HPLC lycopene

A similar system, but with a more hindered porphyrin (tetramesitylporphyrin = tetraphenylporphyrin bearing three methyl substituents in ortho and para positions on each phenyl group), was tested for P-carotene oxidation by molecular oxygen. This system was chosen to slow the oxidation process and thus make it possible to identify possible intermediates by HPLC-DAD-MS analysis. The system yielded the same product families as with lycopene, i.e., (Z)-isomers, epoxides, and P-apo-carotenals, together with new products tentatively attributed to diapocarotene-dials and 5,6- and/or 5,8-epoxides of P-apo-carotenals. The oxidation mechanism appeared more complex in this set-up. 

An interlaboratory study using mixed vegetable reference material showed average relative standard deviations (RSDs) of 23% ranging from 11% for lutein and a-carotene to 40% for lycopene." Triplicate HPLC injections of the same extract showed RSD values of 0% for P-carotene and 6.8% for lutein. ... 

The simplest and cheapest procedure to obtain standards is based on selective extraction followed by crystallization. A method developed to obtain lycopene from tomato residue using factorial experimental design consisted of a preliminary water removal with ethanol, followed by extraction with EtOAc and two successive crys-talhzation processes using dichloromethane and ethanol (1 4), producing lycopene crystals with 98% purity, measured by HPLC-PDA. Using this approach, bixin was extracted with EtOAc from annatto seeds that were previously washed with... 

SFE may also be coupled to GC and HPLC systems [28] for a simple on-line extraction and analysis system. Lycopene is determined in food products such as tomatoes and tomato products, using SFE coupled to HPLC with an HPLC column used for trapping and analysis. The method is short, requires small sample amounts, and has good linearity and sensitivity. Because the entire system is closed, there is little chance for the lycopene to degrade. 

Fig. 2.12. HPLC of the extract at 40°C and 4 000 psi with the entrainer. The identified components are in the order first peak (retention time 2.60min) = reference second peak (retention time 22.69min) = trans-lycopene third peak (retention time 25.22min = /(-carotene. Reprinted with permission from E. Cadoni et al. [36].

It was found that the method is suitable for the extraction of 8.6mg of lycopene (RP-HPLC purity 98.5 per cent) from lOQmg of crude extract of tomato paste containing about 9 per cent of lycopene. The technique has been proposed for the preparative separation of lycopene from tomato paste [38],... 

The effect of temperature on the RP-HPLC behaviour of /(-carotene isomers has been extensively investigated and the results were employed for the separation of carotenoids of tomato juice extract. Carotenoids were extracted from food samples of 2g by adding magnesium carbonate to the sample and then extracted with methanol-THF (1 1, v/v) in a homogenizer for 5min. The extraction step was repeated twice. The collected supernatants were evaporated to dryness (30°C) and redissolved in methanol-THF (1 1, v/v). Separations were performed on a polymeric ODS column (250 X 4.6 mm i.d. particle size 5/.an). The isocratic mobile phase consisted of methanol-ACN-isopropanol (54 44 2, m/m). The flow-rate was 0.8 or 2.0 ml/min. The effect of temperature on the retention times of lycopene and four /(-carotene isomers is shown in Table 2.11. The data indicated that the temperature exerts a considerable influence on the retention time and separation of /(-carotene isomers. Low temperature enhances the efficacy of separation. 

Fig. 2.16. HPLC profile of carotenoids in an extract of vegetable soup. An expansion of the profile from 30 to 39 is shown in the inset (A). Monitored wavelengths were 436, 440, 464, and 409 nm for peaks 9,10,11,12, and 14, respectively, in the inset (A). Peak identification 1 + 1" = all-trans-lutein and cw-lutein 2 = 5,6-dihydroxy-5,6-dihydrolycopene (lycopene-5,6-diol) 3 = j3-apo-8 -carotenal (internal standard) 4 = lycopene 1,2-epoxide 5 = lycopene 5,6-epoxide 6 = 1,2-dimethoxyproly-copene (tentative identification) 7 = 5,6-dimethoxy-5,6-dihydrolycopene 8 = lycopene 9 = pheo-phytin b 10 = neurosporene 11 = (-carotene 12 = pheophytin a 13 = (-carotene 14 = pheophytin a isomer and (-carotene 15 = a-carotene 16 and 16" = all-trans-/fcarotene, cis-/J-carotene 17 and 17" = all-trans- or cA-phytofluene 18 and 18" = all-trans- or cw-phytoene. Reprinted with permisson from L. H. Tonucci et al. [40].

Fig. 2.23. Reversed-phase gradient HPLC profiles of carotenoids in human plasma. A human volunteer was given an oral dose of 5,6-epoxy-/l-carotene (9.1 /imol). Plasma was analysed for carotenoids before (a) and 6h after (b) the oral dose. Peak identification 1, bilirubin 2, lutein 3, zeaxanthin 4, /1-cryptoxanthin 5, 5,6-epoxy-/l-carotene 6, lycopene 7, /1-carotene. The detection wavelength was 445 nm. AU, absorbance unit. Reprinted with permission from A. B. Barua [50],...

Figure F2.2.4 The spectral characteristics of p-carotene (solid line), lutein (long-dashed line), and lycopene (short-dashed line) in an acetonitrile-based HPLC solvent (75 25 5 v/v/v acetoni-trile/methanol/dichloromethane).

