Carotenoid determination in recent marine sediments-practical problems during sample preparation and HPLC analysis

Article history: Received January 2, 2017 Received in revised form March 1, 2017 Accepted April 21, 2017 Available online April 22, 2017 An analytical procedure for the analysis of carotenoids in marine sediments rich in organic matter has been developed. Analysis of these compounds is difficult; the application of methods used by other authors required optimization for the samples studied here. The analytical procedure involved multiple ultrasound-assisted extraction with acetone followed by liquid-liquid extraction (acetone extract:benzene:water 15:1:10 v/v/v) and HPLC analysis. The influence of column temperature on pigment separation and the quantification method were investigated – a temperature of 5 °C was selected for the Lichrospher 100 RP-18e column. The pigments in the sediment extract were quantified using a method based on HPLC analysis (at 450 nm) and spectrophotometric measurements (at 450 nm), and extinction coefficients were determined for standard solutions at this wavelength. It is very important to use the value of the extinction coefficient appropriate to the wavelength at which the detection of carotenoids was carried out. © 2017 Growing Science Ltd. All rights reserved.


Introduction
Carotenoids are a large group of natural compounds (at present ca 700 are known), ubiquitous in the aquatic environment. 1They occur mainly in phytoplankton, macroalgae and bacteria but also, as metabolites of parent carotenoids, in different organisms from other levels of the food chain like zooplankton and fish. 1,2Aquatic basin sediments contain a large variety of carotenoids, which can be used as proxies of plankton and macrophyta occurring in the adjacent waters and of postdeposition conditions. 3,4The analysis of carotenoids is difficult, especially in organic-rich sediments, because they contain not only parent carotenoids and their derivatives, but also a variety of low and high molecular organic compounds, including chloropigments.The most troublesome are separations of compounds with very similar chemical structures, like lutein and zeaxanthin or α-carotene and β-carotene, which differ only in the position of one double bond in the cyclohexyl ring (see Fig. 1).All the steps, i.e. sample collection, extraction, preservation and the chromatographic analysis itself, can affect the results.][9][10] Pigments are extracted with different solvents, i.e. acetone, methanol, ethanol or their mixtures (see Table 1).Sonication is applied to support the extraction process. 5,6,11All these activities should be performed in dim light and as quickly as possible to prevent pigment decomposition.However, a balance between the best possible extraction efficiency and the risk of artefact formation should be maintained.To increase extraction efficiency, liquid-liquid extraction of pigments from an acetone extract to another solvent has sometimes been used. 9here are a number of publications on HPLC methods for determining carotenoids in sediments. 5,16,20- 22RP-HPLC (C18) has mostly been used for this purpose (see Table 2).The mobile phase used by different authors was usually the same, but the gradient system was sometimes changed slightly. 5,22Other factors, such as the mobile phase composition or flow rate, were frequently modified. 6,11The application of methods used by other authors always requires them to be optimized to one's own needs and the available equipment.The choice of appropriate temperature for carotenoid analysis is very important, but column thermostatting is required.Column temperature problems were studied by some authors in the case of phytoplankton, 24,25 and surficial riverine sediment carotenoids 26 , but the results were contradictory.Another important issue is quantification.Some authors calculated pigment concentrations from a calibration curve using HPLC analyses of standards, 6,24,27 others used a method based on additional spectrophotometric measurements of the same but diluted solution as that analysed by HPLC and literature extinction coefficients of particular pigments. 9,11,28e aim of this paper was to develop an analytical procedure (sample preparation, HPLC analysis and quantification method) for determining carotenoids that could be applied to organic-rich sediments, and to indicate the problems to be overcome during the development of this method.The influence of liquid-liquid extraction from an acetone extract to benzene on carotenoid extraction efficiencies was checked.The effect of column temperature on pigment separation was investigated and the different quantification methods were compared.

