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Determination of SeMet and Se(IV) in biofortified

yeast by ion-pair reversed phase liquid
Cite this: J. Anal. At. Spectrom., 2016,
31, 203 chromatography-hydride generation-microwave
induced nitrogen plasma atomic emission
spectrometry (HPLC-HG-MP-AES)
Published on 07 September 2015. Downloaded on 3/25/2019 1:07:45 AM.

Eunice Yañez Barrientos,a Kazimierz Wrobel,a Juan Carlos Torres Guzman,b

Alma Rosa Corrales Escobosaa and Katarzyna Wrobel*a

Selenium biofortified yeast is the most common dietary Se supplement in human nutrition and in farm
animals. Therefore, the production and routine quality control of commercial products are highly
demanded. In this work, a simple and cost-effective procedure is proposed for the determination of
SeMet and Se(IV) in hydrolyzed yeast, consisting of ion-pair reversed phase separation, post-column
hydride generation and Se quantification by atomic emission spectrometry with microwave plasma
sustained by nitrogen (HPLC-HG-MP-AES). Freeze-dried biomass was hydrolyzed with methanesulfonic
acid; chromatographic separation was performed with a mobile phase containing 0.08% v/v
heptafluorobutyric and methanol (92 : 8) at a flow rate of 1 mL min
1; the column effluent was on-line
mixed with an alkaline solution of potassium persulfate (K2S2O8 6% m/v, NaOH 3% m/v), passed through
a reaction coil submerged in a water bath at 60  C, and then 10 M hydrochloric acid was added prior to
hydride generation in the MP-AES multimode sample introduction system (NaBH4 2% m/v, NaOH 0.3%
m/v). The total chromatographic run was accomplished in 5 min and the evaluated on-column
quantification limits were 59 ng Se mL
1 for Se(IV) and 0.52 mg mL
1 for SeMet. The procedure was
Received 14th July 2015
Accepted 2nd September 2015
tested using standardized Seleno Excell® high selenium yeast and then applied for the analysis of
Saccharomyces cerevisiae biofortified under different fermentation and exposure conditions. The
DOI: 10.1039/c5ja00276a
procedure was capable of detecting differences in selenium concentration among cultures and the
www.rsc.org/jaas results were consistent with those obtained while coupling HPLC separation directly to ICP-MS detection.

obtained from cultures of bakers's yeast (Saccharomyces cerevisiae)

1. Introduction that are grown in the presence of sodium selenite (Se(IV)), which
The importance of selenium in human health and nutrition has are then washed to remove residual inorganic selenium and other
long been recognized. Different Se-containing dietary supple- free minerals, and nally the biomass is pasteurized and dried to
ments are in use and among them biofortied yeast is the most obtain a homogenous powder.6,8 Depending on the strain type,
common.1,2 Numerous experimental studies and clinical trials fermentation conditions (medium, pH, temperature, and aera-
have proved the chemopreventive and anti-tumor activity of tion) and Se(IV) concentration, selenized yeast may contain up to
selenized yeast.3–5 Administration of Se-yeast in the form of 3 mg selenium per gram of the dry mass.9 Speciation studies have
different dietary supplements or its incorporation into specic oen been undertaken with the aim to understand the incorpo-
foods has been widely approved, and it was concluded that a daily ration pathways and to fully characterize all selenium compounds
element intake of about 100 mg does not present a safety hazard; in biofortied yeast.10–12 The reported species identity and their
at the same time, the tolerable upper intake level was set at 300 mg abundance have varied for different protocols applied in the
per day.6,7 In addition to the human supplementation, Se-yeast fabrication and analysis of Se-yeast, indicating a need for the
has been accepted as an additive for animal feed, so the overall implementation of quality control schemes.2,13
market demand is quite large.6 Commercial products are The primary organic, bioavailable and benecial selenium
compound in yeast is selenomethionine (SeMet) incorporated
a
into proteins hence its reliable quantication has become the
Chemistry Department, University of Guanajuato, L. de Retana 5, 36000 Guanajuato,
essential part of quality control; it should also be ensured that
Mexico. E-mail: [email protected]; Fax: +52 473 7326252; Tel: +52 473 7227555
b
Biology Department, University of Guanajuato, L. de Retana 5, 36000 Guanajuato,
the residual inorganic selenium has been efficiently eliminated.
Mexico Specically, Se-yeast of good quality does not contain more than