Figure F2.4.1 Liquid chromatography/mass spectrometry (LC/MS) analysis of isomeric carotenes in a hexane extract from 0.5 ml human serum. Positive ion electrospray ionization MS was used on a quadrupole mass spectrometer with selected ion monitoring to record the molecular ions of lycopene, p-carotene, and a-carotene at m/z (mass-to-charge ratio) 536. A C30 HPLC column was used for separation with a gradient from methanol to methyl-ferf-butyl ether. The a -trans isomer of lycopene was detected at a retention time of 38.1 min and various c/ s isomers of lycopene eluted between 27 and 39 min. The all-frans isomers of a-carotene and P-carotene were detected at 17.3 and 19.3 min, respectively.

Standard mixture P-carotene, lycopene, canthaxanthin, lutein, zeaxanthin C-30 Acetone-H20- AgCI04 HPLC/UV-vis (450 nm)/MS/MS/ ESI(+) 0.5, 0.3 pmol for canthaxanthin and p-carotene 97... 

Tomato Lycopene isomers, P-carotene SFE C-30 MeOH-H20- MTBE HPLC/PAD (285, 347, 450 nm) 40... 

Tomato, human prostate Lycopene isomers, a/p-carotene Tomato extraction with acetone-hexane Prostate protein precipitation, extraction with acetone-hexane C-30 MeOH-MTBE- ammonium acetate HPLC/ECD 50 fmol for lycopene 34... 

Human plasma Lycopene isomers, a/p/y-carotene Extraction with hexane, centrifugation, saponification with KOH C-18/C-30 ACN-MTBE HPLC/MS/MS/ APCI(+/-) 11.2 fmol for lycopene 32... 

Human serum Lycopene, lutein, P-carotene, P-crytoxanthin, zeaxanthin Protein precipitation, extraction with n-hexane C-30 MeOH-ACN- THF-BHT HPLC/DAD (450 nm) <0.06 pmol 45... 

Human serum, prostate Lycopene isomers, a/p-carotene Saponification with KOH, extraction with hexane, centrifugation C-30 MeOH-MTBE HPLC/PDA (450 nm)/MS/ APCI(+) 0.93 pmol for lycopene 109... 

HPLC is commonly used to separate and quantify carotenoids using C18 and, more efficiently, on C30 stationary phases, which led to superior separations and improved peak shape.32 4046 An isocratic reversed-phase HPLC method for routine analysis of carotenoids was developed using the mobile phase composed of either methanol acetonitrile methylene chloride water (50 30 15 5 v/v/v/v)82 or methanol acetonitrile tetrahydrofuran (75 20 5 v/v/v).45 This method was achieved within 30 minutes, whereas gradient methods for the separation of carotenoids can be more than 60 minutes. Normal-phase HPLC has also been used for carotenoid analyses using P-cyclobond46 and silica stationary phases.94 The reversed-phase methods employing C18 and C30 stationary phases achieved better separation of individual isomers. The di-isomers of lycopene, lutein, and P-carotene are often identified by comparing their spectral characteristic Q ratios and/or the relative retention times of the individual isomers obtained from iodine/heat-isomerized lycopene solutions.16 34 46 70 74 101 However, these methods alone cannot be used for the identification of numerous carotenoids isomers that co-elute (e.g., 13-ds lycopene and 15-cis lycopene). In the case of compounds whose standards are not available, additional techniques such as MS and NMR are required for complete structural elucidation and validation. 

Figure 4.5 Representative HPLC chromatogram of (a) tomato product lycopene isomers in (b) plasma lyeopene isomers from healthy individuals consuming different lyeopene-containing tomato products for 15 days, demonstrating the presence of isomers in human blood plasma versus the -trans form found in most foods (Hadley et al., 2003).

Often a mass spectrometer is interfaced with an HPLC-PDA system. This technique is especially useful because isobaric species can be chromatographically separated before entering the MS. Interestingly, there are a large number of isobaric species in the field of carotenoids, such as lycopene, [3-carotene, oc-carotene, and y-carotene which all have a parent mass of 536 mu, or (3-cryptoxanthin, oc-cryptoxanthin, zeinox-anthin, and rubixanthin which all have a parent mass of 552 mu. 

Van Berkel and Zhou first tested (3-carotene with ESI positive in 1994 (van Berkel and Zhou, 1994). In this study, a doubly charged molecular ion of (3-carotene was observed as the primary species when triflur-oacetic acid was present in the solution. Van Breemen was the first to utilize ESI as an interface between HPLC and MS to analyze carotenoids (van Breemen, 1995). In this study, ESI operated in negative mode ionized xanthophylls (astaxanthin, (3-cryptoxanthin, and lutein), but did not ionize hydrocarbon carotenes (lycopene and (3-carotene). In contrast, ESI positive produced only [M" ] for all carotenoids in this study, and the addition of halogenated solvents to the post-column effluent greatly enhanced signal intensity (van Breemen, 1995). A later study by Guarantini et al. demonstrated the ability of ESI positive to produce both [M" "] and [M + H]" " for a number of xanthophylls, and these authors attributed the production of the two species to solvent system... 

IMS is a relatively new technique in which ions are separated based on size and shape using an electric field. IMS was utilized by Dong et ah (2010) to separate all-trans -lycopene from cw-lycopene and all-irans -(3-carotene from cw-(3-carotene. Unfortunately, the various cis isomers could not be separated from each other using IMS alone. The authors provided evidence to suggest that cis/trans isomerization of carotenoids occur in-source (ESI positive mode was used in these experiments). Because of this isomerization, it does not appear likely that IMS will replace HPLC as a means of separating geometrical isomers of carotenoids in the near future (Dong et ah, 2010). 

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