Journal of Chromatography 1

8/6/2019 Journal of Chromatography 1

1/15

Journal of Chromatography A, 1217 (2010) 34943502Contents lists available at ScienceDirectJournal of Chromatography A

journal homepage: www.elsevier.com/locate/chroma

Critical assessment of three high performance liquid

chromatography analytical methods for food carotenoidquantificationM. Graa Dias

a, ,, Lusa Oliveira

a, M. Filomena G.F.C. Cames

b, Baltazar Nunes

c, Pieter Versloot

dand Paul J.M.

Hulshofd

a Departamento de Alimentao e Nutrio, Instituto Nacional de Sade Doutor Ricardo Jorge (INSA, I.P.), Av. Padre Cruz,

1649-016 Lisboa, Portugalb CCMM/DQB Faculdade de Cincias da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugalc Departamento de Epidemiologia, Instituto Nacional de Sade Doutor Ricardo Jorge (INSA, I.P.), Av. Padre Cruz, 1649-016

Lisboa, Portugald Division of Human Nutrition, Wageningen University, P.O. Box 8129, 6700 EV, Wageningen, The Netherlands

Received 27 November 2009;

revised 5 March 2010;

accepted 15 March 2010.

Available online 19 March 2010.

Abstract

Three sets of extraction/saponification/ HPLC conditions for food carotenoid quantification were technically and

economically compared. Samples were analysed for carotenoids -carotene, -carotene, -cryptoxanthin, lutein, lycopene,

and zeaxanthin. All methods demonstrated good performance in the analysis of a composite food standard reference

material for the analytes they are applicable to. Methods using two serial connected C18 columns and a mobile phase based

on acetonitrile, achieved a better carotenoid separation than the method using a mobile phase based on methanol and one

C18-column. Carotenoids from leafy green vegetable matrices appeared to be better extracted with a mixture of methanol and

tetrahydrofuran than with tetrahydrofuran alone. Costs of carotenoid determination in foods were lower for the method with

mobile phase based on methanol. However for some food matrices and in the case ofEZ isomer separations, this was not

technically satisfactory. Food extraction with methanol and tetrahydrofuran with direct evaporation of these solvents, and

saponification (when needed) using pyrogallol as antioxidant, combined with a HPLC system using a slight gradient mobile

phase based on acetonitrile and a stationary phase composed by two serial connected C18 columns was the most technically

and economically favourable method.

Keywords: Carotenoids; Food; HPLC ; Method development; Solvent and time reduction

Article Outline

1.

Introduction

2.

Materials and methods2.1.Abbreviations

2.2. Reagents


2/15

2.3. Carotenoid standards

2.4. Samples

2.5.Analytical methods2.5.1. Carotenoid extraction from foods

2.5.1.1. Method 1

2.5.1.2. Method 2

2.5.1.3. Method 32.5.2. HPLC system

2.5.2.1. Method 1

2.5.2.2. Method 2

2.5.2.3. Method 32.5.3. Chromatographic method performance

2.5.4. Statistical analysis

2.5.5. Economic study

3.

Results and discussion3.1. Carotenoid quantification

3.2. HPLC separation conditions

3.3. HPLC system performance

3.4. Economical considerations

4.

Conclusions

Acknowledgements

References

1. Introduction

Carotenoids are phytochemicals biosynthesized by plants, bacteria, algae, and fungi, but animals depend on diet to obtain

them. Humans obtain them mainly through the consumption of fruits and vegetables and their possible beneficial effects have

been well documented in the literature [1] and [2]. A large number of epidemiologic studies have shown inverse correlation

between the consumption of carotenoid-rich fruits and vegetables and the incidence of cancer, cardiovascular, eye and skin

diseases [1] and [2]. Animals absorb carotenoids selectively or unselectively, deposit them as such or slightly altered, or

convert them into vitamin A. Reliability and completeness of a substantial part of current data on food carotenoids still

appears questionable [3]. Bioavailability of carotenoids in humans and the effect of cooking or processing on food

carotenoid content are issues that sometimes give controversial results. This controversy incites for the exclusion of errors

introduced by analytical methods. The inherent difficulty of carotenoid analysis may contribute to this lack of consistency,

mainly because of the large number of these compounds, their likeness and susceptibility to isomerisation and oxidationduring analysis. Besides, considering recent reports highlighting a large percentage of (Z)-isomers in biological fluids and

tissues and different potential antioxidant activity among isomers [4], it has become important to address the significance of

carotenoid isomers and to study the role that (Z)-isomers may play as bioactive compounds. Therefore, it is essential to

improve analytical methods, developing rapid and reliable analytical techniques for the quantitative determination of individual

carotenoids in food matrices. Most frequently analytical methods used in this domain are based on extraction, and

saponification when necessary, followed by reversed phase high performance liquid chromatography (RP- HPLC) . The


3/15

process is usually cumbersome, error prone, time consuming and expensive, limiting per day number of carotenoid analysis in

a laboratory. Hence, more reliable, fast and environment friendly processes of analysis are welcome. The objective of this

study, taking into account technical and economical considerations, is to develop a method to separate and quantify all-E-

isomers of -carotene, -carotene, -cryptoxanthin, lutein, lycopene, and zeaxanthin and (Z)-isomers of -carotene and

lycopene based on two approaches: one performing a good separation and quantification but time and reagent consuming and

the other more economically favourable but with a lower separation potential.

2. Materials and methods

All manipulations were carried out under protection from both ordinary artificial light and direct sunlight and, when feasible,

amber glass was used to avoid carotenoid isomerisation. Extracts were always manipulated under yellow light (Philips lamp

TLD 36W/16 yellow, light transmission 500750 nm, The Netherlands) and within the shortest possible time. When it was

necessary to transport sample extracts under ordinary artificial light, they were completely wrapped in red filter foil

(primary red 106 (Lee Filters, United Kingdom), light transmission of wavelengths between 550 nm and 700 nm).