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1% of inorganic Se whereas SeMet analyzed in an enzymatic or determination of SeMet and Se(IV) by means of a simple and cost-
acid digest accounts for 60–90% of the total selenium;9 these effective procedure is required and for this purpose, ion-pair
specications dictate analytical requirements that should be reversed phase liquid chromatography separation was coupled
met by any procedure employed in quality control. in this work with MP-AES detection via hydride generation for
Beyond any doubt, procedures based on the hyphenation of the rst time (HPLC-HG-MP-AES). For comparative purposes,
gas or liquid chromatography with species-specic or unspecic ICP-MS was used as an alternative element specic detection
isotope dilution – inductively coupled plasma mass spectrom- system, and the results obtained by the two procedures were in
etry – provide accurate and precise quantication results;14–19 good agreement. The proposed procedure enabled for detecting
however, they are still too demanding to be implemented in differences in Se(IV) and SeMet concentrations among yeast
routine analysis. Simplication and miniaturization have been biofortied under different fermentation conditions, which
approached in several studies with an emphasis mainly on the demonstrates its utility in the quality control schemes.
sample pretreatment. In this regard, microwave assisted acid-
or enzymatic digestion as well as ultrasonic enzymatic hydro-
lysis have been explored;18,20 enzymatic hydrolysis was also 2. Experimental
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carried out on a miniaturized microtiter plate.21 On the other 2.1. Apparatus

hand, less sophisticated and cost-effective techniques with An Agilent Series 1050 liquid chromatographic system
respect to ICP-MS have been used as the detection systems for controlled by using Chemstation (Agilent Technologies) was
liquid chromatography.22 To compensate for lower detection used with a Gemini C18 column (50  3 mm, 5 mm) from
power of these techniques and to avoid matrix interference, Phenomenex. The column effluent was transported via PEEK®
post-column hydride generation has been integrated while tubing and typical FIA tubing (0.5 mm i.d.) to the multimode
using atomic uorescence, atomic emission or atomic absorp- sample introduction system (MSIS) of an Agilent 4100 MP-AES
tion spectrometry.22–26 In such an approach, each selenium nitrogen microwave plasma atomic emission spectrometer
species eluting from the column has to be converted to Se(IV), controlled by using Agilent MP Expert Soware (actualized from
because only this species forms hydrogen selenide in the reac- MP-AES 4200). Two T-joints were incorporated between column
tion with sodium borohydride. For the oxidation of organic exit and MSIS for on-line introduction of the oxidizing agent
species, potassium persulfate in an alkaline medium or the and hydrochloric acid, respectively. Aer mixing with an
mixture of hydrogen bromide/potassium bromate can be used, oxidizing agent, a reaction coil (100 cm) was integrated and it
the resulting Se(VI) is usually reduced to Se(IV) by heating with was submerged in a water bath (60  C). The two reagents and
hydrochloric acid 6–10 mol L
1; alternatively, UV irradiation the solution of sodium borohydride were pumped by using a
has oen been employed with addition of alkaline potassium MP-AES peristaltic pump, each of them at the ow rate of 1 mL
iodide for the reduction of Se(VI) to Se(IV).24,26–29 min
1. Tygon peristaltic tubing (0.76 cm i.d.) was used. The
Post-column hydride generation has proved to be a prom- above set-up is schematically presented in Fig. 1.
ising tool for efficient introduction of selenium species into an An Agilent Series 1050 system was coupled directly to induc-
annular-shaped nitrogen microwave induced plasma formed in tively coupled plasma mass spectrometry detector, via a short-
Okamoto cavity.30 More recently, Hammer has developed a length Teon tubing. A model 7500ce ICP-MS (Agilent Technol-
magnetically excited microwave plasma emission source,31 ogies, Tokyo, Japan) with a Meinhard nebulizer and Peltier-
which is now used in a commercially available instrument cooled spray chamber (2  C) was used. The analytical column was
(microwave nitrogen plasma-atomic emission spectrometry, a Polaris C18 column (100  3 mm, 3 mm) from Varian.
MP-AES). In this system, nitrogen is supplied via an air gener-
ator, which drastically lowers the operation cost as compared to
any other atomic spectrometry technique. A robust diatomic gas 2.2. Reagents and samples
(N2) plasma of toroidal shape is generated with a conventional All chemicals were of analytical reagent grade. Deionized water
torch and the introduction of sample aerosol into the core of the (18.2 MU cm, Labconco, USA) and HPLC-grade methanol
plasma occurs through the central channel, in a similar way to (Fisher Scientic, Pittsburgh, USA) were used throughout.
that in an ICP nebulizer and spray chamber conguration. The
feasibility of HPLC-MP-AES coupling for speciation analysis has
recently been demonstrated;32,33 however poor detection power
is an important limitation of this hyphenated technique. In line
with many earlier studies,28,34 post-column hydride generation
might be helpful for enhanced transport efficiency of column
effluent to the plasma and also for the elimination of the
chemical matrix, which is especially troublesome at the most
intense selenium emission line (196.026 nm).
Due to the high demand in the market, many manufacturers
produce Se-yeast and need suitable analytical tools that would be
helpful during optimization of the production process and could
also be used for quality control purposes. Specically, the Fig. 1 General scheme of HPLC-HG-MP-AES coupling.