Results and Discussion
Both wet and freeze-dried sediments were subjected to extraction by different authors (see Table 1).
The time of extraction varied from several minutes 6,18,29 to several hours 4,7,26 and even overnight. 5,11,16,17owever, the influence on extraction efficiency of freeze-drying prior to extraction is not clear.In the case of freeze-dried sediments a 5-10 % content of water was recommended to improve extraction of polar compounds, 4,30 but some authors used acetone for extracting freeze-dried material. 11,13,14ltrasonication is still in routine use in many laboratories and in this work was selected for extracting carotenoids from sediments.We used ultrasound-assisted extraction of wet sediments with acetone for the analysis of carotenoids in Gulf of Gdańsk sediments.This extraction method is used in our Marine Pollution Laboratory (MPL-IO) for chloropigments and has been intercalibrated with other traditional methods of pigment extraction. 9Acetone has been shown to be an effective extraction solvent for wet sediment samples. 30In many methods a small volume of solvent was used (up to 10 ml), after which the extract was analysed by HPLC. 5,11,29In the case of organic-rich sediments a small amount of solvent might be insufficient to extract the whole amount of pigments.To ensure complete extractability in our method, multiple acetone extraction and total volume of solvent (15 -45 ml), larger than that used by other authors, were applied.To reduce the sample volume, liquid-liquid extraction of carotenoids from an acetone extract to benzene was performed (acetone extract:benzene:water -15:1:10 v/v/v).This extraction step speeds up the further evaporation of the solvent and concentrates the analytes, which increases the sensitivity of the method (see Fig. 2).3.
The chromatogram peaks were taller, which means that compounds present in low concentrations (e.g.echinenone, α-carotene) could also be determined.This extraction step did not affect the composition of pigments in the sediment sample.The differences in carotenoid concentrations in a sample before and after liquid-liquid extraction were not statistically significant; this was demonstrated for the most abundant carotenoids (see Fig. 3).Only the major peaks were compared because the values for echinenone and α-carotene in the samples not subjected to liquid-liquid extraction were below the limit of quantification.
It is difficult to analyse all the carotenoid compounds present in a sample in one HPLC run 6,31 , especially in a mixture with chloropigments (which also absorb at 450 nm).Sediment samples contain not only parent carotenoids but also their derivatives (see Fig. 2b), such as cis-zeaxanthin, which is formed by geochemical reduction from diatoxanthin in warm-anoxic conditions, 32 making HPLC separation even more complicated.Therefore, it is important to decide which compounds we wish to quantify first of all; the HPLC method should then be selected accordingly.In this work, the greatest stress was placed on parent pigments, markers of the main phytoplankton groups occurring in the Baltic.These were peridinin and dinoxanthin as markers of dinoflagellates, fucoxanthin (diatoms), diatoxanthin and diadinoxanthin (diatoms and dinoflagellates), alloxanthin (cryptomonads), lutein (green algae), zeaxanthin (cyanobacteria and green algae), canthaxanthin and echinenone (cyanobacteria), α-carotene (cryptomonads) and β-carotene (most algae). 2 Methods developed for seawater are not satisfactorily applicable to sediment extracts.Moreover, the methods used for sediments by other authors (see Table 2) require certain modifications -these may apply to the column (length, diameter or stationary phase), gradient system or column temperature.
The separation of lutein (a pigment of green algae and higher plants) from zeaxanthin (cyanobacteria and green algae) is very important for aquatic environmental studies, as these pigments are useful for analysing taxonomy.The problem of separating lutein from zeaxanthin was noticed already some time ago: 33 these two compounds have very similar retention times and sometimes coelute.Their good separation is evidence that the appropriate analytical method was chosen.The influence of the Lichrospher 100 RP-18e column temperature on the resolution of compounds very similar in chemical structure (lutein/zeaxanthin and α-carotene/β-carotene) was assessed.It was found that the lower the column temperature, the longer the time of analysis, which enhances separation of the mixture's components.Better separation of the lutein/zeaxanthin was achieved using a column thermostatted at 5 °C rather than at 25 °C (see Fig. 4).The resolution of separation at 5 °C between lutein/zeaxanthin (Rs = 1 ± 0.07 for sediment, Rs = 0.79 ± 0.04 for pigment standards) and α-carotene/βcarotene (Rs = 0.96 ± 0.06 for sediment, Rs = 0.65 ± 0.06) was significantly higher (p < 0.05) than at other temperatures.The resolution specifies the selectivity of the stationary phase and column efficiency.The lower resolution of the pigment pair in the standard mixture than in the sediment extract may be because the concentrations of available pigment standards are very low.
For the chromatographic separation of chlorophylls and carotenoids in a phytoplankton extract of seawater Zapata et al. 25 used a column thermostatted at 25 °C, and Jun et al. 26 did likewise for a riverine sediment extract; neither paper gave any explanation why this particular column temperature was applied.Van Heukelem et al. 24 studied different column temperatures to improve separations of phytoplankton pigments from lake water using HPLC; they recommended a higher temperature (60 °C) as optimal for carotenoid separations.These observations do not agree either among themselves or with our results.We chose a column temperature of 5 °C as the most suitable for our purposes.Before injection, the samples were stored in an auto-sampler at the same temperature (5 °C).The lower temperature and darkness prevented pigment decomposition.Reuss and Conley recommended storage of samples in an auto-sampler at an even lower temperature (4 °C). 11It should be emphasized that we chose the column temperature of 5 °C as optimal for the Lichrospher 100 RP-18e column; for other columns, the best temperature may be different.When optimizing a literature method, it is very important to do so with respect to one's own equipment and working conditions.3.
Another problem that arose during the analysis of carotenoids in the sediment extracts was quantification.Some authors have based their calculations on HPLC standard calibration curves, 6,14,27 others have used a method based on an additional spectrophotometric measurement of the same solution as that analysed by HPLC and literature extinction coefficients of particular pigments. 9,11,28The different calculation methods are compared in Fig. 5.
In the case of the spectrophotometric method it is very important to use the appropriate extinction coefficients, i.e. those determined for wavelength at which the detection of carotenoids was carried out.In this work, the chromatograms were recorded and integrated at 450 nm.Table 3 lists the extinction coefficients calculated from absorption measurements of carotenoid standards at 450 nm using the Lambert-Beer law.Particular carotenoids have absorption maxima at different wavelengths, whereas the maximum wavelength depends, for example, on the solvent in which it is determined.Also, the solvent composition during HPLC analysis at the retention time of an analyte is usually different than the solvent for which literature extinction coefficients are available.For example, canthaxanthin has an absorption maximum at 472 nm (in ethanol) 2 , while the extinction coefficient equal to 220 ml•mg -1 •cm - 1 was determined at 466 nm (in petroleum ether) 2 (see Table 3); in this work, the extinction coefficient calculated at 450 nm (in ethanol) was 160 ml•mg -1 •cm -1 .In the spectrophotometric method used in this work both the HPLC chromatogram and absorption spectrum were measured at the same wavelength (450 nm) to minimize errors.
In the case of pigment standards, the concentrations calculated in this way are comparable to the values obtained from calibration curves, while the use of literature extinction coefficients (see Table 3), especially those set for other wavelengths, significantly affects the results (see Fig. 5a).Pigment concentrations in sediments cannot be quantified using HPLC calibration curves.The concentrations of carotenoids in commercial standard mixtures are usually low.Indeed, they were very low (0.77-1.36 mg/l) in the commonly used individual standards that we purchased, whereas the concentrations of these carotenoids in sediment samples were several or even several dozen times higher.Peak areas of carotenoids in sediment extracts were beyond the range of calibration curves.One solution would be to dilute the sediment extract; unfortunately, however, the concentrations of individual pigments in the sediment extracts differ significantly from each other.Diluting a sample could place carotenoids like canthaxanthin, echinenone and α-carotene, present in sediments in low concentrations (see Fig. 2b), beyond the limits of detection.There was no single suitable concentration of sediment extracts for accurately determining all the analytes in a sample, thus enabling the calibration curves of available standards to be used.The analysis of one sample at different concentrations would be time-consuming, especially when a lot of samples have to be analysed.That is why we used the spectrophotometric method of quantification.In this case, it is also important to use the appropriate extinction coefficients, determined for the wavelength at which the detection of carotenoids was carried out (see Figure 5b), as indicated above; the differences are often significant (p < 0.05).

