Food Chemistry 361 (2021) 130173

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Single run analysis of glutathione and its disulfide in food samples by liquid
chromatography coupled to on-line post-column derivatization
Apostolia Tsiasioti , Anastasia-Stella Zotou , Paraskevas D. Tzanavaras *
Laboratory of Analytical Chemistry, School of Chemistry, Faculty of Sciences, Aristotle University of Thessaloniki, GR-54124, Greece

A R T I C L E I N F O A B S T R A C T

Keywords: Glutathione and its disulfide were determined in a single run using liquid chromatography with on-line post-
Glutathione column derivatization and fluorimetric detection (340 nm/425 nm). The analytes were separated using a
Glutathione disulfide reversed-phase column capable of operating at 100% aqueous mobile phase and detected following direct on-line
Post column derivatization
reaction with o-phthalaldehyde (7.5 mmol L− 1) in highly basic medium (0.37 mol L− 1 NaOH). The instrumental
Food samples
and chemical variables were carefully investigated towards high sensitivity and throughput, while special
GSH/GSSG quantification
Liquid chromatography attention was paid to validating potential matrix effects. Glutathione and its disulfide could be selectively
determined with respective LODs of 0.10 and 0.30 μmol L− 1 in the absence of matrix effect (<6%). The
endogenous content of the analytes was accurately determined in various food samples with recoveries ranging
between 80 and 120% in all cases. The proposed method is reliable and promising as a generic analytical tool for
the convenient estimation of the redox status of glutathione in various food matrices.

1. Introduction
Glutathione (GSH) is a low molecular weight tripeptide (cysteine,
glycine, and glutamic acid) extensively found in the cells of animals and
plants (Minich & Brown, 2019; Demirkol & Cagri-Mehmetoglu, 2008).
Due to its highly anti-oxidant properties, GSH protects cells from the
toxic effect of reactive oxygen substances and it plays an important role
in the synthesis of proteins and DNA (Malmezat et al., 2000). In plants
recent studies have presented strong indications on the protective role of
GSH against heavy metal poisoning and on the accumulation of antho­
cyanins (Xiang et al., 2001). GSH is also an important constituent in
grapes and wines and a recent review has highlighted its role in wine­
making (antibrowning, aroma profile, ageing, etc. (Kritzinger et al.,
2013)). Last but not least, GSH has been investigated in the food in­
dustry as an alternative natural antibrowning agent for fresh fruit
(Rojas-Graü et al., 2008; Wu, 2014).
GSH is an extremely sensitive analyte, easily oxidized to its respec­
tive disulfide (GSSG). Sample preparation should therefore not alter the
endogenous GSH/GSSG ratio in real samples and analytical methods
should ideally be capable for the effective estimation of the red/ox status
of GSH in various complicated matrices such as food. For example, the
reliable enzymatic recycling assay (Rahman et al., 2007) that has been
used in broccoli (Raseetha et al., 2013) and mushrooms (Kalaras et al.,
2017) requires two steps for GSH/GSSG analysis (blocking of –SH with a
suitable reagent). In analysis of olives by LC–MS a clean-up step using
solid-phase extraction is necessary prior to measurement (López-Huertas
& Palma, 2020), while simultaneous analysis of GSH and GSSG by HPLC
coupled to coulometric electrochemical detection was only partially
validated (Bayram et al., 2014). A quite significant fraction of the re­
ported analytical protocols for the analysis of GSH/GSSG in food sam­
ples is based on HPLC coupled to pre-column derivatization. Due to the
low absorptivity of the analytes in the UV–Vis region, derivatization has
proven quite useful in terms of sensitivity and stabilization of GSH even
when mass spectrometry is used (Roland & Schneider, 2015; Reinbold
et al., 2008). Pre-column derivatization followed by separation using
HPLC or capillary electrophoresis coupled to UV or fluorescence (FL)
detection has been applied to a variety of foods such as wines (Fra­
cassetti et al., 2011; Webber et al., 2014, 2017), citrus fruits juices
(Kubalczyk & Bald, 2009; Kuśmierek & Bald, 2008) and yeasts (Tsar­
daka et al., 2013). Regardless of the employed derivatizing reagent, the
detection mode and the separation technique, the common character­
istic of all the above procedures is their inability to quantify GSH and
GSSG in a single run. Total GSH is estimated only after reduction of the
disulfide using suitable reagents, introducing additional uncertainty to
the results (reduction efficiency, potential interference of excess
reducing reagent, quantification by difference etc).

