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Best An 1117 HPLC
Best An 1117 HPLC
Key Words
Cheese, Cholecalciferol, Fat-soluble Vitamins, Food Analysis, Food Quality,
Infant Formula, Milk, Milk Powder, Radiostol, Retinol, Tocopherol,
UltiMate 3000 x2 Dual HPLC System, Yogurt
Goal
To develop an efficient high-performance liquid chromatography (HPLC)
method for simple and sensitive determination of retinol (vitamin A),
cholecalciferol (vitamin D3), and tocopherol (vitamin E) in milk-based
nutritionals such as infant formula, adult formula, milk, yogurt, and cheese.
Introduction
Vitamins are a well-known group of compounds that are
essential for human health. These compounds can be
classified into two main groups, water- and fat-soluble.
Retinol (vitamin A), radiostol (vitamin D) and tocopherol
(vitamin E) belong to the fat-soluble group, and play To ensure that these foodstuffs contain the labeled
specific and vital functions in metabolism. Health problems amounts of vitamins, there needs to be a quality control
can be present when these vitamins are either lacking or assay for them. Figure 1 shows the structures of vitamins
in excess. Our intake of vitamins depends on our diet. A, E, and D; in this work these vitamins will be determined
However, even foods that contain the necessary vitamins simultaneously. Reversed-phase HPLC is a technique well
can have reduced vitamin content after storage, processing, suited for vitamin analysis;3-6 however, milk-based
or cooking. Therefore, people take vitamin tablets and/or nutritionals are too complex to use a routine HPLC method
consume milk-based products such as infant formula, for vitamin quantification. For example, the determination
adult formula, milk, yogurt, and cheese to supplement or of vitamin D in milk-based nutritionals is difficult because
incorporate these nutrients into their diet. For example, of the low content and lack of vitamin D stability in
vitamin D is the name given to a series of compounds response to heat, light, oxidation, and the presence of
with antirachitic activity. The most common compounds other milk components (e.g., fat, emulsifiers, amino acids,
having vitamin D activity in foods are ergocalciferol proteins, sterols, and minerals) that can interfere with the
(vitamin D2) and cholecalciferol (vitamin D3).1 Vitamin D2 determination. Emulsifiers cause fat and protein to interact
is primarily found in wild mushrooms, while vitamin D3 is strongly, and successful analytical methods must be capable
primarily present in foodstuffs of animal origin (e.g., cows of disrupting this interaction and release the fat, so that
milk and breast milk); therefore, vitamin D3 is usually the vitamin D can be separated and detected.7 Therefore,
found in milk-based formulas such as infant formula and sample preparation is key to vitamin D determination in
adult nutritionals.2 However, the amount of vitamin D these types of samples. The sample preparation steps for
that is derived from milk in milk-based formulas is usually milk-based nutritionals in the European Union (EU) and
not adequate for the needs of infants, and thus vitamin D3 China regulatory methods each include saponification,
is added to infant formulas. extraction, concentration, dilution, and sample clean-up.2,8
2
Consumables
Figure 1. Structures of vitamins A, E, D2, and D3.
Thermo Scientific Target2 Polypropylene Syringe
Filters (0.45 m, 30 mm, P/N F2502-9)
Thermo Scientific Sun-SRi Luer-Lock Syringe
(Fisher Scientific P/N 14-823-261)
Equipment
Thermo Scientific Dionex UltiMate 3000 Rapid Preparation of Standard Solutions
Separation Dual HPLC system, including: In the EU regulatory method, to calibrate the loss of
DGP 3600RS Dual Ternary Pump System vitamin D in sample preparation, an internal standard
(P/N 5040.0066) with SRD-3600 Integrated Solvent
(I.S.) calibration method is used for the quantitative
and Degasser Rack (P/N 5035.9230)
determination of vitamins A, D, and E. If vitamin D3 is to
WPS 3000TRS Wellplate Sampler, Thermostatted
(P/N 5840.0020) with a 25 L sample loop be determined, vitamin D2 is used as an I.S.2 Vitamin D2 is
(P/N 6820.2415) and a 25 L syringe (P/N 6822.0001) thus used as an I.S. in this work.
TCC 3000RS Thermostatted Column Compartment Stock Standards 1
(P/N 5730.0000) equipped with one 26 p and one
210 p valve Three individual stock standards of vitamin A, E and D3
A 500 L stainless steel sample loop (P/N 6820.2454), are prepared. Dissolve ~0.025 g of vitamins A and D3,
for storage of the portion of the separation of the first and ~0.05 g of vitamin E in 25 mL methanol, respectively.
dimension The concentration of Stock Standard 1 of vitamin D3 is
D AD-3000RS Diode Array Detector (P/N 5082.0020) 1000 mg/L; that of vitamin A is 1250 mg/L, and that of
with 13 L flow cell (P/N 6082.0100) vitamin E is 2050 mg/L.