204 | J. Anal. At. Spectrom., 2016, 31, 203–211 This journal is © The Royal Society of Chemistry 2016
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Stock solutions of selenomethionine (SeMet) and sodium by centrifugation and were washed with deionized water. These
selenite (Se(IV)) were prepared by dissolving respective Sigma samples were denoted as CC10, S10, M10 and R10, respectively.
standards in deionized water. Other Sigma reagents used in this Finally, all samples were lyophilized in a FreeZone 6 Freeze Dry
work were: potassium persulfate, sodium hydroxide, meth- System (Labconco) prior to analysis.
anesulfonic acid, hydrochloric acid, nitric acid, heptauorobutyric
acid (HFBA), sodium borohydride, b-mercaptoethanol and 2.3. Determination of total selenium in yeast by ICP-MS
hydride peroxide. An Agilent Technology mixed internal standard
solution was also used (4 mg L
1 In, 20 mg L
1 Li, 4 mg L
1 Y, An aliquot (25 mg) of each biomass was placed in a poly-
4 mg L
1 Bi, 10 mg L
1 Sc, and 2 mg L
1 Rh). propylene Eppendorf tube, 750 mL of internal standard mix and
Commercial high selenium yeast Seleno Excell®, containing 250 mL of concentrated nitric acid were added, and the sample
1255 mg g
1 Se, was from Cypress Systems, Inc. was heated at 110  C during 1.5 h. Aer cooling to room
Selenium biofortied yeast samples were obtained in the temperature, 500 mL of hydrogen peroxide 30% m/m were added
frame of an on-going study performed at the Department of and the sample was kept at 110  C during 1 h. Finally, the
Biology, University of Guanajuato (Juan Carlos Torres Guzman, volume was adjusted to 1.5 mL, 25 mL were taken and diluted to
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unpublished data). In brief, different cultures of Saccharomyces 5 mL. The ICP-MS operating conditions were as follows: forward
cerevisiae were obtained under a series of fermentation and power 1500 W, plasma gas ow rate 15 L min
1, carrier gas ow
exposure conditions. In the rst approach 2  107 cells mL
1 rate 0.89 L min
1, make-up gas ow rate 0.15 L min
1, sampling
from S. cerevisiae strain (CC) were grown at 28  C during 18 h depth 8 mm, platinum sampling and skimmer cones, dwell
with agitation, in an Erlenmeyer ask containing 50 mL of time 100 ms per isotope, and collision/reaction cell gas He,
minimal medium (6.7 g L
1 yeast nitrogen base without amino 4.5 mL min
1. The isotopes 78Se, 82Se were monitored (115In as
acids, Difco; 40 g L
1 dextrose, BD Bioxon) in the presence of 0, IS). Five point external calibration was performed at element
5, 10, 15, 20 and 30 mg L
1 Se added in form of sodium selenite concentration levels 0–100 mg L
1 with the addition of the
(Sigma-Aldrich). The cells were recovered by centrifugation and internal standard (4.0 mg L
1 In). The linear regression func-
washed with deionized water, these samples were encoded as tions were obtained (r2 > 0.999) and the evaluated instrumental
C0, C5, C10, C20, C30, respectively. The second series of detection limit for 78Se was 25 ng L
1. For accuracy checking,
samples was obtained from S. cerevisiae strains CC, S, M and R Seleno Excell® was analyzed.
(the last three isolated from strain CC by a directed evolution
strategy) that were incubated under the same conditions in a 2.4. Hydrolysis of yeast for speciation analysis
minimal medium in the presence of 10 mg L
1 Se added in form The procedure described previously was used.35 To a 100 mg
of sodium selenite. The obtained yeast cultures were recovered aliquot of the biomass, 2 mL of methanesulfonic acid 4 mol L
1

