Quantitation of chiral amino acids from microalgae by micellar electrokinetic. chromatography and laser induced fluorescence detection

Transcription

1 Quantitation of chiral amino acids from microalgae by micellar electrokinetic chromatography and laser induced fluorescence detection Miguel Herrero 1, Elena Ibáñez 1, Salvatore Fanali 2, Alejandro Cifuentes 1* 1 Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, Madrid, Spain. 2 Institute of Chemical Methodologies (CNR), Area della Ricerca di Roma 1, Via Salaria Km 29, Monterotondo Scalo, Roma, Italy. Running Title: Chiral analysis of microalgae amino acids by MEKC-LIF Corresponding author: Dr. A. Cifuentes, acifuentes@ifi.csic.es; Fax: ; Tel: Keywords: algae, capillary electrophoresis, CE, chiral, enantiomers, food analysis, LIF, MEKC. Abbreviations: D-aa, D-amino acid; L-aa, L-amino acid; OPA, o-phthaldialdehyde; PLE, pressurized liquid extraction; TCA, trichloroacetic acid 1

2 ABSTRACT In this work, a chiral and a non-chiral micellar electrokinetic chromatography with laser-induced fluorescence detection (MEKC-LIF) have been combined to identify and quantify a group of D- and L-amino acids (D/L-aa) in different microalgae samples. The combination of the non-chiral and chiral-mekc-lif methods made easier the identification of the microalgae amino acids, previously derivatized with fluorescein isothiocianate, providing a double proof on the correct detection of these analytes. Three microalgae species, Spirulina platensis, Dunaliella salina and Tetraselmis suecica, were compared in terms of their content in D-Arg, L-Arg, D-Lys, L-Lys, D-Ala, L-Ala, D-Glu, L-Glu, D-Asp and L-Asp. Also, a comparison between two Spirulina platensis samples dried under different conditions (i.e., hot-air or lyophilized) was carried out in order to investigate the effect of the thermal processing on the D/L-aa content. Moreover, two procedures for extraction of amino acids from microalgae (i.e., a classical procedure and pressurized liquid extraction) together with diffferent conditions for amino acids derivatization were studied in order to increase the sensitivity of the whole analytical method. By using the selected chiral-mekc-lif conditions (100 mm sodium tetraborate, 30 mm SDS and 20 mm β- CD at ph 9.7) the main microalgae D/L-aa are separated in less than 25 min with efficiencies up to 840,000 plates/m and good sensitivity (i.e., 330 ng of D-Arg per gram of microalga could be detected by this procedure for a signal to noise ratio of three). Several D-aa were detected in all the microalgae, observing interesting differences in their D/L-aa profiles, what corroborates the usefulness of the chiral-mekc-lif approach to characterize different microalgae species as well as different microalgae drying processes. Moreover, it seems from the chiral-mekc-lif results that pressurized liquid extraction can selectively extract different free amino acids from microalgae. 2

3 1. INTRODUCTION In the last decade, microalgae have become a very interesting natural source for extensive screening of novel compounds with interesting biological activities which may lead to therapeutically useful agents [1,2] or new food ingredients with functional activities [3-7]. In this regard, the development of new analytical procedures able to provide a more systematic chemical characterization of the compounds found in these microalgae and/or in the extracts obtained from them is highly desired. Analysis of chiral molecules is a very useful tool in biological, pharmaceutical, clinical, foods or environmental fields [8]. In this sense, chiral analysis of amino acids is a remarkable methodology that can provide important information allowing a better understanding on the chemistry, nutrition, safety, microbiology, pharmacology, etc. of the organisms in which these molecules are found [9]. For instance, D-Asp and D-Ala seem to be respectively involved in neural transmission in many marine invertebrates [10,11] and osmoregulation [12,13], while D-aa seem to be involved in aging and disease in humans [14,15]. On the other hand, it has been demonstrated that the profile of D/L-aa can be an useful tool for specific characterization of different samples allowing e.g., the detection of food adulteration [9,16-18] or digestibility and nutritional quality of foods [9]. Up to now, HPLC and GC have been the techniques of choice to carry out this type of enantiomeric separations [19-23], providing in some cases unequivocal results. However, these techniques may exhibit some drawbacks because use expensive chiral-columns, the procedures for sample preparation are frequently laborious and time consuming and separations are lengthy 3

