Analysis of Major Carotenoids and Fatty Acid Composition of Freshwater Microalgae

Received:
5 February 2019
Revised:
Analysis of major carotenoids
20 March 2019
Accepted:
14 April 2019
and fatty acid composition of
Cite as: Aline Terra Soares,
Dayane Cristina da Costa,
freshwater microalgae
Armando Augusto
Henriques Vieira,
Nelson Roberto Antoniosi
Filho . Analysis of major
Aline Terra Soares a,∗, Dayane Cristina da Costa a,
carotenoids and fatty acid
composition of freshwater Armando Augusto Henriques Vieira b, Nelson Roberto Antoniosi Filho a
microalgae. a
Heliyon 5 (2019) e01529. Laboratorio de Metodos de Extraç~ao e Separaç~ao (LAMES), Instituto de Química, Universidade Federal de Goias,
doi: 10.1016/j.heliyon.2019. Campus II (Samambaia), 74690-900, Goiânia-GO, Brazil
e01529
b
Laboratorio de Ficologia, Departamento de Botânica, Universidade Federal de S~ao Carlos, S~ao Carlos, SP, Brazil
∗
Corresponding author.
E-mail address: [email protected] (A.T. Soares).
Abstract
Considering the nutraceutical properties, the high commercial value from pigments
and essential lipids and the environmental sustainability, the purposes of this study
were to assess the major carotenoids and fatty acids composition of nine microalgae
species as a source of nutraceutical compounds and as fatty raw material for
biodiesel production. The carotenoid and fatty acid content were analyzed by
high performance liquid chromatography tandem mass spectrometry detection
method with atmospheric pressure chemical ionization mode (HPLC/APCI-MS/
MS) and by high resolution gas chromatography with flame ionization detector
(GC-FID). For the carotenoid analysis, the developed method presented a rapid
response, a good chromatographic separation, higher sensitivity and can provides
more compounds information due the mass spectrum. Among the microalgae
evaluated, Desmodesmus protuberans (10.3 mg g-1), Desmodesmus denticulatus
var. linearis (8.43 mg g-1) and Chlamydomonas planctogloea (7.4 mg g-1) are
good lutein sources. Coelastrum sphaericum (15.29 mg g-1) and Parachlorella
kessleri (22.96 mg g-1) showed high astaxanthin content; the others microalgae
species presents low carotenoid content. In addition, Chlorella zofingiensis
provides high quantities of g-linolenic acid (4.3%). Eicosapentaenoic acid (EPA)
and docosahexaenoic acid (DHA) levels were lower than 1.1 %. Regarding for
https://doi.org/10.1016/j.heliyon.2019.e01529
2405-8440/Ó 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Article Nowe01529
biodiesel production, the promising strains are Coelastrum sphaericum and

Parachlorella kessleri.
Keywords: Natural product chemistry, Analytical chemistry
1. Introduction
Over the last decades, the use of microalgae biomass has gained significant impor-
tance: they are source of a wide range of highly valuable products, including lipids,
pigments, proteins, polysaccharides and phycotoxin, with potential interest for hu-
man consumption as food and as medicines. Pigments and essential fatty acids
have received great attention from the researchers, especially those extracted from
natural sources, as the microalgae. These compounds exhibit pharmaceutical and nu-
traceutical properties and are considered a safe alternative to some existing synthetic
medicines, due to their effectiveness in treating different diseases. In addition, they
have high commercial potential for the production of pharmaceuticals, medicinal,
foods, and cosmetics (Talero et al., 2015; Suleria et al., 2016; Sathasivam et al.,
2017; Tzanova et al., 2017; Wang et al., 2018).
Many microalgae species produce polyunsaturated fatty acids (PUFA), including ei-
cosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are important
compounds for the treatment of different human pathologies (Talero et al., 2015;
Sathasivam et al., 2017). Furthermore, these microorganisms are rich in triacylgly-
cerides, which can be converted into fatty acids methyl esters (FAME) that can be
used as feedstock for biodiesel production. This renewable fuel is less polluting
than petroleum diesel, being a eco-friendly fuel (Menezes et al., 2013; Soares
et al., 2013).
The most important pigments from a commercial and medicinal standpoint are the
carotenoids. They are divided into two groups: (i) carotenes, which are molecules
that have only hydrogen and carbon in their chemical structure, and (ii) xantho-
phylls, which are carotenes oxidized by the presence of oxygen. Both carotenes
and xanthophylls can be found in their free form or esterified to fatty acids (Van
Breemen et al., 2012; Amorim-Carrilho et al., 2014). The qualitative and quantita-
tive analysis of carotenoids is difficult and challenging due to their instability in
the presence of light, heat, acids, and oxygen, as well as their similar structures.
Such compounds are susceptible to oxidation reactions, isomerization, cyclization,
hydrogenation, among others, which leads to their structure modification
(Amorim-Carrilho et al., 2014).
Chromatographic methods are powerful analytical tools to identify and quantify ca-
rotenoids and FAME. Liquid chromatography (LC) is the most used technique to
separate and analyze pigments, in a fast, specific, sensitive, and precise way
2 https://doi.org/10.1016/j.heliyon.2019.e01529
Article Nowe01529
(Rivera et al., 2011). Coupled with ultraviolet/visible (UV/Vis) and mass spectrom-
etry (MS) detector, it presents sensitivity and selectivity in carotenoid analysis for
several matrices. The gas chromatography coupled to flame ionization detector
(GC-FID) is a powerful tool to detect and characterize FAME. These substances
are thermally stable (Soares et al., 2013, 2014) and it is the most applied method
for the FAME analysis in vegetables, fruits, animal and microorganisms
(Cossignani et al., 2011; Menezes et al., 2016; Blasi et al., 2017; Gouveia et al.,
2017).
The present work aimed to identify and quantify the major carotenoids in nine micro-
algae species by an analytical method using mass spectrometry with atmospheric
pressure chemical ionization mode (APCI-MS/MS). Also, determine the FAME
composition by gas chromatography and to predict the potential species as a source
of essential fatty acids and as a feedstock for the production biodiesel.
2. Materials and methods

