UPLC–MS/MS identification of sterol sulfates in marine diatoms

Article abstract of DOI:10.3390/md17010010

Diatoms are unicellular eukaryotic organisms that play a key ecological and biogeochemical role in oceans as major primary producers. Recently, these microalgae have also attracted interest as a promising source of functional products with widespread relevance. Progress in the knowledge of cell and molecular biology of diatoms is envisaged as a key step to understanding regulation of their life cycle in marine environments as well as facilitating their full and profitable exploitation by biotechnological platforms. Recently, we identified sterol sulfates (StS) as regulatory molecules of cell death in the diatom Skeletonema marinoi. As these compounds may have a general role in diatom physiology and chemical signals in aquatic systems, we investigated a suitable tool for their analysis in laboratory and field samples. Herein, we describe a sensitive, fast, and efficient ultra performance liquid chromatography–mass spectrometry (UPLC–MS) method for qualitative and quantitative analysis of StS from crude extract of diatoms and other microalgae. The method was applied to 13 different strains of our collection of marine protists. This first study suggested a species-specific distribution of StS and identified the sulfated derivatives of 24-methylene cholesterol and 24-methyl cholesterol as the most common members in diatoms.

Full text of DOI:10.3390/md17010010

marine drugs
Article
UPLC–MS/MS Identification of Sterol Sulfates
in Marine Diatoms
Genoveffa Nuzzo 1, ,† , Carmela Gallo 1,† , Giuliana d’Ippolito 1,† , Emiliano Manzo 1,
*
Nadia Ruocco 1,2, Ennio Russo 3, Ylenia Carotenuto 3, Maria Costantini 2, Valerio Zupo 2,
Angela Sardo 1 and Angelo Fontana 1
1
Bio-Organic Chemistry Unit, Institute of Biomolecular Chemistry CNR, Via Campi Flegrei 34, Pozzuoli,
80078 Naples, Italy; carmen.gallo@icb.cnr.it (C.G.); gdippolito@icb.cnr.it (G.d.I.); emanzo@icb.cnr.it (E.M.);
nadia.ruocco@gmail.com (N.R.); angela.sardo@icb.cnr.it (A.S.); afontana@icb.cnr.it (A.F.)
Department of Marine Biotechnology, Stazione Zoologica Anton Dohrn, 80121 Naples, Italy;
2
mcosta@szn.it (M.C.); vzupo@szn.it (V.Z.)
Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, 80121 Napoli, Italy;
3
ennio.erre@gmail.com (E.R.); ylenia@szn.it (Y.C.)
*
Correspondence: nuzzo.genoveffa@icb.cnr.it
†
These authors have equally contributed to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 2 December 2018; Accepted: 20 December 2018; Published: 24 December 2018
Abstract: Diatoms are unicellular eukaryotic organisms that play a key ecological and biogeochemical
role in oceans as major primary producers. Recently, these microalgae have also attracted interest as
a promising source of functional products with widespread relevance. Progress in the knowledge
of cell and molecular biology of diatoms is envisaged as a key step to understanding regulation of
their life cycle in marine environments as well as facilitating their full and profitable exploitation by
biotechnological platforms. Recently, we identified sterol sulfates (StS) as regulatory molecules of
cell death in the diatom Skeletonema marinoi. As these compounds may have a general role in diatom
physiology and chemical signals in aquatic systems, we investigated a suitable tool for their analysis
in laboratory and field samples. Herein, we describe a sensitive, fast, and efficient ultra performance
liquid chromatography–mass spectrometry (UPLC–MS) method for qualitative and quantitative
analysis of StS from crude extract of diatoms and other microalgae. The method was applied to
13 different strains of our collection of marine protists. This first study suggested a species-specific
distribution of StS and identified the sulfated derivatives of 24-methylene cholesterol and 24-methyl
cholesterol as the most common members in diatoms.
Keywords: terpene; sterol; diatom; dinoflagellate; sulfotransferase; marine organism; natural product;
secondary metabolite; mass spectrometry; chromatography
1. Introduction
Diatoms are a highly diverse and abundant group of phytoplankton. As photosynthetic organisms,
they contribute to global primary productivity and have a central role in biogeochemical cycling of
carbon, nitrogen and silica [1–3]. Furthermore, diatoms are being investigated for the carbon neutral
synthesis of biofuels and as a source of functional products, including pharmaceuticals, health foods,
and nanomaterials [4].
Although phytoplankton is traditionally composed of passive drifters, many recent reports
have demonstrated that these eukaryotic microbes employ complex mechanisms to sense changes in
environmental cues and activate chemical-based defense [5]. These mechanisms have been mainly
associated with the synthesis of chemical signals (e.g., infochemicals) triggered by biotic and abiotic
Mar. Drugs 2019, 17, 10; doi:10.3390/md17010010
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2019, 17, 10
2 of 10
interactions. For example, when diatoms are subjected to grazing or nutrient stress, cells can
rapidly induce the biosynthesis of lipoxygenase products responsible for the production of apoptotic
polyunsaturated aldehydes (PUAs) and nonvolatile oxylipins [6–12].