* Corresponding author.
E-mail address: ptzanava@chem.auth.gr (P.D. Tzanavaras).

https://doi.org/10.1016/j.foodchem.2021.130173
Received 22 February 2021; Received in revised form 16 May 2021; Accepted 18 May 2021
Available online 21 May 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.
A. Tsiasioti et al.
When it comes to the analysis of complex matrices (e.g. food and
biological material) on-line derivatization in post-column mode (PCD) is
considered as a robust and reliable choice, offering well-documented
advantages in terms of sample preparation and minimization of matrix
related interferences (Zacharis & Tzanavaras, 2013). An overview of
recent HPLC–PCD methods for the determination of GSH (and GSSG) in
various samples is presented in Table 1S (Supplementary Section)
(Zacharis et al., 2013, 2011; Karakosta et al., 2013; Smith et al., 2014;
Özyürek et al., 2012; Li et al., 2011; Pelletier & Lucy, 2004; Robin et al.,
2011). Successful analysis of GSH/GSSG by HPLC–PCD has to overcome
two main “obstacles”; (i) the relative high polarity of the analytes
(separation step) and (ii) the implementation of a PCD chemistry
capable of reacting with both the thiol and its disulfide (derivatization
step). As can be seen in Table 1S, effective HPLC separation of polar GSH
and GSSG can be either achieved by the conventional reversed-phase
mechanism by minimizing the organic fraction in the mobile phase
(up to 100% aqueous in specially designed columns), or by the more
straightforward for polar analytes hydrophilic interaction mechanism
(HILIC).
On the other hand, the determination of GSH/GSSG in a single
chromatographic run does not always seem an easy task. Peletier and
Lucy employed an effective thiol reducing agent (TCEP, tris(2-
carboxyethyl)phosphine) in a first post-column step to reduce GSSG,
followed by an indirect FL chemistry (Pelletier & Lucy, 2004). The
relatively high cost of TCEP is rather unattractive for continuous flow
modes, whereas the kinetics of the reduction under flow conditions
reduce the sensitivity for GSSG (LOD = 4.3 μmol L− 1). Mass spectrom­
etry is in theory capable of determining underivatized GSH/GSSG in a
single run. However post column addition of silver nitrate enhanced the
sensitivity 5-fold in ESI mode ([M + Ag]+ vs [M− H]+) (Robin et al.,
2011). A 250-cm-long C18 column opearated at low flow rate (0.25 mL
min− 1) enabled the use of 50% acetonitrile in the mobile phase,
resulting in a separation cycle of < 10 min. In all other HPLC–PCD ap­
proaches described in Table 1S the determination of GSSG cannot be
accomplished in a single run, since treatment of the sample with a
suitable reducing reagent is required (TCEP, NaBH4, DTT, etc) for the
estimation of total GSH content (GSSG is quantified by difference).
In the present study we propose an HPLC–PCD method for the sep­
aration and direct analysis of GSH and its disulfide in a single run,
avoiding extra sample treatment steps. The analytes are separated iso­
cratically using a reversed-phase column that is stable under 100%
aqueous mobile phases (Prevail®, Alltech), followed by on-line post-
column derivatization with o-phthalaldehyde (OPA) and fluorimetric
detection. In highly alkaline medium (pH > 12) GSH reacts directly with
OPA from both the primary amine and the thiolic group (in the absence
of nucleophilic compounds) to form a tricyclic derivative (Tsikas et al.,
1999). Under the same conditions, the -S–S- bond of the more strongly
retained GSSG is hydrolyzed on-line and the produced GSH reacts with
OPA under the same mechanism (Tsiasioti & Tzanavaras, 2021). The
developed HPLC–PCD method is simple, straighforward, utilizes widely
available instrumentation and reagents and enables the matrix-effect-
free determination of the redox status of GSH in a variety of complex
food matrices (vegetables, wines and grapes).
2. Materials and methods
2.1. Instrumentation
HPLC instrumentation: (i) AS3000 autosampler (Thermo Scientific);
(ii) LC-9A binary pump (Shimadzu, Japan); (iii) RF-551 spectro­
fluorimetric detector operated at high sensitivity (Shimadzu, Japan);
(iv) Elite™ vacuum degasser (Alltech, U.S.); (v) column oven (Jones
Chromatography); (vi) Prevail C18 HPLC column (100 × 4.6 mm i.d., 5
μm, Alltech); (vii) Clarity® software (version 4.0.3; DataApex, Czech
Republic).
Post column instrumentation: (i) Minipuls™ 3 peristaltic pump
2
Food Chemistry 361 (2021) 130173
(Gilson, UK) operated with Tygon pump tubes; (ii) reaction coil heater
(HiChrom Limited); (iii) 200 cm/0.5 mm i.d. PTFE reaction coil.
Other equipment: (i) RF-5301PC batch spectrofluorophotometer
(Shimadzu); (ii) ultrasonic bath (Elma Transonic); (iii) pH meter
(Orion); (iv) centrifuge (Hermle); (v) blender (Ultra Turrax).