2.1. Materials
Methanol, acetonitrile and dichloromethane were purchased from J.T. Baker (To-
kyo, Japan). Ethanol, hexane and sodium sulfate were acquired from Neon Analyt-
ical Reagents (Suzano, Brazil). Deionized water was made using an Elix water
purification system (Millipore, Billerica, MA, USA). The reference standards
trans-canthaxanthin (95%), trans-astaxanthin (97%), b-cryptoxanthin (97%),
trans-lutein (97%), trans-fucoxanthin (95%), trans-b-apo-80 -carotene (apocaro-
tenal - 96%; internal standard), trans-b-carotene (95%) and trans-a-carotene
(95%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polytetra-
fluoroethylene sterile membrane (PTFE e 0.22 mm 25 mm) from Nova Analítica
(S~ao Paulo, Brazil).
2.2. Microalgae sample

The microalgae species evaluated were: Chlamydomonas planctogloea, Eutetramo-
rus fottii, Chlorella zofingiensis, Selenastrum bibraianum, Desmodesmus protuber-
ans, Desmodesmus denticulatus var. linearis, Coelastrum sphaericum,
Parachlorella kessleri and Mougeotia sp. The freshwater microalgae were isolated,
identified, cultivated, lyophilized and courtesy from Federal University of S~ao Car-
los. The culture were grown in borosilicate glass cylindrical bottles containing 1.8 L
of the WC culture medium (Guillard and Lorenzen, 1972) at pH ¼ 7 and 23 1 C
with constant stirring by atmospheric air bubbling enriched with 4% CO2 at 0,1 L
min-1 and lighting of 200 mmol photons m2 s1 with photoperiod (12:12). The bio-
masses were centrifuged, frozen and lyophilized.
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2.3. HPLC conditions and APCI-MS/MS detection

HPLC/APCI-MS/MS (Agilent Technologies, Santa Clara, CA, USA, 1290 Infinity
series) including a quaternary bomb, degasser, autosampler (maintained at 10 C),
column temperature controller and needle wash function operated before and after
the injection for 15 seconds. The detector was 3200 Qtrap linear ion trap quadrupole
mass spectrometry, from Sciex (Ontario, Canada), equipped with a Turbo VTM ion
source operated in the positive APCI ionization mode. Analyst software (Version
1.5.2) was used for data acquisition and MultiQuant software (version 2.1) for
data analysis.
For detection and quantification, the following APCI conditions were applied: (i) ion
source temperature - 450 C; (ii) curtain gas (CUR) - 20; (iii) nitrogen - 20 psi; (iv)
collision gas (CAD) - high; (v) nebulizer current (NC) - 4 mA; (vi) entrance potential
(EP) - 10; (vii) ion source gas (GS1) - 45 L mim1; (viii) channel electron multiplier
(CEM) - 2100. The selected ions were monitored by MRM (multiple reaction moni-
toring) with a dwell time of 150 ms for each Q1-Q3 transitions.
After several trial studies, a mobile phase system of methanol/acetonitrile/water

(84:14:2, v/v/v) (A) and dichloromethane (B) was found appropriate with the
gradient conditions being 100% A and 0% B for 11 min, decreased to 95% A in
18 min, 90% A in 21 min, 85% A in 24 min, 80% A in 27 min, 75% A in 35 min,
50% A in 40 min, 75% A in 41 min, returned to 100% A in 43 min and maintained
for 7 min. The total analysis was 50 min. The injection volume was 20 mL, the col-
umn temperature controller was set at 40 C and the flow rate of 1 mL min-1. The
chromatographic separation was done using YMC C30 column (250 mm 4.6
mm I.D. x 5mm) manufactured by Waters (Milford, MA, USA).
2.4. APCI-MS/MS optimization

Each methanolic analyte solution (10 ppb) in a mixture with eluent A-methanol/
acetonitrile/water (84:14:2, v/v/v) was injected under direct infusion with a syringe
pump coupled a T part to HPLC pump and the APCI source, in order to: (i) confirm
the presence of the molecular ion; (ii) determine the predominant fragments formed
in Q1 and (iii) optimized the compound-dependent parameters. The APCI source
was operated in positive and negative mode and the optimal values (fragmentation
and signal intensity) was selected to optimize the compound fragmentation and
mass spectrometry conditions (CAD, CUR, NC, EP and temperature - item 2.2).
The syringe pump flow rate was 10 mL min-1 and for quaternary HPLC pump was
200 mL min-1. The mass spectrum for each carotenoid was obtained in Q1 MS
scan type (1000 Da s-1) with cycle of 10. Next, the declustering potential (DP), colli-
sion energy (CE) and collision cell exit potential (CXP) for each transition Q1-Q3 of
each standard carotenoid solution was optimized. For the multiple reaction
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monitoring (MRM) method was used a dwell time of 150. The most abundant tran-
sition was selected for quantification and the others for the compounds identification.
2.5. Validation parameters for HPLC/APCI-MS/MS method

