molecules

Review
Chondroitin Sulfate Safety and Quality
Nicola Volpi
Department of Life Sciences, Laboratory of Biochemistry and Glycobiology, University of Modena and Reggio
Emilia, 41125 Modena, Italy; nicola.volpi@unimore.it; Tel.: +39-59-2055543; Fax: +39-59-2055548

Academic Editor: Cédric Delattre




Received: 7 March 2019; Accepted: 9 April 2019; Published: 12 April 2019

Abstract: The industrial production of chondroitin sulfate (CS) uses animal tissue sources as raw
material derived from different terrestrial or marine species of animals. CS possesses a heterogeneous
structure and physical-chemical profile in different species and tissues, responsible for the various
and more specialized functions of these macromolecules. Moreover, mixes of different animal tissues
and sources are possible, producing a CS final product having varied characteristics and not well
identified profile, influencing oral absorption and activity. Finally, different extraction and purification
processes may introduce further modifications of the CS structural characteristics and properties
and may lead to extracts having a variable grade of purity, limited biological effects, presence of
contaminants causing problems of safety and reproducibility along with not surely identified origin.
These aspects pose a serious problem for the final consumers of the pharmaceutical or nutraceutical
products mainly related to the traceability of CS and to the declaration of the real origin of the active
ingredient and its content. In this review, specific, sensitive and validated analytical quality controls
such as electrophoresis, eHPLC (enzymatic HPLC) and HPSEC (high-performance size-exclusion
chromatography) able to assure CS quality and origin are illustrated and discussed.

Keywords: chondroitin sulfate; glycosaminoglycans; osteoarthritis; nutraceuticals; food supplements

1. Chondroitin Sulfate Structure

Hyaluronic acid (HA), keratan sulfate (KS), chondroitin sulfate (CS)/dermatan sulfate (DS) and
heparan sulfate (HS)/heparin are natural macromolecules and the main complex heteropolysaccharides
belonging to the class of glycosaminoglycans (GAGs) [1,2]. Apart from KS, which is composed of
galactose and N-acetyl-d-galactosamine (GalNAc) [3], the backbone of the other GAGs is commonly
represented by typical disaccharide sequences constituted of alternating units of uronic acids, glucuronic
(GlcA) or iduronic (IdoA), and amino sugars, N-acetyl-d-glucosamine (GlcNAc) or GalNAc [4]. KS,
CS/DS and HS/heparin are sulfated macromolecules possessing different degrees of charge density
thanks to the presence of sulfate groups in varying amounts and located in different positions. Moreover,
besides the variability in sulfate groups, these polysaccharides are very heterogeneous in terms of
molecular mass, physicochemical properties, and biological and pharmacological properties [1–4].
CS with different disaccharides and overall carbohydrate backbones may be biosynthesized
possessing various number and position of sulfate groups [4] (Figure 1). Consequently, CS is a largely
heterogeneous GAG formed of alternate and variously sulfated disaccharides of GlcA and GalNAc
linked by β(1→3) bonds. Moreover, the different disaccharides are linked to each other by β(1→4)
bonds. CSA and CSC are constituted by disaccharides sulfated in position C4 or C6 of the GalNAc
residue, respectively, and minor percentages of the monosulfated disaccharide on C2 of GlcA are
possible. Besides the main presence of these two kinds of disaccharide units monosulfated in position
C4 or C6 of GalNAc, disaccharides with a different number and position of sulfate groups can be
present, in various percentages, within the carbohydrate chains [4–6]. Various kinds of disulfated
disaccharides may be present in the CS structure in various amounts also in relation to specific animal

Molecules 2019, 24, 1447; doi:10.3390/molecules24081447 www.mdpi.com/journal/molecules

 Molecules 2019, 24, x FOR PEER REVIEW 2 of 13

C4 or C6 of GalNAc, disaccharides with a different number and position of sulfate groups can be
Molecules 2019, 24, 1447 2 of 13
present, in various percentages, within the carbohydrate chains [4–6]. Various kinds of disulfated
disaccharides may be present in the CS structure in various amounts also in relation to specific animal
sources
sources (as(as
in in cartilaginousororbony
cartilaginous bonyfishes,
fishes,see
see below),
below), such
such as
asthe
thedisaccharide
disaccharidedisulfated in in
disulfated position
position
C2 of GlcA and C6 of GalNAc (the so-called disaccharide D), in position C4 and C6 of
C2 of GlcA and C6 of GalNAc (the so-called disaccharide D), in position C4 and C6 of the GalNAc the GalNAcunit
unit (the disaccharide E) or C2 of GlcA and C4 of GalNAc (the disaccharide B) (Figure 1). Finally, a
(the disaccharide E) or C2 of GlcA and C4 of GalNAc (the disaccharide B) (Figure 1). Finally, a low
low percentage of the non-sulfated disaccharide is almost always present in the CS backbone while
percentage of the non-sulfated disaccharide is almost always present in the CS backbone while the
the fully tri-sulfated disaccharide is generally in trace amount [4–6].
fully tri-sulfated disaccharide is generally in trace amount [4–6].

