Glutathione: methods of sample preparation

science

for chromatography and capillary electrophoresis

Ewelina BŁOŃSKA-SIKORA, Jerzy OSZCZUDŁOWSKI, Zygfryd WITKIEWICZ, Dariusz WIDEŁ - Institute
of Chemistry, Jan Kochanowski University in Kielce

Please cite as: CHEMIK 2012, 66, 9, 929-942

Abbreviations: GSH- reduced glutathone, GSSG- oxidized to the molecule of glutathione. This kind of connection is able to
glutathone, SH- thiol group, K2EDTA- ethylenediaminetetraacetic stabilize some proteins and protect them from oxidative stress.
acid dipotassium salt, K3EDTA- ethylenediaminetetraacetic Glutathionylation regulates also activity of enzymes and process of
acid tripotassium salt, NMR- nuclear magnetic resonance, SDS- transcription. Nucleophosmin involved in assembly of ribosomal
PAGE- sodium dodecyl sulfate polyacrylamide gel electrophoresis, proteins and cyclophilin involved in proteosomal degradadation
MALDI-ToF- matrix-assisted laser desorption/ionization, TCA- of proteins and cytoskeletal proteins belongs to the class of
trichloroacetic acid, NADHP-reduced form of nicotinamide glutathionylated proteins [2].
adenine dinucleotide phosphate, NEM- N-ethylmaleimid, IAA- The antioxidative properties of GSH are especially important
iodoacetic acid, DTT- dithiothreitol, ME- β-mercaptoethanol, for erythrocytes. Red blood cells are highly exposed to free radicals
MBB- monobromobimane, OPA- ortho-phtalaldehyde, BH- mostly because of presence of iron ion, which has different oxidation
borohydride, TBP- tri-n-butylphosphine, TPP- triphenylphosphine, number [3, 4].
TCEP- tris (2-carboxyethyl) phosphine, DTNB- 5,5’-dithiobis- The reduced glutathione/oxidized glutathione ratio (GSH/GSSG) is
(2-nitrobenzoic acid), IA- iodoacetamid, VP- 2-vinylpyridine, signed by R symbol. This is used to evaluate oxidative stress status in
DTPD- 4,4’-dithiopiridine, CMQT- 2-chloro-1-methylquinolinium biological systems, and alterations of this ratio have been demonstrated
tetrafluoroborate, SBD-F- 4- ammonium 7-fluoro-2,1,3- in several diseases and during aging. The value of GSH/GSSG in the
benzoxadiazole-4-sulfonate, ABD-F- 4-aminosulfonyl-7-fluoro- liver in physiological conditions is 300-400, whereas during starvation
2,1,3-benzoxadiazole, DBD-F-4-(N,Ndimethylaminosulfonyl)-7- this ratio decreases to 2. This is very useful tool for determination of
fluoro-2,1,3-benzoxadiazole. damage of different organs and tissues.
In human beings both GSH depletion and growth are linked to
Introduction a number of diseases states but mostly acquired or congenital deficiency
Glutathione (γ-glutamyl-cysteinylglycine) plays a lot of useful is observed. GSH depletions are associated with pathological conditions
functions in human body and therefore determination of this small in organism such as: rheumatoid arthritis, muscular dystrophy, alcoholic
molecule is very important for present-day medicine and pharmacy. liver damage disease, glaucoma, neurodegenerative diseases (Alzheimer
It is synthesized in every procariotic and eucariotic cell because it disease, Parkinson disease, multiple sclerosis), schizophrenia, autism,
takes part in protection against oxidative stress, plays important role arteriosclerosis, diabetes, asthma, chronic obstructive pulmonary [5÷8].
in detoxification and immunity modulation. Thanks to the glutathione Moreover, glutathione concentration measurement represents an
the body is able to protect itself against different infections and cancer important tool for diagnosis of γ-glutamyl cycle disorders. Patients
development, the liver has ability to detoxify heavy metals, toxins and who exhibit deficiency of GSH have low concentration of specific
other xenobiotics and cells are not subject of continuous destruction. enzymes: glutathione synthetase and γ-glutamylocysteine synthetase
Characteristic element of glutathione structure is thiol group (-SH), in erythrocytes and whole organism [2].
which is responsible for biological functions of this compound. Because
of the presence of this group, glutathione can occur in several forms. Sort of matrixes for glutathione determination
The most important forms are: reduced (GSH) and oxidized (GSSG) Glutathione concentration in human beings and animals has been
glutathione. analyzed mostly in plasma, whole blood and erythrocytes, because GSH to
Other widespread forms are: S-nitrosoglutathione and conjugates GSSG ratio in those structures represents sensitive indicator of oxidative
of GSSG and proteins. stress in whole organism. Biological samples have been also collected
Reduced form of glutathione plays the principal role in human body from different tissues and cells such as: gastric mucosa [9] mitochondria
as it protects organism against oxidative stress and negative impact of from lymphocytes and granulocytes [10] human promyelocytic leukemia
oxidants, for example reactive forms of oxygen (hydrogen peroxide, cell line HL-60 [11] lung tissue [12] kidneys, spleen and brain [13].
organic peroxides), egzogenous and endogenous electrophilic Glutathione has been also analyzed with good result in plants tissues for
substances and also oxidized forms of other antioxidants for example example in maize seedlings [14] and in Brassica juncea [15].
vitamins E and C. Molecule of glutathione is also presented in very specific
selenoenzym- glutathione peroxidase, which is the most important Sample preparation
antioxidant protecting organism from hydrogen peroxide and lipid The critical issue during analysis of amino thiols represents sample
peroxides. GSH antioxidative activity appears also in reduction of thiol preparation, because they have huge oxidoreductive activity. Attention
groups of different amino acids and protects them from irreversible must be paid on blood withdrawal, refrigeration during sample
oxidation to sulfonic or sulfinic acid and losing their activities. processing and, for plasma determinations, immediate centrifugation
Moreover, glutathione can be bound to the proteins, leading to of the samples. Long isolation time of erythrocytes from blood,
the formation of glutathionylated proteins [1]. During the process preparation of different tissues and separation of subcellular organelles
of glutathionylation, the thiol groups of amino acids are bounded are accountable for errors in the determination of GSH and GSSG.

