Beta-CAROTENE and LYCOPENE IN PLASMA BY UV/VIS - FAST Code Z64010

Transcription

1 Beta-AROTENE and LYOPENE IN PLASMA BY UV/VIS - FAST ode Z BIOHEMISTRY 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 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 Ed. The major dietary source of lycopene is represented by the tomato (Solanum lycopersicum) which is also called, and its derivatives, in which represents % 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 mg / kg of lycopene, whereas the concentration drops to 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. EUREKA srl LAB DIVISION VAT N info@eurekaone.com Head Quarter: Via Enrico Fermi hiaravalle (AN) ITALY Tel Fax This product fulfills all the requirements of Directive 9/9/E on in vitro diagnostic medical devices (IVD). The declaration of conformity is available upon request. Release N Beta-arotene and Lycopene in plasma - FAST March

2 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.

3 TEHNIAL 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 HPL. Recovery : > 9% Minimum concentration analyzed: µg/l Dynamic Range:. µg/l Normal Range : beta-arotene : µg/l Licopene : µg/l omponents of the kit : All the Reagents are stable years at. Reagent A Deproteinization Solution with Internal Standard, x ml Reagent B Precipitant Solution, x ml Reagent Stabilization Solution, x ml Reagent F Test Solution µg/l, x ml See Warnings Reagent M Mobile Phase, x ml Minimum Instrumental equipment required: Isocratic HPL System with loop of µl Spectrophotometric Detector UV/VIS λ = nm hromatograms Recorder Optional Equipment: Autosampler Operational omputer Blood ollection Procedure : Take ml of blood in a tube with citrate as anticoagulant. Pre-analytical Phase : If you are not analyzed immediately, freeze the sample at -. Stable for months. Stable week at -.

4 PREANALYTIAL PROEDURE Inject µl of Reagent F Test Solution µg/l and verify that the beta-arotene has retention time similar between and minutes. If the Test is all right you can start with the analytical procedure; if not, check the functionality of the analytical system. ANALYTIAL PROEDURE STEP : Samples Preparation Dispense in strictly glass tubes: alibrator Sample Reagent F hemical Standard µl Sample µl Reagent A Deproteinization Sol. µl µl Warning: deproteinize directly on Vortex for at least sec. STEP : Add ml of Reagent B Precipitant Solution Vortex for sec. STEP : entrifuge at. rpm for minutes. STEP : Place the surnatant in a clean strictly glass tubes. STEP : To dryness under a gentle flow of helium or nitrogen and resume with µl of Reagent Stabilization Solution N.B.: at this step the sample is stable days at - INJETION : Inject µl of the solution into the chromatographic system. Release N Beta-arotene and Lycopene in plasma - FAST March

5 Beta-AROTENE and LYOPENE IN PLASMA FAST Warnings REAGENT F : TEST SOLUTION / HEMIAL STANDARD Beta-arotene Lycopene µg/l µg/l SPETROPHOTOMETRI DETETOR PARAMETERS λ GAIN INTEGRATION TIME nm, AUFS sec HPL OLUMN PROTETION To save the analytical column Reverse Phase PURSUIT XRs, x, mm, um the use of Metasaver Precolumn Filter. um ( x pcs), cod. ZA is obligatory. HPL OLUMN ONDITIONING OLUMN LEANING HPL PARAMETERS Install a new analytical Reversed-Phase column (PURSUIT XRs x, mm, µ). Disconnect the detector and flux ml of H O : Methanol ( : v/v ) solution and subsequently ml of H O per HPL, set flow at, ml/min. Don t recycle the washing solutions. Filter the mobile phase with a vacuum system and a suitable filter of, µ. ondition the column with the mobile phase at a flow of, ml/min and discharge the first ml. ondition further on the column for min. also at recycling phase. It is NOT possible to make analysis at recycling phase. If Room Tem. is > is better to preserve the Mobile Phase at among runs. Flux ml of H O and discharge. Flux a solution made of H O : Methanol or Acetonitrile ( : v/v) for min and discharge. When you re-use the column, flux ml of H O before a new conditioning with the Mobile Phase. LOOP µl Recommended Flow rate, ml/min Pressure About bar INTEGRATOR HP 9 / 9 / 9 PARAMETERS ATTENUATION OPERATIONAL OMPUTER PARAMETERS IN ONFORMITY WITH THE SPEIFIATION OF OPERATIONAL OMPUTER SOFTWARE AESSORIES AND ONSUMABLES ODE DESRIPTION PAKAGING Z hemical Standard for beta-arotene and Lycopene x ml ZA Pursuit XRs ( x,mm - um) omplete System PK ZAR Pursuit XRs ( x,mm - um) Repl. PK ZAG hromsep Guard Pursuit XRs ( x,mm - um) Repl. x PK ZA Metasaver Precolumn Filter. um x PK S9U lear glass vials with reduced volume from, ml to ul + caps x PK

6 Beta-AROTENE AND LYOPENE IN PLASMA ( Reference hromatograms )..DATA [PDA-hannel- ] 9.DATA [PDA-hannel- ] O TE A R IS O TE R A Fig. : Test Solution Fig. : Plasma alibrator R.T.. Lycopene R.T.. Internal Standard R.T.. beta-arotene R.T.. Lycopene. µg/l R.T.. beta-arotene. µg/l

7 Beta-AROTENE AND LYOPENE IN PLASMA ( Reference hromatograms ).DATA [PDA-hannel- ].DATA [PDA-hannel- ] IS O TE R A IS E T ARO Fig. : Plasma ontrol Level Fig. : Plasma ontrol Level R.T.. Internal Standard R.T.. Internal Standard R.T.. Lycopene µg/l R.T.. Lycopene µg/l R.T.. beta-arotene µg/l R.T.. beta-arotene µg/l