2.2. Reagents and solutions
All reagents were of analytical grade and were provided by Sigma-
Aldrich, unless otherwise stated. Doubly de-ionized water was pro­
duced by a Milli-Q system (Millipore).
The standard stock solutions of glutathione (GSH) and its disulfide
(GSSG) were prepared daily at 1000 μmol L− 1 in EDTA (5 mmol L− 1).
Dilution in the same solvent was employed to prepare the working so­
lutions. The solution of o-phthalaldehyde (OPA) as post-column deriv­
atization reagent was prepared at a concentration level of 7.5 mmol L− 1
by ultrasonic dissolution of the suitable amount in 5 mL methanol,
following by dilution to 100 mL with doubly de-ionized water. This
solution was typically consumed within 1–2 working days (it is stable for
at least 3 days when stored at 4 ◦ C and protected from light. NaOH so­
lution was prepared at an amount concentration of 0.37 mol L− 1 by
dilution of a 2.0 mol L− 1 stock in water.
The HPLC mobile phase was consisted of 0.1% phosphoric acid in
Milli-Q water. It was prepared daily, including ultrasonic degassing and
filtration under vacuum through 0.45 μm membrane filters
(Whatman®).
2.3. HPLC–PCD procedure
Standard solutions or samples (20 μL injection volume) were injected
in the analytical column and were separated under isocratic elution
(0.1% phosphoric acid) at a flow rate of 0.7 mL min− 1. The column was
thermostated at 50 ◦ C. The separated compounds were mixed down­
stream with the PCD reagents (OPA and NaOH) (0.35 mL min− 1 total
flow rate) forming the OPA-derivatives, on passage through the ther­
mostated reaction coil (200 cm/60 ◦ C). The products were delivered
towards the fluorescence detector and were monitored at 340/425 nm.
It should be noted that during analysis of real samples the column was
periodically flushed with 50–80% acetonitrile for 15 min in order to
remove strongly retained hydrophobic compounds and pevent the
appearance of “ghost peaks”.
2.4. Preparation of food samples
All fresh and frozen vegetable samples (various types of spinach,
peppers, avocado, cucumber, frozen green beans, frozen Brussels sprouts
and fresh asparagus) were purchased from the local market and were
stored as for everyday use. The vegetables were chopped at ca. 1 × 1 cm
pieces using a non-metallic (ceramic) knife and accurately weighed
amounts of 10 g were blended for 1 min in 100 mL of a solution of 1.0
mmol L− 1 EDTA that was kept refrigerated. The resulting suspensions
were extracted ultrasonically for 5 min, centrifuged for 5 min at 4000
rpm and filtered through 0.45 μm syringe filters. Depending on the
concentrations of the analytes in the filtered solutions (estimated
through preliminary analyses), appropriate dilutions were performed, if
needed, in 1.0 mmol L− 1 EDTA. The matrix effect was evaluated using a
pooled sample of various fresh and frozen vegetables (n = 10), following
analogous pretreatment as described above.
Wine samples were also purchased from commercial sources and
were stored bottled in their original packaging until processing. Simple
pretreatment included 5 or 6-fold dilution in 1 mmol L− 1 EDTA
(depending on the labelled ethanol content, see Section 3.4.3), filtration
through disposable syringe filters and direct analysis. For the evaluation
of the matrix effect a pooled wine matrix (n = 5) was prepared using
equal volumes of each wine.
Varieties of commercially available fresh Greek grapes were
A. Tsiasioti et al.
Fig. 1. Graphical depiction of the HPLC–PCD setup; IV = autosampler (V = 20 μL); PP = peristaltic pump (Q = 0.35 mL min− 1); RC = reaction coil (200 cm/0.5 mm
i.d.); FL = fluorimetric detector (340/425 nm); W = waste.
packaged in air-tight plastic bags and stored at –18 ◦ C until analysis.
Pretreatment was carried out according to the procedure described
above for the vegetable samples, with the slight modification of ho­
mogenizing and extracting 20 g grapes in 50 mL EDTA.
3. Results and discussion
In previous studies we examined the OPA-GSH and OPA-GSSG re­
actions under flow conditions (Tsiasioti & Tzanavaras, 2021). Our
findings confirmed and demonstrated that GSH can react on-line with
OPA under both mildly alkaline pH = 8 and highly alkaline medium (pH
> 12) forming a tricyclic fluorescent derivative (Tsikas et al., 1999). On
the other hand, GSSG reacts with OPA, after its on-line hydrolysis/
cleavage of the S–S bond in highly alkaline medium (>0.1 mol L− 1
NaOH), yielding the same derivative. In this study we take advantage of
the above mentioned chemical behavior of GSH/GSSG to explore the
determination of both analytes in a single run following separation by
liquid chromatography coupled to on-line post column derivatization by