4 (usually about 50 min) [21,22]. Also, in some GC methods the derivatizing procedure does not work for some basic amino acids such as arginine [21,22]. To our knowledge, only one work has studied the content of D-aa in microalgae [19]. Namely, in tant work the effect of D-aa content on the growth of Botrydiopsis alpina, Chlorella pyrenoidosa, Chlorella vulgaris, Scenedesmus obliquus, Thalassiosira sp., Nitzschia navis-varingica and Pseudo-nitzschia pungens microalgae was studied by HPLC [19]. However, the HPLC method used only allowed the separation and quantitative determination of D-Asp and D-Ala, requiring very long analysis times (up to 75 min). Similar HPLC method was used later by the same group to study alanine racemase activity in the marine diatom Thalassiosira sp. [20]. Considering these drawbacks and taking into account the economic impact derived from the chiral analysis in many different areas, new analytical procedures able to overcome these limitations could be very useful. In this regard, the remarkable possibilities of capillary electrophoresis (CE) for the separation of chiral compounds were already demonstrated by Gasmman et al. in 1985 [24], and since then this technique has been applied to multiple chiral separations [16,25-27]. However, one of the main limitations of CE is the low sensitivity that the common detectors based on UV absorbance provide, mainly due to the narrow optical pathlength used as detection cell (i.e., the capillary internal diameter). Although some other strategies such as chiral-ce-ms have been proposed [28], the use of laser-induced fluorescence (LIF) together with derivatizing reagents (e.g., o-phthaldialdehyde, OPA, or fluorescein isothiocyanate, FITC) seems to be one of the best alternatives to overcome the sensitivity limitation of CE [29-32]. FITC has frequently been chosen as fluorescent label for amino acids since its excitation wavelength matches the 488 nm light of the argon laser, the derivatives are easily formed, have good electrophoretic properties and generate strong fluorescence signals [29]. In spite of these 4

5 advantages, few papers have been devoted to the chiral separation of FITC-amino acids by CE [17,18,33-35] and, to our knowledge, no MEKC-LIF method has been developed so far to separate and detect the main L- and D-amino acids usually found in microalgae. The goal of this work is to demonstrate the possibilities of MEKC-LIF for the easy, sensitive and fast determination of the main chiral amino acids in different natural microalgae, microalgae extracts and to study the effect of different microalgae processing on their D/L-aa content. 2. MATERIALS AND METHODS 2.1 Chemicals All chemicals were of analytical reagent grade and used as received. β-cyclodextrin (β-cd) from Fluka (Buchs, Switzerland) was used as chiral selector together with sodium dodecyl sulphate (SDS) from Acros Organics (New Jersey, U.S.A.) and boric acid from Riedel-De Häen (Seelze, Germany) for the MEKC running buffer. A water solution containing 5 M sodium hydroxide from Panreac Quimica S.A. (Barcelona, Spain) was used to adjust the ph of the buffers and 0.1 M NaOH was used to rinse the capillary. The buffer was stored al 4º C and warmed at room temperature before use. Distilled water was deionised by using a Milli-Q system (Millipore, Bedford, MA). Fluorescein isothiocyanate (FITC, from Fluka, Buchs Switzerland) was dissolved in acetone analytical grade (Merck, Darmstadt, Germany). Standard L- and D-amino acids were from Sigma (St. Louis MO, USA). Trichloroacetic acid (TCA, from Merck, Darmstadt, Germany) as well as sodium deoxycholate (minimum 97%, Sigma, Madrid, Spain) were used for amino acid extraction. 5

6 2.2 Samples Five different samples from three microalgae species were studied in this work (see Table 1). Air-dried Spirulina platensis was obtained from Algamar S.A. (Pontevedra, Spain) and used for Samples I and III, while lyophilized Spirulina platensis (Sample II) was a kind gift from Dr. Guillermo Garcia-Reina (University of Las Palmas de Gran Canaria, Spain). Dunaliella salina microalga sample (Sample IV) was a kind gift from NBT (Jerusalem, Israel) and Tetraselmis suecica (Sample V) was a kind gift from Dr. Olivia Arredondo (CIBNOR, La Paz, Mexico). Both, Dunaliella salina and Tetraselmis suecica samples consisted on lyophilized microalgae. Sample III was obtained using a pressurized liquid extraction (PLE) procedure as described elsewhere [4]. Briefly, an accelerated solvent extractor ASE 200 (Dionex Corp. Sunnyvale, CA, USA) equipped with a solvent controller was employed. The PLE extraction was performed with ethanol as extraction solvent at 115ºC and 15 minutes at 1500 psi, so that, the solvent was maintained in the liquid state during the whole extraction process. The extractions were performed in 11 ml extraction cells, containing 2.5 g of microalgae. Once the extraction was finished, the extract was immediately cooled with an ice-water bath and the solvent evaporated using a Rotavapor R-200 (Büchi Labortechnik AG, Flawil, Switzerland). Afterwards, the dried extract was re-dissolved in ethanol to a known concentration. For the extraction of free amino acids from samples I, II, IV and V, the microalgae were treated as follows [36]: one ml of 0.37 M TCA was added to mg of dried alga. The mixture was vortexed for 1.5 minutes. Then, 0.2 ml of 3.6 mm sodium deoxycholate was added to make possible the protein precipitation. The mixture was left to stand for 10 min and then, a 15 minutes centrifugation (3000 x g) was carried out. The supernatant was separated and submitted 6

