Fat soluble vitamin detection in Food by LC/MS/MS
Stephen Lock, AB SCIEX, Europe.
]
VITAMINs fat soluble.rdb (Vitamin D3 3): "Linear" Regression ("No" weightin g): y = 3.85e+004 x + 1.5e+005 (r = 0.9986)
2.0e7
1.8e7
1.8e7
1.7e7
1.7e7
1.6e7
1.6e7
Vitamin A
1.5e7
1.4e7
1.3e7
1.3e7
1.2e7
1.2e7
1.1e7
Q1
Q3
RT
ID
DP
EP
CE
1.1e7
A re a , c o u n ts
Mass Spectrometry: Analysis was performed on an AB
SCIEX QTRAP® 5500 LC/MS/MS system.
The source
conditions were a standard set up of curtain gas = 40 psi,
Nebuliser current = 5 kV (positive mode), gas 1 = 30 psi with
the source temperature set at 400 ºC. The MRM conditions
used are shown in Table 1 with the resolution kept at unit for
both Q1 and Q3 and Scheduled MRM™ used to acquire all the
data.
1.0e7
9.0e6
8.0e6
93
4.1
Vitamin A 1
100
11
35
8.0e6
7.0e6
6.0e6
5.0e6
5.0e6
4.0e6
4.0e6
3.0e6
3.0e6
2.0e6
2.0e6
1.0e6
0.0
0.0
0
20
40
60
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Concentratio n, ng/mL
0
VITAMINs fat soluble.rdb (Vitamin K1 1): "Linear" Regressio n ("No" weightin g): y = 3.47e+004 x + 8.29e+004 (r = 0.9999)
269.2
157.2
119.1
4.1
Vitamin A 2
4.1
100
Vitamin A 3
11
100
11
1.10e7
1.7e7
1.05e7
9.00e6
8.50e6
Vitamin K1
1.3e7
8.00e6
5
7.00e6
1.1e7
451.4
451.4
187.1
128
4.7
Vitamin K1 1
4.7
65
Vitamin K1 2
12
65
37
12
1.0e7
15
116
9
6.00e6
9.0e6
5.50e6
5.00e6
8.0e6
4.50e6
7.0e6
451.4
458.4
199.2
136
4.7
Vitamin K1 3
4.7
65
Vitamin K1 IS 1
12
65
45
12
4.00e6
5
116
6.0e6
9
458.4
199.2
4.7
Vitamin K1 IS 2
65
12
45
5
445.4
187.1
4.5
Vitamin K2 1
110
2
33
19
3.50e6
5.0e6
3.00e6
4.0e6
2.50e6
2.00e6
3.0e6
1.50e6
2.0e6
1.00e6
1.0e6
5.00e5
Vitamin (D3)
Cholecalciferol
105
4.5
Vitamin K2 2
0.00
445.4
80.9
4.5
431.4
165.1
4.5
431.4
137.1
437.4
171.1
437.4
397.2
0
9
20
40
60
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Concentratio n, ng/mL
110
2
90
Vitamin K2 3
110
2
71
11
Vitamin E 1
120
9
40
15
4.5
Vitamin E 2
120
9
68
19
4.5
Vitamin E IS 1
120
9
40
15
143.1
4.5
Vitamin E IS 2
120
9
68
19
69.1
4.4
Vitamin D2 1
96
10
51
8
397.2
91.1
4.4
Vitamin D2 2
96
10
83
8
XIC of +MRM (18 pairs): 269.200/92.956 amu Expected RT: 4.1 ID: vitamin A 1 from Sample 61 (meat + 10) of sample test.wiff (Hea...
400.2
69.1
4.4
Vitamin D2 IS 1
96
10
51
8
5.9e6
5.5e6
400.2
91.1
4.4
Vitamin D2 IS 2
96
10
83
8
5.0e6
385.4
90.9
4.4
Vitamin D3 1
115
5
98
9
4.0e6
385.4
367.4
4.4
Vitamin D3 2
115
5
21
17
385.4
259.2
4.4
Vitamin D3 3
115
5
21
17
90.9
4.4
Vitamin D3 IS 1
115
5
98
9
388.4
259.2
4.4
Vitamin D3 IS 2
115
5
21
17
20
40
Max. 3.4e6 cps.
4.5e6
4.05
3.5e6
3.0e6
2.5e6
Meat sample
spiked at 10 ppm
2.0e6
1.5e6
388.4
0
1.0e6
4.24
5.0e5
0.0
3.5
4.0
4.5
5.0
5.5
Time, min
XIC of +MRM (18 pairs): 269.200/92.956 amu Expected RT: 4.1 ID: vitamin A 1 from Sample 60 (meat) of sample test.wiff (Heated ...
0.5
1.0
1.5
2.0
2.5
3.0
6.0
6.5
7.0
7.5
Max. 3.4e5 cps.
4.3e5
4.0e5
Table 2. The MRM conditions used to detect the Fat soluble
vitamins.