2.1. Abbreviations

ACN: acetonitrile, BHT: butylhydroxytoluene, DCM: dichloromethane, EtOH: ethanol, MeOH: methanol, PE: petroleumether, TEA: triethylamine, and THF: tetrahydrofuran.

The terms E (trans) and Z (cis) refer to the geometrical isomers. For convenience, the common carotenoid names have

been used throughout this text, instead of their proper semi-systematic names, and, unless specified, these names refer to

both isomer forms of these carotenoids.

2.2. Reagents

All reagents were of analytical grade or higher; organic solvents for HPLC were HPLC grade; dichloromethane (DCM),

methanol (MeOH), acetonitrile (CAN), and ethanol (EtOH) were from Carlo Erba Reagenti SpA (Italy), tetrahydrofuran (THF)

was from Riedel-de Han, SigmaAldrich (Germany), petroleum ether (PE), anhydrous sodium sulphate, and

butylhydroxytoluene (BHT) were from Panreac Quimica SAU (Spain); triethylamine (TEA), magnesium hydroxide carbonate,potassium hydroxide, ammonium acetate, and pyrogallol were from Merck, KGaA (Germany); hexane was from Fluka

Chimie GmbH (Switzerland) and calcium carbonate from BDH (England). Water with a resistivity of 18.2 M cm (25 C) was

obtained from a Milli-Q Advantage System (Millipore, S.A.S., France).

2.3. Carotenoid standards

All-E-isomers of -carotene, -cryptoxanthin, lutein, lycopene, zeaxanthin, and echinenone were from CaroteNature GmbH

(Switzerland), -carotene was from SigmaAldrich (Germany), and -apo-8-carotenal was from Fluka Chimie GmbH

(Switzerland). All standards were provided in ampoules containing 1 mg except -carotene (5 mg) and -apo-8-carotenal

(5 g flasks). Standard solutions were prepared in DCM and concentrations were measured with a spectrophotometer

(Thermo Scientific Evolution 300), after evaporation and re-dissolution in the appropriate solvent. Extinction coefficient

, wavelength and solvents pertaining to each of the specified compounds were as follows [5]: lutein2550, 445 nm

and zeaxanthin2540, 450 nm, both in ethanolic solution; -cryptoxanthin2460, 451 nm, lycopene3450, 472 nm, -

carotene2800, 444 nm, and -carotene2560, 450 nm, all in hexane solution. Individual working solutions of each

carotenoid were injected in the HPLC system on the day of the preparation for purity check (ratio of peak area of the

individual carotenoid to sum of all the chromatogram peak areas) and the concentrations of the standard solutions were

corrected accordingly. Six levels of working calibration solutions with a mixture of the six carotenoids (concentrations 0.05


4/15

5 g/mL, evenly distributed) were injected daily to build the calibration curves by the weighted least squares method. (Z)

Isomers of -carotene and lycopene were quantified using extinctions coefficients of their counterparts all-(E)-isomers. All

standard solutions were stored in the dark, under nitrogen, at 70 C. Calibration standards, except lycopene at 0.05 g/mL

(own data not published), showed to be stable for 6 months under these conditions.

2.4. Samples

Fruits and vegetables were grouped in three main classes according to their colour/carotenoid composition. Two different

food items representing each class were analysed: leaf beet (Beta vulgaris L. sp. vulgaris convar. cicla L. Alef.) and galega

kale (Brassica oleracea L. var. acephala D.C.) representing the green fruits and vegetables, containing mainly lutein and -

carotene; pumpkin (Curcubita pepo L. var. styriaca Greb.) and tomato (Lycopersicon esculentum M.) representing the

yellow/red fruits and vegetables, containing mainly hydrocarbon carotenoids; peach (Prunus persica L.) and orange (Citrus

sinensis L.) representing the yellow/orange fruits, containing a complex carotenoid profile including carotenol esters. Leaf

beet, peach, and orange were collected from the market in The Netherlands, in 2006, pumpkin and tomato were produced

in Portugal (central region), in 2008, and kale was harvested in Portugal (central region), in March 2009 (planted the

previous year). Samples from The Netherlands were preserved frozen at 70 C, under nitrogen during 30 months;

pumpkin was harvested six months before the analysis and it was preserved at room temperature; tomato was harvested six

months before the analysis, it was immediately peeled with boiling water, slightly drained, grinded and preserved in a

freezer at 20 C according to Portuguese household practices. Each sample of 1 kg minimum was composed by

representative edible parts of at least three fruits/plants, and the non-edible parts (peel, seeds, and thick midribs) were

discarded. In addition, a standard reference material (SRM) and an in-house control sample (baby food, vegetables

with egg, Olvarit, Nutricia, The Netherlands) were analysed, the first to monitor the method trueness and the second to

check the method trueness daily. The SRM (NIST 2383) was composed of the following ingredients: orange juice, infant

formula, corn, rice flour, creamed spinach, carrots, papaya juice, tomato paste, beef, macaroni, wheat flour, non-fat milk,

romano cheese, soya protein, onion powder, green pepper, celery oil, and oregano oil; the control sample was composed

of tomato, peas, carrots, potato, water, skimmed milk, egg, corn oil, and flour. The SRM had the following carotenoid

certified concentrations, in mg/100 g (reported as-received): total -carotene, 0.083

0.016; total -carotene, 0.312

0.063;

-cryptoxanthin (includes esters), 0.138

0.031; lutein (includes esters), 0.116

0.033; zeaxanthin (includes esters),

0.086

0.014; and carotenoid reference composition: trans--carotene, 0.085

0.024; trans--carotene, 0.240

0.080; 13-

+ 15-cis--carotene, 0.0321

0.0071; trans-lycopene, 0.63

0.12; total-lycopene, 0.70

0.15. The baby food control

sample, analysed by another laboratory, showed the following concentrations, in mg/100 g: -carotene, 0.11; -carotene,

0.43; -cryptoxanthin, not detected; lutein, 0.095; total-lycopene, 1.6; zeaxanthin, 0.0059.