Figure 1. Structure of the disaccharides present in chondroitin sulfate backbone constituted of

Figure 1. Structure
d-glucuronic acid and of the disaccharides presentlinked
N-acetyl-d-galactosamine in chondroitin
by β(1→3) sulfate
bonds.backbone constituted
The various of D-
disaccharides
are linked each other by β(1→4) linkages. Minor percentages of very rare disaccharides may alsoare
glucuronic acid and N-acetyl- D-galactosamine linked by β(1→3) bonds. The various disaccharides have
linkedgroup
a sulfate each other by β(1→4)
in position linkages.
C3 of Minor percentages
the glucuronic of verygroup.
acid. Ac, acetyl rare disaccharides may also have a
sulfate group in position C3 of the glucuronic acid. Ac, acetyl group.
Besides the above-illustrated CS structures, a peculiar CS polysaccharide known as CSB or DS is
known Besides
in nature,theformed
above-illustrated CS structures,
of disaccharide units ofaIdoA
peculiar
andCS polysaccharide
GalNAc known asinCSB
mainly sulfated or DS C4
position
withis aknown
minorinand
nature, formed
variable of disaccharide
content units of IdoA
of the disulfated and GalNAc
disaccharide mainly C4
in position sulfated in position
of GalNAc and C4
C2 of
with a minor and variable content of the disulfated disaccharide in position C4 of GalNAc and C2 of
IdoUA [4]. However, the biosynthetic processes generally produce highly modified oligosaccharide
IdoUA [4]. However, the biosynthetic processes generally produce highly modified oligosaccharide
domains separated by regions having a low-degree of structural modifications introducing further
domains separated by regions having a low-degree of structural modifications introducing further
heterogeneity. Consequently, the CS and DS chains may be hybrid polymers of low modified (CS) and
heterogeneity. Consequently, the CS and DS chains may be hybrid polymers of low modified (CS)
highly modified (DS) domains [4]. The sulfation heterogeneity introduces a great variability in the
and highly modified (DS) domains [4]. The sulfation heterogeneity introduces a great variability in
position of sulfateofgroups
the position sulfateand in CS charge
groups and in density.
CS chargeMoreover, theMoreover,
density. different number of disaccharide
the different number units
of
forming the CS polymer and the overall molecular weight generated are other key
disaccharide units forming the CS polymer and the overall molecular weight generated are other factors influencing
key
its properties. Finally,
factors influencing itsthe combination
properties. Finally,ofthe
thecombination
different sulfated disaccharides
of the different sulfatedand oligosaccharide
disaccharides and
oligosaccharide
domains producesdomains
a huge produces
number of a huge number of heterogeneous
heterogeneous CS polymersCS polymers possessing
possessing various,and
various, specific
specific and
specialized specialized
biological and biological and pharmacological
pharmacological functions [1,2].
functions [1,2].

