LC Technical Note

Analysis of Vitamins D2 and D3 in Foods

LT170

GL Sciences Inc.

Vitamin D in foods includes vitamin D2 (calciferol), which is found in vegetable foods such as mushrooms, and vitamin D3(cholecalciferol), which is found in animal foods such as powdered milk, meat, rice, egg, and fish. Other active ingredients
include the metabolites 25-hydroxyvitamin D3, 1α, and 25-dihydroxyvitamin D3.
The vitamin D content in foods is small, typically several micrograms per 100 grams, which makes analysis difficult.
Pretreatment requires removal of contaminants, hydrolysis to remove lipids, followed by solvent extraction. The extracts
obtained as described in the Technical Note No33 are then concentrated and purified by preparative LC followed by a twostage HPLC analysis.
In this report, we have been able to purify samples after solvent
Structural Formula
extraction using size-exclusion chromatography (SEC) with an Inertsil Diol
H3C
CH3column, and then directly introducing the sample onto an analytical column
CH3
CH3
using a heart-cut method. With this method samples can be analyzed in aCH
single step without purification and concentration by LC separation.
2H3C(K. Kanno)
Purification byAnalysis byGeneral analysis of preparative LCHPLCHOVitamin DVitamin D2

PreparativeLCSample
In this Technical
Note
Vitamin D analysis

HPLC

H3C
CH3
CH3
CH2

Preparative LC withoutpurification

H3C

HOVitamin D3

Examples: Analysis of vitamin D standards
After the sample is injected onto the pretreatment column for purification,
the valve is switched at the time of elution of the vitamin D peak which isinjected onto an ODS separation column using a heart-cut method.

Structures are created using Chemistry 4-D Draw which is provided
By ChemInnovation Software, Inc.

mAU
0.4

Area

0.60.8

1. D2 (Calciferol)(0.02 mg/L)
2. D3 (Cholecarciferol) (0.02 mg/L)

2-0.2 0.0

0.2

1Concentration (mg/mL)

01020Time (min)30

HPLC conditions
Columns
Analytical column:Inertsil ODS-P (5 μm, 250 x 4.6 mm I.D.)
Pretreatment column :Inertsil Diol (5 μm, 250 x 7.6 mm I.D.)
Temperature:40 ˚CDetector:UV 265 nm
Injection volume:300 μLFlow rate
Main column:1.5 mL/min
Pretreatment column :1.5 mL/ min (0-7 minutes, 25 minutes to)
0.5 mL/min (7-25 minutes)

Calibration curve(0.02 mg/L -2.0 mg/L)

Example of valve switching timing (depending on the pretreatment column)
0-5.5 minute position
5.5-6.3 minute position 1
6.3 minutes to position 0Mobile phase
Pretreated column acetonitrile
Analytical column acetonitrile

GL Sciences LC Technical Note
Flow diagram
In the example below, the valve is switched between 5.5 minutes and 6.3 minutes* during which the vitaminD2 and D3 peaks elute from the pretreatment column, only during the elution of the target components arethey injected onto the analysis column using a heart-cut method.

0 min to 5.5 min
Purification on a SECcolumn
6.3 min to 35 min
Analysis on an
analytical column

UV Detector

Pump

Waste liquid

Auto Sampler

Column Oven

Pump

Valve switching

5.5 min -6.3 min*samples purified
on a SEC column
are sent to theanalysis column(heart-cutmethod)UV Detector

Pump

Waste liquid

Auto Sampler

Column Oven

Pump

*The valve switching timing using the heart-cut
method varies and must be set according to thepretreatment column selected.

Chromatogram using a pretreatment column
only

2Powdered milk
samples

Driedshiitake
mushroom
samples

Reference
Standard0Using a heart-cut method, only the peaks
eluting from 5.5 minutes to 6.3 minutes*
were introduced onto the analysis column.

46Time (min)8101202

46Time (min)810

120246Time (min)81012

GL Sciences LC Technical Note
Examples of vitamin D pretreatment in foods
Sample- Dried shiitake mushroom 0.2 g and powdered milk 1.0 g

Saponification-1% (w/w) (3 mL in sodium chloride)
1% (w/w) pyrogallol-ethanol solution 10 mL
60% (w/w) potassium hydroxide solution 5 mL
- 70 ˚C, 60 min- Cooling

Extraction-1% (w/w) sodium chloride solution 19 mL- Mixture of ethyl acetate-n-hexane (1:9, v/v) 15 mL- Shaking

Two liquid①- Aqueous layer- Organic layer- Repeat (1)Combine organic layers

Concentration-Concentration under vacuum, at not more than 40 ˚CConstant volume-A mixture of acetonitrile and methanol (9:1, v/v) 1 mL (for samples other than dried shiitake mushroom)-A mixture of acetonitrile and methanol (9:1, v/v) 5 mL (dried shiitake mushroom)

Filtration-GL Chromatodisk non-aqueous 13N 0.45 μm

HPLC

Example of real sample analysis
Commercial powdered milk

1.
2.

Vitamin D2Vitamin D3

42 ND

mAU
22.0-2

STD

mAU-2

18

101 ND

200mAU
22STDmAU

Vitamin D2Vitamin D3

4

Processed sample volume: 1.0 g

1.
Commercial dried shiitake
2.
mushroom
Processed sample volume: 0.2 g20181020Time (min)1.0Time (min)Magnification

00.0

Magnification

01020Time (min)30*For samples that are different from the samples detailed in Technical Note No 33.
*Some samples may not be able to separate from contaminants.

02040Time (min)60

GL Sciences LC Technical Note
Pretreatment column
Inertsil Diol

5 μm, 250 x 7.6 mm I.D. Cat.No. 5020-05666

Columns for both water-based and organic solvent-based SEC. The molecular weight exclusion limit is about 10,000, which issuitable for the separation of compounds with molecular weights of several hundreds to several thousands.

Analytical column
Inertsil ODS-P 5 μm, 250 x 4.6 mm I.D. Cat.No. 5020-02002

Cap with vial/septum
1.5 mL screw cap vial (brown) set 9-425500 sets Cat. No. 1030-54247
1.5 mL screw cap vial (brown) set 9-425100 sets Cat.No. 1030-54128

GL Sciences disclaims any and all responsibility for any injury or damage which may be caused by this data directly or indirectly.
We reserve the right to amend this information or data at any time and without any prior announcement.

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Shinjuku-ku, Tokyo,163-1130, Japan
Phone: +81-3-5323-6620
Fax: +81-3-5323-6621
Email: world@gls.co.jp
Web: www.glsciences.com

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De Sleutel 95652 AS Eindhoven
The Netherlands
Phone: +31 (0)40 254 95 31
Email: info@glsciences.eu
Web: www.glsciences.eu

International Distributors

Visit our Website at www.glsciences.com/distributors

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Phone: +86 (0)21-6278-2272
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Torrance, CA 90503
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Fax:310-265-4425
Email: info@glsciencesinc.com
Web: www.glsciencesinc.com