A homogeneous sample of each of the foods was carefully obtained by means of a homogenizer/mixer knife mill

(Grindomix GM200, Retsch). Each homogeneous sample was divided into different glass containers, carefully capped

under inert atmosphere, and immediately frozen at 70 C. All manipulations were carried out under protection from both

ordinary artificial light and sunlight.

2.5. Analytical methods

Three sets of different extraction/saponification/ HPLC conditions, methods 1, 2 and 3, were compared. Method 1 was

based on the analytical methods described in Refs. [6], [7], [8] and [9]; method 2 was adapted from the method described in

Ref. [10]; method 3 was developed based on methods 1 and 2.

On the day of the analysis, after homogenization, approximately 2 g of each of the foods was weighed in triplicate on an

analytical balance to deci-milligram and analysed. The general method consisted of extraction, concentration of the extract

in a rotary evaporator, drying under a stream of nitrogen, dissolving the solid residue in the mobile phase, RP- HPLC


5/15

separation, identification, and quantification by external calibration. For conversion of carotenol esters to their parent

hydroxycarotenoids, a saponification step was done before injection into the HPLC system; this step also eliminates lipids

and chlorophylls. From our laboratory experience, although hydrocarbons carotenoids may resist to saponification,

xanthophylls could be lost during this step and subsequent washing; furthermore saponification appreciably extends the

time of analysis. Therefore saponification was included only when it was strictly necessary, i.e., in the analysis of materials

containing lipids (SRM and in-house control sample) and carotenol esters (SRM, and the fruits, peach and orange).Since, for the analysed samples, the used HPLC systems separate chlorophylls from the carotenoids under study it was

not necessary to destroy them.

Method performance was verified in each analytical sample by recovery tests for methods 1 and 3 (method 2 did not

separate the internal standard from some carotenoids under study). Each analytical sample was spiked, at the beginning

of the procedure, with a carotenoid not present in the sample: -apo-8-carotenal or echinenone, the last one only when

samples contain chlorophylls (chlorophylls co-elute with -apo-8-carotenal under the HPLC conditions used). Internal

standard recovery was calculated based on the ratio between peak chromatogram areas obtained for the spiked sample

and for the individual internal standard solution, corrected for the same concentration.

2.5.1. Carotenoid extraction from foods

2.5.1.1. Method 1

Magnesium hydroxide carbonate to neutralize any organic acids and internal standard (-apo-8-carotenal for fruits or

echinenone for green vegetables), were added to each analytical sample (2 g, in triplicate), followed by four repeated

extractions, each time with 50 mL of a 1:1 (v/v), THF and MeOH mixture, using a DI 25 basic batch high-performance

homogeniser (IKA-WERKA GMBH) at 13,500 rpm during 1 min followed by vacuum-filtration. After adding 50 mL of 10%

NaCl solution, exhaustive liquidliquid extraction (three times) was done to transfer analytes to a petroleum ether phase

which was evaporated at 35 C in a rotary evaporator system (composed by a vacuum controller V-850, a rotavapor R-210,

a heating bath B-491 and a vacuum pump V-700 from Bchi GmbH, Switzerland, and a minichiller, from Huber GmbH,

Germany), releasing the vacuum with nitrogen flow. All solvents used contained BHT, at 0.01% (w/v), as antioxidant. The

residue was carefully dissolved in 0.5 mL of DCM and then diluted to 5.0 mL with mobile phase without DCM, filtered

through a 0.45 m PVDF syringe filter and injected into the HPLC system. For fruits, 2.0 mL of this solution was mixed

with 1.0 mL of methanol containing 0.1% (w/v) BHT, and saponified with 1.0 mL of 20% (w/v) methanolic KOH solution, for

1 h at room temperature, in the dark and under nitrogen. After addition of 20 mL of 10% sodium chloride solution, extraction

with petroleum ether was done at least three times. Thereafter, the organic phase was washed with water until neutral pH,

verified by phenolphthalein solution, and evaporation was performed as described above. The residue was treated as the

one obtained before saponification but reconstituted to a total volume of 2.0 mL. If the analyte concentration was beyond the

highest point of the calibration curve, further dilution was done.

2.5.1.2. Method 2

Each analytical sample (2 g, in triplicate) added with 4.0 g of anhydrous sodium sulphate desiccant and 0.5 g of calciumcarbonate (to neutralize any organic acids) was extracted four times (until the extract was colourless) with 30 mL THF

containing 0.01% BHT, using a DI 25 basic batch high-performance homogeniser (IKA-WERKA, GMBH) at 24,000 rpm,

three times for 15 s, waiting 15 s between homogenisations. The extracts were filtered through filter paper (Whatman no.

