1

Quantitation of chiral amino acids from microalgae by micellar electrokinetic

chromatography and laser induced fluorescence detection

Miguel Herrero1, Elena Ibáñez1, Salvatore Fanali2, Alejandro Cifuentes1*

1 Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain.

2 Institute of Chemical Methodologies (CNR), Area della Ricerca di Roma 1, Via Salaria Km

29,300 - 00016 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: +34 91 5644853; Tel:

+34 91 561 88 06

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

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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.

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

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 path-

length 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

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.

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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 0.150 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

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 2.

2.4 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

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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 N2 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

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 (SAA) together with the

lower FITC signal (SFITC) was selected. As can be observed in Table 3, the higher the proportion

of sample in the mixture, the higher the SAA/SFITC 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

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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.

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

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

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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 r2 values up to 0.999 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

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

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

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

AGL2005-05320-C02-01 and AGL2005-06726-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.

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[20] Yokoyama, T., Tanaka, Y., Sato, M., Kan-No, N., Nakano, T., Yamaguchi, T., Nagahisa, E.,

Fisheries Sci. 2005, 71, 924–930.

[21] Erbe, T., Brückner, H., Z. Lebensm. Unters Forsch A. 1998, 207, 400-409.

[22] Erbe, T., Brückner, H., Eur. Food Res. Technol. 2000, 211, 6-12.

[23] Bruckner, H., Langer, M., Lupke, M., Westhauser, T., Godel, H., J. Chromatogr. A 1995,

697, 229-245.

[24] Gassman, E., Kuo, J. E., Zare, R. N., Science 1985, 230, 813-814.

[25] Chankvetadze, B., Capillary Electrophoresis in Chiral Analysis, John Willey & Sons,

Chichester, England, 1997.

[26] Hernández-Borges, J., Rodríguez-Delgado, M. A., García-Montelongo, F. J., Cifuentes, A.,

Electrophoresis 2005, 26, 3799-3813.

[27] Cifuentes, A., Electrophoresis 2006, 27, 283-303.

[28] Simó, C., Rizzi, A., Barbas, C., Cifuentes, A., Electrophoresis 2005, 26, 1432-1441.

[29] Cheng, Y. F., Dovichi, N. J., Science 1988, 242, 562-564.

[30] Issaq, H. J., Chan, K. C., Electrophoresis 1995, 16, 467-481.

[31] Wan, H., Blomerg, L. G., J. Chromatogr. A 2000, 875, 43-88.

[32] Engstroem, A., Andersson, P. E., Jossefsson, B., Pfeffer, D., Anal. Chem. 1995, 67, 3018-

3022.

[33] Nouadge, G., Nertz, M., Couderc, F., J. Chromatogr. A 1995, 716, 331-334.

[34] Pérez-Ruiz, T., Martínez-Lozano, C., Bravo, E., Chromatographia 2000, 52, 599-602.

[35] Jin, L. J., Rodriguez, I., Li, S. F. Y., Electrophoresis 1999, 20, 1538-1545.

[36] Campanella, L., Russo, M.V., Avino, P., Anali di Chimica 2002, 92, 343-352.

[37] Carlavilla, D., Moreno, M.V., Fanali, S., Cifuentes, A. Electrophoresis 2006, 27, 2551-2557.

19

[38] Richmond A., in: Borowitzka, M.A., Borowitzka, L.J., Microalgal biotechnology. Cambridge

University Press. Cambridge, UK, 1988, p.85-121.

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.

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).

22

Table 2. Linear range and calibration curves for the quantitative determination of the ten D- and

L- amino acids used in this work.

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.

(%) RSDn=3 Amino

acid

Concentration rangea) [μg/ml]

Equationb) Correlation coefficient (r2) Area Analysis

time D-Arg 2.5 – 0.313 y = 94353x + 640 0.998 3.3 0.3

L-Arg 5.0 - 0.0625 y = 68932x – 4085 0.995 3.0 0.7

D-Lys 2.5 – 0.0625 y = 28098x – 731 0.999 2.4 0.3

L-Lys 2.5 – 0.0625 y = 29610x – 1191 0.999 1.7 0.6

D-Ala 3.33 – 0.0417 y = 110417x – 3539 0.995 6.9 0.8

L-Ala 3.33 – 0.083 y = 57225x – 3710 0.997 8.4 0.6

D-Glu 4.167 – 0.417 y = 50306x – 4191 0.993 6.8 1.2

L-Glu 4.167 – 0.167 y = 53099x – 925 0.999 8.2 1.6

D-Asp 2.5 – 0.0625 y = 40920x + 213 0.995 5.4 0.8

L-Asp 2.5 – 0.0625 y = 47621x - 2183 0.999 7.3 1.2

23

Table 3. Amounts of sample and FITC solution tested to optimize the derivatization procedure

and amino acid signal (SAA )/FITC signal (S FITC) ratio obtained.