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GSH undergo non-enzymatic autoxidation at pH >7, and enzymatic Only a few methods (e.g. NMR) do not demand deproteinization.
conversion of GSH. This step is mediated by γ-glutamyl transpeptidase Acidification, addition of an organic solvent, such as acetonitryl,
which exhibit optimal activity at neutral pH. That compound has large acetone or methanol, and ultrafiltration are used for protein
oxidoreductive abilities because of huge reactivity of –SH group. elimination [2]. Organic solvents are preferable during the analysis
The measurement of GSSG concentration, especially in plasma with mass spectrometer [19]. Ultrafiltration is a useful method to
also demands caution and special sample preparation. After withdrawal remove proteins because it does not require the addition of acids
GSSG is degraded by proteolysis during less than 2 minutes [19]. or organic solvents that can affect the separation, derivatization and
Refrigeration during sample pretreatment and protein precipitation detection. The separation of GSH and GSSG can also be achieved
can minimize oxidation and proteolysis. by applying membrane filtration in combination with centrifugal
Of course, the differences in sample preparation stages depends microconcentration [20].
on type of the tissue, form of glutathione and methodology of analysis, Typically, to obtain plasma from blood samples it is necessary to
but there are some common bases for all methods. Sample preparation acidify blood immediately after venipuncture [21]. Acidification causes
divides into several stages however each method does not have to precipitation of proteins including undesirable enzymes disturbing in
contain all of them: analyze.
• sample collection and its protection Acidification also gives possibility to measure total glutathione
• precipitation of proteins concentration in each tissue by releasing free glutathione from
• blocking of free thiol groups, using alkaline or acidic reagents protein-GSH adducts [1]. There are two helpful techniques used in
• reduction of disulfides determination of glutathione conjugates with proteins (glutathionylated
• derivatization proteins) SDS-PAGE and MALDI-ToF analysis. However, they are
• Sample collection. qualitative or semiquantitative, what represents the main disadvantage
The most important aspect during this procedure is to protect of these procedures, thus the concentrations of the various
samples from coagulation process speeded up by calcium ions as protein–GSH adducts sometimes cannot be determined [2].
a cofactor of the blood clotting cascade. This undesirable process is Precipitation has been handled with several acids such as
reduced by adding to fresh collected blood the small amount of chelating trichloroacetic acid (TCA), trifluoroacetic acid, perchloric acid,
reagents such as K2EDTA, K3EDTA or heparin. Blood samples can be sulfosalicylic acid, picric acid, metaphosphoric acid, yielding a clear,
also collected to special tubes with chelator, protecting blood from protein-free supernatant after centrifugation. Sulfosalicylic acid and picric
coagulation induced by Ca2+ or thrombocytes during the contacts with acid are usually used at a concentration of 5% w/v, and metaphosphoric,
plastic or glass surface of test tubes. That substances also entrap many chlorine (VII) and trichloroacetic acids at a concentration of 1% w/v.
transition metals (including Fe2+) avoiding some oxidative reactions. However the major problem in such determination is oxidation of
Glutathione is usually studied in plasma, which contains only 0.5% thiols frequently leading to an overestimation of disulphides [13]. TCA
of the blood content, whereas erythrocytes contain 99.5%. Plasma seems to be the most useful deproteinization acid because it has been
concentration of GSH reflects concentration of this compound in observed that only 3-4 % of GSH are oxidized within 20 h at 0°C after