4.26
3.5e5
I n te n s it y , c p s
3.0e5
Vitamin A (trans-Retinol)
RESULTS
Vitamin E (α-Tocopherol)
Figure 1. The chemical structures of the Fat soluble vitamins.
XIC of +MRM (18 pairs): 269.200/92.956 amu Expecte...
I n t e n s it y , c p s
I n t e n s it y , c p s
4.89
Vitamin E
1.0e4
5000.0
Hexane (15 mL) was added and the samples vigorously shaken and
vortex-mixed for 20 s three times with the tubes kept cool between
mixing. At the end of the hexane extraction the tubes were centrifuged
(3000 rpm, 5 minutes). The top layer (3 ml) was taken and evaporate
to dryness. The sample was reconstituted with acetonitrile (0.5 ml) and
vortexed (30 s), sonicated (5 mins) and vortexed (30 s) and then this
extract was diluted with 50% aqueous acetonitrile (2 ml). This sample
preparation was designed to detect vitamins at levels found in foods
(0.1 – 50 ppm) and for lower levels a SPE extraction has also been
developed to detect these vitamins but this is not discussed in this
presentation.
Blank Meat
sample
1.5e5
0.0
0.0
3.89 4.06
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Time, min
4.5
5.0
5.5
6.0
6.5
7.0
Vitamin A
Figure 4. The opposite
figure
shows
an
experiment where a
sample of minced meat
bought from a
local
shop
had
been
extracted and analysed
(bottom pane). A subset
of the same sample was
then spiked at 10 ppm
with a mixture of the fat
soluble vitamins and
analysed. The results
clearly
shows
the
detection of A, D, E and
K in the spike although
the meat was not
completely
blank.
10ppm was used as this
is the level at which
some pet foods are
spiked with vitamin A
and below the level of
vitamin E additives.
CONCLUSIONS
0.0
1.0
4.0
5.0
Time, min
XIC of +MRM (18 pairs): 385.400/90.900 amu Expecte...
2.0
3.0
6.0
7.0
1.0
2.0
3.0
4.0
5.0
Time, min
XIC of +MRM (18 pairs): 397.221/69.100 amu Expecte...
Max. 6.4e5 cps.
6.0
7.0
Max. 3.1e5 cps.
4.7e5
8.0e5
4.0e5
4.56
6.0e5
Vitamin D3
2.0e5
4.55
3.0e5
2.0e5
Vitamin D2
1.0e5
0.0
1.0
4.0
5.0
Time, min
XIC of +MRM (18 pairs): 451.400/187.100 amu Expect...
2.0
3.0
6.0
7.0
1.0
2.0
3.0
4.0
5.0
Time, min
XIC of +MRM (18 pairs): 445.400/187.100 amu Expect...
Max. 1.2e6 cps.
5.15
1.21e6
6.0
7.0
Max. 5.4e5 cps.
4.73
5.0e5
I n t e n s it y , c p s
1.00e6
I n t e n s it y , c p s
3.95
5.0e4
3.0e5
2.0e5
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Concentration, ng/mL
1.0e5
0.0
Sample preparation: Sample (1 g) was mixed with methanol (1 mL)
containing pyrogallol (3%, w/v) and 50% aqueous methanol was added
(3 mL) together with internal standard solution (concentration 1ppm,
100 µL). The sample was sonicated (10 minutes) and shaken or roller
mix for a further 20 minutes. KOH was then added and the sample
vortex mixed. Saponification, to break up the fats and release the
vitamins, was then performed at 40 ºC for 30 min during which the
samples were briefly vortex mixed. The samples were then cooled and
acidified with a solution of HCL
2.0e5
4.0e5
4.54
1.5e4
4.0e5
Max. 5.1e5 cps.
4.04
5.0e5
2.0e4
I n t e n s it y , c p s
MATERIALS AND METHODS
Max. 2.4e4 cps.
4.79
2.4e4
I n t e n s it y , c p s
Traditionally individual methods have been used to screen for each
vitamin so one method that is capable to screen for all fat soluble
vitamins would be beneficial.
Here we present some new data
acquired by LC/MS/MS with a screening method which contains
Vitamin K1, K2, D2, D3, A and E. Detection of these vitamins has been
shown to be at the low ppb level by LC/MS/MS and reverse phase
chromatography using positive mode atmospheric pressure chemical
ionisation. The mass spectrometry methods utilises Scheduled MRM™
and a small particle size HPLC column and some food samples were
obtained and then extracted by and analysed by LC/MS/MS to show
the applicability of this method to routine sample analysis.
2.5e5
1.0e5
XIC of +MRM (18 pairs): 431.400/165.100 amu Expect...
60
Figure 3. The above figures are typical calibration lines that
can be produced in APCI mode when analysing fat soluble
vitamin standards. All calibration lines were linear over the
range tested (1 – 250 ppb) and had ‘r’ values over 0.99.