Table 1 Instrumental operating conditions of the HPLC-HG-MP-AES system

Chromatographic conditions

Column Gemini C18 (50  3 mm, 5 mm), Phenomenex

Mobile phase Heptauorobutyric acid 0.08% m/v : methanol (92 : 8)
Flow rate 1 mL min
1
Injection volume 100 mL

On-line treatment of the column effluent and hydride generation

Oxidizing agenta K2S2O8 6% m/v + NaOH 3% m/v, 1 mL min
1
Reaction coil 100 cm long, 0.4 mm i.d., 60  C
Acidicationa HCl 10 mol L
1, 1 mL min
1
Hydride generationa NaBH4 2% m/v + NaOH 0.3% m/v, 1 mL min
1

MP-AES detection
Nitrogen 140 psi
Air injection ow rate Low
Nebulizer/spray chamber MiraMist Teon®/multimode sample introduction system
Nebulizer pressure 165 kPa
Viewing position 12
Acquisition mode Continuum
Integration time 5s
Stop time/post time 20 min/2 min
Detection wavelength 196.026 nm
Background correction Off-peak le + right
Number of pixels 3
a
These solutions were carried via a MP-AES peristaltic pump.

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were added and the mixture was heated at reux during 16 h into the chromatographic system. For the recovery experiment,
(120  C). The obtained hydrolyzate was evaporated and the 250 mL aliquot of the re-suspended Selen Excell® digest was
residue was re-suspended in 10 mL of HFBA 0.08% m/v for taken, 500 mL of Se(IV) 750 mg Se L
1 and 500 mL of SeMet 2.5 mg
MP-AES detection and in 10 mL of HFBA 0.1% m/v for ICP-MS. Se L
1 were added and the volume was brought to 10 mL with
For each sample, hydrolysis was carried out in triplicate. It is HFBA; by so doing the standard addition corresponded to
noteworthy that methanesulfonic acid hydrolysis was selected 150 mg Se g
1 of Se(IV) and 500 mg Se g
1 of SeMet in the
due to the efficient release of Se–methionine from proteins, as biomass. All results are presented as mg of selenium per gram of
demonstrated elsewhere.35,36 the freeze-dried biomass.

2.5. Selenium speciation by HPLC-HG-MP-AES 3. Results and discussion

Separation was achieved on a reversed phase column using 3.1. Method development
heptauorobutyric acid as an ion-pair reagent. Prior to hydride
Direct introduction of Se(IV) or SeMet standard solution into
generation, the column effluent was mixed with an alkaline
MP-AES was tested and by so doing, selenium at a concentration
solution of potassium persulfate, heated in a water bath at 60  C
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of 5 mg L
1 was not even detected, which conrmed the need