2. Chondroitin
2. Chondroitin SulfateActivity
Sulfate Activity
CSCSis ais natural
a natural biomacromolecule abundantly
biomacromolecule abundantly distributed
distributedininvirtually
virtuallyallallinvertebrates
invertebrates andand
vertebrates
vertebrates (and (and consequently
consequently in in humans)
humans) and
and involved
involved inin manybiological
many biologicalprocesses
processes[1,5,7,8].
[1,5,7,8]. Based
Based on
on its organism-to-organism
its organism-to-organism and tissue-to-tissue
and tissue-to-tissue structural
structural diversity
diversity in length
in chain chain length and sulfation
and sulfation patterns,
patterns, CS provides specific biological functions at molecular, cellular and organ level
CS provides specific biological functions at molecular, cellular and organ level such as cell adhesion, such as cell
celladhesion,
division andcell differentiation,
division and differentiation,
morphogenesis, morphogenesis,
organogenesisorganogenesis
and neural networkand neural network
formation [9–11].
formation [9–11]. Moreover, CS is particularly abundant in all mammalian connective
Moreover, CS is particularly abundant in all mammalian connective tissues, especially in the cartilage, tissues,
especially
skin, in the ligaments
blood vessels, cartilage, skin, blood vessels,
and tendons ligaments
with different and tendons
structure with
and level different structure
of sulfation and
[12]. In addition
level of sulfation [12]. In addition to its conventional structural roles in the composition of
to its conventional structural roles in the composition of extracellular matrix and formation of organs
extracellular matrix and formation of organs and tissues, such as cartilages and bones, recent
and tissues, such as cartilages and bones, recent evidence demonstrates that CS chains are involved in
evidence demonstrates that CS chains are involved in important biological functions in inflammation,
important biological functions in inflammation, infection and wound repair [7–11].
infection and wound repair [7–11].
The biological effects of CS are related to its ability to interact with a wide variety of other
bio(macro)molecules including (but not limited to) matrix molecules, growth factors, protease inhibitors,
cytokines, chemokines, adhesion molecules and pathogen virulence factors via aspecific/specific sulfated
saccharide domains within the chains [11–13]. In fact, along with aspecific interactions due to its
numerous negative charges provided by the sulfate and carboxyl groups, CS is involved in specific
Molecules 2019, 24, 1447 3 of 13

binding to bioactive molecules for the presence of peculiar functional domain structures which are
formed by combinations of the various disaccharide units [11–13] (see Figure 1).
Osteoarthritis (OA) is a global issue affecting all known countries and ethnicities with huge
incidence and economic impact [14]. OA particularly affects the older population producing variable
degrees of limitation of movement and major activities of daily living. Moreover, OA is expected to
dramatically rise in the future, in particular for the population above 70 years of age [14–16], also
related to the observed increase of obesity in western populations [17,18]. Current treatments are
mainly based on pain reduction [19] and treatments having the aim to reduce the progression of
the disease, such as the S/DMOADs (structure/disease modifying anti-osteoarthritis drugs) [20,21].
CS inhibits the extracellular proteases involved in the metabolism of connective tissues, and it is
able to stimulate proteoglycans production by chondrocytes as well as reduce the cartilage cytokine
production but also to induce apoptosis of articular chondrocytes [8,22]. Furthermore, CS increases the
intrinsic viscosity of the synovial liquid and, in bones, it accelerates the mineralization process and
repair [8,22]. Overall, CS has a key role in articular and bone metabolism by controlling cartilaginous
matrix integrity and bone mineralization. Consequently, with others, treatment with CS is reported
to improve OA symptoms and to reduce the disease. In fact, in many published studies, CS has
demonstrated to be a symptomatic slow acting drug for the treatment of OA (SYSADOA) and to have
disease modifying properties in long-term treatments (so-called DMOAD) (see [20–24] for reviews).

3. Safety and Quality Concerns of Chondroitin Sulfate

CS, according to its nature, must be extracted from animal sources and submitted to purification
processes for commercial purposes [5,6]. Actual commercial production of CS is based on terrestrial
raw material, from bovine, porcine, and chicken, or marine sources such as cartilaginous fish, sharks
and skate, but also bony fishes ([5,6,25] and personal information). Moreover, a mix of all these
sources is possible, producing a CS final product with mixed characteristics and properties. The use of
possible not controlled raw animal material (tissues, bones and cartilages but also soft organs) poses
the problem of a final product with not controlled structure and poor reproducibility along with the
not well identified origin with the consequence of variable grade of purity, biological effects, presence
of contaminants, clinical efficacy and safety [5,6,26].
Besides the source material, manufacturing processes, the presence of contaminants and other
natural biomolecules, as well as many other factors, contribute to the quality, structure and properties of
the final product influencing CS overall biological, nutraceutical and pharmacological capacities. In fact,
as well known, CS possesses a complex structure strictly depending on the tissue, organ and species
but also on the age of the animals [5,6]. Moreover, the biological and nutraceutical properties may vary
with the structure as well as the oral absorption that is influenced by the different CS physicochemical
properties [27–29]. Furthermore, due to the extractive origin of CS and to the different purification
processes applied, the presence of bacteria, virus or prions cannot be excluded [30]. Additionally,
other various natural bioactive (macro)molecules may be present in different content as contaminants
in CS final extracts [5,6], and voluntary adulteration by artificial compounds is also known. Finally,
a restriction use related to religious issues is possible (see below for these CS concerns). Overall,
depending on the source origin and on the manufacturing processes, variable CS final products for
structure, quality and activity may be produced (Table 1) [5,6,26].
Molecules 2019, 24, 1447 4 of 13

Table 1. Main general characteristics and properties of animal CS for commercial purposes.