whole organism [1]. Glutathione analyzing in human plasma brings the addition of TCA to the sample [11]. The restoration of neutral-
some difficulties and improper sample preparing may completely alkaline pH in acidified samples also leads to a rapid decrease of thiol
alter analytical data. Because of to the fact that GSH concentration in concentration if no previous treatment with thiol-masking agents has
red blood cells is significantly higher (500 times) than in plasma this is been performed.
necessary to pay attention to protecting samples from haemolysis of
erythrocytes. Even dissolution of 1% of red blood cells may drastically Reduction of disulphides
increase plasma concentration of GSH. The determination of free plus bound GSH and other low molecular-
The second important aspect both during blood preparation mass aminothiols, the reduction of the disulfide bonds formed between
and sampling other tissues and organs is protecting them from high them and other thiols or proteins needs to be accomplished [19].
temperature. A drastic decrease of GSH concentration has been Precursor of GSH -cysteine can be detected only after reduction of all
observed during samples storage in room temperature as an effect of disulfides in biological sample Reduction can be achieved by: chemical
oxidation to GSSG even about several dozen percent after 5 minutes reaction (adding reductants), electrolysis or enzymatic reactions
from blood collecting. The temperature of samples storage depends (adding glutathione reductase or NADPH to the sample) [2, 22]. There
on time from preparation to chromatographic processing. Samples are are a variety of chemical reductants and the selection of the reducing
frozen from -20°C to -80 °C. reagent is very important for assay performance. For example, some
reductants can react with labeling reagents what is a source of wrong
Precipitation of proteins results. Reduced glutathione can undergo reoxidation process before
The essential stage during the samples collection is elimination of derivatization procedure what often leads to overestimation of GSSG
some undesirable enzyme, for example γ- glutamyl transpeptidase, and incorrect results.
which is responsible for glutathione catabolism. To avoid complete This phenomenon can be avoided with thiol-masking agents,
decomposition of analyzed thiol compounds it is necessary to remove such NEM [11, 23-27] or IAA [19, 28]. Oxidation is also catalyzed
immediately this enzyme and also other proteins disturbing in analyze. by metal ions (especially iron and cooper), therefore using chelating
The tissues with high concentration of γ-glutamyl transpeptidase such agents like EDTA 1,10 fenantrolin [2] or deferoxamine [11] during
as: kidneys and spleen should be frozen immediately after collection sample collection is required. For some kind of analysis, it is necessary
and homogenization, whereas the tissues with low concentration to remove completely the excess reducing agent before further
of this enzyme such as liver, spleen and brain do not have to be modification, for example by gel filtration or acid precipitation. The
homogenized. They should be frozen after collecting and storage reducing agents cannot also cross-react with derivative reagents
at -20 °C. Direct injection of biological samples into the HPLC or during the next stage of analysis what often leads to wrong results of
CE system is not recommended [19]. For example, the presence of analysis. Reducing agent must be also compatible with the thiol-specific
proteins in injected samples during CE analysis may led to adsorbing derivatization agent [29].
them on the capillary wall and affecting migration time, peak shape A quantitative elimination of reducing agent can be difficult
and detection response [20]. to obtain with acid precipitation however the disadvantage of gel

