Beta-CAROTENE and LYCOPENE IN PLASMA BY UV/VIS - FAST – Code Z64010
BIOCHEMISTRY
The carotene is a provitamin terpene contained in many fruits and vegetables to give them a color
ranging from yellow to red. Are particularly rich fruits such as apricots, persimmons, melons,
peaches, oranges and vegetables like carrots, tomatoes, yellow squash, red peppers, but also
green leafy vegetables such as spinach, broccoli, the turnips and chicory. E 'consists of 8 isoprene
units cyclization to extremes and exists in two forms called alpha and beta (see illustration). Betacarotene is the most common form in nature. Arriving in the gut, is converted into vitamin A in part,
a substance essential for healthy skin and eyes, and the rest is absorbed and stored as such.
Lycopene is a non-polar alkyl compound made up of only hydrogen and carbon, in the group of
carotenoids. Is a food additive, identified by the initials E160d.
The major dietary source of lycopene is represented by the tomato (Solanum lycopersicum) which
is also called, and its derivatives, in which represents 60% of the total carotenoid content. The
lycopene content is influenced by the level of ripeness of the tomato, it was estimated that in fact,
ripe red tomatoes are 50 mg / kg of lycopene, whereas the concentration drops to 5 mg / kg in
maize varieties. Other natural sources of lycopene are melon, guava and pink grapefruit. The
concentration of lycopene in human serum is closely related to prolonged intake of these
commodities. Moreover, the bioavailability of the compound appears to be higher in heat-treated
products (eg tomato sauce) compared to raw products.
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Release N° 002
Beta-Carotene and Lycopene in plasma - FAST
1
March 2010
The average concentration of lycopene in plasma varies widely in different populations, reflecting
the consumption of tomato and its derivatives. The distribution in body tissues is not uniform but
correlated with the presence of lipids: Lycopene is most abundant in adipose tissue, testes and
seminal fluid, adrenals, liver, prostate and breast. Lycopene Being a lipophilic substance , its
absorption is related to the presence of fat in the diet. The cooking of foods may increase the
bioavailability due to dissociation of protein complexes in which it is incorporated or the dispersion
of crystalline aggregates of carotenoids. Intestine, in the presence of bile acids, lycopene is
dissolved, embedded in micelles and absorbed by the mucous membrane for passive transport.
The intact molecule can be incorporated into chylomicrons and transported within the lymphatic
system. It seems that there are transport proteins specific to this compound, but which are
transported by lipoproteins, especially low-density lipoprotein (LDL).
Although it has activity of the precursor of vitamin A, it was so exceptional antioxidant, by virtue of
its structure Achile the number of conjugated double bonds and its high hydrophobicity.
In general, carotenoids are effective antioxidants because of their effectiveness as a scavenger
(literally scavenging) of free radicals. Among the carotenoids lycopene appears to be the most
efficient oxygen quencher, thanks to the presence of two additional double bonds compared to the
structure of other carotenoids. Lycopene, like other carotenoids, has assets of cancer prevention.
Early research in this direction have been stimulated by epidemiological studies which have shown
a relationship between consumption of fruits and vegetables in general and decreased risk of
certain cancers. Several published studies give the lycopene's ability to reduce the risk of prostate
cancer in humans and experimental studies on mice suggest that has the ability to suppress the
growth of breast cancer cells. The anticancer action of lycopene was investigated also at
gastrointestinal, endometrial and skin.
2
TECHNICAL FEATURES
Principle of the Method :
This method allows to determine plasma concentrations of beta-carotene and lycopene. A rate of two
reagents are added plasma precipitation and, after centrifugation, the supernatant is transferred into the test
tube and injected into HPLC.
Recovery :
> 95%
Minimum concentration analyzed:
20 µg/L
Dynamic Range:
20 – 10.000 µg/L
Normal Range :
beta-Carotene : 100 – 600 µg/L
Licopene :
100 – 600 µg/L
Components of the kit :
All the Reagents are stable 3 years at 2 –8 °C.
Reagent A – Deproteinization Solution with Internal Standard, 1 x 60 ml
Reagent B – Precipitant Solution, 1 x 100 ml
Reagent C – Stabilization Solution, 1 x 10 ml
Reagent F – Test Solution 1000 µg/L, 1 x 10 ml
See Warnings
Reagent M – Mobile Phase, 2 x 500 ml
Minimum Instrumental equipment required:
Isocratic HPLC System with loop of 50 µl
Spectrophotometric Detector UV/VIS λ = 453 nm
Chromatograms Recorder
Optional Equipment:
Autosampler
Operational Computer
Blood Collection Procedure :
Take 3 ml of blood in a tube with citrate as
anticoagulant.
Pre-analytical Phase :
If you are not analyzed immediately, freeze the
sample at -20 °C. Stable for 3 months.
Stable 1 week at 2-8 °C.
3
PREANALYTICAL PROCEDURE
Inject 100 µl of Reagent F – Test Solution 1000 µg/L and verify that the beta-Carotene has retention time
similar between 6 and 8 minutes. If the Test is all right you can start with the analytical procedure; if not,
check the functionality of the analytical system.
ANALYTICAL PROCEDURE
STEP 1 : Samples Preparation
Dispense in strictly glass tubes:
Calibrator
400 µl
Reagent F – Chemical Standard
Sample
Reagent A – Deproteinization Sol.
600 µl
Sample
400 µl
600 µl
Warning: deproteinize directly on Vortex for at least 30 sec.
STEP 2 : Add 1 ml of Reagent B – Precipitant Solution
Vortex for 20 sec.
STEP 3 : Centrifuge at 4.000 rpm for 10 minutes.
STEP 4 : Place the surnatant in a clean strictly glass tubes.