Exp. Sample (μl)a) FITC solution (μl)b) SAA/S FITC 1 100 200 0.065 2 150 200 0.115 3 200 200 0.159 4 200 150 0.194 5 200 100 0.286

a) Sample concentration: 2.1 mg/ml.

b) FITC concentration: 3.75 mM FITC in acetone.

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.

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 (% RSDn=3).

d) Relative percentage of D-aa calculated as 100 x D-aa/(D-aa+L-aa)

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 250.3b)

(3.1)c) 2.5b)

(9.7)c) 0.98d) 119.8

(3.0) 217.3 (2.6)

64.4 693.6 (3.1)

188.6 (3.3)

21.4 3361.9 (4.0)

210.0 (6.7)

5.9 102.3 (3.1)

221.5 (4.1)

68.4

II 67.9 (1.6)

n.d. - n.d. n.d. - 275.8 (16.1)

89.6 (2.9)

24.7 4840.6 (3.2)

182.2 (5.4)

3.6

110.2 (1.1)

82.3 (9.1)

41.5

III 823.3 (1.9)

44.5 (2.8)

5.1 1370.7 (4.3)

n.d. - 3337.2 (18.1)

1189.2 (6.3)

26.3 597.5 (2.9)

n.d. - n.d. n.d. -

IV 518.8 (9.8)

133.5 (7.2)

20.5 n.d. n.d. - 1602.1 (8.4)

179.8 (9.8)

10.1 714.0 (5.4)

261.4 (9.3)

26.8 176.7 (13.6)

5.6 (5.9)

3.1

V 697.4 (2.3)

3.6 (10.7)

0.52 n.d. n.d. - 3419.8 (4.1)

74.9 (5.6)

2.1 721.6 (5.4)

145.2 (6.7)

16.8 111.1 (6.1)

18.1 (5.8)

14.0

25

A

D,L-Lys

D,L-Arg

D,L-Ala

D,L-GluD,L-Asp

10 15 200

200

400

600

800

1000

RFU

Time (min)

*

*

10 15 20

0

50

100

150

200

250

300

RFU

Time (min)

B

D-Arg

43 5

L-Ala

*L-Arg

D-Ala

*

D-GluL-Glu

D-AspL-Asp

L-Lys

D-Lys

1

2

A

D,L-Lys

D,L-Arg

D,L-Ala

D,L-GluD,L-Asp

10 15 200

200

400

600

800

1000

RFU

Time (min)

*

*

A

D,L-Lys

D,L-Arg

D,L-Ala

D,L-GluD,L-Asp

10 15 200

200

400

600

800

1000

RFU

Time (min)

*

*

10 15 20

0

50

100

150

200

250

300

RFU

Time (min)

B

D-Arg

43 5

L-Ala

*L-Arg

D-Ala

*

D-GluL-Glu

D-AspL-Asp

L-Lys

D-Lys

1

2

10 15 20

0

50

100

150

200

250

300

RFU

Time (min)

B

D-Arg

43 5

L-Ala

*L-Arg

D-Ala

*

D-GluL-Glu

D-AspL-Asp

L-Lys

D-Lys

1

2

Figure 1.

26

Figure 2.

27

18 19 20 21 220

50

100

150

200

250

300

350

400

450

Time (min)

RFU

0

50

100

150

200

250

300

350

400

450

RFU

D-AspD-Glu

C

B

A

18 19 20 21 220

50

100

150

200

250

300

350

400

450

Time (min)

RFU

0

50

100

150

200

250

300

350

400

450

RFU

18 19 20 21 220

50

100

150

200

250

300

350

400

450

Time (min)

RFU

0

50

100

150

200

250

300

350

400

450

RFU

D-AspD-Glu

C

B

A

Figure 3.

28

10 15 20

10 15 2010 15 20

II

D-Arg

L-ArgD-Ala

L-Ala

D-Glu

L-Glu

D

10 15 200

200

400

600

800

D-Arg

D-Ala

L-Ala

D-Glu

L-Glu

D

10 15 200

200

400

600

800

II

D-Arg

D-Ala

L-Ala

D-Glu

L-Glu

D

10 15 200

200

400

600

800

D-Arg

D-Lys

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

10 15 200

200

400

600

800

L-Lys

D-Glu

0

200

400

600

800

1000

1200

D-ArgL-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

10 15 200

200

400

600

800

1000

1200

D-ArgL-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

II

-

L-Glu

II

-

L-Glu

D-GluD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-AspD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-AspD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-AspD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-Asp