Continuing our research on ecologically and physiologically relevant compounds in marine
habitats [11
growth control and cell death in the marine bloom-forming diatom Skeletonema marinoi [17]. In this
microalga, we identified three different StS (cholesterol, dihydrobrassicasterol, and -sitosterol sulfate)
–16], we recently reported that the production of sterol sulfates (StS) was linked to
β
whose concentrations changed along the growth curve and induced apoptosis-like mechanism in a
dose-dependent manner, suggesting that these molecules play a role as intracellular mediators in
physiological and ecological functions of diatoms. Programmed cell death is considered as one of the
major causes for control of algal blooms; thus StS alone or in synergy with other molecules might
determine the duration of the growth curve of these microalgae and affect the seasonal succession of
phytoplankton species in marine environments. On the other hand, inhibition of StS synthesis increases
the number of algal cells [17] and is a potential mechanism for multiplying algal biomass to be used
in biotechnological applications or as a starting material for the extraction of functional compounds.
In this view, a fast and sensitive liquid chromatography–mass spectrometry (LC–MS) method for
the identification and quantification of StS may represent a suitable analytical technique to support
studies in monitoring seasonal dynamics of diatom populations as well as find direct and general
application in the development of diatom-based platforms for biotech applications. For this purpose,
we present herein a new sensitive and fast ultra-performance liquid chromatography–electrospray
ionization tandem mass spectrometry (UPLC–ESI-MS/MS) for StS investigation in microalgae and
other biological samples. The method was set up using synthetic StS synthesized by sulfation of
commercially available sterols. The reliability of the analytical approach was validated on S. marinoi
and confirmed on marine photoautotrophic protists, including dinoflagellates and other diatoms.
2. Results
UPLC–ESI-MS/MS was used to identify StS composition directly from crude organic extracts.
The chromatographic method was based on a biphenyl UPLC column and a gradient of MeOH in water
that allowed the elution of StS as a sharp peak in the range between 4.5 and 7 min over a total run
time of 20 min (Figure 1). Chromatographic conditions were optimized in several trials to achieve good
resolution and symmetrical peak shapes. The UPLC system was coupled to a high-resolution Q-Exactive
benchtop hybrid quadrupole-orbitrap mass spectrometer equipped with a high-resolution electrospray
ionization (HR-ESI) probe operating in negative ionization mode. Combining the advantages of high
resolution (70 k), accurate mass measurements (typically below 2 ppm), and tandem mass analysis,
the method permitted the identification of StS at very low concentrations with a detection limit of 20 pg.
Except for cholesterol sulfate, other sterol sulfates were not easily available in the market.
For this reason, we built a small library of StS containing synthetic standards prepared by
sulfation of the commercial sterols 24-methylene cholesterol, desmosterol, campesterol, brassicasterol,
dihydrobrassicasterol, stigmasterol, fucosterol, and β-sitosterol. As reported in Scheme 1, sulfation
was carried out by reaction of 1 mg of each sterol with triethylamine–sulfur trioxide complex in
N,N-dimethylformamide as polar aprotic solvent. To obtain the sodium salt, the reaction mixture
was passed through the cation exchange resin DOWEX 50WX8 (sodium form, Na+) in MeOH [18].
The synthesis was quantitative and, after purification by silica gel followed by reversed-phase
high-performance liquid chromatography (RP-HPLC), recovery of the sulfated products gave an
average yield of 65%.

Mar. Drugs 2019, 17, 10
3 of 10
Figure 1. LC–MS chromatograms of synthetic standards of sterol sulfates under the analytical
conditions described in this work. ( ) desmosterol sulfate; ( ) cholesterol sulfate; ( ) 24-methylene
cholesterol sulfate; ( ) brassicasterol sulfate; ( ) dihydrobrassicasterol sulfate; ( ) campesterol sulfate;
) fucosterol sulfate; (H) stigmasterol sulfate; ( -sitosterol sulfate. Values near each peak represent
retention time and molecular mass as obtained by ion extract mode.
A
B
C
D
E
F
(
G
I) β
In order to establish the recovery yield of StS and to perform quantitative analysis of natural
products, commercially available [25,26,26,26,27,27,27-D7]-cholesterol-sulfate (CHOS-d) was added
as internal standard to the microalgal pellet prior to extraction. The calibration curve for the ratio of
peak area–concentration of CHOS-d showed a linear relationship over a concentration range of 1 to
100 ng/mL, that is in the range of the physiological occurrence of StS in the microalgal samples [17,18].
The regression equation was y = 4721x, and the correlation coefficient (r2) was 0.9933, indicating
good linearity (Figure 2). Deviation from the expected concentration ranged from 97.4% to 102.0%,
and the coefficients of variation were all within 5%. Similar results were also observed with other
synthetic sterol sulfates (data not shown), thus corroborating the reliability of the method performance.