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filtration is regeneration of disulfides unless the buffer is kept at low than TCEP. They are also more reactive toward small-molecule [19].
pH. Furthermore, gel filtration could not be used during working with The esterification increases membrane permeability, allowing the
partially aggregated material [2, 30]. esters to penetrate phospholipid bilayers [2].

Thiol-containing reductants Derivatization

This group contains such reductants as: dithioerythritol, Every procedure of thiols analyzing except for vivo assays and those
dithiothreitol (DTT) and β-mercaptoethanol (ME).They are the based on electrochemical and tandem mass spectrometry, require
most often used reductants in aminothiols analysis, highly specific. derivatization. Derivatization of GSH usually increases the method
However the one of disadvantage of this group is that they can specificity and sensitivity. Derivatization reagent should react rapidly
cross-react with detection agents such as monobromobimane (MBB) and specifically with thiol group at lowest possible temperature and
or o-phthalaldehyde (OPA), leading to the formation of interfering weakly acidic pH to prevent oxidation of the analytes. Chromophore
fluorescent compounds [2, 19]. or fluorophore added to an analyte decrease the limits of detection
The other inconvenience is fact, that at optimal for that reactions or quantitation. Increase in sensitivity subsequent to derivatization
pH value above 7 oxidation can occur. The reagents are also sensitive result in improvement in chromatography delivers more analyte into
to oxidation so they must be protected from oxygen and maintain with the detector in a narrower chromatographic peak. Improvement in
metal chelating agents. sensitivity is only one function of analytical derivatization, but also
ME belongs to weak reagents and must be used at very high for the stabilization of thiols, ionization responses or induction of
concentration. ME is mainly used in the presence of sodium dodecyl a charge [20]. Derivatives are usually more lipophilic than the analyte
sulfate [2]. This reagent was used as a redactor in many applications: what is especially important for small molecules which are invariably
UV, fluorometric, CE, LCMS. DTT is stronger reducing agent than hydrophilic [33].
ME, used mostly in spectrophotometric techniques. An important
advantage of this reagent is its membrane permeability, which gives Reagents for UV-VIS detection
the possibilities of using it as a reductant in cellular systems. Ultraviolet detection is commonly used technique in high
N-acetyl-cysteine ethyl ester is strong disulfide bond-reducing performance liquid chromatography and capillary electrophoresis.
thiol. This reagent was used for the quantitative analysis of GSH and Analytical methods using colorimetric reagents and UV absorbance
GSSG in the cytosol of red blood cells as GSH-OPA derivative with detection are less sensitive, but simpler as compared with fluorescent
fluorometric detection [31]. or electrochemical detection. There are a number of thiol-reactive
Sodium and potassium borohydride (BH) are a very strong, highly reagents, commercially available or made in laboratories which react
reactive reducing agents and that is why their solutions should be with thiol functional group to produce UV absorbing derivative.
prepared immediately before use. They are also unstable in aqueous The compounds most commonly used are: maleimide-type
solution. Use of sodium borohydride may lead to problems with reagents to which N-ethylmaleimide (NEM) belongs: iodoacetic acid
derivatization reaction because of pH control difficulties [28, 29]. (IAA) and iodoacetamid (IA), yielding thioethers [19]. NEM is also used
Reduction of protein disulfides with high concentration of that reagents in combination with glutathione reductase-coupled enzymatic recycling
