Open-column and thin-layer chromatographic techniques have been extensively discussed in earlier reviews (Davies 1976, Gross 1991). High performance liquid chromatography (HPLC) is a rapid, reproducible and highly sensitive technique used with wide varieties of stationary phases, mobile phase systems and detectors for determination of carotenoids in tomato and tomato products. The standard method for determination of carotenoids fails to resolve cis-isomers (AOAC 1995). Both C18 and C30 stationary phase columns are used, but the former allows only partial separation of cis-isomers, whereas the latter provides a higher resolution of cis-isomers. Research (2000-2006) on HPLC methods with their conditions used in the determination of carotenoids and/or lycopene in tomato and tomato products is summarized in Table 4, while the earlier HPLC methods have been reviewed by Shi and Le Maguer (2000).
Identification
Carotenoids are usually identified by their chromatographic and spectrophotometric behavior. The absorption spectrum of each chromatographically pure pigment is recorded and compared with values from the literature. For identifying the geometric isomers, photoisomerization is induced by illuminating authentic standards of
B. Stephen Inbaraj and B.H. Chen151
Extraction HPLCa column Mobile phase/flow rate/detector References
0.2 g spray-dried tomato C18 (250 ¥ 4.6 mm i.d., Acetonitrile/methanol/2-propanol (44:54:2, v/v/v); Anguelova and powder with 40 mL tetrahydro- 5 mm) Vydac column flow rate 1 mL/min; PDAb Warthesen (2000) furan/methanol (1:1, v/v)
2 g tomato paste extracted with C18 (150 ¥ 3.9 mm i.d., Methanol/tetrahydrofuran/water (67:27:6, v/v/v); Baysal et al. (2000) hexane/ acetone/absolute alcohol/ 5 mm) Waters Symmetry flow rate 1.5 mL/min; PDAb
toluene mixture (or) 53 g tomato column paste extracted with supercritical
fluid CO2 at 55°, pressure 300 bar, flow rate 4 kg/h
5 g tomato pulp extracted with C30 (250 ¥ 4.6 mm i.d., Gradient elution using methanol/methyl Tiziani et al. (2006) 50 mL methanol and filter cake with 3 mm) YMC column with tert-butyl ether (95:5, v/v for initial 5 min and
50 mL acetone/hexane (1:1, v/v) after C30 guard column changed to 30:70, v/v for 55 min); flow rate
homogenizing with 1 g CaCO3, 4 g 1 mL/min; PDAb
Celite and 50 mL methanol for 1 min and filtered
150 g tomato/tomato juice/50 g C18 (250 ¥ 4.6 mm i.d., Linear gradient using acetonitrile/methanol/ Takeoka et al. (2001) tomato paste homogenized with 5 mm) Rainin Dynamax dichloromethane/hexane (85:10:2.5:2.5 to
10% Celite, 10% MgCO3 and column with a guard 45:10:22.5:22.5% at 40 min); flow rate 250 mL tetrahydrofuran, followed column 0.8 mL/min; PDAb
by extraction in 250 mL tetrahydrofuran
10 g fresh/processed tomato C30 (250 ¥ 4.6 mm i.d., multi-step linear gradient using 15 to 50% Nguyen et al. (2001) homogenized with 50 mL 3 mm) polymeric column methyl-tert-butyl ether in methanol for 55 min;
methanol, 1 g CaCO3 and with a C30 guard column flow rate 1 mL/min; PDAb 3 g Celite followed by extraction
with acetone/hexane (1:1, v/v)
1-10 g tomato pericarp extracted C30 (250 ¥ 4.6 mm i.d., Methyl-tert-butyl ether/methanol/ethyl acetate Ishida et al. (2001) with 20 mL dichloromethane after 3 mm) YMC column (40:50:10, v/v/v); flow rate 1 mL/min; PDAb
Contd.
Tomatoes and Tomato Products methanol, 0.01% ethylenediamine
tetraacetic acid and butylated hydroxyanisole (1-10 mg)
2 g tomato seeds and skins C18(2) (150 ¥ 4.6 mm i.d., Gradient elution using A: methanol/0.2 M Rozzi et al. (2002) extracted by sonication with 20 mL 3 mm) Phenomenex ammonium acetate (90:10, v/v) and B: methanol/
chloroform for 30 min (or) Luna column 1-propanol/1.0 M ammonium acetate Supercritial fluid CO2 at 32 to 86°, (78:20:2, v/v/v); electrochemical detector pressure 138 to 483 bar, flow
rate 2.5 mL/min
10 g tomato slurry extracted by C30 (250 ¥ 4.6 mm i.d., Methyl tert-butyl ether/methanol (7:3, v/v); Dewanto et al. (2002) shaking with a mixture of 5 mL 3 mm) YMC column flow rate 1 mL/min; UV-VISc at 471 nm
chloroform 3 mL acetone and 15 mL hexane for 5 min
0.5 g freeze-dried tomato C30 (250 ¥ 4.6 mm i.d., Linear gradient using A: methanol-water Gómez-Prieto et al.
extracted with supercritical 5 mm) Develosil UG (96:4 v/v) B: methyl tert-butyl ether from (2003) fluid CO2 at 0.25–0.90 g/mL column at 20° 83(A):17(B) to 33(A):67(B) at 60 min;
density, temperature 40°, flow rate 1 mL/min; PDAb
flow rate 4 mL/min
8 g tomato juice agitated with C30 (250 ¥ 4.6 mm i.d., Gradient elution with A: 1-butanol/ acetonitrile Lin and Chen (2003) 0.2 g MgCO3 and 40 mL 5 mm) YMC column (30:70, v/v) and B: methylene chloride; flow
ethanol/hexane (4:3, v/v) rate 2 mL/min; PDAb
for 30 min
2 g tomato juice/sauce/soup/ C30 (250 ¥ 4.6 mm i.d., Methanol/methyl-tert-butyl ether; flow rate Seybold et al. (2004) baked tomato slices 5 mm) YMC column at 23∞ 1.3 mL/min; PDAb
homogenized with 400 mg MgO, and 500 mL echinenone followed by extraction with 35 mL methanol/
tetrahydrofuran (1:1, v/v) in ice for 5 min
Contd.