and then hydrochloric acid was added, as shown in Fig. 1. In the
for post-column hydride generation. Se-yeast contains a low
MP-AES multimode sample introduction system, the column
concentration of Se(IV) which is efficiently converted to H2Se in
effluent was introduced through the lower channel, sodium
the presence of sodium borohydride, so a desired sensitivity
borohydride was pumped through the upper channel and
enhancement was expected for this species in the proposed
nebulization gas was introduced perpendicularly. The chro-
system. On the other hand, SeMet is the most abundant Se
matographic, hydride generation and MP-AES instrument
compound and improving the detection power for this species
operating conditions are given in Table 1. The chromatographic
was less critical. As briey described in the Introduction,
run and the detection system were started simultaneously; the
quantitative conversion of SeMet to Se(VI) and then to Se(IV) for
raw data from continuous acquisition were saved as a Microso
hydrogen selenide generation is not straightforward and
Excel le for further treatment using GRAMS 5.0 (Thermo
requires relatively harsh chemical conditions with prolonged
Scientic). For each chromatogram, binominal ltration and
heating, sonication and/or UV irradiation.24,26–29 To simplify this
extrapolation (32 points) were applied, followed by Fourier
step, the direct oxidation of SeMet to Se(IV) was tested by using
ltration 97%. For external calibration, eight solutions con-
an alkaline solution of sodium persulfate,29 expecting incom-
taining Se(IV) 0; 0.25; 0.5; 1.0; 2.0; 3.0; 4.0; 5.0 mg Se L
1and
plete but reproducible conversion.
SeMet 0; 2.5; 5; 10; 20; 30; 40; 50 mg Se L
1 were used. The
The conversion of SeMet to Se(IV) was examined using a set-
re-suspended yeast samples (10 mL) were ltered (0.22 mm
up presented in Fig. 1, but without a chromatographic column.
Whatman lters) prior to their injection into the chromato-
An aliquot (100 mL) of SeMet standard solution (5 mg Se L
1)
graphic system. For the recovery experiment, the Seleno Excell®
was repeatedly injected into the ow system, while varying the
digest was evaporated and re-suspended in 5 mL of HHBA
following parameters: concentration of potassium persulfate
0.08% m/v. Two aliquots of 1 mL were taken, one was diluted to
(2–8% m/v), length of the reaction coil (50–150 cm), temperature
a 1 : 1 ratio with HFBA and the second was spiked with 500 mL of
(20–80  C), concentration of hydrochloric acid (6–10 mol L
1) and
SeMet 40 mg Se L
1, 100 mL of Se(IV) 40 mg Se L
1 and brought
concentration of sodium borohydride (1–4% m/v). For
to 2 mL with HFBA. Addition of two standards corresponded to
comparative purposes, the same conditions were also tested for
200 mg Se g
1 of Se(IV) and 1000 mg Se g
1 of SeMet in the yeast
Se(IV), 5 mg Se L
1. The selection criteria applied were: (1) the
biomass.
highest possible analytical signal and (2) the lowest possible
standard deviation obtained for replicate injections; in these
2.6. Selenium speciation by HPLC-ICP-MS experiments the analytical signal was acquired as the peak area
Separation was achieved on a reversed phase Polaris C18 under manually selected MP-AES operating conditions listed in
column; isocratic elution with HFBA 0.1% m/v : methanol Table 1. As expected, the most important variables were those
(95 : 5) was carried out at the ow rate of 1 mL min
1 and the related to the oxidation step; in the absence of potassium per-
injection volume was 20 mL. ICP-MS operating conditions were sulfate, SeMet did not produce any signal whereas the highest
the same as those for total Se determination except for the and the most reproducible signal was obtained using 6% m/v
carrier gas ow rate of 0.75 L min
1 and make-up gas ow rate persulfate. At this concentration, the Se(IV) signal was 20.7%
of 0.10 L min
1. The isotopes 78Se and 82Se were monitored. For lower as compared to that obtained without the addition of
external calibration, a series of mixed Se(IV) and SeMet standard persulfate. It is noteworthy that when standard solution was
solutions was used, both species at the concentrations of 0, 10, introduced directly into MP-AES, Se(IV) 5 mg L
1 was not
20, 50, 100, 250, and 500 mg Se L
1. Linear regression functions detected. Furthermore, the analytical signal of SeMet gradually
were obtained for peak area measurements, yielding good augmented when temperature and the length of reaction coil
linearity (r2 > 0.999). The on-column detection limits (78Se) were were increased; however the signal improvement was accom-
0.76 mg Se L
1 for Se(IV) and 0.38 mg Se L
1 for SeMet, respec- panied by peak broadening and also by sensitivity loss for Se(IV)
tively. The re-suspended yeast samples were 40 times diluted so the compromise conditions had to be adopted. It should be
and ltered (0.22 mm Whatman lters) prior to their injection stressed that the addition of an oxidizing agent aer the elution