The calibration curves were prepared based on internal standardization method using
apocarotenal as internal standard (IS) at 10 mg mL-1 in methanol. For lutein, astax-
anthin, a-carotene and cryptoxanthin the curves were prepared at concentrations of
5, 10, 20, 30, 40, 50 and 60 mg mL-1; canthaxanthin with the concentrations of 1, 2.5,
5, 10, 20, 25 and 30 mg mL-1; fucoxanthin at concentrations of 1; 2.5; 5; 10; 20; 25
and 35 mg mL-1; and for b-carotene with the of concentrations of 5; 10; 20; 40; 60; 80
and 100 mg mL-1. Each standard solution was injected in triplicate, and the mean
peak area for each concentration was used. The standard curves were prepared by
plotting concentration ratio (standard versus internal standard) against its area ratio.
The regression equation and correlation coefficient (r) for APCI-MS/MS was used
the MutiQuant software, version 2.1 (Sciex, Framingham, USA). (Anvisa, 2002;
Thompson et al., 2002; INMETRO, 2010; AOAC, 2012; Magnusson and
€
Ornemark, 2014; Ravisankar et al., 2015).
The limit of detection (LOD) and the limit of quantitation (LOQ) were determined
using three low concentrations of the carotenoid standards. Each concentration was
injected into HPLC system three times. The concentrations for each analyte are
demonstrated at Table 1. Each standard curve was prepared by plotting concentra-
tion against area. LOQ and LOD were calculated using Eqs. (1) and (2). S is the stan-
dard deviation of the intercepts and s corresponds to the average value of the slope:
s
LOQ ¼ 10 x ð1Þ
S
s
LOD ¼ 3 x ð2Þ
S
The intra-day variability was carried out by five injections at the same day with the
standard solutions at concentration of 4.0 mg mL-1 to b-carotene and lutein, 9.0 mg
Table 1. Carotenoids concentrations for LOD and LOQ determination.
Carotenoids Concentration (mg mLL1)
Fucoxanthin 0.65e1.25e2.5
Astaxanthin 4.0e5.0e6.0
Lutein 2.0e3.0e4.0
Canthaxanthin 0.27e0.5e1.0
Cryptoxanthin 5.0e7.0e9.0
a-carotene 5.0e7.0e9.0
b-carotene 2.0e3.0e4.0
Article Nowe01529
mL-1 to a-carotene and cryptoxanthin and 1.0; 2.5; 6.0 mg mL-1 to canthaxanthin,
fucoxanthin and astaxanthin, respectively. Similarly, the inter-day variability was
performed by measuring carotenoid concentrations on the 1st, 2nd and 3rd day
with three determinations each day for a total nine determinations, using the standard
concentration of 20 mg mL-1. The precision assays were calculated according to Eq.
(3) (Taverniers et al., 2004). RSD is the relative standard deviation, SD is the stan-
dard deviation and X is the determined average concentration:
SD
RSD ¼ *100 ð3Þ
X
2.6. Pigments extraction

The carotenoids were extracted from microalgae according to Soares et al. (2016).
Approximately 500 mg of microalgae biomass was weighted in an erlenmeyer flask
and 10.0 mL of the hexane:ethanol mixture (1:1) were added. This mixture was left
in ultrasonic bath for 40 minutes at room temperature (23 C 2 C). After that, 7.5
mL of Na2SO4 10% aqueous solution was added, shaken by hand for 30 seconds and
left to rest until phases were separated. The upper phase, rich in carotenoids, was
collected and saved in a round-bottom flask. This procedure was repeated for four
times with the residue within erlenmeyer, totaling five extractions. The combined
upper phases were evaporated in a rotatory evaporator (Quimis - Q344b2, Diadema,
Brasil). The extracted was dissolved in 2.0 mL of methanol and filtered in a PTFE
sterile membrane for HPLC analysis.
2.7. Quantification of FAME content produced by direct

transesterification of the microalgal biomass
Direct transesterification of the biomass of microalgae samples was performed ac-
cording Soares et al. (2014). The organic phase (upper) containing the FAME was
collected and analyzed using gas chromatography. The chromatographic analysis
of the FAME composition used an Agilent 7890 Gas Chromatograph equipped
with a flame ionization detector (FID) and a split/splitless injector, using a DB-
WAX capillary column (30 m 0.25 mm 0.25 mm). The oven was operated
with an initial temperature of 70 C, heated at 10 C min1 until 240 C, remaining
at this temperature for 13 min, and then heated 5 C min1 to 250 C. The injector
temperature was maintained at 310 C with an injection volume of 2 mL, in the split
mode, with a split ratio of 10:1. The detector temperature was kept at 310 C.
Hydrogen was used as the carrier gas at a linear velocity of 42 cm s1, and nitrogen
was used as the auxiliary make-up gas at 20 mL min-1. The FAME were identified by
direct comparison with oil samples of known composition (soybean, canola, and
crambe) through the injection of FAME reference standards (Nu-ChekPrepÒ,
code 17A and 19A) and analyses via high-resolution gas chromatography coupled
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with mass spectrometry (HRGC-MS), as described in previous paper by Soares et al.