(1,4 mol/l) is complete after few minutes, while with the lower method [34-36], fluorescence detection and mass spectrometry for the
concentrations even 30 min. measurement of GSSG as the thiol-masking agent. The application of
BH is used in quantitative reduction of protein disulfides with or NEM presents difficulties due to the inhibition of glutathione reductase
without the presence of denaturing agents and also in reduction of by NEM. The reagent excess must therefore be eliminated either by
protein disulfides in whole cell extracts. The main advantage of BH is solvent extraction or by solid-phase extraction [19].
that excess reagent can be removed easily by the addition of acid or Reactions with NEM are typically performed at concentration
acetone. The solutions containing BH tend to foam but this problem 1 M and below pH 7 or at pH values above 7 if incubation times are
can be solved by adding surface active compounds, for example octanol prolonged to more than 2 hours. The instability in alkaline media
or hexanol [2,19]. caused by hydrolysis of the maleimide ring and the fact that some
low-molecular thiol adducts with NEM can undergo intramolecular
Trialkylphosphines transamidation to cyclic forms at pH values above 9 [2]. NEM also binds
This group has more advantages than reducing agents described amino groups at pH >7.5, even if more slowly as compared with thiol
above. Ttri-n-butylphosphine (TBP), triphenylphosphine (TPP) and functionalities and that is why during long incubations at alkaline pH,
tributylphosphine and tris (2-carboxyethyl) phosphine (TCEP) or if a derivatization agent of the amino group, such as 1-fluoro-2,4-
are strong reducing agents, active even in low concentration. dinitrobenzene is used the excess of NEM must be removed before the
Trialkylphosphines do not react with other functional groups of alkalization of the medium [20, 22]. Giustarini et all [37] developed the
amino acids furthermore they are unreactive with thiol alkylating method based on the analysis of GSH conjugate with NEM. Blood
and derivatization agents. However they are quite reactive toward samples treated with NEM were stable at −20 °C for 90 days.
iodoacetic acid, iodoacetamide and NEM even at low pH, thus the IA and IAA are also used as the thiol-masking agents for the
reactions with phosphines must be carried out in a step separate measurement of GSSG. These alklylating agents react irreversibly
from alkylation [2, 22]. with aminothiols in a nucleophilic substitution reaction formed
TCEP is the most popular reagent in this group used in analysis. Its S-carboxymethyl and S-carboxamidomethyl derivatives. They are
use may provide more reproducible assays than with TBP and TPP. This water-soluble and can be prepared at concentrations of 1 and
is water-soluble and nonvolatile reagent, used mostly for low-molecular 0.5 M, respectively at pH 8. They must be protected from light during
disulfide bonds and surface-exposed protein disulfides reduction. The storage. Unfortunately at neutral or alkaline pH they react with the
advantage of this reagent in comparison with thiol-containing reductants hydroxyl group of tyrosine, the amino group of lysine and imidazole
is its higher resistance to oxidation catalyzed by metal ions. However, group of histidine. Besides, at low pH both reagents react with the
TCEP can interfere with certain disulfide containing derivatizing agents, sulfur atom of methionine. However, the SH group reacts much faster
including 5, 5’-dithiobis-2-nitrobenzoic acid (DTNB) [19]. This reagent than any other group in the undesirable reactions. For example, IAA
was used by many authors in different analysis [9, 29, 32]. Mono-, di- reacts with histidine 1000-fold slower than with cysteine. Thus, the
and trimethyl ester analogs are more reactive at lower pH and lipophilic alkylation procedure assuming the use of these reagents seems to be