206 | J. Anal. At. Spectrom., 2016, 31, 203–211 This journal is © The Royal Society of Chemistry 2016
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of Se(IV) was not considered in order to keep the ow system as

simple as possible (persulfate, hydrochloric acid and borohy-
dride solutions were delivered by using the MP-AES peristaltic
pump). Other variables (hydrochloric acid and borohydride
concentrations) were less critical and had a similar effect on the
signals of two Se species. The nally selected parameters are
given in Table 1 and in Fig. 2, typical records for three succes-
sive injections are presented for Se(IV) and SeMet. For Se(IV)
5 mg L
1, the average peak area was 58 299 (RSD ¼ 2.2%) and
for this same concentration of SeMet, the average peak area was
8182 (RSD ¼ 2.6%). For ve between-day replicates the respec-
tive relative standard deviations were 2.6% and 3.1%. The signal
of SeMet was about seven times lower than that of Se(IV) but it
presented acceptable repeatability both in successive injections
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and for between-days measurements. Fig. 3 Typical HPLC-HG-MP-AES chromatograms obtained for cali-
Based on many earlier studies,12,37 anion exchange and ion- bration solutions (0; 0.25; 0.5; 1.0; 2.0; 3.0; 4.0; 5.0 mg Se L
1for Se(IV)
pair reversed phase separations were considered and in the rst and 2.5; 5; 10; 20; 30; 40; 50 mg Se L
1 for SeMet).
approach, the possible effect of the mobile phase composition
on the analytical signal of SeMet and Se(IV) was examined in the
FIA system. Phosphate buffer at pH 6.8 (25–100 mmol L
1), using the Se signal obtained for the lowest calibration standard
typically used in anion exchange separations, caused important for each selenium species.38 The obtained calibration DLs were
depression of the SeMet signal even at the lowest concentration 18 mg Se L
1 for Se(IV) and 0.16 mg Se L
1 for SeMet; the
tested (about 30% decrease). On the other hand, hepta- respective QL values were 59 mg Se L
1 and 0.52 mg Se L
1. To
uorobutyric acid (0.05–0.2% m/v) did not affect the selenium evaluate method DLs and QLs, the baseline was acquired from
signal for any species; therefore it was decided to use a reversed the chromatogram of control biomass (C0) and this sample was
phase column. It was also veried that up to 15% v/v of meth- analyzed aer the addition of the lowest calibration standard of
anol could be safely added to the mobile phase without dete- the two species. The obtained DL and QL for Se(IV) were 3.7
rioration of analytical signals. Isocratic separation was carried mg Se g
1 and 11.9 mg Se g
1, respectively, whereas for SeMet
out on a Gemini C18 (50  3 mm, 5 mm) column under the these values were 32 mg Se g
1 and 104 mg Se g
1. For ve
conditions listed in Table 1; Se(IV) was eluted with a retention between-day replicates, the lowest RSD for analytical signals of
time of 1.16  0.05 min and SeMet 2.97  0.07 min; the total both species was obtained in the concentration range of 10–40
chromatographic run was 5 min. mg Se L
1; <1.5% for SeMet and <2.9% for Se(IV). The above
analytical parameters were suitable for the application of the
3.2. Method validation proposed procedure in the analysis of Se-yeast digests.
The procedure was tested by analyzing standardized Seleno
In Fig. 3, typical chromatograms obtained for the calibration Excell® yeast, which had been evaluated and determined to be
solutions are presented. For six independent calibration Generally Recognized As Safe (GRAS) in accordance with the
processes, the linear regression functions were: ASe ¼ Federal Food, Drug, and Cosmetic Act.7 This product was used
2862.8cSe(IV)
238.6 and ASe ¼ 451.78cSeMet + 644.18 with r2 > by Clark et al. in the rst clinical trial that demonstrated the
0.998 (ASe – peak area; cSeMet/cSe(IV) – concentrations as mg potential of Se-yeast in cancer prevention.39 Total Se determined
Se L
1). The detection limits (DLs) and quantication limits in HNO3/H2O2 digested biomass was 1289  33 mg Se g
1 (n ¼ 3),
(QLs) were evaluated based on the signal-to-noise ratio; the in agreement with the 1255 mg Se g
1 reported by the manu-
criterion of three and ten standard deviations was adopted facturer. The concentration found in methanesulfonic acid
hydrolyzate was 1293  39 mg Se g
1, conrming efficient
solubilization of all selenium forms in this sample. In Fig. 4a
two chromatograms are presented that were obtained for Seleno
Excell® and for the same sample aer standard addition, and
the respective quantication results are given in Table 2. Of
note, a more complex composition of yeast hydrolyzate as
compared to the calibration solutions caused a slight decrease
of the retention times for both species (compare Fig. 3 and 4).
For further accuracy checking, methanesulfonic hydrolyzate
of Seleno Excell® was also analyzed by the HPLC-ICP-MS
procedure as described in Section 2.6. Two chromatograms
corresponding to the spiked and non-spiked sample are shown
Fig. 2 Successive FIA signals acquired for Se(IV) and SeMet (5 mg Se L
1 in Fig. 5a, whereas the quantication results are included in
each) under hydride generation conditions listed in Table 1. Table 2. The elution of several minor species other than Se(IV)