OPA in highly alkaline medium (Fig. 1).
The starting values of the main HPLC and PCD variables were: MP =
0.2% phosphoric acid (pH = 2.0), Q(MP) = 0.7 mL/min, c(OPA) = 5
mmol L− 1, c(NAOH) = 1.0 mol L− 1, T(RC) reaction coil = 50 ◦ C, l(RC) =
200 cm and Q(PCD) = 0.25 mL min− 1 for each stream. The amount
concentrations of GSH and GSSG were 10 μmol L− 1.
3.1. Chromatographic conditions
GSH and GSSG are polar compounds with low retention on reversed-
phase columns. The use of highly aqueous mobile phases could be a
viable alternative for the efficient retention and separation of such
compounds (Gritti et al., 2020). For this reason, the Prevail C18 column
was selected as, according to the manufacturer, it can be combined with
mobile phases ranging from 100% aqueous to 100% organic. Due to the
presence of carboxyl groups in the analytes, acidic pH is suitable to
suppress their ionization and enhance the retention. The mobile phase
also contained 1 mmol L− 1 EDTA, to avoid potential oxidation of GSH by
traces of metallic ions present in the chromatographic system (Tang
et al., 2003).
Preliminary comparative studies demonstrated that phosphoric acid
under isocratic elution achieved better retention and chromatographic
efficiency compared to acetic acid and formic acid (data not shown). As
expected, GSH was eluted first, followed by the more hydrophobic GSSG
(it is comprised of two GSH molecules). The effect of the volume fraction
of phosphoric acid on the chromatographic behavior of the analytes was
studied in the range of 0.05–0.2%. The experimental results demon­
strated that the retention times of both analytes remained practically
unaffected with the increase of the acid. A practical level of 0.1% was
selected for subsequent studies.
3
Food Chemistry 361 (2021) 130173
The investigation of the potential effect of the column temperature
proved that it could be a critical factor in terms of resolution and sep­
aration cycle. Temperature increase affects eluent viscosity leading to
more efficient diffusion of hydrophobic components between the eluent
and medium thereby controlling resolution (Dolan, 2002). The column
temperature was therefore examined at four different levels (30, 40, 50
and 60 ◦ C). As can be observed in Fig. 1S (Supplementary Section),
variation of the column temperature within the selected range affected
mainly the more hydrophobic GSSG. A value of 50 ◦ C was selected for
further experiments, as it provided a satisfactory resolution factor of >
5.0, reasonable duration of the separation cycle and did not “stress” the
column at the upper limit of its specifications for temperature.
Under the preferred HPLC conditions, GSH was eluted at 4.8 min,
GSSG at 8.9 min and the resolution factor (Rs) was 7.5.
3.2. Post-column derivatization
Under the selected chromatographic conditions, the main chemical
and instrumental variables for the PCD reaction were investigated: the
concentration of OPA and NaOH, the flow rate of the reagents, the
temperature and the length of the reaction coil. All the above factors
were individually investigated (one-at-a-time) and all experiments were
carried out using a mixture of GSH and GSSG at an amount concentra­
tion of 10 μmol L− 1 each. The experimental results can be summarized as
follows:
(i) The alkalinity of the reaction medium proved to be a significant
factor as it affects both the cleavage of the S–S bond of GSSG and
the reaction of GSH with OPA. The experimental results are
presented in Fig. 2S (Supplementary Section). In the case of GSH,
variation of NaOH in the range of 0.1–2.0 mol L− 1 resulted in a
decrease in sensitivity; the phenomenon was less pronounced in
the range of 0.1–0.37 mol L− 1. This behavior should be more or
less expected as previous flow-based studies indicated that the
reaction of GSH with OPA is favored in the pH range of 8–12
(Tsiasioti & Tzanavaras, 2021). On the other hand, GSSG was
affected in a different manner, since the role of alkalinity is dual.
The sensitivity increased>10-fold in the range of 0.1 to 0.5 mol
L− 1 NaOH, where the cleavage of the S–S bond predominates.
For higher NaOH amount concentrations the GSH-OPA reaction
predominates and the trend is analogous to reduced GSH. As can
be seen in Fig. 2S, a concentration of 0.37 mol L− 1 provides a
viable compromise for both analytes.
(ii) The effect of the concentration of the derivatizing reagent was
investigated in the range of 2.5–15 mmol L− 1. As can be seen in
Fig. 3S (Supplementary Section), OPA concentrations higher than
7.5 mmol L-1 offer practically “steady state” conditions in terms of
sensitivity for both compounds. The latter value has been selected
A. Tsiasioti et al. Food Chemistry 361 (2021) 130173