The characteristic fragment at m/z 97 in the MS/MS spectra was associated with the loss of the
sulfate group and was used as a diagnostic marker for the unambiguous identification of StS in
complex mixtures.

Mar. Drugs 2019, 17, 10
4 of 10
Scheme 1. Synthesis of sterol sulfates by direct sulfation of commercially available sterols.
Figure 2.
) Calibration curve and (
fragmentation of deuterated cholesterol sulfate used as internal standard.
Electrospray ionization mass spectrometry (ESI-MS) response to sterol sulfates.
(
A
B) diagnostic loss of sulfate from sterol sulfates as exemplified by
The above method was first applied for the analysis of S. marinoi as proof of concept and
then tested on other eight diatom strains including five benthic (Nanofrustulum shiloi, Cylindrotheca
closterium, Cocconeis scutellum, Phaeodactylum tricornutum, and Diploneis sp.) and three planktonic species
(Cyclotella cryptica, Thalassiosira rotula, and Thalassiosira pseudonana). In addition, we also assessed, for
the first time, the presence of StS in dinoflagellates, namely, Heterocapsa sp., Scripsiella sp., Amphidinium
massarti, and Amphidinium carterae, which are known to biosynthesize a specific family of sterols known
as dinosterols. In agreement with our previous report [17], the analysis of S. marinoi confirmed the
presence of cholesterol, dihydrobrassicasterol, and β-sitosterol sulfates and revealed the presence of two
additional minor compounds—24-methylene cholesterol sulfate and fucosterol sulfate—representing 2.9
and 0.5% of the total StS content, respectively (Figure 3). Among the other protists, all diatom species
(N. shiloi, C. closterium, C. scutellum, Diploneis sp., T. rotula, P. tricornutum, T. pseudonana, and C. cryptica)
showed significant level of sterol sulfates, whereas these compounds were not detectable in dinoflagellates
(Heterocapsa sp., Scripsiella sp., A. massarti, and A. carterae).
Figure 3. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis of sterol sulfates
in S. marinoi. (
sulfate; ( ) 24-methylene cholesterol sulfate; (
-sitosterol sulfate. Percentage of each sterol sulfate is reported on the left part of each spectrum,
whereas the corresponding molecular ion is shown on the right.
A
) Total ion current chromatogram in the range between 460 and 500 m/z; (
B) cholesterol
C
D
) dihydrobrassicasterol sulfate; ( ) fucosterol sulfate;
E
(F) β

Mar. Drugs 2019, 17, 10
5 of 10
In agreement with the literature about diatom sterols [19], UPLC–MS of diatom extracts revealed that
the most common metabolites were the sulfated derivatives of 24-methylene cholesterol and 24-methyl
cholesterol. Moreover, like in plants, StS were generally present in diatoms as mixtures of more
components that mostly differed in the substitution at C-24 of the side chain by methyl, ethyl, and propyl
residues. The good correlation between intraspecific composition of the pools of sterols and sterol sulfates
shows that sulfation can occur without selection of the substrates. This may imply the occurrence of a
single class of sulfotransferase for the sulfation of this family of compounds [18]. Interestingly, we detected
single products in only two species, namely, C. closterium and Diploneis sp., that contained desmosterol
sulfate and brassicasterol sulfate, respectively (Table 1; Supplementary Table S1).
Table 1. Distribution of sterol sulfates in the diatom species under analysis in this study. The method
does not allow to discriminate among epimers.
Molecular
Formula
Compounds
Calculated MS
Measured m/z
Species Distribution
C. closterium
P. tricornutum
S. marinoi
−
−
Desmosterol sulfate
Cholesterol sulfate
C27H43O4S
C27H45O4S
463.2887
465.3044
463.2896
465.3047
S. marinoi
C. cryptica
C. scutellum
P. tricornutum
T. pseudonana
T. rotula
−
24-methylene cholesterol sulfate
C28H45O4S
477.3044
477.3043
N. shiloi
Brassicasterol sulfate
(24β-methyl-22-dehydrocholesterol)
−
−
C28H45O4S
C28H47O4S
477.3044
479.3200
477.3052
479.3205
C. scutellum
Diploneis sp.
S. marinoi 1
C. Cryptica 1
T. pseudonana
T. rotula
24-methyl cholesterol sulfate
Fucosterol sulfate
S. marinoi
N. shiloi
−
−
C29H47O4S
C29H49O4S
491.3200
493.3357
491.3201
493.3361
β-Sitosterol sulfate
(24β-ethyl cholesterol)
S. marinoi
1
Dihydrobrassicasterol sulfate, as characterized after purification and NMR analysis of the natural product.