II

-

L-Glu

II

-

L-Glu

D-Glu

0

200

400

600

800

1000

1200

D-ArgL-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

10 15 200

200

400

600

800

1000

1200

D-ArgL-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

II

-

L-Glu

II

-

L-Glu

D-GluD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-AspD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-AspD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-AspD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-Asp

II

-

L-Glu

II

-

L-Glu

0

100

200

300

400

500

600

D-Arg

L-Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

10 15 200

100

200

300

400

500

600

D-Arg

L-Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

IVIVArg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

IVIV

0

100

200

300

400

500

600

D-Arg

L-Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

10 15 200

100

200

300

400

500

600

D-Arg

L-Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

IVIVArg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

IVIV

0

100

200

300

D-Arg

L-Arg

L-Lys

D-Ala

L-Ala

L-Glu

10 15 200

100

200

300

L-Glu

IIIIIIIIIIII

0

100

200

300

D-Arg

L-Arg

L-Lys

D-Ala

L-Ala

L-Glu

10 15 200

100

200

300

L-Glu

IIIIIIIIIIII

10 15 200

200

400

600

800

1000

1200

D-Arg

L-Arg

L-Asp

D-Ala

L-Ala

D-Glu

L-Glu D-Asp

10 15 200

200

400

600

800

1000

1200

D-Arg

L-Arg

L-Asp

D-Ala

L-Ala

D-Glu

L-Glu D-Asp

VV

L-AlaL-AlaL-AlaL-Ala

VV

10 15 200

200

400

600

800

1000

1200

D-Arg

L-Arg

L-Asp

D-Ala

L-Ala

D-Glu

L-Glu D-Asp

10 15 200

200

400

600

800

1000

1200

D-Arg

L-Arg

L-Asp

D-Ala

L-Ala

D-Glu

L-Glu D-Asp

VV

L-AlaL-AlaL-AlaL-Ala

VV

L-Asp

10 15 20

10 15 2010 15 20

II

D-Arg

L-ArgD-Ala

L-Ala

D-Glu

L-Glu

D

10 15 200

200

400

600

800

D-Arg

D-Ala

L-Ala

D-Glu

L-Glu

D

10 15 200

200

400

600

800

II

D-Arg

D-Ala

L-Ala

D-Glu

L-Glu

D

10 15 200

200

400

600

800

D-Arg

D-Lys

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

10 15 200

200

400

600

800

L-Lys

D-Glu

0

200

400

600

800

1000

1200

D-ArgL-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

10 15 200

200

400

600

800

1000

1200

D-ArgL-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

II

-

L-Glu

II

-

L-Glu

D-GluD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-AspD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-AspD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-AspD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-Asp

II

-

L-Glu

II

-

L-Glu

D-Glu

0

200

400

600

800

1000

1200

D-ArgL-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

10 15 200

200

400

600

800

1000

1200

D-ArgL-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

II

-

L-Glu

II

-

L-Glu

D-GluD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-AspD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-AspD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-AspD-Arg

L-Arg D-Ala

L-Ala

D-Glu

L-Glu

D-AspL-Asp

II

-

L-Glu

II

-

L-Glu

0

100

200

300

400

500

600

D-Arg

L-Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

10 15 200

100

200

300

400

500

600

D-Arg

L-Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

IVIVArg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

IVIV

0

100

200

300

400

500

600

D-Arg

L-Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

10 15 200

100

200

300

400

500

600

D-Arg

L-Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

IVIVArg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

Arg

D-Ala

L-Ala

D-Glu

L-Glu

D-Asp

L-Asp

IVIV

0

100

200

300

D-Arg

L-Arg

L-Lys

D-Ala

L-Ala

L-Glu

10 15 200

100

200

300

L-Glu

IIIIIIIIIIII

0

100

200

300

D-Arg

L-Arg

L-Lys

D-Ala

L-Ala

L-Glu

10 15 200

100

200

300

L-Glu

IIIIIIIIIIII

10 15 200

200

400

600

800

1000

1200

D-Arg

L-Arg

L-Asp

D-Ala

L-Ala

D-Glu

L-Glu D-Asp

10 15 200

200

400

600

800

1000

1200

D-Arg

L-Arg

L-Asp

D-Ala

L-Ala

D-Glu

L-Glu D-Asp

VV

L-AlaL-AlaL-AlaL-Ala

VV

10 15 200

200

400

600

800

1000

1200

D-Arg

L-Arg

L-Asp

D-Ala

L-Ala

D-Glu

L-Glu D-Asp

10 15 200

200

400

600

800

1000

1200

D-Arg

L-Arg

L-Asp

D-Ala

L-Ala

D-Glu

L-Glu D-Asp

VV

L-AlaL-AlaL-AlaL-Ala

VV

L-Asp

Figure 4