Conclusions
The sediments from the Gulf of Gdańsk contained a variety of carotenoids, typical of both marine and freshwater organisms, and their derivatives.A Lichrospher 100 RP-18e column temperature of 5 °C was selected for the parent pigments, which are markers of the phytoplankton groups abundant in the Baltic.Resolution at this temperature was better than at the other temperatures studied.Liquidliquid extraction from an acetone extract to benzene during sample preparation concentrates the analytes, which increases the method's sensitivity.
The pigments in the sediment extract were quantified using a method based on spectrophotometric measurements and extinction coefficients.It is very important to use the appropriate extinction coefficients values, i.e. determined for the wavelength at which the detection of carotenoids was carried out.

Pigment extraction
The carotenoids from a sediment sample were extracted according to the procedure used for chloropigments by the Marine Pollution Laboratory -Institute of Oceanology, Polish Academy of Sciences (MPL-IO). 34A frozen sediment sample (~3 g) was placed in a glass centrifuge tube and left in the dark to thaw (~5 min).The water was removed by centrifugation (6 min, 2500 rpm).The sample was flushed with 15 ml acetone, mixed, sonicated (2 -3 min), centrifuged again and the extract decanted.The extraction was repeated until the supernatant was colourless (max.3 times).The acetone extracts were transferred to a separating funnel for liquid-liquid extraction in an acetone extract:benzene:water system (15:1:10 v/v/v).The benzene layer was transferred to a glass vial, evaporated to dryness in a stream of argon and kept frozen (-20°C) until HPLC analysis.The extracted sediment was dried at 60°C and weighed.The pigment content was calculated per dry sediment weight.
To assess the influence of liquid-liquid extraction on carotenoid analysis, the following experiment was performed (in triplicate).The acetone extract of the sediment sample (30 ml) was divided into two portions.One (15 ml) was extracted in an acetone extract:benzene:water system (15:1:10 v/v/v), then evaporated and dissolved in a smaller volume of acetone (1 ml) and analysed using HPLC, whereas the other (15 ml) was analysed without the re-extraction step.The carotenoid concentrations of both subsamples were determined and the results compared (Fig. 3).The separations were carried out according to the procedure described in the HPLC analysis section (Lichrospher 100RP-18e column, column temperature 5°C).