To confirm the assignment of unknown StS, due to the little difference in the retention time
between structural isomers with the same m/z, analyses in coelution with presumed standard were
also performed (data not shown). The LC–MS data do not provide information on the configuration
of the substituents at C-24, thus the method cannot be applied to distinguish stereoisomers, such as
dihydrobrassicasterol and campesterol sulfate, that differ only in the α- or β-orientation of the methyl
group (Table 1). For S. marinoi and C. cryptica, identification of dihydrobrassicasterol sulfate compound
was achieved after purification and structure elucidation by NMR of the natural product (Table 1).
Besides this, the analysis of different microalgae delineated a species-specific composition of StS, thus
suggesting a good chemotaxonomic and biochemical support.
The presence of CHOS-d as internal standard also allows quantitation and assessment of recovery
yields. For this reason, in addition to the qualitative analysis, the UPLC–MS method is suitable for
the quantification of sterol sulfates in natural samples. As proof of concept, Table 2 reports the total
amount of StS in the cluster composed of the benthic diatoms C. closterium, N. shiloi, C. scutellum,
and Diploneis sp. In order to compare the different species, concentration of StS in micrograms were
normalized per milligram of carbon as measured by CHNS elemental analysis on dry biomass. It is
worth noting that StS are minor metabolites, and the amount produced is steadily increased from the
beginning to the end of the growth curve [17]. For this quantitative study, the species subjected to
analysis were obtained from cell pellets harvested during the declining phase.

Mar. Drugs 2019, 17, 10
6 of 10
Table 2. Quantitative analysis of sterol sulfates in the benthic diatoms Cylindrotheca closterium,
Nanofrustulum shiloi, Cocconeis scutellum, and Diploneis sp. Sterol sulfate content is expressed as g/mg
of carbon (dry weight). Each sample was analyzed in triplicate. Means ± standard error is reported.
µ
Diatom Species
Compounds
µg StS/mg C
C. closterium
Desmosterol sulfate
Total StS
Brassicasterol sulfate
Fucosterol sulfate
Total StS
26.21 ± 2.6
26.21 ± 2.6
5.94 ± 1.0
N. shiloi
60.69 ± 7.3
66.63 ± 5.2
10.09 ± 1.3
3.91 ± 0.1
C. scutellum
Diploneis sp.
24-methylene cholesterol sulfate
Brassicasterol sulfate
Total StS
14.00 ± 1.45
11.03 ± 2.9
11.03 ± 2.9
Brassicasterol sulfate
Total StS
3. Discussion
Sterol sulfates are natural products that, before our report in diatoms [17], have been described
in many marine invertebrates [20 28]. The molecules represent a class of chemical mediators
–
biosynthesized by transfer of a sulfo moiety (SO3H−) to the secondary hydroxyl function at C-3
of sterols. The reaction is catalyzed by a broad family of enzymes named sulfotransferases that have
a recognized role in the metabolism of many organisms, including mammalians and plants [18].
The conversion of sterols to StS gives new biological functions and changes the chemophysical properties
of the organic compounds. However, although sulfation has also been used as derivatization for
the detection and quantification of apolar sterols by LC–MS analysis [29], all methods available for
quantification suffer from several technical limitations (ionization, chromatographic resolution, sensibility,
and reproducibility). Thus, the development of a new UPLC–MS method for the study of StS appears
useful not only in the study of the physiology of marine diatoms but even in other research fields
including biomedical applications where qualitative and quantitative analysis of these metabolites
is important as potential disease biomarkers [30–32]. Although cholesterol 3-sulfate, at low levels,
can be the main sulfolipids in many cell types, it has been reported to have a variety of signaling
and regulatory functions toward many aspects of lipid metabolism, inflammatory responses, and cell
proliferation through its actions on nuclear receptors [33]. To the best of our knowledge, even in the
most comprehensive LC–MS methods proposed to date, analysis requires complex solvent mixtures and
the use of additives for pH control to improve peak shape, resolution, and ionization of the charged
lipids [32]. On the other hand, the method reported here showed a satisfactory chromatographic
resolution, sensitivity, and ionization of this molecules by the use of a elution gradient of MeOH in water,
thus drastically simplifying the procedure of solvent preparation and elution of the metabolites.
The study of the levels of StS in the two major groups of marine photoautotrophic eukaryotic
microbes underlines their presence in diatoms, with differences in concentration and composition among
the different species, as well as suggests their absence in dinoflagellates. This is an intriguing aspect
that deserves further investigation. In fact, dinoflagellates contain a very specific pool of sterols, named
dinosterol and its analogs, which are chemically characterized by the presence of a methyl group at
C-4. The absence of the corresponding sulfates may indicate the absence of specific sulfotransferases as
well as the occurrence of biochemical mechanisms that can replace the physiological role of StS in these
eukaryotic cells.
In conclusion, we have developed a simple and robust LC–MS/MS method that is suitable but
not limited to the qualitative and quantitative analysis of StS in marine samples. The platform allows
the study of a large number of samples in a relatively short time. In particular, the method has been
designed for the analysis of field samples and, in our opinion, can provide a very useful tool to study
marine phytoplankton communities.