54) at normal pressure. Combined filtrates were evaporated to near dryness in the rotary evaporator referred for method 1,

releasing the vacuum with nitrogen flow, and the residue was re-dissolved in a mixture of THF:MeOH (1:1), transferred to an

appropriate volumetric flask and made up to volume with the same solvent mixture and 25 L was injected into the HPLC


6/15

system. Orange, peach, NIST SRM-2383 and baby food control samples were saponified, as follows: 2 mL of the

solutions obtained above were transferred to glass tubes, evaporated to dryness and 1.5 mL of 5% ethanolic KOH solution

containing pyrogallol (0.2%) was added. The tubes were purged with nitrogen, and the content mixed for 3 h on a reciprocal

shaker at 250 rpm (KS-15, Edmund Bhler GmbH). After addition of 1.5 mL water, the mixture was extracted three times

with 3 mL hexane. The hexane layers were evaporated in a dry block heater under nitrogen. The residue was dissolved in

2.0 mL THF:MeOH (1:1, v/v) and 25 L was injected into the HPLC system.

2.5.1.3. Method 3

Each analytical sample (2 g, in triplicate) added with 1.0 g of magnesium hydroxide carbonate to neutralize any organic

acids and internal standard (-apo-8-carotenal for fruits or echinenone for green vegetables) was extracted four times with

50 mL MeOH:THF (1:1, v/v) containing BHT (0.01%), using a DI 25 basic batch high-performance homogeniser (IKA-

WERKA GMBH) at 13,500 rpm for 1 min and the liquid phase was separated from the solid phase by vacuum filtration. The

combined filtrates were evaporated to near dryness in the rotary evaporator referred to in method 1, releasing the vacuum

with a flow of nitrogen. The residue was carefully re-dissolved in 0.55.0 mL of DCM and then diluted to 5.050.0 mL with

mobile phase without DCM, depending on analyte concentration, and 50 L were injected into the HPLC system. Orange,

peach, NIST SRM-2383 and baby food control samples were saponified, as follows: 2 mL of the solutions obtainedabove were transferred to glass tubes, evaporated to dryness and 0.75 mL of 0.4% methanolic solution of pyrogallol and

0.75 mL of 10% methanolic KOH solution of potassium hydroxide were added. The tubes were purged with nitrogen, and

the content mixed for 1 h on a reciprocal shaker (KS-15, Edmund Bhler GmbH) at 250 rpm. After addition of 1.5 mL water,

the mixture was extracted three times with 3 mL petroleum ether. The petroleum ether layers were evaporated in a dry block

heater. The residue was dissolved in 0.2 mL of DCM and then diluted to 2.0 mL with mobile phase without DCM.

2.5.2. HPLC system

A HPLC system (Waters Corp., USA), composed by a 600 Controller and quaternary Pump, a 717plus Auto Sampler, a

2998 Photodiode Array Detector and Empower software, was used.

Mobile phases were filtered through a 0.45 m PTFE membrane filter and degassed in an ultrasonic bath. All sample

extracts were filtered through 0.45 m PVDF syringe filters before injection into the HPLC system. A 0.5 m in-line filter

was used to protect HPLC columns. The in-line filter was used instead of a guard column because a reduction of

resolution was observed with the latter, possibly due to the extra volume introduced by additional fittings and worst guard

column packaging.

Stainless-steel frits, tubing and connectors throughout the chromatographic system were replaced, when feasible, by PEEK

(poly ether ether ketone) materials to minimize carotenoid degradation.

Sample carotenoid extracts were identified using the HPLC system software by comparing their retention time (RT) and

visible absorption spectra (max (usually three peaks) and spectral fine structure, defined as the ratio of the peak heights

between absorption bands, as a percentage (%III/II) taking the minimum between the two bands as baseline [11]) with those

of carotenoid standards. Sample peak purity was checked using the HPLC system software. Quantification was done

by external standards calibration, based on peak areas. Internal standard recovery was used to correct sample carotenoid

concentration in methods 1 and 3. For samples requiring saponification, hydrocarbon carotenoid content was determined

from the results obtained before saponification and xanthophylls content was determined from the results obtained after

saponification.

2.5.2.1. Method 1


7/15

Two serial connected analytical columns, a Waters Spherisorb ODS2 PEEK lined (Alltech, cat. no. 8161), 100 4.6 mm

(5 m particle size, 80 pore diameter, 12% carbon load) connected to a reversed phase Vydac (cat. no. 201TP54)

containing silica polymerically modified with C18, 250 4.6 mm (5 m particle size, 300 pore diameter, 8% carbon load)

were used. The mobile phase was acetonitrile:methanol:dichloromethane, 75:20:5, v/v/v, containing 0.1% BHT and 0.05%

triethylamine; methanol contained 0.05 M ammonium acetate. Flow rate was 1.5 mL/min, injection volume 50 L and stop

time 35 min (except for orange, which was 105 min due to its complex carotenoid pattern). Chromatograms were obtainedby monitoring elution at 450 nm.

2.5.2.2. Method 2

A reversed phase Vydac 218TP54 column (250 4.6 mm) containing silica polymerically modified with C18 (5 m particle

size, 300 pore diameter, 8% carbon load) was used. Separations were achieved using a mixture of MeOH:THF (92.5:7.5,

v/v) with 0.1% of triethylamine. The flow rate was 1.0 mL/min and total run time per sample was 40 min (except for

orange, which was 105 min). Chromatograms were obtained by monitoring elution at 450 nm.