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Fig. 5 HPLC-ICP-MS chromatograms of yeast hydrolyzates (meth-

anesulfonic acid): (a) (—) Seleno Excell and (——) the same sample after
standard addition (details given in Section 2.5). (b) (—) Sample C10 and
(——) sample C20.
Fig. 4 HPLC-HG-MP-AES chromatograms of methanesulfonic acid
hydrolyzates of yeast: (a) (—) Seleno Excell and (——) the same sample
after standard addition (details given in Section 2.5). (b) (—) Sample C10
and (——) sample C20.
3.3. Application for the analysis of different yeast cultures
A series of S. cerevisiae cultures were obtained in the Biology
Department of the University of Guanajuato as shortly described
and SeMet is observed in Fig. 5a, in agreement with many in Section 2.2. The results of Se determination in yeast digested
earlier reports.9,10 Based on HPLC-ICP-MS results, the sum of with HNO3/H2O2 and hydrolyzed with methanesulfonic acid are
SeMet and Se(IV) in the non-spiked sample accounted for 81.6% shown in Fig. 6. For the rst experiment (samples C0–C30), total
of total acid-digested selenium, whereas for HPLC-HG-MP-AES acid-digested Se increased accordingly as the Se(IV) concentra-
this percentage was 82.3%. Previously reported data for Seleno tion added to the growth medium increased, whereas in meth-
Excell indicated 84% SeMet and below 1% of Se(IV) as referred to anesulfonic acid hydrolyzed samples this tendency was much
total Se.7,9 Recovery results obtained for the two procedures aer less marked. It is noteworthy that starting from the Se(IV)
standard addition were in the range of 98.7–104.1% (Table 2); concentration of 15 mg L
1 (C15), the yeast biomass was pink,
highly consistent speciation results obtained for the control likely revealing the presence of elemental selenium. Therefore,
sample by two different procedures demonstrates the suitability the difference observed between two treatments was indicative
of the HPLC-HG-MP-AES system for the determination of SeMet of the amount of Se0 in the sample and the Se concentration in
and Se(IV) in Se-biofortied yeast. methanesulfonic acid hydrolyzate corresponding to the total of
other than Se0 element forms incorporated into the biomass. In

Table 2 Determination of SeMet and Se(IV) in Seleno Excell® and percentage recoveries obtained in the method of standard addition by the
proposed HPLC-HG-MP-AES procedure (1) and by HPLC-ICP-MS (2) (means and standard deviations based on n ¼ 3)

SeMet Se(IV)

Added, Found  Recovery, Added, Found  Recovery,

Procedure mg Se g
1 SD, mg Se g
1 % mg Se g
1 SD, mg Se g
1 %

1 0 1044  42 0 16.5  1.5

1000 2085  49 104.1 200 221  18 102.2
2 0 1036  37 0 15.5  1.4
500 1529  46 98.7 150 169  15 102.6