Mar. Drugs 2019, 17, 10
7 of 10
4. Materials and Methods
4.1. General
Methanol and water, LC/MS grade, were from MercK (Darmstadt, Germany).
[25,26,26,26,27,27,27-D7]-cholesterol sulfate standard (CHOS-d) and cholesterol sulfate were
purchased from Sigma-Aldrich (Milan, Italy). Reference sterol sulfates were synthesized in-house by
chemical derivatization of sterols (24-methylene cholesterol, desmosterol, campesterol, brassicasterol,
dihydrobrassicasterol, stigmasterol, fucosterol, and
β-sitosterol) that were purchased from
Sigma-Aldrich (Milan, Italy). HPLC for synthetic StS standard purification were performed on a
JASCO system (PU-2089 Plus quaternary gradient pump equipped with a MD-2018 Plus photodiode
array detector and Sedex 85 high-sensitivity LT-ELS detector). UPLC–MS analysis was performed on
Q-Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA)
equipped with an Infinity 1290 UHPLC System (Agilent Technologies, Santa Clara, CA, USA).
4.2. Chemical Derivatization of Sterols to Sterol Sulfates
0.01 mmol of each sterol was dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and sulfur
trioxide-triethylaminecomplex (0.04 mmol) was added to the solution. The reaction mixture was stirred
at 35 ◦C under argon atmosphere for 1 h. After quenching by 0.2 mL MilliQ water, the mixture was
purified on silica gel column to remove the excess of triethylamine–sulfur trioxide. The resulting StS
was dissolved in MeOH and mixed with 0.5 g Dowex50WX8 in the sodium form. After stirring for 5 h
at room temperature, the sample was filtrated on paper, and the process was repeated on the solution.
Sodium salt of StS was dried by rotary evaporator as yellowish powder. Sample purification was
finally achieved on analytical reversed-phase column (Phenomenex, C-18 Luna 4.6
×
250 mm, 100 Å)
using a gradient of CH3OH/H2O in agreement with Reference [17]. Elution was carried out by a flow
rate of 1 mL min−1, and StS were detected by an evaporative light-scattering detector (evaporation
◦
◦
temperature 45 C, nebulization temperature 60 C, N2 flow 1.4 mL min−1). Pure products were
suspended in MeOH and diluted to obtain a MS sample of 5 µg/mL. For the analysis, 5 µL of this
solution was injected under the LC–MS conditions described below.
4.3. Microalgal Material
Skeletonema marinoi (CCMP 2092 purchased from Bigelow Laboratories) (CCMP331) was used
to validate the method, whereas five benthonic (Nanofrustulum shiloi, Cylindrotheca closterium,
Cocconeis scutellum, Diploneis sp., and Phaeodactylum tricornutum), three planktonic diatoms
(Cyclotella. cryptica, Thalassiosira rotula, and Thalassiosira pseudonana), and four dinoflagellates
(Heterocapsa sp., Scripsiella sp., Amphidinium massarti, and Amphidinium carterae) were analyzed for
identification of StS.
−2
◦
S. marinoi was grown in f/2 medium [34] at 100
µ
mol (photons) m
s
−1, at 20
±
2 C, with 14:10
h (light/dark) photoperiod and pH 7 by 50 mM Mops. C. cryptica, T. rotula, and T. pseudonana were
◦
grown in f/2 medium [34] at 200
µ
mol (photons) m−2
s
−1, at 20
±
2 C, with 14:10 h (light/dark)
photoperiod and gently bubbled with steril air, whereas P. tricornutum was grown in f/2 in the same
condition of light and temperature reported for the diatoms described above.
N. shiloi, C. closterium, C. scutellum, and Diploneis sp. were grown in 17-cm glass Petri dish [35
]
plates in f/2 medium at 140 µmol m−2 s−1 and 18 ± 1.0 ◦C, with a 12:12 h (light/dark) photoperiod.
Dinoflagellates (Heterocapsa sp., Scripsiella sp., A. massarti, and A. carterae) were cultured in K
medium at 22.0
glass Fernbach bottles, each containing 1 L of culture.
±
0.5 ◦C, under a 14:10 h light/dark regime and at 100 mol m−2 s−1 in sterile 1.8 L
µ
All diatoms were harvested at late exponential-early stationary phase by cent◦rifugation in a
swing-ou◦t Allegra X12R (Beckman Coulter Inc., Palo Alto, CA, USA) at 2300 g, 4 C, for◦10 min
1800 g, 4 C). Algal pellets were immediately frozen in liquid nitrogen and stored at
−
80 C until

Mar. Drugs 2019, 17, 10
8 of 10
chemical analyses. Cell growth (cells mL−1) was estimated daily using a Bürker counting chamber
(Merck, Leuven, Belgium) (depth 0.100 mm) under an inverted microscope.