2.5.2.3. Method 3

Two serial connected analytical columns, a Waters Spherisorb ODS2 PEEK lined (Alltech, cat. no. 8161), 100 4.6 mm(5 m particle size, 80 pore diameter, 12% carbon load) connected to a reversed phase Vydac (cat. no. 201TP54)

containing silica polymerically modified with C18, 250 4.6 mm (5 m particle size, 300 pore diameter, 8% carbon load)

were used. Before HPLC mobile phase was established, different combinations of conventional organic solvents, ACN,

MeOH, DCM, THF, and isopropanol were examined for the separation of the mixture of the carotenoids under study,

including internal standards. The best mobile phase achieved was composed by ACN (eluent A), and MeOH:DCM, 80:20

(v/v) (eluent B); besides eluent B contained 0.04% BHT, 0.2% TEA, and 0.04 M ammonium acetate. Eluents A and B were

used in a combination of isocratic and linear gradient conditions. Pumps, respectively A (eluent A) and B (eluent B) were

programmed according to the following sequence (values in %), 014 min: 67:33, 17 min: 75:25, 26 min: 73:27, 27 min:

67:33. Flow rate was 1.5 mL/min, injection volume 50 L and run time 30 min (except for orange, which was 105 min).

Considering that the absorption maxima forE-isomers, in the eluent used, were 448 nm for -carotene and lutein, 455 nmfor -carotene, -cryptoxanthin and zeaxanthin, and 473 nm for lycopene, chromatograms were obtained by monitoring

elution at 450 nm, except for lycopene which were obtained by monitoring elution at 473 nm (absorption maximum).

2.5.3. Chromatographic method performance

Chromatographic method performance was evaluated using four parameters: retention factor, related to the analyte

distribution between the mobile phase and the column stationary phase; resolution, which measures the separation between

two peaks in a chromatogram; tailing factor, which measures the peak symmetry; theoretical plate number, which measures

the column performance. There are no advises about these parameters for food analysis. However the Food and Drug

Administration established recommendations for the pharmaceutical industry, in general, the values being for the retention

factor and resolution >2, the tailing factor 2, and the theoretical plate number >2000 [12].

2.5.4. Statistical analysis

The statistical comparison of data was performed for each carotenoid by two-way analysis of variance, to evaluate the

effects of the factors involved, method and matrix, on dependent variable, carotenoid concentration. Inter-day uncontrolled

effects were minimized (always same operator, same equipments, same reagents, same standards, same calibration curve,

and same room temperature) as it was not feasible to assay the three methods for each matrix at the same day. To

determine differences among analytical methods, post hoc tests, Tukey's HSD, Scheffe's, and Bonferroni's were applied.


8/15

When differences among methods were significant one-way analysis of variance was applied for each matrix. Differences

were considered significant at the 0.05 level. SPSS version 17.0 software for Windows was used.

Trueness [13] was quantified, analysing a certified reference material (NIST 2383), through -score (

), where Clab and Ccert are the laboratory and assigned analyte concentration

values, respectively, ulab is the laboratory combined standard uncertainty, assumed similar to ucert (more unfavourablesituation), and ucert is the combined standard certified reference material uncertainty. ucert is not reported in the Certificate of

Analysis, and was estimated as half of the expanded certified reference material uncertainty, U). The method is considered

unbiased ifz-score values are between 2 and 2 (approximately the 95% confidence interval). Z-score was also used to

quantify bias between carotenoid concentrations obtained in our laboratory and in another laboratory for baby food control

sample, considering each laboratory's combined standard uncertainty as half of the expanded certified reference material

uncertainty (most unfavourable situation).

Carotenoid content, of the different items, obtained by each method was calculated from the mean of the three replicates

with the 95% confidence limit ( , where t is obtained from the t-distribution, and s is the standard deviation).

2.5.5. Economic study

Economical costs were analysed for the three methods under study, considering the analysis of twelve analytical samples

with the saponification step, a manpower price of 20 euro/h, and a residue elimination of 6.3 euros by kg (as an

approximation it was considered an effluent with a density of 0.9). HPLC equipment and laboratory facilities were not

considered since they are almost the same for all methods and they were also affected to other laboratory work. HPLC

columns were not taken into account because it was verified that the system with one column had half the lifetime of the

system with two columns. Concerning the laboratory work, for each of the three methods three hypotheses were

considered. Hypothesis 1 refers to HPLC analysis of sample extracts only after saponification, without analysis of the

extracts obtained before saponification. Hypothesis 2 considers HPLC analysis of sample extracts before and after

saponification in two different days corresponding to the days where they were obtained. Hypothesis 3 refers to HPLC

analysis of sample extracts before and after saponification at the second day of analysis. In the last hypothesis extracts

obtained before saponification are stored 24 h before injection in the HPLC system but as demonstrated in a previous

work [8], extracts in mobile phase are stable at least for 24 h at 4 C.

3. Results and discussion

3.1. Carotenoid quantification

The three methods were able to successfully quantify all-E-isomers of -carotene, -carotene, -cryptoxanthin, lycopene,

lutein and zeaxanthin in nearly all the matrices studied. Occasional difficulties occurred in the quantification of all-E--

carotene in tomato by method 2 and all-E-zeaxanthin in leaf beet by method 1; spectral analysis of the peaks observed at -

carotene and zeaxanthin retentions times, respectively, revealed compounds or compound mixtures that cannot be

identified as such, possibly due to unresolved matrix interferences. Methods 1 and 3 enabled the separation among three Z-

isomers and all-E-isomer of lycopene, and between Z-isomers and all-E-isomer of -carotene. Method 2 enabled the

separation between Z-isomers and all-E-isomer of -carotene but did not allow the separation between Z-isomers and all-E-

isomer of lycopene. Chromatograms of baby food sample extracts illustrative of differences among methods are

presented in Fig. 1.


9/15

Fig. 1.

Baby food carotenoid elution profile obtained by three methods, plotted at 450 nm except when assigned 473 nm.