7 to another 1 h centrifugation process (4500 x g). Again, the supernatant was collected and employed for the derivatization procedure. 2.3 Standard solutions. Quantitative values were assessed by injecting (in triplicate) five different concentrations of the standard solutions containing L- and D-amino acids. The calibration curves were calculated plotting corrected peak area (i.e., area/time) versus amino acid concentration (expressed as μg/ml) and the results are given in Table Derivatization procedure. The FITC derivatization procedure was optimized as described below. The selected procedure consisted of mixing an aliquot of 625 μl of the sample (from I to V, see Table I) with 1100 μl of water and 3 ml of 355 mm sodium tetraborate buffer at ph 10. This mixture was adjusted to ph 10 by adding 1 M sodium hydroxide. Water was added till a final volume equal to 5 ml. For amino acid standards derivatization, a 625 μl aliquot was mixed with 1375 μl of water and 7 ml of 355 mm sodium tetraborate buffer at ph 10. Again, this mixture was adjusted to ph 10 by adding 1 M sodium hydroxide and water was added till a final volume equal to 10 ml. 200 μl of these final solutions were mixed with 100 μl of a 3.75 mm FITC solution in acetone. The reaction took place overnight in darkness at room temperature. After derivatization, samples were diluted with water prior to their injection in the MEKC-LIF. 2.5 MEKC-LIF conditions 7

8 Analysis of L- and D-amino acids was carried out in triplicate using a P/ACE 2100 CE apparatus from Beckman Instruments (Fullerton, CA, USA) equipped with an Ar + laser at 488 nm (excitation wavelength) and 520 nm (emission wavelength) also from Beckman Instruments to detect FITC-amino acids. Bare fused-silica capillary was purchased from Composite Metal Services (Worcester, England). The capillary dimensions were 50 cm to the detection window, 57 cm total length and 50 μm i.d. and was thermostated at 30.0 ºC. Injections were made at the anodic end using N 2 pressure of 0.5 p.s.i. (3.45 kpa) for 3 s and the applied voltage was 20 kv. The P/ACE 2100 CE instrument was controlled by a PC running the System GOLD software from Beckman. Before first use, new capillaries were preconditioned by rinsing with 0.1 M NaOH for 30 min. The washing protocol between runs was optimized to obtain adequate reproducibility, selecting the following conditions: at the beginning of each run, the capillary was rinsed with 0.1 M NaOH for 1 min, followed by 2 min with Milli-Q water, and then equilibrated with the running buffer (100 mm sodium tetraborate, 30 mm SDS and 20 mm β-cd at ph 9.7) for 5 min. At the end of the day, the capillary was rinsed with Milli-Q water for 10 min and then nitrogen was passed for 2 min. 3. RESULTS AND DISCUSSION A group of five chiral amino acids (Arg, Lys, Ala, Glu and Asp) was selected to carry out this study. This selection was based on the fact that these five amino acids seem to be the majority in other microalgae (namely, Botrydiopsis alpina, Chlorella pyrenoidosa, Chlorella vulgaris, Scenedesmus obliquus, Asterionella sp., Nitzschia navis-varingica, Pseudo-nitzschia pungens and Thalassiosira sp.) as demonstrated by Yokoyama et al. using HPLC [19]. Moreover, it can 8

