HPLC analysis, of carotenoids

A.M. Pupin, M.J. Dennis and M.C.F. Toledo, HPLC analysis of carotenoids in orange juice. Food Chem. 64 (1999) 269-275. 

D. Application HPLC Analysis of Carotenoids and Carotenoid Esters in Red Bell Pepper (Ref. 66)... 

Fig. m.5 Isocratic HPLC analysis of carotenoid standards. Condition 5- mi x 250-mm x 4.6-mm Vydac 201TP column, 90 10 methanol/acetonitrile mobile phase, 1.0 ml/min flow rate, visible detection at 450 nm, and column temperature 25°C [84]... 

Other Human Tissues. Details of extraction procedures and isocratic as well as RP-HPLC analysis of carotenoids present in various human tissues have been summarized by Parker (218) and by Schmitz et al. (219). Cis and trans isomers of carotenoids may have different biological activities. Thus the isomeric composition of lycopene and P-carotene was determined in the serum of healthy volunteers and in seven human tissues obtained by autopsy soon after death, using RP-HPLC on a Merck 5-p.m C18 end-capped column with a solvent mixture of CH3OH/CH3CN/CH2CI2/H2O) (7/7/2/0.16) and a photodiode array detector (220). 

G Zachariev, I Kiss, J Szabolcs, G Toth, P Molnar, Z Matus. HPLC analysis of carotenoids in irradiated and ethylene oxide treated red pepper. Acta Aliment 20 115-122, 1991. 

HART J D and SCOTT K J (1995) Development and evaluation of an HPLC method for the analysis of carotenoids in foods and the measurement of carotenoid content of vegetables and fruits commonly consumed in the UK. Food Chem. 54(1) 101-111. 

Mercadante A.Z., Rodriguez-Amaya, D.B., and Britton, G., HPLC and mass spec-trometric analysis of carotenoids from mango, J. Agric. Food Chem., 45, 120, 1997. 

Scott, K. et al.. Interlaboratory studies of HPLC procedures for the analysis of carotenoids in foods. Food Chem., 57, 85, 1996. 

The HPLC analysis of milkweed, the food-plant source for Monarch butterflies, demonstrates that it contains a complex mixture of carotenoids including lutein, several other xanthophylls, xanthophyll epoxides, and (3-carotene, Figure 25.3b. There is a component in the leaf extract that is observed to elute near 8min, which has a typical carotenoid spectrum but is not identical to that of the lutein metabolite observed at near the same retention time in the extracts from larval tissue. 

Mercadante AZ, Rodriguez-Amaya DB and Britton G. 1997. HPLC and mass spectrometric analysis of carotenoids from mango. J Agric Food Chem 45 120—123. 

Another study employed both TLC and HPLC for the analysis of carotenoids of Calendula officinalis L. TLC separation of all E(trans)-a,3-carotene, cryptoxanthin, zeaxanthin and lutein was performed on a silica layer using petroleum ether-j-propanol-CIICI, (90 10 70 v/v). The same carotenoid pigments were separated by RP-HPLC using an ODS column (250X4 mm, i.d.). The organic modifiers were methanol, THF and ethyl octane. The flow rate was 1 ml/min, pigments were detected at 440 nm [20],... 

QUANTITATIVE CAROTENOID COMPOSITION (PG/CELL), OBTAINED BY HPLC ANALYSIS, OF H. PLUVIALIS CELLS IN THE STATIONARY PHASE IN CULTURES WITH DIFFERENT CONCENTRATIONS OF NITRATE (G/L), ACETATE AND MALONATE (% W/V)... 

Scott, K.J. 1992. Observation on some of the problems associated with the analysis of carotenoids in foods by HPLC. Food Chemistry 45 357-364. 

The use of supercritical fluid chromatography for carotene separation has been examined and optimized, especially in regard to temperature, pressure, and organic modifiers in the supercritical fluid (71). With an RP column it was possible to resolve an a-carotene-cis isomer from an all-trans carotene as well as two cis isomers of /3-carotene from an all-trans /3-carotene. As with HPLC, only polymeric C,8 columns were able to resolve the cis isomers of a- and /3-carotene from the all-trans isomers. Supercritical fluid chromatography offers the advantage not only of an efficient separation but also of fast analysis. Indeed, the use of SFC with ODS-based columns for the analysis of carotenoid pigments affords a threefold reduction of analysis time compared to HPLC (72). The elution order of carotenoids and their cis isomers was found to be the same as in RP-HPLC. The selectivity of the system could further be increased by adding modifiers (e.g.,... 

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. 

Mass spectrometry data is often paired with UV-Visible spectra, NMR, or HPLC retention time for carotenoid identification. In addition, HPLC coupled with MS as a detector has been reported to be 100 times more sensitive than PDA for detection and quantification of some carotenoids (van Breemen, 1995 van Breemen et ah, 1996). While mass spectrometry can be a powerful tool, it should be noted that the analysis of carotenoids (which are nonvolatile, thermally labile, and inherently unstable) presents a special challenge to the mass spectrometry analyst. An overview of common MS components and techniques is provided in Chapter 2 (techniques not previously mentioned are briefly described below). 

Lietz, G. Henry, C.J.K. 1997. A modified method to minimise losses of carotenoids and tocopherols during HPLC analysis of red palm oil. Food Chem. 60 109-117. 

In an excellent review of variables influencing the analysis of carotenoids by HPLC, Scott [768] noted that some parameters had adverse effects on the system, First, stainless steel frits seemed to lead to lowered responses for 8-cryptoxanthin and a- and / -carotene. Replacement of the frits with metal-free frits increased responses, but the replacement of stainless steel column tubing and injector tubing did not increase response. This is most likely a result of the large disparity in contact surface areas between the frits and the tubing. Storage of lycopene samples in 0.1% BHT (antioxidant)-preserved chloroform vs. chloroform without BHT showed considerably less degradation with time. The same results were found for BHT-preserved THF vs. unpreserved THF. Optimization of extraction procedures is also addressed in this paper. 

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