(2014).
3. Results and discussion

3.1. APCI-MS/MS optimization and HPLC development
To investigate the MS/MS performance with APCI ionization source for detection
and quantification of carotenoids, preliminary studies were done using the direct
infusion method of the system by injecting each standard solution in a full scan
acquisition, in both positive and negative modes. The data showed that the signal
output in positive mode analyses was better than negative mode. Additionally,
low in-source fragmentation was observed in negative mode, compared with their
corresponding spectra in positive mode. Thus, the subsequent MS/MS experiment
and compound parameters were optimized in positive mode. The mass spectra in
the positive mode from the carotenoids are demonstrated on Supplementary
Material.
During the analysis, the MS/MS parameters for CUR, CAD, NC, and EP barely alter
the protonated molecular ion signal intensity, thus the values were 20, high, 4 mA,
and 10, respectively. The source temperature ranged from 400 to 450 C; however,
most carotenoids presented better signal intensity at 450 C, and this value was used
for all analyzes. The resulting transitions (Q1-Q3) per analyte and respective settings
optimized to the parameters of DP, CE, and CXP are given in Table 2. The quanti-
tative and qualitative analyzes were carried out in MRM mode, by monitoring the m/
z transitions to the precursor ion. The most intense signal was used for the quantifi-
cation and the less intense transitions for the compound confirmation.
After the MRM mode optimization via infusion, the chromatographic separation
condition was adjusted. The chromatographic separation was developed using a
standard carotenoid mixture and comparing the separation for the substances stud-
ied. Initially a gradient solvent system of methanol/acetonitrile/water (84:14:2, v/
v/v) and methylene chloride developed by Soares et al. (2016) was used to sepa-
rate the carotenoid mixture using a C30 column. For this, four different flow rates
were evaluated (0.4, 0.6, 0.8, and 1 ml min-1). The peak chromatographic separa-
tion and the analysis time were influenced by the flow rate. Using a low flow rate,
the peak separation was impaired and the time analysis was longer; the peaks res-
olutions were also affected. For further separation, a flow rate of 1.0 ml min-1 was
used.
Next, the effect of column temperature was studied at 10, 20, and 40 C. The
maximum column temperature recommended by the manufacturer is 40 C, and
this showed better separation efficiency for the investigated carotenoids. On the other
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Table 2. Positive ion APCI tandem mass with MRM parameters of the Q1/Q3
transitions. Da ¼ daltons.
Carotenoids (Molecular Retention Q1 Mass Q3 Mass DP CE CXP

Mass - Da) Time (min) (Da) (Da)
Fucoxanthin (658.906) 7.31 659.500 567.200 46.0 26.0 9.0

659.500 581.600 52.0 27.0 8.0
659.500 641.600 40.0 20.0 8.0
Astaxanthin (596.838) 15.29 597.600 379.200 66.8 17.0 5.0
597.600 473.200 30.0 32.0 5.0
597.600 505.000 47.0 28.0 5.0
597.600 561.500 62.0 16.0 5.0
597.600 579.100 43.0 21.0 5.0
Lutein (568.871) 17,37 569.200 551.300 87.0 20.0 19.0
551.300 533.400 63.4 20.0 7.0
551.300 495.500 54.6 20.0 7.0
551.300 459.300 47.4 26.2 4.8
551.300 429.000 53.3 23.1 4.8
Apocarotenal (416.638) 21.36 417.300 333.200 50.0 20.0 8.2
417.300 347.300 59.0 23.0 3.2
417.300 399.200 60.0 14.0 4.4
Canthaxanthin (564.840) 23.11 565.600 427.400 64.2 18.0 18.8
565.600 413.000 67.3 23.9 4.7
565.600 363.400 44.8 21.7 6.8
565.600 361.100 59.9 23.8 3.5
565.600 459.200 55.6 25.0 17.0
Cryptoxanthin (552.872) 29.42 553.500 535.400 77.0 16.9 5.9
553.500 385.000 88.9 18.0 9.0
553.500 331.000 58.8 16.2 14.0
553.500 347.200 66.8 23.9 5.8
a-carotene (536.873) 34.64 537.600 177.300 55.0 30.0 8.0
537.600 321.100 45.5 26.3 20.9
537.600 347.200 67.5 29.2 35.0
537.600 413.400 56.4 25.7 22.0
b-carotene (536.873) 37.45 537.600 177.300 55.0 30.0 8.0
537.600 321.100 57.8 28.0 8.0
537.600 413.500 66.5 27.0 3.0
hand, the best carotenoid separation obtained by Huck et al. (2000) with a Phenom-
enex C18 column (250 2 mm, 5 mm) in isocratic gradient was at a temperature of
21 C. In these column and gradient conditions, at temperatures higher than 34 C,
the zeaxanthin and lutein were not easily separated.
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In addition, the gradient of the mobile phase was evaluated for the separation of the
standard carotenoid mixture. The optimal proportion decreased an elution time to 50
minutes; comparing with a previous study (Soares et al., 2016), the chromatography
conditions analysis reduced 10 minutes, which saves solvent. Fig. 1 shows the chro-
matogram obtained, the carotenoids peaks were adequately resolved.
3.2. HPLC/APCI-MS/MS validation parameters