for further experiments taking also into account the reasonable
consumption of the reagent under the continuous flow PCD
mode.
(iii) As depicted in Fig. 4S (Supplementary Section), the effect of the
reaction temperature is more pronounced for the two-step
chemistry of GSSG. Increase of the temperature of the reaction
coil in the range of 30–70 ◦ C resulted in a ca. 2.5 fold improve­
ment in sensitivity for the analysis of GSSG, indicating that the
cleavage of the S–S bond is favored kinetically at elevated
temperatures. The value of 60 ◦ C was finally selected, as it offered
adequate sensitivity combined with lack of bubble formation in
the flow lines and the flow-through cell of the fluorimetric
detector.
(iv) The total flow rate of the PCD reagents should always be
considered during optimization of HPLC–PCD methods, as it af­
fects both the reaction time and the dispersion of the zones of the
analytes through the mixing ratio of the mobile phase and PCD
reagents. As can be seen in Fig. 5S (Supplementary Section) the
total flow rate of the PCD steams had a more marked effect on
GSH, resulting in a decrease of ca. 50% in the examined range of
0.25 to 0.65 mL min− 1. This behavior can be attributed to three
main factors: (i) the decrease of the mobile phase to reagents
ratio, (ii) reduction of the reaction time and (iii) increase of the
alkalinity of the reaction medium. A practical total flow rate of
0.35 mL min− 1 was selected in terms of sensitivity, flow stability
and reagents consumption.
(v) Finally, drastic improvement in the peak areas was obtained by
increasing the reaction coil length in the range of 0–200 cm. At
the selected value of 200 cm, 2-fold and 8-fold sensitivity im­
provements were recorded for GSH and GSSG, respectively.
Longer coils did not offer significant improvement apart from
prolonging the analysis cycle.
the curves, the percent residuals were satisfactory, ranging between
–13.0% and + 6.0% for GSH and –14.3% and + 12.2% for GSSG.
The LODs were calculated from the standard deviations of the in­
tercepts approach, based on the following equation;
LOD = 3 × SDb/s
where SDb is the standard deviation of the intercept and s is the slope of
the regression line. The LODs were found to be 0.10 μmol L− 1 and 0.30
μmol L− 1 for GSH and GSSG, respectively. The LOQ was set to be the
lowest concentration level of the calibration curves and was found to be
0.25 μmol L− 1 for GSH and 1.0 μmol L− 1 for GSSG. Taking into account
the sample preparation described in the Experimental Section, the pro­
posed method can accurately quantify GSH and GSSG in real samples at
levels starting from 2.5 and 10 nmol g− 1, respectively.
In order to evaluate the within-day precision, successive injections
(n = 8) at 5.0 μmol L− 1 of both analytes were conducted. The RSD values
of peak areas were satisfactory and were calculated to be 0.9% and 1.3%
for GSH and GSSG, respectively. Τhe RSD values of the retention times
were calculated, as well, and found to be 0.03% for GSH and 0.08% for
GSSG. The between-day precision was evaluated by obtaining inde­
pendent calibration curves at different working days (n = 6). The RSD of
the regression slopes was 4.7% for GSH and 2.8% for GSSG (Table 2S in
Supplementary Section).
The robustness of the PCD method was examined by small deliberate
variations of the following post-column parameters; flow rate of PCD
reagents, temperature of the reaction coil, amount concentrations of
OPA and NaOH. As can be seen in Table 3S (Supplementary Section), the
percent recoveries were satisfactory in all cases, ranging between 96.2
and 101.7% for GSH and 95.5–108.6% for GSSG.
3.4. Application to real samples