4.4. Sample Preparation for LC/MS Analysis
For each microalgae, wet cell pellet (~50 mg) was suspended with 250
µ
L of MeOH. After sonication
◦
for 5 min, the sample was centrifuged at 1000 g for 10 min at 4 C, and the organic phase was recovered.
The pellet was re-extracted with MeOH, the supernatant was combined in vial, and the organic solvent
was removed under nitrogen stream. Each extract was finally suspended in LC–MS-grade MeOH
and diluted to obtain a MS sample of 50
µg/mL of which 5 µL were injected for LC–MS analyses.
For quantitative analysis, 1 g of d7-cholesterol sulfate dissolved in MeOH was added to each 10 mg of
µ
diatom pellet (wet biomass) before extraction. For each run, peaks of sterol sulfates were extract
to verify the presence of the diagnostic fragmentation m/z 97 in the MS/MS spectra due to the
loss of the sulfate group and to compare the retention time to the deuterated standard (m/z 472.3;
see Supplementary Table S1). Experiments were performed in triplicate.
4.5. Ultra Performance Liquid Chromatography/High Resolution Mass Spectrometry (UPLC/HRMS)
Chromatographic separations were achieved on Kinetex Biphenyl 2.6
µ
m, 150
×
2.1 mm column
(Phenomenex, Italy), at 28 ◦C. Eluent A was water, and eluent B was MeOH. After 2 min of isocratic
condition with 70% of B, the elution program consisted of a gradient from 70 to 85% B in 8 min, then to
100% B in 5 min, and holding at 100% B for 5 min more. A post-run equilibration step of 5 min was
included prior to each analysis. The flow rate was 0.3 mL µL,
min−1. The injection volume was 5
·
and the autosampler was maintained at 10 ◦C. MS analyses were carried out on Q-Exactive hybrid
quadrupole-orbitrap mass spectrometer in ESI negative mode. Source parameters were as follows:
◦
spray voltage of 3.0 kV, capillary temperature of 320 C, S-lens RF level of 45, auxiliary gas temperature
◦
of 320 C, sheath gas flow rate of 40, auxiliary gas flow rate of 30. Full MS scans were acquired over the
range 200–600 with a mass resolution of 70,000. The target value (AGC) was 1
×
106 and the maximum
allowed accumulation time (IT) was 100 ms. For the data-dependent MS/MS (ddMS2) analyses, a Top
10 method was used. The 10 most intense peaks were selected for fragmentation with a stepped
normalized energy of 20–25–30 eV. AGC was 1 × 105 with IT 75 ms and 17,500 mass resolution.
Linearity,
calibration curve,
and limit of detection were evaluated on
[25,26,26,26,27,27,27-D7]-cholesterol sulfate standard (CHOS-d).
The calibration curve was
obtained by spiking increasing concentrations of standard solution prepared in MeOH, including the
following points: 100, 50, 10, 5, and 1 ng/mL. Each sample was analyzed in triplicate.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/17/1/10/s1,
Table S1: Distribution of diatom species examined: comparison of StS retention time with deuterated standard
(m/z 472.3) and synthetic derivatives, Figure S1–S6: Chromatograms of diatoms analyzed.
Author Contributions: Work design, experimental planning, and data analysis: G.N., C.G., G.d., and A.F.; LC–MS
analysis and validation, G.N.; strain production and species analysis: C.G., N.R., M.C., E.R., Y.C., A.S., and V.Z.;
chemical synthesis: E.M.; draft preparation: G.N.; data interpretation, review, and critical editing, A.F. All the
authors participated in the proofreading of the manuscript.
Funding: This work has been supported by the project SIBAFEQ (PON01_02740) for the biotechnological
exploitation of marine diatoms within the frame of the National Operational Program of Research and Innovation
and MIUR-PRIN2015 grant “Top-down and bottom-up approach in the development of new bioactive chemical
entities inspired on natural products scaffolds” (Project No. 2015MSCKCE_003). The authors are very grateful to
Dr. Lucio Caso for the technical support with the algal cultures.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Nelson, D.M.; Treguer, P.; Brzezinski, M.A.; Leynaert, A.; Queguiner, B. Production and dissolution of
biogenic silica in the ocean—Revised global estimates, comparison with regional data and relationship to
biogenic sedimentation. Glob. Biogeochem. Cycles 1995, 9, 359–372. [CrossRef]

Mar. Drugs 2019, 17, 10
9 of 10
2.
3.
4.
Bidle, K.D.; Azam, F. Accelerated dissolution of diatom silica by marine bacterial assemblages. Nature 1999
397, 508–512. [CrossRef]
Benoiston, A.S.; Ibarbalz, F.M.; Bittner, L.; Guidi, L.; Jahn, O.; Dutkiewicz, S.; Bowler, C. The evolution of
diatoms and their biogeochemical functions. Phil. Trans. R. Soc. B 2017, 372, 20160397. [CrossRef] [PubMed]
Bozarth, A.; Maier, U.G.; Zauner, S. Diatoms in biotechnology: Modern tools and applications. Appl.