Variable and generally not defined and heterogeneous source of extraction

Possible cross-contamination between sources of different origin
Possible presence of bacteria, virus and/or prions
High content of proteins that are not characterized (up to 5–10%). Some of these proteins may have
allergenic potential able to develop immune reactions
Variable content of immunogenic keratan sulfate and other natural biopolymers
Variable purity
Heterogeneous structure and physicochemical properties. Variable molecular mass, polydispersity and
charge density
Process of extraction generally not controlled causing possible modifications of the CS structure, such as
desulfation and/or depolymerization
Possible intentional adulteration by artificial (macro)molecules
Possible batch-to-batch variability
In general, no evaluation of any biological activity

CS from various sources has a carbohydrate backbone formed of disaccharides with sulfate groups
in different percentages and positions (see Figure 1) producing polymers possessing different charge
densities. Furthermore, due to the biosynthetic processes, CS macromolecules with different grades of
polymerization may be generated with various molecular masses and polydispersity related to specific
tissues, organs and species. Because of these structural variations, and the further possible presence of
specific oligosaccharide sequences (and purity, see below) of the preparations for therapy applications
or in nutraceuticals, CS may have different biological properties and capacities [5,26]. According to
this structural variability, different and peculiar activities have been reported depending on the CS
structure [5]. Finally, bioavailability and pharmacokinetic have been reported to change depending on
CS structural properties and origin when orally administered during therapy [27–29].
Extraction and purification processes may further produce CS preparations having different
structures introducing modifications of the macromolecular characteristics and chemical properties by
controlled as well as undesired depolymerization, desulfation and/or chemical modifications [6].
Another key point is related to the accurate evaluation of the CS content and purity when the
preparation is produced for therapeutic or nutraceutical applications. In fact, the CS content and
purity may change according to the manufacturing processes and/or tissue sources. Depending on the
purification protocols, the extracts may have a variable content and purity of CS due to the presence of
polluting inorganic or organic side-products (Table 1).
All the above-mentioned aspects pose a serious problem for the pharmaceutical or nutraceutical
Companies having the necessity to declare the real origin of the active ingredients and the label
traceability of CS from the raw material up to the finished products. Moreover, an accurate label
indication of the CS content is of paramount importance to assure to the final consumer a real
dose administration also considering the possible intentional adulteration by potentially dangerous
(macro)molecules (see below) mimicking CS structure and properties.

3.1. Natural Contaminations

As already mentioned, commercial CS produced from animal sources requires long and complex
procedures of extraction and purification with the aim to eliminate or reduce the content of the other
(macro)molecules present as natural contaminants [26] (Figure 2). At the same time, contamination of
CS may be accidentally caused by the same extraction and purification processes if not well controlled.
Obviously, as more steps of purification are adopted having differential capacities in the elimination of
natural contaminants of various nature, CS final products with higher quality may be obtained. On the
Molecules 2019, 24, 1447 5 of 13

other hand, the production of high-quality CS of pharmaceutical grade generally requires high costs of
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production and24,
2019, 24, xx FOR
FOR PEER
quality PEER REVIEW
REVIEWcontrols.
analytical 55 of
of 13
13

Figure
Figure
Figure 2.
2. Natural
2. Natural
Natural contaminations
contaminations eventually
eventually
contaminations present
present
eventually in
in chondroitin
in chondroitin
present sulfate
sulfate preparations
sulfate preparations
chondroitin and
and responsible
preparations and
for responsible
reduced for
title reduced
and title
possible and possible
side-effects. side-effects.
responsible for reduced title and possible side-effects.

3.1.1. Polysaccharides
3.1.1. Polysaccharides
3.1.1. Polysaccharides
Depending
Depending
Depending on on
the the
on the animal
animal source
animal source and
and on
source on
andthe the
the extraction
onextraction and
and purification
and purification
extraction procedures
procedures
purification that
that generally
procedures that
aregenerally
complex are
andcomplex
require and require
long time, long
CS time, CS
extracts extracts
and finaland final
products products
for for commercial
commercial purposes
purposes
generally are complex and require long time, CS extracts and final products for commercial purposes are also
are also
polluted polluted with variable percentages of other polysaccharides that are co-extracted with
are also polluted with variable percentages of other polysaccharides that are co-extracted with CS its
with variable percentages of other polysaccharides that are co-extracted with CS CS
during
during
during its
preparation preparation
its(Figure 3). (Figure
preparation (Figure 3).
3).