3.3. Validation of the method The developed HPLC–PCD method has been applied to a variety of
food samples including various vegetables, wines and fresh grapes.
The proposed HPLC–PCD method was validated in terms of linearity,
limit of detection (LOD), limit of quantification (LOQ), within-day and 3.4.1. Evaluation of the potential matrix effects
between-day precision and robustness. Due to the complexity of the selected matrices, the evaluation of the
Linearity was evaluated by cumulative calibration curves including potential matrix effects was evaluated separately for vegetables, wines
all available data points from the analyses of aqueous standards that and grapes.
were obtained on different working days during validation. In this way, Six matrix-matched calibration curves (3 for GSH and 3 for GSSG)
potential between-day variations are compensated. Linearity was in the were constructed using pooled samples of vegetables, wines and grapes,
range of 0.25–10.0 μmol L− 1 for GSH and 1.0–16.0 μmol L− 1 for GSSG. respectively. Following pretreatment, each pooled sample was spiked
The regression equations and the regression coefficients for GSH and with elevated concentrations of the analytes in the range of 1.25–10.0
GSSG were as follows; μmol L− 1 for GSH and 2.5–15.0 μmol L− 1 for GSSG. The percent matrix
effect was calculated by comparison of the aqueous and matrix-matched
A = 1076.8 (±5.98) × c(GSH) − − 24.427 (±33.62), r2 = 0.9979 calibration curves. As can been seen in Table 4S (Supplementary Sec­
tion), the percent matrix effects for GSH/GSSG were − 0.14% and +
A = 374.97 (±4.86) × c(GSSG) + 289.94 (±40.09), r2 = 0.9922 5.74% (vegetables), − 2.36% and + 2.92% (wines), − 4.38% and +
2.15% (grapes) respectively.
where A = is the peak area and c the concentration of the GSH and GSSG All above mentioned values confirm the direct applicability of the
respectively in μmol L-1. Taking into account the cumulative character of proposed method following simple processing and using the aqueous
calibration curves for quantification.
Table 1
Analysis of various vegetable samples. 3.4.2. Potential oxidation of GSH during sample preparation
Vegetables GSH (nmol g− 1) (SD) GSSG (nmol g− 1) (SD) When analyzing GSH in real samples, potential oxidation during
Fresh spinach 293.4 (±28.8) <LOQ sample preparation should always be of concern. For this reason we
Packed spinach 288.6 (±10.1) <LOQ performed a series of experiments in order to evaluate if GSH is oxidized
Baby spinach 479.2 (±37.3) <LOQ at this stage of the analytical cycle. It should be noted that due to the
Frozen spinach 22.4 (±0.1) 34.8 (±2.6) sensitivity and selectivity of our method the pretreatment of the samples
Green pepper 254.8 (±3.0)
is simple, it is based on a single step and EDTA is employed to avoid
<LOQ
Red pepper 107.1 (±0.7) <LOQ
Avocado 233.2 (±17.1) 89.1 (±2.8) oxidation by trace metallic ions.
Cucumber 132.5 (±6.6) <LOQ A pooled sample of vegetables was processed as follows; (i) extrac­
Frozen green beans 8.8 (±2.4) <LOQ tion of GSH/GSSG was carried out by the proposed procedure and the
Frozen brussels sprouts 219.9 (±17.7) 108.4 (±8.7)
extract was analyzed by the HPLC–PCD method; (ii) in a second
SD = standard deviation from three independent analyses (n = 3). experiment, NEM (N-ethylmaleimide, 1 mmol L− 1) was added to the