9 be expected that the chiral-mekc-lif profiles obtained by using the ten enantiomers proposed in this work can provide enough information to characterize the microalgae investigated. 3.1 Method development and figures of merit. Previously to assess the suitability of the chiral-mekc-lif methodology proposed, the derivatization procedure was investigated in order to achieve the maximum amino acid signal with the lowest interferences from FITC. It had to be done due to the use of FITC as probe produces a high number of interfering fluorescent compounds in the sample. To do so, different volumes of sample and FITC solutions were used to carry out the derivatization reaction, testing five different combinations as indicated in Table 3. The reaction products were analyzed by CE- LIF and the reaction mixture that gave the higher amino acid signals (S AA ) together with the lower FITC signal (S FITC ) was selected. As can be observed in Table 3, the higher the proportion of sample in the mixture, the higher the S AA /S FITC ratio. Thus, the final selected reaction mixture consisted on 200 μl of sample and 100 μl of FITC solution. As mentioned before, to our knowledge, no chiral-mekc-lif analysis has been reported to analyze chiral amino acids from microalgae. Considering the complexity of these natural microorganisms and, consequently, the complex electrophoretic profiles expected from them a double MEKC-LIF approach was proposed. Namely, both a non-chiral-mekc-lif method and a chiral-mekc-lif method were applied considering that in this way a more complete information on the composition of these real samples should be obtained. In order to find suitable non-chiral-mekc-lif separation conditions for the selected group of amino acids (i.e., Arg, Lys, Ala, Glu, Asp), similar MEKC conditions to those developed for 9

10 vinegar amino acids were tested [37], but in this case without chiral selector (i.e., the running buffer tested was 100 mm sodium tetraborate, 30 mm SDS at ph 9.7). To study these initial MEKC conditions, a standard mixture of the five amino acids was derivatized with FITC under the reaction conditions mentioned above and injected. The results are given in Figure 1A, showing that under these conditions both adequate signals and resolutions are achieved for the five amino acids in less than 25 min. Moreover, as can be seen in Figure 1A it was possible to separate all the amino acids peaks from the several interfering FITC peaks (marked with an asterisk) coming from the derivatization reagent. The peak identification was carried out by injecting each amino acid or FITC separately. To get adequate chiral-mekc-lif conditions, different concentrations of β-cd as chiral selector were added to the running buffer observing that the best chiral resolution for the five L/D amino acids was obtained by using 20 mm β-cd. Therefore, the following enantioselective buffer was selected: 100 mm sodium tetraborate, 30 mm SDS, 20 mm β-cd at ph 9.7. As it can be observed in Figure 1B, the separation of the ten chiral amino acids can be performed under these conditions in less than 25 min. Interestingly, besides the peaks corresponding to the D/L-amino acids, two more peaks per amino acid appeared marked as 1 to 5 in Figure 1B. By injecting each amino acid separately, it was possible to conclude that these peaks correspond to impurities (either, from the original standards or from the derivatization reaction). Namely, the peaks marked as 1, 2, 3, 4 and 5 correspond to impurities from Arg, Lys, Ala, Glu and Asp, respectively. Moreover, as can be deduced from the double-peak obtained for each impurity (each couple coming out with the same height), it can be hypothesized that these compounds also correspond to enantiomers that are separated by this chiral-mekc-lif procedure. 10

11 Once the ability of the non-chiral and chiral-mekc-lif methods to separate the selected amino acids was assessed, a microalga sample (Sample I from Table 1) was analyzed using the same methods to confirm if the separation conditions were also suitable for these real samples or if, on the contrary, other compounds, including other amino acids, were interfering. As mentioned, the real sample was analyzed in a first step using the non-chiral conditions (Figure 2A) and then by the chiral-mekc-lif method (Figure 2B, note that the y-axis is different in Figure 2A and B). As can be observed in Figure 2A, the five amino acids were adequately separated and easily identified in this microalga sample thanks to the less complex electrophoretic profile obtained using the non-chiral-mekc-lif method. Besides, as it can be observed in the Figure 2B, the adequate separation of the ten amino acids was also possible under these conditions for this real sample. Moreover, the combined application of the non-chiral and chiral-mekc-lif methods provided, apart of the required information on the D/L-aa composition, a double proof on the correct identification of the investigated chiral amino acids. Namely, characterization and identification of each amino acid was carried out in all real samples by adding increasing amounts of D/L standard amino acids to the derivatized sample and running their subsequent non-chiral or chiral-mekc-lif analysis as shown in Figure 3. Interestingly, as can also be seen in Figures 2A, 2B and 2C, new peaks were detected in the real sample electrophoregram, mainly in the area from 12 to 17 min as it is shown in the enlarged area shown by Figure 2C, that did not correspond to any of the investigated standard compounds, and possibly corresponding to other amino acids present in the sample. In order to demonstrate this point, standards of other different amino acids were coinjected with the sample, observing under the non-chiral conditions of Figure 2A that Met, Pro, Phe and His matched some of these peaks. However, it was not possible to completely identify their enantiomers 11