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thiol-specific. The disadvantages of IA in comparison with IAA are that The benzofurazans: ammonium 7-fluoro-2,1,3-benzoxadia-
the reagent is membrane impermeable and is inappropriate for thiols zole-4-sulfonate (SBD-F), 4-aminosulfonyl-7-fluoro-2,1,3-benzo-
determination in vivo and it can form undesired products [2, 19]. xadiazole (ABD-F) and 4-(N,Ndimethylaminosulfonyl)-7-fluoro-
Another alkylating agent is 2-vinylpyridine. This compound in 2,1,3-benzoxadiazole (DBDF) are representatives of this group.
contrast to NEM does not inhibit glutathione reductase and that is The activity of these reagents is in this order: DBD-F > ABD-F >
why is used as the alkylating agent in the DTNB–glutathione reductase SBD-F but both the selectivity to thiols and the solubility in aqu-
recycling assay. The optimum value of pH for VP is between 5 and 8 and eous solution are opposite: SBD-F > ABD-F > DBD-F [20]. The
the reactions require high excess of this reagent and long incubation unreacted reagents and its hydrolysis products are non-fluorescent
times [2]. Recently, 1-methyl-2-vinylpyridinium trifluoromethane the thiol adducts are stable. Thus, they exhibit excellent sensitivity
sulfonate and 1-methyl-4-vinylpyridinium trifluoromethane sulfonate and specificity towards sulfhydryl, resulting in no interfering reagent
have attracted attention as thiol-masking agents but they were used peaks [19]. SBD-F is the most popular reagent in this group used
only in spectrophotometric assays for analyzing thiols [19 ]. in different analysis [9]. The drawbacks of using SBD-F are: a long
5,5’-Dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman’s reagent) derivatization time and high temperature requirements (1h 60°C).
invented by Ellman in 1959 has been still the most common used Thiol–SBD-F derivatives are stable for at least 8 h when protected
reagent for the quantification of thiols in pre-column reactions from light [48]. In contrast, ABD-F offers fast and quantitative re-
[20]. It reacts with the thiolate anion in a thiol–disulfide exchange action under mild conditions, and is therefore suitable for reactions
reaction, resulting in the formation of the yellow derivate. DTNB prior to CE [19]. Their advantage is that they do not cross-react
is widely utilized for the derivatization of GSH and GSSG in the with phosphines; thus, disulfide reduction and thiol derivatization
classical spectrophotometric, enzymatic recycling or GSH-recycling can take place in the same step [2, 49, 50]. They have also much
assay developed by Tietze [34]. It is based on the reduction of GSSG higher selectivity to thiols than bimane. The drawbacks are facts
to GSH with glutathione reductase in the presence of NADPH and that benzofurazans react very slowly and that is why derivatization
formation of the colored product 5-thionitrobenzoate [19]. DTNB with this group requires drastic conditions (pH 9.5; 60°C for1h that
has been employed in several studies [35, 40, 41]. Alternative are likely to pose a risk of reoxidation of GSH [2]. The derivatives
application of DTNB using the reagent for post-column reaction with a benzofurazan structure are: 4-(N-acetylaminosulfonyl)-7-
was shown by Nozal et al. [42]. A lot of evidence has accumulated fluoro-2,1,3-benzoxadiazole, 4-(N trichloroacetylaminosulfonyl)-
that many protein sulfhydryls give an incomplete reaction with 7-fluoro-2,1,3-benzoxadiazole and 7-chloro-N-[2-(dimethylamino)
Ellman’s reagent, even during prolonged assay times. Reiner et al. ethyl]-2,1,3-benzoxadiazole-4- sulfonamide [51].
[43] solved this kinetic problem by including cystamine as Bimanes are found practical reagents which react rapidly with
a “mediator” between the protein sulfhydryl and Ellman’s reagent. analyzed thiols. One example of this group is monobromobimane
Another representative of this group are 4, 4’-dithiodipyridine which reacts rapidly, but not specifically, with thiols at pH 8.0 at room
(DTDP) and 2, 2-ditiopyridine which reacts with thiols in an exchange temperature [48].