4
A. Tsiasioti et al. Food Chemistry 361 (2021) 130173

On the basis of these findings, a series of experiments was carried out

in order to try to resolve the peak of GSSG from the endogenous inter­
ference. Based on the 100% aqueous mobile phase of the HPLC step, the
only parameter that could provide sufficient alteration on the chro­
matographic selectivity was thought to be the column temperature. As
already identified during optimization experiments (see Section 3.1 and
Fig. 1S) the column temperature has a considerable impact on the
retention of GSSG, while the role of column temperature on controlling
selectivity in liquid chromatography is very well documented in the
literature (Dolan, 2002). Indeed, as can be seen in the series of chro­
matograms of Fig. 9S (Supplementary Section), lowering the column
temperature to 40 ◦ C (compared to the 50 ◦ C that was initially selected
during optimization), provided sufficient separation between GSSG and
the interfering peak (Rs > 1.5).
The effect of the lowering of the column temperature on the quan­
tification of GSH and GSSG was evaluated by recovery experiments of
Fig. 2. Representative chromatograms of (A) frozen spinach and (B) fresh baby
spinach; the fresh baby spinach was diluted 10-fold.
aqueous standard mixtures processed using the cumulative calibration
curves described in Section 3.3. In all analyzed mixtures the recoveries
were better than 90–110%, being within the evaluated range of residuals
extraction solution in order to block the –SH group of GSH. Three in­
reported in the validation experiments.
dependent extractions were performed in each case. Analysis of the
The three asparagus samples were therefore analyzed and the results
resulting extracts revealed no variations in the levels of GSSG (<10%),
confirmed GSH levels of > 1000 nmol g− 1 (1300–1600 nmol g− 1). As
indicating no oxidation of GSH in the absence of NEM.
mentioned above, no GSSG was quantified in any of the samples.
Analogous accuracy experiments were also carried out for asparagus and
3.4.3. Determination of GSH/GSSG in vegetables
the respective recoveries ranged between 103.4 and 107.6% (GSH) and
The selected vegetables were various types of spinach (included
89.2 and 94.8% (GSSG).
fresh, frozen, packaged and baby spinach), fresh red and green peppers,
avocado, cucumber, frozen green beans, frozen Brussels sprouts and
3.4.5. Determination of GSH/GSSG in wines and grapes
fresh asparagus. All samples were purchased from the local market and
Prior to application of the proposed HPLC–PCD method to wine
prepared as described in the experimental section.
samples, it was necessary to evaluate the effect of the ethanolic content
In order to evaluate the reproducibility of the sample preparation,
of the samples on the chromatographic behavior of the analytes. It is
each sample was processed in triplicate. As shown in Table 1, GSH was
more or less well-known that if the diluent of the samples is more highly
quantified in all examined vegetables at concentration levels ranging
eluotropic than the mobile phase, the analytes may be expected to
between ca. 10–500 nmol g− 1. The lowest levels of GSH were detected in
behave differently as they enter the column, causing fronting or splitting
frozen green beans, and the higher in fresh baby spinach (for the analysis
issues when the analytes elute from the end of the column (Layne et al.,
of fresh asparagus, see discussion in the related section). A graphical
2001; Catchpoole et al., 2006). Standard solutions of GSH and GSSG
comparison of the GSH/GSSG content in two representative spinach
were prepared at various volume fractions of EtOH (0–15% v/v) and
samples (fresh baby spinach and frozen spinach) is shown in the overlaid
analyzed. These series of experiments are critical, as the results will
chromatograms of Fig. 2. The analysis results indicate that (at least in
dictate the necessary dilution factor of the wine samples prior to injec­
the selected samples) fresh spinach contains significantly higher
tion. As can be seen in the representative chromatograms in Fig. 10S
amounts of GSH compared to frozen. On the other hand, GSSG was
(Supplementary Section), the peak of GSSG is severely affected by the
quantified only in the frozen spinach indicating potential oxidation of
diluent of the sample, whereas the less strongly retained GSH seems to
GSH during processing (Shalaby et al., 2014). GSSG was identified in all
be rather unaffected. These findings are in perfect accordance with
samples but only in three of them at quantifiable levels of > 10 nmol g− 1
theoretical studies using 100% aqueous mobile phases in HPLC (Loeser
(frozen spinach, avocado and frozen brussels sprouts). As can be seen in
& Drumm, 2006). A volume fraction of EtOH of 2.5% is perfectly
Table 1, the GSH/GSSG (redox) ratios were ca. 2–3 in fresh avocado and
tolerable by the proposed HPLC–PCD method, indicating a necessary
frozen beans, and < 1 in frozen spinach.
5–6 fold dilution factor prior to analysis.
The accuracy was validated by spiking experiments in the vegetables
Another important parameter that was investigated was the potential
samples at concentration levels of 25 and 50 nmol g− 1 for GSH and
effects of the presence of sulfites in wines on the GSH/GSSG ratio. Sulfite
GSSG. As can be seen in Table 5S (Supplementary Section), the re­
is commonly used as a preservative and anti-microbial agent in wine­
coveries ranged between 93.0 and 108.3% for GSH and 91.4 and 116.0%
making, with reported concentrations of up to 30 mg L− 1 (Kontaxakis
for GSSG, confirming the applicability of the method in real samples.
et al., 2020). Although the anti-oxidant effects of sulfite will be in
Representative chromatograms of blank and spiked avocado and frozen
“equilibrium” in the bottled samples, it could affect the results of ac­
Brussels sprouts are depicted in Figs. 6S and 7S (Supplementary Section)
curacy studies when GSH and GSSG are spiked. Additionally, sulfite is a
respectively.
common nucleophilic reagent that is employed in OPA derivatization
reactions of primary amines as an alternative to the odorous 2-mercap­
3.4.4. Analysis of fresh asparagus
toethanol; in this sense it should be examined as a potential interference.
Asparagus is one of the most GSH-rich vegetables (Demirkol et al.,
Mixtures of GSH (2.5 μmol L− 1) and GSSG (10 μmol L− 1) were therefore
2004). However, analysis of three different asparagus samples (domestic
spiked with elevated concentrations of sulfite in the range of 10–100 mg
and imported) revealed an interfering peak that partially overlapped
L− 1. The mixtures were analyzed (i) immediately after mixing (t = 0
with GSSG. Although asparagus was included in the pooled sample
min) and (ii) after 60 and 180 min. Representative overlaid chromato­
during matrix effect studies (see Section 3.4) its effect was “diluted” in
grams in Fig. 11S (Supplementary Section) showed that (i) sulfite is not
the presence of the other vegetables and the interfering peak co-eluted
detectable by the selected PCD chemistry even at the highest level of
with GSSG. Fig. 8S (Supplementary Section) depicts representative
100 mg L− 1 and (ii) sulfite levels higher than 50 mg L− 1 can almost
overlaid chromatograms of blank and spiked asparagus samples (no
totally reduce GSSG within ca. 180 min. Based on these findings, the
endogenous GSSG was detected in all blank asparagus samples).
spiked samples during accuracy experiments were analyzed