12 using the enantioselective conditions of Figure 2B since frequent peak overlapping had place among the peaks migrating between 12.5 and 17 min under these conditions. Since the ten chiral amino acids selected for this study were correctly separated by the proposed chiral-mekc-lif method a characterization of its main figures of merit was carried out. Namely, using these chiral-mekc-lif conditions, the D/L-amino acids were separated in less than 25 minutes as can be seen in Figures 1 and 2 with efficiencies ranging from 370,000 plates/m for L-Asp to 720,000 plates/m for D-Arg for the standards. Also, the limits of detection (LOD), calculated considering a signal/noise ratio equal to three, were ranging from 30.8 nm for L-Lys to 8.9 nm for L-Arg. This method also provided high efficiencies and good sensitivities when it was applied to microalgae samples. Namely, efficiencies up to 840,000 plates/m were obtained for L-Arg, while the LOD values were as low as 330 ng of D-Arg per gram of microalga. Given the good figures of merit in terms of analysis speed, efficiency and sensitivity of this chiral-mekc-lif procedure for standards and real samples, the same separation conditions were maintained to continue with the quantitation of the chiral amino acids. 3.2 Quantitation of D/L-amino acids in different microalgae samples. Figure 4 shows the chiral-mekc-lif electropherograms obtained from the five microalgae samples studied in this work (see Table 1). Their amino acids identification was done as described above (i.e., analysing each sample under the chiral and non-chiral conditions). From a first sight, it could be concluded from Figure 4 that the most abundant chiral amino acid in Spirulina platensis samples (I and II) is L-Glu, while L-Ala is the most abundant in the Dunaliella salina and Tetraselmis suecica samples (IV and V). Interestingly, it can also be observed that by means of the pressurized liquid extraction (PLE) with ethanol from Spìrulina 12

13 platensis (Figure 4.III), a different profile is obtained compared with the most classical extraction of free amino acids from the same sample (Figure 4.I). In order to properly compare the L- and D-amino acids contents in these different microalgae samples, quantitation of the L- and D-amino acid contents was carried out by using the chiral- MEKC-LIF method. To do that, calibration curves were obtained for the ten chiral amino acids considered in this work injecting in triplicate five different concentrations in the range indicated in Table 2. In general, as can be seen in Table 2, good linearity coefficients were obtained for all the amino acids with r 2 values up to for L-Asp, L-Glu, L- and D-Lys. Moreover, as can also be observed in Table 2, this method provided good reproducibility values for both the corrected peak area (with RSD values up to 8.4% for L-Ala) and the analysis time (with RSD values up to 1.6% for L-Glu). All the microalgae samples were then injected (also in triplicate) and their D/L-amino acids contents determined using the calibration curves given in Table 2. The amounts determined for each amino acid in these real samples are given in Table 4. From the analysis of the quantitative results given in Table 4, some conclusions can be reached: the presence of D- and L-amino acids was detected in all the samples studied. Moreover, the relative amount (and presence) of the ten chiral amino acids was clearly different for all the samples. Thus, the comparison of the four different microalgae samples submitted to the same drying process and free amino acids extraction process (II, IV and V) showed a significant different pattern which can be useful to characterize different microalgae species. While in the Spirulina platensis samples (II) the most abundant amino acid is L-Glu, in Dunaliella salina (Sample IV) and Tetraselmis suecica (Sample V) the most abundant was L-Ala, corroborating the results mentioned above. Taking into account only the Spirulina platensis samples, comparing the results from Sample I and II it 13

14 could tentatively be infered some influence from the microalga drying process on its D/L-amino acids profile, as can be deduced comparing the results from Sample I (dried using hot air) and Sample II (lyophilized). Thus, the amount of both L- and D- free amino acid is clearly higher in the Spirulina platensis sample obtained using hot air (Sample I) than in that obtained using a lyophilization process (Sample II). Freeze-drying, also called lyophilization, is well-known for being a soft drying process that can keep the integrity of aqueous samples better than using high temperatures (like e.g., hot-air drying). Besides, the protein content of Spirulina platensis is very high (ca. 70% of its weight) being phycobiliproteins the most abundant group [38]. Interestingly, it has been demonstrated that these phycobiliproteins can easily degradate at temperatures higher than 50ºC [7], what could explain the higher free amino acids content observed in Sample I dried using hot-air. However, in spite of these interesting results some other factors should also be considered. For instance, it is well known the great importance of the growth conditions in the chemical composition of the different microalgae [38]. Since the origin of the samples I and II was not exactly the same, these amino acids content differences could also be due to different growth conditions. Several differences were also observed when two different extraction procedures were applied to the same Spirulina platensis sample to obtain free amino acids. Namely, a classical procedure to extract amino acids (Sample I in Table 1) and a pressurized liquid extraction (PLE) procedure using ethanol (Sample III in Table 1) were compared. The use of PLE provided both a clear decrease of the extraction of negative amino acids (D- and L-Glu and D- and L-Asp) and a general increase for Arg, Lys and Ala, as can be seen in Table 4. This could be explained, among other reasons, by the variation of the constant dielectric of the solvent under the extraction conditions (ethanol at 111ºC and 1500 psi), that could bring about a better matching between the polarity of Arg, Lys and Ala and the polarity of the solvent under the mentioned 14