Compounds identification: 1all-E-lutein; 2all-E-zeaxanthin; 4equinenone; 6all-E-lycopene; 7Z-lycopene; 8Z-

lycopene; 9Z-lycopene; 10all-E--carotene; 11all-E--carotene; 12Z--carotene. Chromatographic conditions as

referred in Section 2.View Within Article

Analysis of the SRM, NIST 2383 (results presented in Table 2), gave z-scores between 2 and 2, attesting the good

performance of the three methods for carotenoids determination. Also, for baby food control sample, previously

characterized in another laboratory by method 2, gave similar results to those obtained by the external laboratory (| z-

score| < 2).

The carotenoid content of the eight samples analysed for method comparison is listed in Table 1.

Table 1. Carotenoid content of fruit, vegetables, and composite foods by three methods.

Carotenoid content (mg/100 g)

E--carotene

E--carotene

Total--carotene

E--cryptoxanthin

E-luteinE-lycopene

Total-lycopene

E-zeaxanthin

Orange

Method 1 ND0.061 0.0

10 a

0.061 0.0

10 a

0.64 0.1

9 a

0.0783 0

.0080 a

ND ND0.065 0.

016 aMethod 2 ND

0.0335 0.0065 b

0.0335 0.0065 b

0.544 0.098 a

0.0279 0.0015 b

ND ND0.0619 0.0086 a

Method 3 ND0.043 0.015 b

0.043 0.015 b

0.663 0.081 a

0.056 0.013 c

ND ND0.098 0.027 b

Peach

Method 1 ND0.101 0.021 a

0.102 0.022 a

0.033 0.013 a

0.132 0.021 a

ND ND0.059 0.064 a


10/15


11/15

Carotenoid content (mg/100 g)

E--carotene

E--carotene

Total--carotene

E--cryptox

anthin

E-luteinE-lycope

ne

Total-lycope

ne

E-zeaxant

hin

0077 b .00064 b .00064 b 015 b

Tomato

Method 1 ND0.17 0.10a

0.17 0.10a

ND0.1020 0.0073 a

6.7 1.0a

8.44 0.74 a

ND

Method 2 ND0.513 0.075 b

0.607 0.090 b

ND ND NS8.34 0.87 a

ND

Method 3 ND0.255 0.029 a

0.255 0.029 a

ND0.0771 0.0063 b

9.05 0.90 b

9.6 1.1b

ND

ND: not detected (detections limits: -carotene0.0009 mg/100 g, -cryptoxanthin0.0006 mg/100 g, lycopene

0.0008 mg/100 g, zeaxanthin0.0008 mg/100 g); NSnot separated; when letters (a c) are different for the same matrix and

analyte means that the difference between methods was statistically significant (p < 0.05).

Two-way analysis of variance revealed that there are no differences for carotenoids -carotene and zeaxanthin analysed by

the three methods, both present at low to medium levels. For all-E-isomer of -carotene, -cryptoxanthin and lutein, and

total-lycopene, the same statistical analysis evidenced matrix/method interaction and post hoc tests showed that method 2

gives the lowest concentrations, except for lycopene, for which both methods, 2 and 1, give the lowest values. Lower

concentrations obtained when method 2 was applied were possibly related to the impracticality of using internal standards

(co-elution with carotenoids under study). For the same method, major discrepancies, not explained by absence of recovery

corrections were observed for -carotene and lutein in leafy green vegetable matrices. These differences are probably

associated with the extraction solvent. Matrix extraction solvent in method 2 was THF alone, which is less efficient in

disrupting carotenoid protein bond, than the mixture of MeOH/THF used in methods 1 and 3. Despite the fact that a higher

homogeniser rate was used in method 2, some very small green matrix particles remained in the kale filtrate cake.

The application of one-way analysis of variance by matrix, tacking into account replicate analysis, followed by post hoc tests

when differences were relevant, showed for all matrices that method 3 is almost always in the group giving the higher

carotenoid content; exceptions were lutein in NIST SRM 2383, kale, leaf beat, and tomato, and -carotene and zeaxanthin

in the in-house baby food control sample. Notwithstanding, as p-values for these carotenoids/matrices are in the vicinity of

0.05, if day-to-day analytical method variance was taken into account (methods applied on different days) method 3 would

probably be in the group that gives the higher carotenoid content for the studied matrices. Further differences checking

based on measurement uncertainties obtained in a previous work for method 1

Filtration using a vacuum system resulted in drastic time reduction compared to room pressure filtration. Concerning the use

of anhydrous sodium sulphate before evaporation to dry organic phase it was not observed any decrease in terms of

evaporation time or residual liquid. Comparing methods 1 and 3 extractions, it was verified that method 1 partition in

petroleum ether (corresponding to an increase of solvent consumption) was dispensable. Method 3 showed an

improvement in mean internal standard recoveries both with and without saponification, increasing from 70% to 83%, and

from 89% to 94%, respectively.

3.2. HPLC separation conditions


12/15

Method 2 mobile phase based on MeOH showed adequate retention and separation characteristics for the all-E-carotenoids

under study, except for lycopene, for which Z-forms co-eluted with all-E. Besides it did not separate internal standards, also

base line stabilization along elution was difficult due to THF. Nevertheless, if there is no intention to separate all-E- from Z-

isomers of lycopene or to apply the method to matrices containing these carotenoids, method 2 mobile phase could be a

good choice concerning economical reasons, providing the analyst is aware of method limitations. Mobile phase based on

ACN enabled the separation of the referred carotenoid isomers, and also allowed a better separation of the carotenoidsunder study among themselves and from the internal standards. Besides, it avoids erroneous identification induced by

matrix interferences with the same retention times as the carotenoids under study (e.g. chromatograms obtained by method

2 for peach and orange presented compounds at lycopene retention time). In addition, for viscosity reasons, an organic

phase based on acetonitrile instead of methanol has the benefit of permitting the operation at higher flow rates with

acceptable backpressure levels, and to work with two in series columns.