reaction. DTDP is more sensitive and reactive thiol detection agent This reagent enables relatively high fluorescence emission and
than DTNB and it can be used at lower pH (≥ 4.5 instead of at pH 8.0), allows detection even in small concentration of analytes. The drawbacks
however resistance to hydrolysis is lower [2, 19, 20]. Due to its small are that the reagent itself and the hydrolysis products are fluorescent
size, amphiphilic nature, and lack of charge, DTDP quickly reacts and undergoes photooxidation Moreover they co-react with thiol-
with poorly accessible protein sulfhydryls [43]. containing reductants, other thiols and phosphines [2, 53]. It was used
As a derivatization reagent for UV detection, 2-chloro-1- in determination of glutathione in plasma [29, 37] whole blood and
methylquinolinium tetrafluoroborate (CMQT) is used for the saline [53] myoblasts in human multinucleated muscle fibers [54],
determination of different forms of plasma thiols: cysteine, red blood cells [55], mitochondria [56], human endothelial cells [57]
cysteinylglycine, glutathione and homocysteine. The CMQT- and astrocytes [58].
HPLC-UV method provides quantitative information on total, free Ortho-phthalaldehyde (OPA) can react with both the sulphydryl
and protein-bound thiols based on assays with derivatization after and amino group of thiols to form a highly fluorescence derivative [2].
reduction of whole plasma or its acid-soluble and acid-precipitated OPA exhibits no native fluorescence, but during the reaction of OPA
fractions. Samples were reduced with sodium borohydride with a thiol generates a thiol-2-alkyl-substituted isoindole which is
[44, 45] or tris (2-carboxyethyl) phosphine [29].This reagent was also highly fluorescent. It is monitored with an excitation wavelength of
used for determination of glutathione and other thiols in urine [46] 348 nm and an emission wavelength of 450 nm. OPA reacts with thiols
and saliva [47]. in thiol-selective and heterobifunctional reactions. In the thiol-selective
The derivatization reagents commonly used for determination OPA reaction this is necessary to use amine as a co-reagent prior to
of endogenous and exogenous thiols are: 2-halopyridinium and the derivatization reaction to achieve selective derivatization of thiols.
2-haloquinolinium salts which react rapidly with thiols in slightly alkaline Glycine, 2-aminoethanol, 2-mercaptoethanol, N-acetylcysteine,
water solution to form stable S-pyridinium or 3-mercaptopropionic acid or ethanethiol are commonly used as the
S-quinolinium derivatives. Reactions with these reagents enable co-reagents [19]. In the heterobifunctional reactions OPA reacts
multi thiol measurement within 1–15 min at room temperature [20]. directly with the compounds containing both thiol and amine groups
such as: GSH and γ-Glu-Cys without addition of the co-reagent [59].
Fluorescent thiol reagents However, co-reagents added to aminothiols direct the OPA reaction
Derivatization with fluorescent thiol reagents is widely used in selectively towards thiols [19]. GSSG cannot react with OPA to form
chromatographic or electrophoretic separation as a sensitive method fluorescent derivative but after its hydrolysis at pH 12 it is possible to
for the detection of thiols. Fluorescent detection allows much lower obtain fluorescent derivative [25]. Reactions with this reagent require
detection limits than UV. Reagent must form GSH adducts with mild reaction conditions and enable determination of some amino
fluorescence yield to permit the measurement of GSH at picomolar acids. However, simultaneous measurement of multiple aminothiols
amounts or less. Fluorophores that react with the thiol group are the with OPA involves alkylation of free thiols. OPA also has the advantage
most selective but they do not allow the detection of GSSG. Reagents of a short derivatization time at room temperature [39].
targeting the amino group allow the simultaneous fluorimetric Optimal pH for derivatization with this reagent is between 9.5 and
determination of GSH and GSSG, even if less selectively [19]. 12 [13, 60÷63].