15 PLE conditions, giving as a result a higher extraction of these compounds. This effect could be used to selectively enrich (or reduce) the PLE extracts with determined compounds obtained from these natural sources. Regarding the content in D-amino acids, their presence was confirmed in all the samples as can be seen in Table 4. In this sense, it is interesting to corroborate the good reproducibility also observed in general for these real samples, as can be deduced from the %RSD values given in Table 4. Moreover, the proportion of D-amino acid (given as %D in Table 4) was different for the five samples what seems to corroborate the usefulness of this chiral-mekc-lif procedure to characterize different microalgae species and microalgae processing. Thus, %D values were clearly different depending on the microalga species as can be deduced comparing these values for the three different microalgae studied in this work (namely, Spirulina platensis, Dunaliella salina and Tetraselmis suecica, corresponding respectively to Samples II, IV and V in Table 4). The same can be applied to the %D values obtained for the two different microalga processing, hot-air vs. lyophilization, corresponding respectively to Sample I and II in Table 4. Moreover, the %D values of Table 4 also corroborate the aforementioned selectivety brought about by the PLE extraction regarding the removal of the most negative amino acids (Glu and Asp) as can be deduced comparing their %D values from Sample III with those obtained using the classical extraction process (Sample I). CONCLUDING REMARKS In this work, the good possibilities of a new approach combining a non-chiral together with a chiral-mekc-lif method have been demonstrated to identify D/L amino acids from different microalgae samples. Quantitation of the chiral amino acids is carried out using the 15

16 enantioselective MEKC-LIF method, which provides the separation of 10 D/L-amino acids in less than 25 min with efficiencies up to 840,000 plates/m and LODs down to 8.9 nm for standards and 330 ng of free amino acid per gram of sample. Results on D/L-amino acids contents from the different samples demonstrate that this method can be an interesting tool able to differentiate microalgae species (Spirulina platensis, Dunaliella salina and Tetraselmis suecica) as well as microalgae processing (lyophilization vs. hot-air drying). Moreover, the method also shows the possibilities of applying pressurized liquid extraction to selectively extract microalga free amino acids compared with a more classical procedure. Acknowledgements M.H. would like to thank Ministerio de Educación y Ciencia for a FPI grant. This study has been supported by a CSIC-CNR Project (2004IT0037). Authors are also grateful to the AGL C02-01 and AGL C04-02 Projects (Ministerio de Educacion y Ciencia) and the S-505/AGR-0153 Project (Comunidad Autonoma de Madrid, CAM) for financial support of this work. 16

17 REFERENCES [1] Baker, J.T., Hydrobiologia 1984, 116, [2] Moore R.E., Patterson, M.L., Carmichael, W.W., in: Fautin D.G. (ed.), Biomedical importance of marine organisms. California Academy of Sciences, San Francisco, CA, USA [3] Herrero, M., Ibáñez, E., Señoráns, J., Cifuentes, A., J. Chromatogr. A 2004, 1047, [4] Herrero, M., Martín-Álvarez, P.J., Señoráns, F.J., Cifuentes, A., Ibáñez, E., Food Chem. 2005, 93, [5] Herrero, M., Ibáñez, E., Cifuentes, A., J. Sep. Sci. 2005, 28, [6] Simó, C., Herrero, M., Neusüß, C., Pelzing, M., Kenndler, E., Barbas, C., Ibáñez, E., Cifuentes, A., Electrophoresis 2005, 26, [7] Herrero, M., Simó, C., Ibáñez, E., Cifuentes, A., Electrophoresis 2005, 26, [8] Armstrong, D. W., Chang, C. D., Li, W. L., J. Agric. Food Chem. 1990, 38, [9] Friedman, M., J. Agric. Food Chem. 1999, 47, [10] D Aniello, A., Giuditta, A., J. Neurochem. 1977, 29, [11] D Aniello, A., Giuditta, A., J. Neurochem. 1978, 31, [12] Matsushima, O., Katayama, H., Yamada, K., Kado, Y., Mar. Biol. Lett. 1984, 5, [13] Preston, R.L., Comp. Biochem. Physiol. 1978, 87B, [14] Helfman, P.M., Bada, J.L., Zigler, J.L., Nature 1977, 268, [15] Mann, E.H., Sandhouse, M.E., Burg, J., Fisher, G.H., Science 1983, 220, [16] Simó, C., Barbas, C., Cifuentes, A., Electrophoresis 2003, 24, [17] Simó, C., Barbas, C., Cifuentes, A., J. Agric. Food Chem. 2002, 50, [18] Simó, C., Martín-Álvarez, P. J., Barbas, C., Cifuentes, A., Electrophoresis 2004, 25,