The chromatography separation by HPLC/APCI-MS/MS demonstrated that the
techniques can be used to identify and quantify carotenoids, due to excellent
response for each component, with well separated chromatographic peaks, which
indicated a strong selectivity of the carotenoids. In addition, the chromatographic
conditions ensure good specificity, as each pigment identification was not affected
by the presence of the other compounds, because a small amount of cis compounds
can be reliably detected.
For MS/MS detector, the trans astaxanthin and cis canthaxanthin peaks are overlap-
ping at 15.62 min; however, they could be differentiated using the mass spectrum
extracted from this peak. It is possible to identify two peaks with different spectrums,
one in 15.62 min characteristic of astaxanthin and another in 15.9 min of canthaxan-
thin. When the trans canthaxanthin peak was extracted at retention time of 23 min,
Fig. 1. Chromatogram obtained by HPLC/APCI-MS/MS in positive mode from the carotenoid standards
mixture. 1 ¼ trans-fucoxanthin (50 ppm/); 2 ¼ trans-astaxanthin (100 ppm); 3 ¼ trans-lutein (100 ppm);
IS ¼ internal standard (86 ppm); 4 ¼ trans-canthaxanthin (40 ppm); 5 ¼ trans-cryptoxanthin (100 ppm);
6 ¼ trans-a-carotene (100 ppm); 7 ¼ trans-b-carotene (120 ppm).
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three more peaks were shown in small amount, with retention time of 15.9, 17.85,
25.98 min. These three little peaks, presenting the characteristic ions fragments iden-
tified of trans canthaxanthin as well as the same Q1/Q3 transitions, indicate the pres-
ence of cis isomer forms of canthaxanthin and one cis compound overlapping with
trans astaxanthin peak. The mass spectrometry selectivity is higher for multi-
compound analysis, especially when using the MRM mode, even for analytes that
co-elute (Elsinghorst et al., 2011) because each analyte is confirmed by more than
one Q1/Q3 transition.
The values for LOD and LOQ for each carotenoid are showed in Table 3, along with
the Q1/Q3 MRM transitions. Canthaxanthin showed the lowest LOD and LOQ, fol-
lowed by fucoxanthin and lutein. Using the MS/MS, the LOD and the LOQ for most
analytes were <0.29 mg mL-1 and <0.97 mg mL-1, respectively, with the exception
of cryptoxanthin.
The intra- and inter-day precision for each analyte was determined according to Eqs.
(3) and (4), and are shown in Table 3. The inter-day precision was evaluated on three
different days, and the repeatability by five same day injections. For the tandem MS
detector, the inter-day precision was higher than intra-day for a-carotene, fucoxan-
thin, and lutein. According to Anvisa (2002), the RSD parameters cannot be higher
than 5%, while AOAC (2012) determines the RSD acceptability as a function of the
analyte concentration level. Hence, for an analyte in a concentration ranging from 1
Table 3. Limit of detection (LOD), quantification (LOQ), intra-day and inter-day

precision, calibration curves and the correlation coefficients (r) for the working
range of the seven carotenoids obtained by HPLC/APCI-MS/MS in the positive
mode (Q1-Q3 highest intensity transition).
Carotenoid LOD LOD Intra-day Inter-day Linearity

precision precision
mg mLL1 CC* RSD** CC RSD curve r
Fucoxanthin 0.016 0.055 2.48 1.32 20.59 1.42 y ¼ 0.02059x þ 0.03555 0.9995
(659.5e641.6)
Astaxanthin 0.202 0.672 6.21 4.77 21.17 3.97 y ¼ 0.00426x þ 0.39878 0.9900
(597.6e379.2)
Lutein (569.2e533.4) 0.082 0.272 4.06 2.13 21.12 3.31 y ¼ 0.01042x e 0.01398 0.9989
Canthaxanthin 0.004 0.014 1.03 2.77 19.52 1.64 y ¼ 0.02103x þ 0.52746 0.9904
(565.6e363.4)
Cryptoxanthin 0.473 1.432 9.04 1.16 20.92 0.91 y ¼ 0.00712x þ 0.00945 0.9998
(553.5e347.2)
a-carotene (537.6e177.3) 0.290 0.968 8.96 1.32 19.81 3.06 y ¼ 0.00173x e 0.00236 0.9952
b-carotene (537.6e413.1) 0.034 0.113 3.93 3.91 20.75 2.73 y ¼ 0.00059x þ 0.00027 0.9956
*
Concentration calculated (CC, mg mL1).
**
Relative standard deviation (RSD, %).
Article Nowe01529
ppm to 10 ppm, a RSD between 11% and 7.3% is acceptable. For concentrations be-
tween 10 ppm and 100 ppm, the maximum accepted value is between 7.3% and
5.3%, respectively (Taverniers et al., 2004). The RSD values found for the caroten-
oids are lower than the limits specified by the two parameters (ANVISA and
AOAC). Therefore, the RSD value obtained for intra- and inter-day guarantees
the precision of the quantification method for a-carotene, astaxanthin, b-carotene,
canthaxanthin, cryptoxanthin, fucoxanthin, and lutein.
MS-MS/APCI calibration curve and the correlation coefficient (r) are reported in
Table 3. The concentration working range chosen for the carotenoids shows a
very strong linear relation with the analytical signal, with values of r than 0.99 for
MS-MS/APCI. The AOAC (2012) recommend a correlation coefficient >0.99, ac-
cording to Anvisa (2002), the linear correlation coefficient must be 0.98 and
INMETRO (2010) determines r 0.90. Similar values were found for astaxanthin
(r ¼ 0.9981) and canthaxanthin (r ¼ 0.9988) (Tzanova et al., 2017).
3.3. Quantitation of carotenoids in selected microalgae

