivalent.
  3. Cyclohexane, HPLC grade (Fisher Scientific, P/N C6204) or equivalent.
  4. Ethanol, HPLC grade (Fisher Scientifc, P/N A995-4) or equivalent.
  5. Butylated hydroxytoluene (BHT) (Acros Organics, P/N AC219830500) or equivalent.
  6. -Tocopherol, (Sigma-Aldrich, P/N T3251-5G) or equivalent.
  7. -Tocopheryl acetate (Sigma-Aldrich, P/N A00498) or equivalent.
  8. All-trans-retinol, (Toronto Research chemicals, P/N R252000 or equivalent). Retinol should be kept under inert gas and stored at -80 °C.
  9. Retinyl acetate, (Sigma-Aldrich, P/N R4632-1G or equivalent).


Equipment
  1. Vortex mixer (Fisher Scientific).
  2. Tabletop centrifuge (Fisher Scientific 225).
  3. Refrigerated microcentrifuge (Fisher Scientific AccuSpin‱ Micro 17R).
  4. UV-visible spectrophotometer.
  5. HPLC system from Waters Corporation, Acquity H, equipped with a degasser, a quaternary pump, a refrigerated autosampler, a UV diode array detector and a fluorescence detector.
  6. CentriVap Concentrator (LabConco) equipped with a Labconco‱ VACUUBRAND‱ Diaphragm Vacuum Pump and a Labconco‱ CentriVap‱ -105 Cold Trap.


Reference standards stock solutions
Note
Vitamins are light sensitive. Keep them protected from the light by using amber containers and working in subdued light.

Vitamin Sock Solution
A stock solution of a mixture of a-tocopherol 160 Parts per Million (PPM) and retinol 16 Parts per Million (PPM) in EtOH will be provided to the participating laboratories. Concentrations have been confirmed by UV-spectrophotometry before preparing the mixture

Internal standards mixture in EtOH
A mixture of a-tocopheryl acetate 20 Parts per Million (PPM) and retinyl acetate0.5 Parts per Million (PPM) in ethanol

Retinyl acetate 1,000 ppm in acetonitrile
Using a 10 mL volumetric flask, weigh 10 mg of retinyl acetate and complete the volume to 10 mL with acetonitrile.

Reference standards working solutions
Calibration working standard in acetonitrile
Mixtures of a-tocopherol and retinol in acetonitrile are prepared at the following concentrations (ppm) in acetonitrile:
ABCDEFGH
CAL1 CAL2 CAL3 CAL4 CAL5 CAL6 CAL7
a-Tocopherol 0.25 0.50 1.0 2.0 4.0 8.0 16
Retinol 0.025 0.05 0.1 0.2 0.4 0.8 1.6
Theworking standard solutions are stored at -20 °C

Sample Preparation
Sample Preparation
Weigh out 1 g of serum in a 15 ml polypropylene tube.

Add 100 µL of the internal standard spike mix containing a-tocopheryl acetate 20 ppm and retinyl acetate 0.5 ppm in ethanol. Vortex briefly.

Add 2 mL of ethanol with 0.04% BHT to precipitate the proteins.

Add 4 mL of cyclohexane with 0.04% BHT.

Vortex for 00:00:30 .

Centrifuge the tubes at 3200 x g for 00:10:00


Using a pipette, remove 2 mL from the top layer (cyclohexane) and transfer in a disposable borosilicate glass tube.
Evaporate the cyclohexane vacuum using the vacuum concentrator (Labconco) at 35 °C (minimum temperature settings). It takes about 1 hour to evaporate 20 samples.

Reconstitute the residue in 0.2 mL of acetonitrile, vortex 20 seconds.
Transfer the extract into a 0.5 mL polypropylene microcentrifuge tube.
Centrifuge for 10 minutes at 13,000 g and +4 °C.
Transfer 50 µL to a HPLC amber vial equipped with a polypropylene insert. Keep the remaining extract at -20 °C.

The sample is ready for HPLC analysis
Instrument Methods Parameters for Analysis
Autosampler refrigerated at +4 °C.
Mobile phase: Methanol 100% (isocratic mode).
Flow rate: 1.5 ml/minutes.
Injection volume: 20 mL with needle wash with acetonitrile.
UV Detection:
Retinol and retinyl acetate: 325 nm.
a-tocopherol: 294 nm
a-Tocopheryl acetate: 285 nm
Fluorescence detection:
Retinol and retinyl acetate: excitation wavelength 325 nm and emission wavelength 470 nm.
a-tocopherol: Excitation wavelength 295 nm and emission wavelength at 330 nm
Sample Analysis
Open Empower 3 (icon E on the desktop).
Load the method used to run the samples.
Start the system: purge the lines if necessary, start the pump, the autosampler refrigeration and turn on the detectors.
Fill out the sample list by indicating:
The vial number
Description of the sample
The method’s name
Datafile name
Load the standards, the internal standard solution diluted twice to mimic the concentration in the extracted sample and the samples on the autosampler tray.
Start the sequence by clicking on the “Start” button.
Data analysis
Load the Empower 3 method to process data.
External calibration is used as the internal standards are not present in the calibration standards. The calculated sample concentration will not take the recovery into account.
The integration parameters have been set-up from the standards (UV and fluorescence signals).
Enter the sample dilution (sample weight divided by
Linear calibration should be used with a weighing factor of 1/x2.
Export the data into an Excel spreadsheet by selecting.
Calculate the recovery for each sample based by comparing the peak area of the internal standards in the samples and the peak area of an internal standards mixture.
Calculate the vitamins concentrations in each sample.
Protocol references
Merck index, Eighth edition for the Molar Extinction Coefficient values of a-tocopherol and retinol in ethanol.