18 [19] Yokoyama, T., Kan-No, N., Ogata, T., Kotaki, Y., Sato, M., Nagahisa, E., Biosci. Bitechnol. Biochem. 2003, 67, [20] Yokoyama, T., Tanaka, Y., Sato, M., Kan-No, N., Nakano, T., Yamaguchi, T., Nagahisa, E., Fisheries Sci. 2005, 71, [21] Erbe, T., Brückner, H., Z. Lebensm. Unters Forsch A. 1998, 207, [22] Erbe, T., Brückner, H., Eur. Food Res. Technol. 2000, 211, [23] Bruckner, H., Langer, M., Lupke, M., Westhauser, T., Godel, H., J. Chromatogr. A 1995, 697, [24] Gassman, E., Kuo, J. E., Zare, R. N., Science 1985, 230, [25] Chankvetadze, B., Capillary Electrophoresis in Chiral Analysis, John Willey & Sons, Chichester, England, [26] Hernández-Borges, J., Rodríguez-Delgado, M. A., García-Montelongo, F. J., Cifuentes, A., Electrophoresis 2005, 26, [27] Cifuentes, A., Electrophoresis 2006, 27, [28] Simó, C., Rizzi, A., Barbas, C., Cifuentes, A., Electrophoresis 2005, 26, [29] Cheng, Y. F., Dovichi, N. J., Science 1988, 242, [30] Issaq, H. J., Chan, K. C., Electrophoresis 1995, 16, [31] Wan, H., Blomerg, L. G., J. Chromatogr. A 2000, 875, [32] Engstroem, A., Andersson, P. E., Jossefsson, B., Pfeffer, D., Anal. Chem. 1995, 67, [33] Nouadge, G., Nertz, M., Couderc, F., J. Chromatogr. A 1995, 716, [34] Pérez-Ruiz, T., Martínez-Lozano, C., Bravo, E., Chromatographia 2000, 52, [35] Jin, L. J., Rodriguez, I., Li, S. F. Y., Electrophoresis 1999, 20, [36] Campanella, L., Russo, M.V., Avino, P., Anali di Chimica 2002, 92, [37] Carlavilla, D., Moreno, M.V., Fanali, S., Cifuentes, A. Electrophoresis 2006, 27,

19 [38] Richmond A., in: Borowitzka, M.A., Borowitzka, L.J., Microalgal biotechnology. Cambridge University Press. Cambridge, UK, 1988, p

20 FIGURE LEGENDS Figure 1. MEKC-LIF electropherograms of a standard mixture of ten D/L-amino acids (A) without and (B) with chiral selector in the running buffer. Sample: Standard mixture of FITC derivatized amino acids injected for 3 s at 0.5 psi. Peaks marked with an asterisk correspond to FITC impurities. Peaks marked as 1, 2, 3, 4 and 5 correspond to impurities from Arg, Lys, Ala, Glu and Asp, respectively. Separation conditions: Running buffer: 100 mm sodium tetraborate, 30 mm SDS at ph 9.7. (B: plus 20 mm ß-cyclodextrin). Capillary: 57 cm total length, 50 cm detection length and 50 μm i.d.. Running voltage: 20 kv. LIF detection: Ar + laser at 488 nm (excitation wavelength) and 520 nm (emission wavelength). Figure 2. MEKC-LIF electropherograms from Spirulina platensis microalga (A) without and (B) with chiral selector in the running buffer. Injected sample: FITC derivatized Sample I injected for 3 s at 0.5 psi. All the other conditions as in Figure 1. Figure 3. Chiral-MEKC-LIF electrophoregrams from Spirulina platensis microalga (A); the same sample injected in A plus 10 µg/ml of D-Asp and 10 µg/ml of D-Glu (B); and the same sample injected in B plus 10 µg/ml of D-Asp and 10 µg/ml of D-Glu (C). All the conditions as in Figure 1B. Figure 4. Chiral-MEKC-LIF electrophoregrams of the D/L-amino acids profile from the five microalgae samples investigated in this work (see Table 1). All the conditions as in Figure 1B. 20