Microalgae are a good source of bioactive compounds with anti-oxidant potential,
such as carotenoids, that work as pharmacological molecules within therapeutic
treatment/prevention of diseases (Talero et al., 2015; Suleria et al., 2016). In this
context, new species of microalgae have been investigated as a natural source of me-
dicinal substances. This study evaluated the carotenoid composition of nine micro-
algae. The calibration curves from MS-MS/APCI were used to quantify the
carotenoids in microalgae (Table 4). The main sources of lutein are Chlamydomonas
planctogloea, Desmodesmus protuberans and Desmodesmus denticulatus var. linea-
ris, with lutein content in microalgae biomass of 7.4, 10.53, and 8.46 mg g-1, respec-
tively. These species can be used as a source of this pigment. Currently, Marigold
flowers are the commercial source most used for the lutein production. The lutein
production from dried flowers of Marigold Calendula officinalis varies from 0.04
to 0.301 mg g-1 (Lin et al., 2015). Therefore, these microalgae can produce around
35 to 263 times more lutein. Nevertheless, no carotenoid profile was found for these
microalgae species in the literature. Taking as reference the same genus Desmodes-
mus, in Soares et al. (2016) when the carotenoids were extracted with hexane:etha-
nol, the lutein yield was 17% of the total carotenoids in the microalga biomass. When
the biomass saponification was carried out with potassium hydroxide followed by
solvent extraction, the lutein amount was 47.4%. Xie et al. (2016) also using the
basic saponification, and the lutein content accounted for about 50% of total
carotenoids.
Parachlorella kessleri, Coelastrum sphaericum and Chlorella zofingiensis are a nat-

ural source of astaxanthin. Although the astaxanthin is predominantly in the ester
form, C. sphaericum presents astaxanthin in free form, which, based on the weight
12
Table 4. Carotenoids composition in selected microalgae species obtained by HPLC/APCI-MS/MS in the positive mode.
Microalgae Content (mg gL1)
Lutein Astaxanthin Cryptoxanthin Fucoxanthin Canthaxanthin b-carotene a-carotene
Chlamydomonas planctogloea 7.4 0.14 0.99 - 1.49 - -

Eutetramorus fottii 1.72 - - - 0.38 - -
Chlorella zofingiensis 0.49 5.65 0.11 - 0.18 0.29 0.09
Selenastrum bibraianum 1.73 0.41 1.34 0.41 0.03 0.16 0.08
Desmodesmus protuberans 10.53 - - - 0.14 - -
Desmodesmus denticulatus var. linearis 8.46 - - 0.07 - 0.40 0.21
Coelastrum sphaericum 2.75 15.29 0.42 - 0.21 0.11 -
Parachlorella kessleri 1.40 22.96 0.26 - - - -
Mougeotia sp. 1.56 3.48 0.73 0.92 - 0.14 -
Article Nowe01529
Article Nowe01529
of lyophilized biomass, corresponds to 6.47 mg g-1. The commercial microalgae

Haematococcus pluvialis, which is the main source used to produce astaxanthin, ex-
hibits 5% of free astaxanthin (Ranga et al., 2009). The ester form was confirmed by
the presence of the protonated molecular ion for the fatty acid palmitic (m/z 257.2)
and oleic (m/z 283.1) in the mass spectrum. The highest yield of astaxanthin was
from the microalga P. kessleri, almost 23 mg g-1. Considering only the astaxanthin
and lutein production based on the weight of biomass, the astaxanthin yield is around
16 times higher than lutein; however, Minhas et al. (2016) mentioned that astaxan-
thin productivity per day of P. kessleri is about two times higher than lutein produc-
tivity. The astaxanthin content in C. zofingiensis showed 5.65 mg g-1 per biomass,
similar to that reported by Pelah et al. (2004), who mentioned that the cultivation
of C. zofingiensis under stress conditions may accumulate 70% of astaxanthin and
30% of canthaxanthin and that the estimated ratio between canthaxanthin and astax-
anthin content was 0.558. Here, this ratio was 0.032, because the microalga was
cultivated under low light irradiance and low salinity content. Compared with
shrimp samples, raw and cooked shrimp present astaxanthin content of 0.42 and
0.664 mg g-1, respectively (Sanches-Silva et al., 2013). In eggs of rainbow trout spe-
cies, the astaxanthin content was 0.17 103 mg g-1 (Tzanova et al., 2017). Thus,
the microalgae studied in this work are promising astaxanthin sources.
The other microalgae species had low yield of carotenoids, but among the species
evaluated, S. bibraianum (Fig. 2) had the highest content of cryptoxanthin (1.34
mg g-1); Chlamydomonas planctogloea for canthaxanthin (1.49 mg g-1) and Mou-
geotia sp. for fucoxanthin (0.92 mg g-1). Lutein has a zeaxanthin isomer. The lutein
spectrum was differentiated from its isomer by the intensity between the molecular
protonated ion m/z 569 and the fragment m/z 551. For lutein, the m/z 551 fragment is
more abundant than the protonated molecular ion m/z 569, whereas zeaxanthin ex-
hibits the opposite profile (Rivera et al., 2011).
Regarding to carotenes, the levels of a-carotene and b-carotene were below of 0.40
mg g-1. Considering the marine microalga Dunaliella salina, which is used for the b-
carotene commercial production, it produces on average 5e10 mg per gram of dry
weight (Deli et al., 2014), in this sense the microalgae evaluated in this study pre-
sents low yield in these carotenoids.
3.4. FAME analysis from the microalgae