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homogenizing with 500 mL ethanol a C18 guard column at 25° tomato varieties; flow rate 1 mL/min; PDAb
5 g tomato puree with 120 mL C30 (250 ¥ 4.6 mm i.d.) Methanol/methyl-tert-butyl ether/ethyl acetate Qiu et al. (2006) hexane/acetone/ethanol (2:1:1, v/v/v) YMC column) at 24° (50:40:10, v/v/v); flow rate 1.5 mL/min; UV-VISc
3 g of tomato extracted by stirring C18 (300 ¥ 2 mm i.d., Methanol/acetonitrile (90:10 v/v) plus 9 mM Barba et al. (2006) with 100 mL hexane/acetone/ 10 mm) mBondapack triethylamine; flow rate 0.9 mL/min; UV-VISc at
ethanol (50:25:25, v/v/v) for 30 min column with a C18 475 nm precolumn (20 ¥ 3.9 mm
i.d., 10 mm) at 30°
250 mg raw/cooked tomato C18 (250 ¥ 4.6 mm i.d., Methanol/acetone (90:10, v/v); flow rate Mayeaux et al. (2006) extracted in acetone/ethanol/ 5 mm) Discovery column 0.8 mL/min; PDAb
hexane mixture of 1 mL each (vortexed for 30 s)
aHPLC, high performance liquid chromatography; bPDA, photodiode array; cUV-VIS, ultraviolet-visible.
anticipated carotenoids and subjecting to HPLC analysis under the same condition as that of unknown carotenoid extract. By comparing the retention time and absorption spectrum, the geometric isomer can be tentatively determined. A cis-isomer differs from the all-trans-isomer in its adsorption affinity and absorption spectrum. The absorption maximum of a cis-isomer shifts to lower wavelength compared to its all-trans counterpart. The di-cis-isomers may shift to a lower wavelength than mono-cis-di-cis-isomers. The identification of carotenoids based on spectral characteristics has been well described in the literature (Davies 1976, Goodwin 1981). Further confirmation of identified carotenoids can be made by performing cochromatography test using isomerized and non-isomerized carotenoid standards.
A photodiode array detector (PDA) coupled with HPLC provides online monitoring of absorption spectrum of carotenoid peaks at multiple wavelengths. However, it fails to provide complete structural and stereochemical information of carotenoids. Mass spectrometer enables the quantification and elucidation of carotenoid structure on the basis of molecular mass and characteristic fragmentation pattern. Figure 4A shows the negative-ion atmospheric pressure chemical ionization (APCI) product ion tandem mass spectrum of lycopene molecular ion radical (m/z 536.43) (van Breemen 2005). Though lycopene, a-carotene and b-carotene show similar molecular ion peaks, lycopene can be selectively monitored using its characteristic fragment ion of m/z 467 obtained by elimination of terminal isoprene group, while a-carotene and b-carotene fail to form this fragment ion because of their terminal ring groups. b-Carotene gives a characteristic fragment ion m/z 444 because of loss of toluene, as illustrated in a positive-ion APCI spectrum (Fig. 4B) (Lacker et al. 1999). Structurally significant fragment ions of a-carotene are m/z 480 and 388, which correspond to the retro-Diels-Alder fragment ion, [M-56]+., and a loss due to both toluene and retro-Diels-Alder fragmentation [M-92-56]+., respectively (van Breemen et al. 1996). The collision-induced dissociation mass spectrum of lutein was found to show fragment ions of m/z 551, 459 and 429 due to the losses of water [MH-18], both water and toluene [MH-18-92] and the terminal ring [MH-140], respectively (van Breemen et al. 1996). However, mass spectrometer fails to distinguish among stereoisomers.
High resolution nuclear magnetic resonance (NMR) spectroscopy with higher magnetic field strength (800-900 MHz) and the application of two-dimensional pulse sequences assist in elucidating the double-bond stereochemistry, which is the key area for identification of most carotenoids and their isomers. Tiziani et al. (2006) recently profiled carotenoids in a lipophilic mixture of tomato juice by a combination of
homonuclear and heteronuclear 2D NMR techniques, and 1H and 13C NMR assignments for all-trans-lycopene, 5-cis-lycopene, 9-cis-lycopene, 13-cis-lycopene, all-trans-b-carotene and 15-cis-phytoene have been reported.
Fig. 4 Mass spectrum of (A) lycopene and (B) b-carotene (van Breeman 2005, Lacker et al.
1999). (A) negative-ion APCI (atmospheric pressure chemical ionization) product ion tandem mass spectrum of the lycopene molecular ion radical of m/z 536.43 (note the m/z 467 fragment ion due to elimination of an isoprene group in lycopene does not occur for a- and b-carotenes);
(B) positive-ion APCI mass spectrum of b-carotene.
Other non-destructive methods including Fourier transform-Raman spectroscopy, attenuated total reflection infrared spectroscopy, and near infrared spectroscopy were also recently employed for determination of lycopene and b-carotene in tomato and tomato products, and the most appropriate statistics for calibration models was shown by infrared spectroscopy (Baranska et al. 2006, Pedro and Ferreira 2005).