HPLC analysis 4.3.1. HPLC set, mobile phase and detection
A sediment sample, previously evaporated to dryness, was dissolved in acetone and injected (50 µl) into the column through a guard column.The HPLC system (Knauer, Germany) consisted of an autosampler (Optimas), three pumps (Smartline 100), a degasser (Smartline), a column thermostat (Smartline) and a detector (DAD K-2800).
The column was a Lichrospher 100RP-18e (250 x 4 mm, 5 µm, 100 Å; Merck, Germany) with a guard column (Lichrospher 100RP-18e, 4 mm x 4 mm; Merck, Germany); this same system had previously been used for chloropigment analysis by Szymczak-Żyła and Kowalewska. 34The mobile phase and gradient system was a modified version of that used by Chen et al. 5 The gradient programme (1ml/min) began isocratically with mobile phase A (85:15 methanol:0.5 M ammonium acetate, aq.v/v), which was then ramped to 100% B (90:10 acetonitrile:water v/v) for 4 min.and to 25% B and 75% C (100% ethyl acetate) over the next 34 min.The gradient was returned to 100% B during 4 min, and ramped to 100 % A for another 4 min.Finally, the solvent was run isocratically for 4 minutes with 100% A. The whole analysis lasted 50 minutes.During the HPLC analysis, absorption spectra were collected from the 360-700 nm range.

The influence of column temperature
The carotenoid separations obtained for four different column temperatures (5 °C, 10 °C, 15 °C and 25 °C) for the mixture of pigment standards and sediment extracts were compared (Fig. 4).The analyses were carried out according to the procedure described in the HPLC analysis section (in triplicate; Lichrospher 100RP-18e column).

Pigment identification and quantification
The chromatograms (recorded at 450 nm) were integrated using ClarityChrom Software ver.5.0.2.Individual carotenoids were identified on the basis of retention times and absorbance spectra compared with individual pigment standards.The carotenoid concentrations in a sample were calculated in the same way as those of chloropigments 35  where: c1, c2 -carotenoid concentrations in µg/g and nmol/g, respectively; E450 -extinction measured spectrophotometrically at 450 nm (UV 1800 Shimadzu, Japan); %A450 -% of the pigment peak area in the total area of all integrated peaks of the HPLCDAD chromatogram recorded at 450 nm; v -volume of acetone solution prepared for HPLC analysis (ml); D -dilution factor (dilution of the solution prepared for HPLC, for spectrophotometric measurement);  -extinction coefficient for the carotenoids (ml•mg -1 •cm -1 ); l -optical path length (cm); w -weight of dried sediment (g); Mm -molecular mass.
The extinction coefficients of the carotenoids were calculated from absorption measurements at 450 nm of carotenoid standards using the Lambert-Beer law (Table 3) and were compared with the values gleaned from the literature. 2 The extinction coefficients from the literature and calculated for the individual standards used in this work were used to calculate carotenoid concentrations.The results are presented in Table 3.
This quantification method was compared with the one based on calibration curves.The calibration curves (peak area as a function of concentration) were determined for particular carotenoids: peridinin, fucoxanthin, diadinoxanthin, diatoxanthin, lutein, zeaxanthin, canthaxanthin, echinenone and βcarotene.The calibration curves were prepared for five different concentrations; the calibration curve for β-carotene is exemplified in Fig. 6.

Statistical analysis
The results were statistically processed using STATISTICA 12.5 software (StatSoft, Poland): Student's t-test and one-way analysis of variance (ANOVA) were used.

Table . 1
. Methods of carotenoid extraction from sediments

Table . 2
. HPLC methods for the determination of carotenoids in sediment extracts

Table 3 .
Absorption maxima, extinction coefficients and molecular masses of individual parent carotenoids 2 calculated from absorption measurements of carotenoid standards at 450 nm using the Lambert-Beer law (this work) ** according to literature data2