The chromatogram obtained by GC-FID of the microalga C. planctogloea is demon-
strated in Fig. 3, while the fatty acid composition of the nine species of microalgae is
shown in Table 5. The chromatographic conditions that were established made it
possible to highlight and separate fatty acid methyl esters with 14e24 carbon atoms
such as those found in vegetable oils and animal fats in analyses lasting up to 30 mi-
nutes. However, the fatty acid content of conventional vegetable oils and animal fats,
Article Nowe01529
Fig. 2. Chromatogram obtained by HPLC/APCI-MS/MS in positive mode from the microalgae Selenas-
trum bibraianum. 1 ¼ trans-lutein; 2 ¼ astaxanthin; IS ¼ internal standard; 3 ¼ cis-lutein; 4 ¼ cryptox-
anthin; 5 ¼ a-carotene; b-carotene and canthaxanthin ester; 6 ¼ fucoxanthin ester and canthaxanthin
ester.
and even of microalgae of the same class varies qualitatively and quantitatively
(Cossignani et al., 2017; D’Alessandro et al., 2018; Bertoldi et al., 2019).
The essential fatty acids are precursors of prostaglandin E1, a major active biological
compound required to reduce inflammation and blood pressure (Ronda and Lele,
Fig. 3. Chromatogram obtained by GC/FID from Chlamydomonas planctogloea by direct biomass trans-
esterification. Legend: peak 1: C14:0/ peak 2: C16:0/ peak 3: C16:1 c9/ peak 4: C16:2 c7,10/ peak 5:
C17:0/ peak 6: C16:3 c6,9,12/ peak 7: C16:4 c6,9,12,15/ peak 8: C18:0/ peak 9: C18:1 c9/ peak 10:
C18:1 c11/ peak 11: C18:2 c9,12/ peak 12: C18:3 c9,12,15/ peak 13: C20:0/ peak 14: C20:1 c9.
Table 5. FAME composition obtained by direct transesterification of microalgae biomass grown in WC medium.
15
FAME Fame composition (%)

Chlamydomonas Eutetramorus Chlorella Selenastrum Desmodesmus Desmodesmus Coelastrum Parachlorella Mougeotia

planctogloea fottii zofingiensis bibraianum protuberans denticulatus sphaericum kessleri sp.
C14:0 3.5 1.2 0.6 2.4 1.4 1.9 0.5 6.6 2.3
C16:0 22.4 27.5 31.9 23.4 28.0 21.9 31.6 14.7 32.8
C16:1 c9 11.5 2.8 1.6 0.9 2.4 1.8 0.4 0.3 2.6
C16:2 c7,10 2.4 7.8 1.6 1.1 1.6 1.5 0.3 1.0 1.6
C17:0 0.7 - 1.7 0.3 0.6 - - 0.3 -
C16:3 c6,9,12 2.5 9.9 0.8 0.6 2.0 4.3 2.1 0.6 4.3
C16:4 c6,9,12,15 8.3 0.3 6.5 7.1 8.3 4.5 0.9 0.3 0.4
C18:0 1.1 1.7 0.9 1.6 3.6 2.7 2.7 3.8 1.6
C18:1 c9 16.7 8.2 16.0 27.4 21.6 33.6 38.0 44.0 16.2
C18:1 c11 5.6 - 9.9 - - - - - -
C18:2 c9,12 5.4 22.3 12.3 11.3 7.9 6.7 11.5 13.3 11.2
C18:3 c6,9,12 - - 4.3 0.2 0.9 0.7 0.4 0.2 1.1
C18:3 c9,12,15 19.7 18.1 9.7 20.4 19.0 16.8 7.6 11.2 25.1
C18:4 c6,9,12,15 - - 1.5 2.2 2.7 2.4 1.9 1.2 0.8
C20:0 0.1 0.2 - - - 0.1 1.0 0.3 -
C20:1 c9 0.1 - - - - 0.6 - 1.8 -
C20:5 c5,8,11,14,17 - - - - - 0.4 1.0 0.4 -
C22:6 c4,7,10,13,16,19 - - - 1.1 - 0.1 - - -
C24:0 - - 0.7 - - - 0.1 - -
SFA 27.8 30.6 35.8 27.7 33.6 26.6 35.9 25.7 36.7
MUFA 33.9 11 27.5 28.3 24.0 36 38.4 46.1 18.8
DUFA 7.8 30.1 13.9 12.4 9.5 8.2 11.8 14.3 12.8
Article Nowe01529
TUFA 22.2 28 14.8 21.2 21.9 21.8 10.1 12 30.5
PUFA 8.3 0.3 8.0 10.4 11.0 7.4 3.8 1.9 1.2
Legend: SFA is the sum of the contents of saturated fatty acids; MUFA is the sum of the contents of mono-unsaturated fatty acids; TUFA is the sum of the contents of tri-unsaturated fatty acids;
PUFA is the sum of the contents of polyunsaturated fatty acids.
Article Nowe01529
2008). An exemple of these acids are: gamma linolenic acid (GLA - C18:3 cis
6,9,12), eicosapentaenoic acid (EPA - C20:5 u3) and docosahexaenoic acid
(DHA - C22:6 u3). The highest amount of GLA was found in C. zofingiensis
(4.3%), for EPA in C. sphaericum (1%) and DHA in S. bibraianum (1.1%). Howev-
er, among the evaluated microalgae, only C. zofingiensis can be a promise from the
standpoint of metabolic supplementation.
Considering the environmental sustainability, like biodiesel production, taking ac-