Thermo Scientific Dionex Chromeleon Chromatog-
raphy Data System (CDS) software, version 7.2 Stock Standards 2
Thermo Scientific RT Stirring Hotplate Dilute 200 L of each Stock Standard 1 to 10 mL with
(P/N SP136320-33Q) methanol, respectively. The concentration of Stock
Standard 2 of vitamin D3 is 20 mg/L, that of vitamin A is
25 mg/L, and that of vitamin E is 41 mg/L.
Connect UV detector
0.00 50 0 50
and the column on the
first dimension, for the
1_2
determination of vitamins
15.00 A and E, and clean-up
of vitamin D
1_6 Store the elution from 10 0 90
15.75 1_10 15.75 to 16.2 min in the
100 0 0 500 L sample Loop
19.00
100 0 0
20.00
0 100 0 Connect UV detector and
25.00 the column on the
1_2 1_10 second dimension,
27.00 50 50 for the determination
of vitamin D 60 40 0
28.00
29.00
50 0 50
Complete the analysis,
30.00 1_6 1_2 the valves return to their 0 100 0
original positions
Waste
Waste
Column 2
Pump 2
Pump 1 DGP3600
Autosampler
6-Port 10-Port
Valve Valve Column 1 Autosampler
UV Detector
Optimization of the On-Line Two-Dimensional when using acetonitrile and methanol, the commonly used
HPLC Method HPLC mobile phase solvents. Moreover, use of a small
Having different retention mechanisms in the first and column in the first dimension is beneficial to reducing
second dimensions is important to the construction of a elution volume, as well as decreasing any solvent incom-
successful on-line two-dimensional HPLC method. Normal patibility. Therefore, the Acclaim 120 C8 (3 m, 2.1
phase (NP) as the first dimension and reversed-phase (RP) 100 mm) and Accucore Polar Premium (C18, 2.6 m,
as the second dimension is ideal. However, for the on-line 3.0 150 mm) columns are used for the first and second
two-dimensional HPLC system, solvent incompatibility of dimensions, respectively. Figures 3 and 4 show chromato-
the two dimensions can result in the analytes not being grams of a mixed standard of vitamins A, D2, D3 and E
retained in the second dimension. Using a micro column and an infant formula sample (vitamin D2 already added
(e.g., 1 10 mm) in the first dimension and a solvent as an I.S.), respectively, using the specified two-dimensional
evaporation interface were reported to eliminate the HPLC method. On the first dimension (retention time
problem of solvent incompatibility.11-13 However, the use 020 min), vitamins A and E are separated (Figures 3A
of the micro column for first dimension and a typical and 4A), while vitamin D peak in the infant formula
format column for the second dimension decreases sample cannot be discriminated, although it is observed in
detection sensitivity;11,12 and analytes may be lost due to the mixed vitamin standard. In the second dimension
evaporation in the vacuum assisted dynamic solvent (retention time 2030 min), chromatograms of the infant
evaporation interface.13 formula sample (Figure 4B) show efficient elimination of
interferences, and baseline resolution between vitamins D2
Therefore, in this work, reversed-phase is used for both
and D3 with a peak resolution of 2.2. Therefore, vitamins
dimensions to decrease the effects of solvent incompat-
A and E can be determined in the first dimension, and
ibility. Compared to the C18 stationary phase, the C8
vitamin D can be determined in the second dimension.
stationary had weak retention for the fat-soluble vitamins
10
A B
100
3
1
2
5 a
mAU mAU 23
b Figure 3. Chromatograms of (A) a mixed
50 c
standard vitamins A, D2, D3, and E resolved
using the on-line two-dimensional HPLC
c 4 method, and detected at (a) 263 nm,
2/3
b 0 (b) 296 nm, and (c) 325 nm in the first
1 2/3 4
a 23 dimension (020 min) and the second
-10 dimension (2030 min), and (B) enlarged
-2
0.0 20 40 20 25 30 chromatograms of the second dimension
min min (2030 min). Peaks: 1- VA , 2- VD2, 3- VD3, 4- VE.
190 10
4
A B
1
mAU mAU
23
100 5 a
Figure 4. Chromatograms of (A) an infant
23 b
formula with vitamin D2 added as an I.S.
c resolved using the on-line two-dimensional
HPLC method, and detected at (a) 263 nm,
c (b) 296 nm, and (c) 325 nm in the first
1
b 0 dimension (020 min) and the second
1
a dimension (2030 min), and (B) enlarged
-20 -2
0 20 40 20 25 30 chromatograms of the second dimension
min min (2030 min). Peaks:, 1- VA, 2- VD2, 3- VD3, 4- VE.
7
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