5
A. Tsiasioti et al. Food Chemistry 361 (2021) 130173

Table 2 (GSSG).
Analysis of wine samples by the proposed HPLC–PCD method. On the other hand, GSSG was predominant in all analyzed domestic
Wine Samples GSH (μg L− 1) (SD) GSSG (μg L− 1) (SD) grapes, ranging between 336 and 512 nmol g− 1 (see Table 3). The GSH
content was significantly lower ranging between 18 and 232 nmol g− 1.
Rose Sample 1 1850 (±25) N.D
Rose Sample 2 950 (±15) ND The experimental results were also satisfactory in terms of accuracy (see
White Sample 1 < LOQ ND Table 7S in Supplementary Section) with the recoveries being in the
White Sample 2 1300 (±20) ND range of 98.6–113.6% (GSH) and 82.7–106.4% (GSSG).
White Sample 3 580 (±12) ND Representative chromatograms from the analysis of wines and grapes
ND = not detected. can be found in Fig. 3.
SD = standard deviation from three independent analyses (n = 3).
4. Conclusions

Table 3 In this study we propose a new HPLC–PCD method for the simulta­
Analysis of grapes samples by the proposed method.
neous determination of GSH and GSSG. The developed method allows
Grapes GSH (nmol g− 1) (S.D.) GSSG (nmol g− 1) (SD) the simultaneous determination of both analytes in a single run, offering
Sample 1 29 (±0.5) 502 (±8.9) considerable advantages over analogous approaches where either
Sample 2 21 (±0.3) 336 (±5) blocking of GSH or reduction of GSSG is required. Additional interesting
Sample 3 49 (±0.7) 427 (±2.6) features include; (i) the analytes are isostatically separated in reversed-
Sample 4 18 (±0.6) 511 (±2.5)
phase mode using 100% aqueous mobile phase in<12 min; (ii) the se­
Sample 5 232 (±2.1) 403 (±12)
lective derivatization of GSH and GSSG with OPA in highly alkaline
SD = standard deviation from three independent analyses (n = 3). medium offers a selective analytical scheme with sensitivity at the low
micromolar level; (iii) endogenous GSH and GSSG could be determined
in various complicated matrices without matrix effect and with mini­
mum sample preparation, yielding satisfactory accuracy and precision;
(iv) when/if required, chromatographic selectivity can be controlled by
control of the column temperature; (v) GSH/GSSG levels in real samples
were in good agreement with previous reports (Demirkol et al., 2004;
Demirkol and Cagri-Mehmetoglu, 2008; Kritzinger et al., 2013; Zacharis
et al., 2011).

CRediT authorship contribution statement

Apostolia Tsiasioti: Methodology, Investigation, Validation,

Writing - original draft. Anastasia-Stella Zotou: Methodology, Writing
- review & editing. Paraskevas D. Tzanavaras: Conceptualization,
Methodology, Supervision, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial

interests or personal relationships that could have appeared to influence
the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.foodchem.2021.130173.

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