count that European legislation (European Committee, 2008) establishes a
maximum of 12% for linoleic acid and 1% for polyunsaturated acids (PUFA) (EN
14214). In addition to linolenic acid, g-linolenic acid (C16:3 cis 6,9,12), C16:3
and C17:3 may also be present in microalgae species (Soares et al., 2013;
Menezes et al., 2016). Since these are similar to linolenic acid, they need to be taken
into consideration. The 12% limit should include the sum of the levels of tri-
unsaturated fatty acids rather than linolenic acid levels alone. C. sphaericum and
P. kessleri have PUFA levels of 3.8% and 1.9%, respectively, also, TUFA levels
of 10.1% and 12.0%, respectively, which gives these microalgae a fatty acid compo-
sition similar to that accepted by the European community. Also, these species pre-
sented a considerable amount of MUFA, 38.4% and 46.1%, respectively. To improve
fatty acid composition, studies modifying culture conditions to reduce polyunsatu-
rated acid content should be conducted.
Of the nine microalgae studied, E. fottii has demonstrated biodiesel production po-
tential, with 0.3% of PUFA content. However, there is still a need to induce a reduc-
tion in the TUFA yield. This microalga showed the highest C16:3 content. Unlike,
C. planctogloea, C. zofingiensis, S. bibraianum, D. protuberans and D. denticulatus
are composed with PUFA yield higher than 7.4% of its total fatty acids. Analysis
shows a high concentration of C16:4 cis 6,9,12,15 and C18:4 cis 6,9,12,15 was
also found. These microalgae strains are not a suitable for biodiesel production.
4. Conclusion
The MS/MS-APCI detector is rapid and sensitive detector to determine and differen-
tiate carotenoids, as well as has high specificity, due the mass spectrum obtained
from the chromatographic peaks, which allows the elucidation of compound struc-
ture, even for peaks that co-elute. Among the evaluated microalgae species, for
pigment production, Desmodesmus protuberans, Desmodesmus denticulatus var.
linearis and Chlamydomonas planctogloea are good sources for lutein and Coelas-
trum sphaericum and Parachlorella kessleri for astaxanthin. Considering the bene-
fits that the essential fatty acids offer to human health, Chlorella zofingiensis
provides high levels of g-linolenic. According to their fatty acids profiles and aiming
at biodiesel production, the microalgae Coelastrum sphaericum and Parachlorella
Article Nowe01529
kessleri are the most promising among the species of microalgae analyzed, by higher
due to the composition of fatty acids that are forthcoming with the specifications laid
down by the standard EN 14214.
Declarations
Author contribution statement
Aline Soares: Conceived and designed the experiments; Performed the experiments;
Analyzed and interpreted the data; Wrote the paper.
Dayane C. da Costa: Performed the experiments.
Armando Vieira: Contributed reagents, materials, analysis tools or data.
Nelson R. A. Filho: Conceived and designed the experiments; Analyzed and inter-
preted the data; Contributed reagents, materials, analysis tools or data.
Funding statement
This work was supported by the Ministry of Science, Technology and Innovation
(MCTI) (FINEP (Agreement No. 01.10.0457.00) and CNPq (Case No. 407556/
2013-3). Nelson R. A. Filho was supported by CAPES and CNPq (Case No.
312019/2013-0).
Competing interest statement

The authors declare no conflict of interest.
Additional information
Supplementary content related to this article has been published online at https://doi.
org/10.1016/j.heliyon.2019.e01529.
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Analysis of Major Carotenoids and Fatty Acid Composition of Freshwater Microalgae

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biodiesel production, the promising strains are Coelastrum sphaericum and

Keywords: Natural product chemistry, Analytical chemistry

2. Materials and methods

2.2. Microalgae sample

2.3. HPLC conditions and APCI-MS/MS detection

After several trial studies, a mobile phase system of methanol/acetonitrile/water

2.4. APCI-MS/MS optimization

2.5. Validation parameters for HPLC/APCI-MS/MS method

Table 1. Carotenoids concentrations for LOD and LOQ determination.

Carotenoids Concentration (mg mLL1)

2.6. Pigments extraction

2.7. Quantiﬁcation of FAME content produced by direct

with mass spectrometry (HRGC-MS), as described in previous paper by Soares et al.

3. Results and discussion

Carotenoids (Molecular Retention Q1 Mass Q3 Mass DP CE CXP

Fucoxanthin (658.906) 7.31 659.500 567.200 46.0 26.0 9.0

3.2. HPLC/APCI-MS/MS validation parameters

Table 3. Limit of detection (LOD), quantiﬁcation (LOQ), intra-day and inter-day

Carotenoid LOD LOD Intra-day Inter-day Linearity

mg mLL1 CC* RSD** CC RSD curve r

3.3. Quantitation of carotenoids in selected microalgae

Parachlorella kessleri, Coelastrum sphaericum and Chlorella zoﬁngiensis are a nat-

Microalgae Content (mg gL1)

Lutein Astaxanthin Cryptoxanthin Fucoxanthin Canthaxanthin b-carotene a-carotene

Chlamydomonas planctogloea 7.4 0.14 0.99 - 1.49 - -

of lyophilized biomass, corresponds to 6.47 mg g-1. The commercial microalgae

3.4. FAME analysis from the microalgae

FAME Fame composition (%)

Chlamydomonas Eutetramorus Chlorella Selenastrum Desmodesmus Desmodesmus Coelastrum Parachlorella Mougeotia

Considering the environmental sustainability, like biodiesel production, taking ac-

Dayane C. da Costa: Performed the experiments.

Armando Vieira: Contributed reagents, materials, analysis tools or data.

Competing interest statement

Anvisa, 2002. Resoluç~ao RE no899 e Guia para validaç~ao de metodos analíticos e

AOAC, 2012. Guidelines for Single Laboratory Validation of Chemical Methods

Guillard, R.R.L., Lorenzen, C.J., 1972. Yellow-green algae with chlorophyllid C. J.

INMETRO, 2010. Orientaç~

Rivera, S., Vilar

Talero, E., García-Mauri~

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