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detection: Comparison with two other methods. Electrophoresis. 2000,
28(18), 3277–3283. on Preparative and Industrial
70. Araujo A., Saraiva, M., Lima, J.: Determination of total and oxidized glu-
tathione in human whole blood with a sequential injection analysis system.
Talanta. 2008, 74(5), 1511-1519.
Chromatography and Allied Techniques
(SPICA 2012)
30 September – 3 October 2012

Brussels, Belgium, Europe

Ewelina BŁOŃSKA-SIKORA is a graduate of the Medical University of
Lublin (Faculty of Pharmacy, 2008). Currently, she is a Ph.D. student of the
Institute of Chemistry, Jan Kochanowski University in Kielce where she deals
with the analysis of glutathione in human body. She has written 6 articles in the Since 26 years, this Symposium is the event to
scientific press and she is the author or co-author of three posters at national
and international conferences. attend to follow the state of the art in the field
Tel.: 63317189, e-mail: [email protected]
of Preparative and Industrial Chromatography.

Workshops will be proposed on the opening

Jerzy SZCZUDŁOWSKI – Ph.D., graduated from the Jagiellonian day, Sunday September 30th where worldwide
University. He is the author of 30 articles in international and Polish journals,,
several dozen lectures and communications on domestic and foreign scientific experts will share their experience to introduce
conferences, . The Council Member of Editorial of the Aparatura Badawcza
i Dydaktyczna journal. The member of Editorial in the Polish Normalization and describe the main fields of Preparative
Committee. The reviewer applications in Operational Programme
Innowacyjna Gospodarka. Currently, he performs function of the vice- Chromatography. During the symposium, the
director of Institute of Chemistry at Jan Kochanowski University in Kielce.
The scientific practice: Sweden, University of Umea - Department of Public 3 days sessions will present the latest innovations
Health and Environmental Studies, Environmental Chemistry and National
Defense Research The Establishment (1993) as well as the industrial trends at development and

production scales. The covered topics will range

from process development to industrial applications

Zygfryd WITKIEWICZ – Director of the Institute of Chemistry at Jan
Kochanowski University in Kielce. He also works for Military Technical
of the purification techniques. The latest advances
Academy in Warsaw. Professor is the chief editor of Aparatura Badawcza
i Dydaktyczna journal, edited by COBRABiD. He is the president of
in process modelling and innovative processes
Technical Committee responsible for Air Quality in Polish Normalization
Committee and a member of The Analytical Chemistry Committee in
(ie. multicolumn technologies) will be highlighted,
Polish Academy of Science and a member of the Chromatographic Analysis
commission, a member of PTChem, IUPAC and The Chromatographic
keeping the regulatory, environmental and economic
Society. He published over 250 papers and 7 books. He is co-author of
21 patents. Professor received the Cwiet Medal granted by the Russian aspects into consideration. The evolution of the
Chromatographic Association.

stationary phases will also be part of the program

as well as the latest trends in membrane, extraction

Dariusz WIDEŁ – M.Sc., graduated from the Mathematics and Sciences
Faculty at Jan Kochanowski University of Humanities and Sciences in Kielce
and other purification technologies.
(chemistry, 2009). He works on the assistantship in Physical Chemistry
Department at Institute of Chemistry, UJK. He is accomplishing the doctoral
thesis ,, Application of electromigration and chromatographic method in Web Site: http://www.icsc2011.fr/
waters and drinks analysis” under the direction of Prof. Zygfryd Witkiewicz.

942 • nr 9/2012 • tom 66