Microbiol. Biotechnol. 2009, 82, 195–201. [CrossRef] [PubMed]
,
5.
6.
Vardi, A. Cell signaling in marine diatoms. Commun. Integr. Biol. 2008, 2, 134–136. [CrossRef]
Miralto, A.; Barone, G.; Romano, G.; Poulet, S.A.; Ianora, A.; Russo, G.L.; Buttino, I.; Mazzarella, G.; Laabir, M.;
Cabrini, M.; et al. The insidious effect of diatoms on copepod reproduction. Nature 1999, 402, 173–176. [CrossRef]
Ribalet, F.; Wichard, T.; Pohnert, G.; Ianora, A.; Miralto, A.; Casotti, R. Age and nutrient limitation enhance
polyunsaturated aldehyde production in marine diatoms. Phytochemistry 2007, 68, 2059–2067. [CrossRef]
Barreiro, A.; Carotenuto, Y.; Lamari, N.; Esposito, F.; d’Ippolito, G.; Fontana, A.; Romano, G.; Ianora, A.;
Miralto, A.; Guisande, C. Diatom induction of reproductive failure in copepods: The effect of PUAs versus
non volatile oxylipins. J. Exp. Mar. Biol. Ecol. 2011, 401, 13–19. [CrossRef]
7.
8.
9.
Ianora, A.; Bastianini, M.; Carotenuto, Y.; Casotti, R.; Roncalli, V.; Miralto, A.; Romano, G.; Gerecht, A.;
Fontana, A.; Turner, J.T. Non-volatile oxylipins render some diatom blooms more toxic for copepod reproduction.
Harmful Alga 2015, 44, 1–7. [CrossRef]
10. Fontana, A.; D’Ippolito, G.; Cutignano, A.; Romano, G.; Lamari, N.; Gallucci, A.M.; Ianora, A. LOX-induced
lipid peroxidation mechanism responsible for the detrimental effect of marine diatoms on zooplankton grazers.
Chembiochem 2007, 8, 1810–1818. [CrossRef]
11. Fontana, A.; d’Ippolito, G.; Cutignano, A.; Miralto, A.; Ianora, A.; Romano, G.; Cimino, G. Chemistry of
oxylipin pathways in marine diatoms. Pure Appl. Chem. 2007, 79, 481–490. [CrossRef]
12. d’Ippolito, G.; Nuzzo, G.; Sardo, A.; Manzo, E.; Gallo, C.; Fontana, A. Lipoxygenases and lipoxygenase
products in marine diatoms. Methods Enzymol. 2018, 605, 69–100. [PubMed]
13. Cimino, G.; Ciavatta, M.L.; Fontana, A.; Gavagnin, M. Metabolites of marine opisthobranchs: Chemistry and
biological activity. In Bioactive Compounds from Natural Sources; Tringali, C., Ed.; Taylor & Francis: London,
UK, 2001; pp. 579–637.
14. Marthi, R.; Fontana, A.; Uriz, M.J.; Cimino, G. Quantitative assessment of natural toxicity in sponge: Toxicity
bioassay versus compound quantification. J. Chem. Ecol. 2003, 29, 1307–1318. [CrossRef]
15. Mollo, E.; Fontana, A.; Roussis, V.; Polese, G.; Amodeo, P.; Ghiselin, M.T. Sensing marine biomolecules: Smell,
taste, and the evolutionary transition from aquatic to terrestrial life. Front. Chem. 2014, 2, 92. [CrossRef] [PubMed]
16. Fontana, A.; Ciavatta, M.L.; D’Souza, L.; Mollo, E.; Naik, C.G.; Parameswaran, P.S.; Cimino, G. Selected
chemo-ecological studies of marine opisthobranchs from Indian coasts. J. Indian Inst. Sci. 2001, 81, 403–415.
17. Gallo, C.; d’Ippolito, G.; Nuzzo, G.; Sardo, A.; Fontana, A. Autoinhibitory sterol sulfates mediate programmed cell
death in a bloom-forming marine diatom. Nat. Commun. 2018, 8, 1292–1303. [CrossRef] [PubMed]
18. Gallo, C.; Nuzzo, G.; d’Ippolito, G.; Manzo, E.; Sardo, A.; Fontana, A. Sterol sulfates and sulfotransferases in
marine diatoms. Methods Enzymol. 2018, 605, 101–138.
19. Rampen, S.W.; Abbas, B.A.; Schouten, S.; Sinninghe Dams, J.S. A comprehensive study of sterols in marine diatoms
(Bacillariophyta): Implications for their use as tracers for diatom productivity. Limnol. Oceanogr. 2010, 55, 91–105.