When developing method 3, a mobile phase for the HPLC system based on methanol was considered, owing to

economical and carotenoid solubility reasons. Since THF, although stabilized with BHT, has unfavourable handling

characteristics and is incompatible with PEEK, tentative replacement by isopropanol, a solvent with a polarity near THF and

more environment, economical and technical friendly was done, however complete separation of all carotenoids was poor or

even absent. The similarity among the compounds under study makes it difficult to achieve simultaneously an isocratic

separation in a reasonable time frame with acceptable chromatographic characteristics. An alternative approach was the

gradient elution. The best separation under the mentioned conditions was achieved with the mobile phase referred for

method 3. This mobile phase showed the particular advantage of improving the separation in the range between lutein and

echinenone; in this range and for some food matrices, compounds other than those under study could cause

interferences, furthermore, -apo-8-carotenal internal standard has sometimes impurities that could co-eluted with

zeaxanthin and less frequently also with lutein. For reproducibility reasons, the first condition of method 3 mobile phase

program was tested along all separation but one of the three lycopene Z-isomers co-eluted with all-E--carotene.

Solvent mismatch between mobile phase and injection solution usually resulted in peak distortion that is more prevalent for

early eluting compounds. The composition of the injection solvent is a compromise between good solubility of carotenoids,

compatibility with mobile phase and no deformation of peaks. As described in [14], interaction between carotenoids,

injection solvent and mobile phase may cause multiple peaks, leading to misinterpretations. Therefore, the sample diluent

consisting of mobile phase would be the best choice to avoid misshaped peaks. However, the analytes under study have

low solubilities in mobile phases based on ACN. To solve this problem a first solubilisation of the analytes in

dichloromethane was done, with a further dilution in mobile phase without dichloromethane, with a final DCM concentration

lower than 20%; higher DCM concentrations resulted in lutein splitting and drastically lowered separation between lutein and

zeaxanthin. Method 2 sample diluent did not cause peak distortion and comparatively to methods 1 and 3 reduces work

with re-dissolution of dry extracts.

3.3. HPLC system performance

Results from chromatographic method performance evaluation are summarized in Table 2. Data were obtained from theanalysis of a standard carotenoid mixture at a concentration of 2 g/mL.

Table 2. System suitability parameters of the chromatographic methods.

All-E--carotene

All-E--carotene

All-E--cryptoxanthin

All-E-lutein

All-E-lycopene

All-E-zeaxanthin


13/15

All-E--carotene

All-E--carotene

All-E--cryptoxanthin

All-E-lutein

All-E-lycopene

All-E-zeaxanthin

Method 1Retention factor, k 7.26 10.4 2.41 0.75 4.66 0.86Resolution (L/Z)a, Rs 0.91Tailing factorb, Tf 1.25 1.15 1.33 0.91 1.40 1.00Theoretical plates, N3095 4495 1913 1810 2583 1915

Method 2k 4.93 5.47 3.56 2.43 7.81c 2.53(L/Z) Rs 0.95Tf 1.25 1.12 1.07 0.98 1.50c 0.98N 5587 5090 4483 5728 3260c 7491

Method 3k 7.79 9.75 2.96 0.91 5.58 1.07

(L/Z) Rs 1.17Tf 1.17 1.18 1.25 1.02 1.14 1.07N 2768 3554 2058 1950 3337 2003

a L/Z: lutein/zeaxanthin; resolution values for other individual analytes are greater than 2.6.b Measures taken at 5% of peak height.c Total-lycopene.

Retention factors for lutein and zeaxanthin in methods 1 and 3 were below 2; notwithstanding they are near 1, the minimum

generally acceptable in pharmaceutical industry to avoid interferences with unretained material. Resolution between lutein

and zeaxanthin is below the recommended value of 2 (baseline separation for two similar perfectly shaped Gaussian peaks

is observed with Rs = 1.5); the best resolution (Rs = 1.17) was obtained for method 3. To improve the separation between

lutein and zeaxanthin, the option would be to choose different stationary phases for xanthophylls and carotenes. However

the need of two HPLC systems increases costs. In food samples, zeaxanthin is the least abundant among the

carotenoids studied and usually with considerably lower levels than lutein, enabling a base line separation in almost all

food matrices. Tailing factors ranged from 0.91 and 1.50; the higher value was obtained for lycopene with method 2 which

did not separate all-E- from Z-isomers, the back half-width being affected by Z-isomers. Plate number changes with the

inverse of the square of peak width, so smaller plate numbers mean broader peaks and consequently less resolution.

Method 2 presented the best plate number for all carotenoids, which is possibly related to lower injection volume (half).

3.4. Economical considerations

Results obtained for hypotheses 1, 2 and 3 are presented in Table 3.

Table 3. Costs of carotenoid analysis, per 12 samples, in euro.

Method 1 Method 2 Method 3

Reagents, including standardsHypothesis 1 163 93 155


14/15

Method 1 Method 2 Method 3

Hypothesis 2 258 132 243Hypothesis 3 214 106 189

Consumable material 50 50 50Manpower 395 280 260Reagents elimination 28 17 25TotalHypothesis 1 636 440 490Hypothesis 2 731 479 578Hypothesis 3 687 453 524

Considering that hypothesis 1 could conduct to the worst technical results and that hypothesis 2 involves costs that could

not be technically justifiable (6% more for methods 1 and 2, and 8.5% for method 3), hypothesis 3 will be chosen. As shown

in Table 3, the major contribution for carotenoids HPLC analysis cost is manpower, therefore, efforts should be applied to

reduce this item contribution; method 3 compared to method 1 represents a 34% reduction in work hours and a 24%

reduction in total cost.