21 Table 1. Description of the five samples studied in this work including the microalga species, microalga drying process and amino acids extraction procedure. Sample Microalga species Microalga drying process Extraction of free amino acids I Spirulina platensis Hot-air Classical extraction a) II Spirulina platensis Lyophilization Classical extraction III Spirulina platensis Hot-air PLE b) IV Dunaliella salina Lyophilization Classical extraction V Tetraselmis suecica Lyophilization Classical extraction a) Extraction performed according to the procedure by Campanella et al. [36]. b) Extraction performed by pressurized liquid extraction (PLE) with ethanol at 111ºC, 1500 psi for 15 minutes. (see Section 2.2 for details). 21

22 Table 2. Linear range and calibration curves for the quantitative determination of the ten D- and L- amino acids used in this work. Concentration (%) RSD n=3 Amino range a) Equation b) Correlation acid coefficient (r 2 ) Area Analysis [μg/ml] time D-Arg y = 94353x L-Arg y = 68932x D-Lys y = 28098x L-Lys y = 29610x D-Ala y = x L-Ala y = 57225x D-Glu y = 50306x L-Glu y = 53099x D-Asp y = 40920x L-Asp y = 47621x a) five different concentrations were used for each calibration curve and each concentration was injected in triplicate. b) y = amino acid corrected peak area; x = amino acid concentration given in μg/ml. 22

23 Table 3. Amounts of sample and FITC solution tested to optimize the derivatization procedure and amino acid signal (S AA )/FITC signal (S FITC ) ratio obtained. Exp. Sample (μl) a) FITC solution (μl) b) S AA /S FITC a) Sample concentration: 2.1 mg/ml. b) FITC concentration: 3.75 mm FITC in acetone. 23

24 Table 4. L- and D-amino acids content (given in μg/g) a) determined in the five different microalgae samples studied in this work. Arg Lys Ala Glu Asp Sample L- (RSD) D- (RSD) % D L- (RSD) D- (RSD) % D L- (RSD) D- (RSD) % D L- (RSD) D- (RSD) % D L- (RSD) D- (RSD) % D I b) (3.1) c) 2.5 b) (9.7) c) 0.98 d) (3.0) (2.6) (3.1) (3.3) (4.0) (6.7) (3.1) (4.1) 68.4 II 67.9 (1.6) n.d. - n.d. n.d (16.1) 89.6 (2.9) (3.2) (5.4) (1.1) 82.3 (9.1) 41.5 III (1.9) 44.5 (2.8) (4.3) n.d (18.1) (6.3) (2.9) n.d. - n.d. n.d. - IV (9.8) (7.2) 20.5 n.d. n.d (8.4) (9.8) (5.4) (9.3) (13.6) 5.6 (5.9) 3.1 V (2.3) 3.6 (10.7) 0.52 n.d. n.d (4.1) 74.9 (5.6) (5.4) (6.7) (6.1) 18.1 (5.8) 14.0 a) μg of D- or L-aa per g of microalgae. When necessary samples were conveniently diluted till achieving a final concentration within the linear range given in Table 2. b) Average values from three replicates given in μg/g. c) Relative standard deviation (% RSD n=3 ). d) Relative percentage of D-aa calculated as 100 x D-aa/(D-aa+L-aa) 24

25 1000 D,L-Arg A 800 D,L-Ala RFU 600 D,L-Lys 400 * D,L-Glu D,L-Asp 200 * Time (min) B D-Ala L-Ala RFU D-Arg L-Arg * D-Glu D-Lys L-Lys 2 * L-Glu D-Asp L-Asp Time (min) Figure 1. 25

26 Figure 2. 26

27 D-Glu D-Asp C 300 RFU 250 B 250 RFU A Time (min) 0 Figure 3. 27

28 I L-Glu L-Lys 400 D-Lys L-Ala 200 D-Ala D-Asp L-Arg D-Glu L-Asp D-Arg L-Lys L-Arg D-Arg III L-Ala D-Ala L-Glu II L-Ala L-Glu D-Glu L-Glu - D-Arg L-Arg D-Ala D-Asp L-Asp D-Glu L-Ala D-Ala L-Arg D-Arg IV L-Glu D-Asp D-Glu L-Asp L-Ala V L-Arg D-Ala L-Glu D-Arg D-Glu D-Asp L-Asp Figure 4 28