[CrossRef]
20. McKee, T.C.; Cardellina, J.H.; Riccio, R.; D’Auria, M.V.; Iorizzi, M.; Minale, L.; Buckheit, R.W. HIV-inhibitory
natural products. 11. Comparative studies of sulfated sterols from marine invertebrates. J. Med. Chem. 1994
37, 793–797. [CrossRef]
,
21. Patil, A.D.; Freyer, A.J.; Breen, A.; Carte, B.; Johnson, R.K. Halistanol disulfate B, a novel sulfated sterol from the
sponge Pachastrella sp.: Inhibitor of endothelin converting enzyme. J. Nat. Prod. 1996, 59, 606–608. [CrossRef]
22. Rudi, A.; Yosief, T.; Loya, S.; Hizi, A.; Schleyer, M.; Kashman, Y. Clathsterol, a novel anti-HIV-1 RT sulfated
sterol from the sponge clathria species. J. Nat. Prod. 2001, 64, 1451–1453. [CrossRef] [PubMed]
23. Chen, B.; Shen, X.R.; Kong, J.L. Arenicolsterol A, a novel cytotoxic enolic sulfated sterol from a marine
annelid Arenicola cristata. Chin. J. Chem. 2005, 23, 599–602. [CrossRef]
24. Yang, S.W.; Buivich, A.; Chan, T.M.; Smith, M.; Lachowicz, J.; Pomponi, S.A.; Chu, M. A new sterol sulfate,
Sch 572423, from a marine sponge, Topsentia sp. Bioorg. Med. Chem. Lett. 2003, 13, 1791–1794. [CrossRef]
25. DiGirolamo, J.A.; Li, X.-C.; Jacob, M.R.; Clark, A.M.; Ferreira, D. Reversal of fluconazole resistance by
sulfated sterols from the marine sponge Topsentia sp. J. Nat. Prod. 2009, 72, 1524–1528. [CrossRef] [PubMed]

Mar. Drugs 2019, 17, 10
10 of 10
26. Morinaka, B.I.; Pawlik, J.R.; Molinski, T.F. Amaroxocanes A and B: Sulfated dimeric sterols defend the
caribbean coral reef sponge Phorbas amaranthus from fish predators. J. Nat. Prod. 2009, 72, 259–264. [CrossRef]
[PubMed]
27. Festa, C.; De Marino, S.; Dauria, M.V.; Bifulco, G.; Renga, B.; Fiorucci, S.; Zampella, A. Solomonsterols A
and B from Theonella swinhoei. the first example of C-24 and C-23 sulfated sterols from a marine source
endowed with a PXR agonistic activity. J. Med. Chem. 2011, 54, 401–405. [CrossRef] [PubMed]
28. Sepe, V.; Bifulco, G.; Renga, B.; D’Amore, C.; Fiorucci, S.; Zampella, A. Discovery of sulfated sterols from
marine invertebrates as a new class of marine natural antagonists of farnesoid-x-receptor. J. Med. Chem. 2011
,
54, 1314–1320. [CrossRef]
29. Casanovas, A.; Hannibal-Bach, H.K.; Jensen, O.N.; Ejsing, C.S. Shotgun lipidomic analysis of chemically
sulfated sterols compromises analytical sensitivity: Recommendation for large-scale global lipidome analysis.
Eur. J. Lipid Sci. Technol. 2014, 116, 1618–1620. [CrossRef]
30. Gouveia, M.J.; Brindley, P.-J.; Santos, L.L.; Correia da Costa, J.M.; Gomes, P.; Vale, N. Mass spectrometry
techniques in the survey of steroid metabolites as potential disease biomarkers: A review. Metabolism 2013
62, 1206–1217. [CrossRef]
,
31. Sánchez-Guijo, A.; Oji, V.; Hartmann, M.F.; Schuppe, H.C.; Traupe, H.; Wudy, S.A. High levels of oxysterol
sulfates in serum of patients with steroid sulfatase deficiency. J. Lipid Res. 2015, 56, 403–412. [CrossRef]
32. Sánchez-Guijo, A.; Oji, V.; Hartmann, M.F.; Traupe, H.; Wudy, S.A. Simultaneous quantification of cholesterol
sulfate, androgen sulfates, and progestagen sulfates in human serum by LC–MS/MS. J. Lipid Res. 2015
,
56, 1843–1851. [CrossRef]
33. Strott, C.A.; Higashi, Y. Cholesterol sulfate in human physiology: What’s it all about? J. Lipid Res. 2003, 44, 1268–1278.
[CrossRef] [PubMed]
34. Guillard, R.R.L.; Ryther, J.H. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and
Detonula confervacea Cleve. Can. J. Microbiol. 1962, 8, 229–239. [CrossRef] [PubMed]
35. Raniello, R.; Iannicelli, M.M.; Nappo, M.; Avila, C.; Zupo, V. Production of Cocconeis neothumensis
(Bacillariophyceae) biomass in batch cultures and bioreactors for biotechnological applications: Light
and nutrient requirements. J. Appl. Phycol. 2007, 19, 383–391. [CrossRef]
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).