In t e n s ity , c p s
Vitamin D2
(Ergocalciferol)
Vitamin E
7.50e6
1.2e7
6.50e6
445.4
Vitamin K1 (Phylloquinone)
80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
Concentration, ng/mL
1.00e7
0.0
Vitamin K2 (menaquinone)
60
1.5e7
15
31
40
9.50e6
5
41
20
VITAMINs fat soluble.rdb (Vitamin E 1): "Linear" Regression ("No" weighting): y = 2.15e+004 x + 1.16e+004 (r = 0.9993)
1.8e7
1.4e7
269.2
9.0e6
6.0e6
1.6e7
269.2
1.0e7
7.0e6
1.0e6
CXP
Vitamin D3
1.5e7
1.4e7
A re a , c o u n ts
The final fat soluble vitamin is Vitamin D and this can be ingested as
cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2) and the body
can also synthesize it from cholesterol when sun exposure is sufficient.
Vitamin D2 can actually be synthesized in adequate amounts by all
humans from sunlight, however when sun exposure is low and vitamin
D in the diet is absent diseases can occur namely, e.g. rickets the
childhood form of osteomalacia. So like other vitamins, vitamin D is
added to staple foods such as milk to avoid disease due to deficiency.
VITAMINs fat soluble.rdb (Vitamin A 1): "Linear" Regression ("No" weighting): y = 3.75e+004 x + 8.21e+004 (r = 0.9996)
1.9e7
1.9e7
A re a , c o u n ts
Vitamins are needed for healthy growth and these include a group of fat
soluble vitamins including A, D, E and K. Vitamin A is important, it is a
source of metabolites which are important growth factors and also in
the form of retina it is needed by the eye for both low-light and colour
vision. Vitamin E is again a group of vitamins of similar structure.
There are eight different forms of vitamin E, of which γ-tocopherol is
common in the diet and found in for example margarine but is not
absorbed as efficiently by people as α-Tocopherol, the most biologically
active form of vitamin E which is the second most common form of
vitamin E (found in for example wheat germ) and is the form normally
used as an additive either in it’s native form or as the acetate ester.
Vitamin E is an antioxidant that stops the production of reactive oxygen
species formed when fat undergoes oxidation and helps reduce cancer
risks from such species which can cause DNA damage. Vitamin K is
also a group of structurally similar, fat-soluble vitamins that are needed
for the post-translational modification of certain proteins required for
blood clotting and is involved in metabolic pathways of bone and other
tissue. The two naturally occurring forms are vitamin K1 and vitamin
K2.[2]
Chromatography: Samples were separated by reversedphase HPLC on a small particle size C18 column (Kinetex C18
50x 2.1mm, 2.6 µm, Phenomenex) at 600 µl/min using a
Shimadzu UFLC System over a 7.5 minute gradient from 20%
methanol in water to 100% methanol. The column temperature
was maintained at 60 ºC and an injection volume of 50 µL was
used.
A re a , c o u n ts
INTRODUCTION
Vitamin K1
5.00e5
4.0e5
3.0e5
Vitamin K2
2.0e5
REFERENCES
1.0e5
0.00
This work has clearly demonstrated that Fat soluble vitamins can
be detected using APCI and LC/MS/MS analysis. Although the
responses seen for each vitamin vary with the weakest being
vitamin E, these responses actually align with the RDA of these
vitamins in food so the approach of a multi vitamin assay by
LC/MS/MS is valid with detection levels of some vitamins below 1
ppb. In this original work a method to develop the native vitamins
has been developed and the detection limit in food will depend
mainly on the extraction techniques used. For low level vitamins
e.g. vitamin D, a solid phase extraction may be required (which has
been developed but not discussed in this presentation) while for
high level vitamins such as A and E the simple saponification and
liquid / liquid extraction is sufficient. In future work the ester forms
of vitamin A and E are planned to be added to this method as these
are often the forms at which the vitamins are used as additives2.
0.0
1.0
2.0
3.0
4.0
Time, min
5.0
6.0
7.0
1.0
2.0
3.0
4.0
Time, min
5.0
6.0
7.0
Figure 2. The 20 ppb solvent standard above is a mixture of
the fat soluble vitamins. In this figure you can see that the
response for each vitamin varies with Vitamin E the weakest
with 20ppb it’ s limit of detection (LOD). However vitamin E’s
recommended daily does (RDA) is a 1000 x higher at 12 mg
compared to the most active vitamin, vitamin D, whose RDA is
5 µg while vitamin A RDA is 800 µg and vitamin K is 75 µg1.
These RDA are set in Europe by the European Commission
(Commission Directive 2008/100/EC). Therefore the vitamin E
level will be over a 1000 times higher than vitamin D in food so
it’s LOD can be set a lot higher. In this method vitamin D’s
LOD, in both the D2 and D3 form, is below a ppb.
1. http://www.food.gov.uk/multimedia/pdfs/labellingfoodsupplement
s.pdf.
2. Directive 2002/46/EC of the European Parliament.
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