Reagents are the second major cost contributing factor with a high impact, since mobile phases are organic. Considering

that currently acetonitrile price is 10 times higher than 2 years ago (for method 3, contribution to the total price changed

from 1.6% to 15.7%), all efforts to reduce ACN consumptions are important. Method 3 saves up 18% of this solvent, when

compared to method 1. A simple way of reducing reagent costs in future is to reuse mobile phase by using isocratic

methods, when possible. Although this used phase contains analytes, after a good effluent homogenisation, they are

present at constant concentration, so they do not generate peaks in the chromatogram and, besides, concentrations are

below the detection limit. Notwithstanding, some precautions need to be considered; as DCM is the most volatile component

and ACN the least, and as contamination by samples will increase from batch to batch, the mobile phase could not be

recycled infinitively and the waste container must be as sealed as possible. In spite of recycling adding labour costs,

environmental concerns may be more important. Since columns used had 4.6 mm internal diameter, reducing mobile phase

consumption, hence reagent costs, could also be easily obtained through reducing column diameter. To obtain the same

retention time, a ratio of 5 between the square diameters (corresponding to a 2.1 mm internal diameter column) will reduce

the flow rate by the same factor. Injection volume should be reduced accordingly.

Concerning consumable material, all samples were filtered through a 0.45 m filter before being introduced in the HPLC

autosampler tray. Each filter is expensive (1 euro or more) adding a significant cost (4.6% for method 3). It was verified

that a centrifugation is enough to settle out any particles with the advantage of avoiding problems related to carotenoid

adsorption in the filter that could be dissimilar for different concentrations.

4. Conclusions

The need for technical and economical improvement of qualitative and quantitative carotenoid analysis, determined the

detailed study of some existing methods (summarized as methods 1 and 2) and the development of another one (method

3).

The three evaluated methods showed good performance, which was demonstrated through the results obtained from the

analyses of the Standard Reference Material, NIST 2383. Nevertheless, two of the studied methods presented some

inconveniences that should be taken into account.


15/15

Method 1 (matrix extraction with MeOH and THF, partition to petroleum ether, BHT as saponification antioxidant, and

HPLC isocratic method based on ACN and two serial connected C18 columns) was the least favourable in terms of time

and solvent consumption and also when saponification is needed.

Method 2 (matrix extraction with MeOH, pyrogallol as saponification antioxidant, and HPLC isocratic method based on

MeOH and one C18 column), despite saving 44% on reagent costs, showed poor separation (e.g. all-(E) lycopene from the

(Z) isomers), did not enable the use of internal standards due to co-elution, and could not thoroughly extract some greenvegetable matrices.

Method 3 (matrix extraction with MeOH and THF, pyrogallol as saponification antioxidant, and HPLC gradient method

based on ACN, and two serial connected C18 columns) seems to be the most efficient when considering analytical

performance, for all food matrices studied, concerning extraction, saponification, and separation of carotenoids. It showed

the best technical/economical performance for quantification of -carotene, -carotene, lycopene, and theirZ isomers, and

also -cryptoxanthin, lutein and zeaxanthin, enabling the use of internal standards, -apo-8-carotenal and echinenone.

When compared with method 1, method 3 internal standard recovery, with saponification, improved from 70% to 83% and

reagents, manpower and consumable material costs were reduced by 24%.

Acknowledgements

M. Graa Dias is grateful to INSA, I.P. for providing the research fellowship BIC 01/2003-II and to EuroFIR project (funded

under the EU 6th Framework Food Quality and Safety Programme, Project contract No. FOOD -CT-2005-513944) for

providing the training grant A2005/6-5.

References

[1] N.I. Krinsky and E.J. Johnson, Mol.Aspects Med.26 (2005), p. 459. Article | PDF (542 K) | View Record in Scopus |

Cited By in Scopus (130)

[2] W. Stahl and H. Sies, Biochim.Biophys.Acta1740 (2005), p. 101. Article | PDF (123 K) | View Record in Scopus |

Cited By in Scopus (116)

[3] D.B. Rodriguez-Amaya, J. Food Compost.Anal.13 (2000), p. 641.Abstract | PDF (108 K) | View Record in Scopus

| Cited By in Scopus (13)

[4] V. Bhm, N.L. Puspitasari-Nienaber, M.G. Ferruzzi and S.J. Schwartz, J.Agric. Food C hem.50 (2002), p. 221. Full

Text via CrossRef| View Record in Scopus | Cited By in Scopus (93)

[5] Marina I. Heinonen, V. Ollilainen, Eeva K. Linkola, Pertti T. Varo and Pekka E. Koivistoinen, J.Agric. Food C hem.37

(1989), p. 655. Full Text via CrossRef| View Record in Scopus | Cited By in Scopus (119)

[6] D.J. Hart and K.J. Scott, Food C hem.54 (1995), p. 101. Article | PDF (1294 K) | View Record in Scopus | Cited By

in Scopus (357)

[7] K.J. Scott, P.M. Finglas, R. Seale, D.J. Hart and I. Froidmont-Grtz, Food C hem.57 (1996), p. 85. Article | PDF

(726 K) | View Record in Scopus | Cited By in Scopus (37)

[8] M. Graa Dias, M. Filomena G.F.C. Cames and L. Oliveira, Food C hem.109 (2008), p. 815.

[9] M. Graa Dias, M. Filomena G.F.C. Cames and L. Oliveira, Food C hem.113 (2009), p. 808.

[10] P.J.M. Hulshof, C. Xu, P. van de Bovenkamp, Muhilal and C.E. West, J.Agric. Food C hem.45 (1997), p. 1174.

Journal of Chromatography 1

Documents

Transcript of Journal of Chromatography 1