STEP 5 : To dryness under a gentle flow of helium or nitrogen and resume with100 µl of Reagent C –
Stabilization Solution
N.B.: at this step the sample is stable 2 days at 2-8 °C
INJECTION :
•
Inject 50 µl of the solution into the chromatographic system.
Release N° 002
Beta-Carotene and Lycopene in plasma - FAST
4
March 2010
Beta-CAROTENE and LYCOPENE IN PLASMA – FAST – Warnings
REAGENT F : TEST SOLUTION / CHEMICAL STANDARD
1000 µg/L
1000 µg/L
Beta-Carotene
Lycopene
SPECTROPHOTOMETRIC DETECTOR PARAMETERS
λ
GAIN
INTEGRATION TIME
453 nm
0,001 AUFS
10 sec
HPLC COLUMN PROTECTION
To save the analytical column Reverse Phase PURSUIT XRs C18, 50 x 4,6 mm, 3 um the use
of Metasaver Precolumn Filter 0.5 um (1 x 10 pcs), cod. ZA6005 is obligatory.
HPLC COLUMN CONDITIONING
Install a new analytical C18 Reversed-Phase column (PURSUIT XRs C18 50 x 4,6 mm, 3 µ).
Disconnect the detector and flux 30 ml of H2O : Methanol ( 20 : 80 v/v ) solution and
subsequently 30 ml of H2O per HPLC, set flow at 1,5 ml/min. Don’t recycle the washing
solutions. Filter the mobile phase with a vacuum system and a suitable filter of 0,22 µ. Condition
the column with the mobile phase at a flow of 1,5 ml/min and discharge the first 30 ml. Condition
further on the column for 30 min. also at recycling phase. It is NOT possible to make analysis
at recycling phase. If Room Tem. is > 20° C is better to preserve the Mobile Phase at 2 – 8° C
among runs.
COLUMN CLEANING
Flux 30 ml of H2O and discharge. Flux a solution made of H2O : Methanol or Acetonitrile (20 : 80
v/v) for 30 min and discharge. When you re-use the column, flux 30 ml of H2O before a new
conditioning with the Mobile Phase.
HPLC PARAMETERS
50 µl
1,5 ml/min
About 80 bar
LOOP
Recommended Flow rate
Pressure
INTEGRATOR HP – 3394 / 3395 / 3396 PARAMETERS
ATTENUATION
5
OPERATIONAL COMPUTER PARAMETERS
IN CONFORMITY WITH THE SPECIFICATION OF OPERATIONAL COMPUTER SOFTWARE
ACCESSORIES AND CONSUMABLES
CODE
DESCRIPTION
PACKAGING
Z64020
Chemical Standard for beta-Carotene and Lycopene
2 x 5 ml
ZA6001050C046 Pursuit XRs C18 (50 x 4,6mm -3 um) Complete System
1 PK
ZA6001050R046 Pursuit XRs C18 (50 x 4,6mm -3 um) Repl.1
ChromSep Guard Pursuit XRs C18 (50 x 4,6mm -3 um)
ZA6001CG3
Repl.3
ZA6005
Metasaver Precolumn Filter 0.5 um
Clear glass vials with reduced volume from 1,5 ml to 15 ul +
S29307U
caps
1 PK
5
1 x 3 PK
1 x 10 PK
1 x 100 PK
Beta-CAROTENE AND LYCOPENE IN PLASMA
( Reference Chromatograms )
50
mAU
50
04 1.0001.DATA [PDA-Channel-1 ]
mAU
48
46
191.DATA [PDA-Channel-1 ]
45
44
42
40
40
38
36
35
34
32
30
30
28
26
25
24
22
E
N
E
T
O
R
A
C
20
20
18
16
15
E
N
E
T
O
R
A
C
14
12
10
E
N
E
P
O
CI
L
10
8
SI
5
6
E
N
E
P
O
CI
L
4
2
0
0
-2
-5
-4
-6
RT [min]
-8
RT [min]
0
0.5
1
1.5
Fig. 1 :
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
9
9.5
0
10
Test Solution
R.T. 4.1 Lycopene
R.T. 6.0 beta-Carotene
0.5
1
Fig. 2 :
6
1.5
2
2.5
3
3.5
4
4.5
Plasma Calibrator
R.T. 2.7 Internal Standard
R.T. 3.3 Lycopene
R.T. 6.5 beta-Carotene
5
5.5
6
6.5
1.500 µg/L
1.500 µg/L
7
7.5
8
Beta-CAROTENE AND LYCOPENE IN PLASMA
( Reference Chromatograms )
50
mAU
50
181.DATA [PDA-Channel-1 ]
45
45
40
40
35
35
30
30
25
25
20
20
E
N
E
T
O
R
A
C
15
10
E
N
E
P
O
CI
L
SI
5
121.DATA [PDA-Channel-1 ]
mAU
15
E
N
E
T
O
R
A
C
10
0
0
-5
-5
E
N
E
P
O
CI
L
SI
5
RT [min]
0
0.5
1
Fig. 3 :
1.5
2
2.5
3
3.5
4
4.5
Plasma Control Level 2
R.T. 2.7 Internal Standard
R.T. 3.4 Lycopene
R.T. 6.5 beta-Carotene
5
5.5
6
6.5
7
7.5
RT [min]
8
0
0.5
Fig. 4 :
500 µg/L
500 µg/L
7
1
1.5
2
2.5
3
3.5
4
4.5
Plasma Control Level 1
R.T. 2.7 Internal Standard
R.T. 3.4 Lycopene
R.T. 6.5 beta-Carotene
5
5.5
6
6.5
350 µg/L
350 µg/L
7
7.5
8