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Paper JAAS

in Fig. 4b and in Fig. 5b, respectively. The concentration-

dependent chromatographic signals of Se(IV) and SeMet are
clearly observed in both systems; however, a more sensitive
HPLC-ICP-MS procedure allowed for the detection of several
other, minor Se compounds (similarly as in Fig. 5a). Quanti-
cation results for SeMet and Se(IV) are summarized in Table 3,
these data show good agreement between two different specia-
tion systems which reaffirms the validity of the procedure
proposed here. It should be stressed that the percentage amount
of individual species and of their sum with respect to total
HNO3/H2O2 digested Se were also consistent between the two
procedures (Table 3). On the other hand, the concentrations of
Fig. 6 Total selenium concentrations found in HNO3/H2O2 digests
and methanesulfonic acid hydrolyzates of different yeast cultures. SeMet and Se(IV) varied, depending on the growing and exposure
conditions applied. In the rst series, SeMet was relatively stable
Published on 07 September 2015. Downloaded on 3/25/2019 1:07:45 AM.

within a range of 10–20 mg Se L
1 for Se(IV) in the medium;

the second experiment, varying growth conditions were used however, increasing concentrations of Se(IV) in the medium were
while keeping the same concentration of Se(IV) in the medium accompanied by gradual buildup of this species in yeast. In the
(10 mg L
1), none of these cultures presented pink color. It can second series, CC10 was obtained under the same growth and
be observed that for the series of samples R10, M10, N10 and exposure conditions as for C10 in the rst series; total Se
CC10, no important differences in Se concentrations were found concentrations and speciation results were consistent for these
between two treatments and the results were similar to those two samples, indicating good repeatability of the microbiolog-
obtained for Seleno Excell® (Fig. 6), which indicates efficient ical experiment and also good repeatability of analytical results.
incorporation of the element in yeast. Speciation analysis in the The samples R10 and N10 corresponded to two different yeast
yeast methanesulfonic acid hydrolyzates was carried out by strains, lower efficiency of Se incorporation was expected in
HPLC-HG-MP-AES and by HPLC-ICP-MS; typical chromatograms these cultures and indeed the lowest total Se and SeMet were
obtained for two different cultures (C10 and C20) are presented found (Table 3 and Fig. 6). Finally, the M10 sample contained

Table 3 Determination of SeMet and Se(IV) in S. cerevisiae cultures obtained under different growth and exposure conditions by two different
speciation procedures (HPLC-HG-MP-AES; HPLC-ICP-MS)

SeMet Se(IV)

Mean  Mean  % Se in SeMet +

Yeast culture SD mg Se g
1 % Se in biomass SD mg Se g
1 biomass Se(IV) in hydrolyzate, % Se

HPLC-HG-MP-AES
C0 nd — nd — —
C5 322  25 73.3 58.1  2.7 13.2 99.5
C10 1035  41 73.5 98.1  4.2 7.0 88.7
C15 1044  37 33.1 202  5 6.4 92.2
C20 1078  42 81.0 675  37 1.3 92.6
C30 969  23 15.1 989  39 15.5 85.5
R10 979  21 71.7 108  10 7.9 95.3
M10 1275  31 81.5 129  13 8.2 94.8
N10 791  32 54.6 139  18 9.6 70.7
CC10 1041  39 71.7 114  10 7.9 86.5
Excell 1044  42 81.0 16.5  1.5 1.3 82.0

HPLC-ICP-MS
C0 7.3  0.4 60.5 nd — 62.6
C5 318  23 72.5 59.7  2.8 13.7 98.9
C10 1038  34 73.7 96.7  3.7 6.9 88.7
C15 1056  42 33.5 210  5 6.7 93.6
C20 1082  33 19.8 663  32 12.1 92.1
C30 976  21 15.3 973  41 15.2 85.1
R10 964  24 70.6 105  9 7.7 93.6
M10 1281  38 81.9 121  11 7.7 94.7
N10 802  35 55.4 133  16 9.2 71.1
CC10 1050  42 72.4 110  8 7.6 86.9
Excell 1036  37 80.4 15.5  1.4 1.2 81.4

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JAAS Paper

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