Figure 3. Natural
Figure polysaccharides eventually present in
in chondroitinsulfate
sulfate preparations and the
Figure 3.
3. Natural
Natural polysaccharides
polysaccharides eventually
eventually present
present in chondroitin
chondroitin sulfate preparations
preparations and
and the
the
consequences on the chondroitin quality and title.
consequences on the chondroitin quality and title.
consequences on the chondroitin quality and title.

HAHA in particular, and DS with its carbohydrate backbonebackbone madeofofiduronic iduronic acid, have been
HA in in particular,
particular, and and DSDS withwith itsits carbohydrate
carbohydrate backbone made made of iduronic acid, acid, havehave been
been
detected
detected in in
raw raw materials
materials and and formulations
formulations and and
reported reported
in in scientific
scientific
detected in raw materials and formulations and reported in scientific papers [31] due to their
papers papers
[31] due [31]
to due
their to their
ubiquitous
ubiquitous
presence presence
in tissues
ubiquitous presence from in
in tissues
which from
tissues CS iswhich
from CS
generally
which CS is generally
generally produced.
isproduced. Moreover,
produced. Moreover,
KS has been
Moreover, KS
KS has
has been
been detected
detected in many
detected
in
batchesmany batches of CS produced from shark cartilage, averaging 16% of
in many batches of CS produced from shark cartilage, averaging 16% of the total GAGs [32,33] (Figure 4).
of CS produced from shark cartilage, averaging 16% of the the total
total GAGs
GAGs [32,33]
[32,33] (Figure
(Figure
4).
4). Indeed,
Indeed, as KSas
Indeed, is KS
as KS is
is involved
involved involvedin thein the
the structure
instructure
structure of
of the
of the the proteoglycan
proteoglycan
proteoglycan aggrecan
aggrecan
aggrecan located
located
located ininallall
in all cartilages,
cartilages,ititithas
cartilages,
has
beenhas been
been detected
detected detected
in extracts in
in extracts
extracts and
and finished
and finished finished preparations
preparations
preparations of variousof
of various
origin origin
various origin with
along along
alongCSwith
with CS
CS extracted
extracted extracted
from the
from
samefrom the
the same
same
cartilages cartilages
A total[34].
cartilages
[34]. of 15A
[34]. total
total of
Asamples, of 15
15 samples,
samples,four
including including
including
samples four
of samples
four samples
CS of
of CS
CS as
as laboratory laboratory
as reagents,
laboratoryone
reagents,
of CSone
reagents,
sample as asample
one sample of
of CS
food additive CS as
as aand
a food
food tenadditive
additive
samples and
andof ten
ten samples
samples
dietary of
of dietary supplements
dietarycontaining
supplements supplements CS, containing
containing CS,
were examinedCS,
were
for were examined
examined
the presence for
of KSfor by the presence
theusing
presence of
specific KS
of KS by using specific
by using specific
immunodiffusion immunodiffusion
andimmunodiffusion and enzyme-linked
and enzyme-linked
enzyme-linked immunosorbent assay
immunosorbent
(ELISA). Apart fromassay
immunosorbent assay
the three(ELISA).
(ELISA).
samples Apart
Apartof CSfrom
fromas the
the three
three samples
laboratory samples
reagents, of CS
ofall as
as laboratory
CSsamples laboratory
were found reagents,
reagents, all
all
to contain
samples
samples were
were found
found to
to contain
contain varying
varying amounts
amounts of
of KS
KS [34].
[34]. Finally,
Finally, itit is
is worth
worth mentioning
mentioning that
that KS
KS
varying amounts of KS [34]. Finally, it is worth mentioning that KS possesses immunogenic capacities
possesses immunogenic capacities able to
to develop immune reactions [35]. Depending on the animal
ablepossesses
to develop immunogenic
immune reactions capacities ableDepending
[35]. develop on immune reactions
the animal raw[35]. Depending
material used for onCS theextraction,
animal
raw
raw material
material used
used for
for CS
CS extraction,
extraction, HS
HS may
may also
also be
be present
present due
due to
to its
its comparable
comparable physicochemical
physicochemical
HS may also be present due to its comparable physicochemical properties with CS, in particular for its
properties
properties with
with CS,CS, in in particular
particular for for its
its nature
nature of of sulfated
sulfated heteropolysaccharide
heteropolysaccharide [12]. [12].
nature of sulfated heteropolysaccharide [12].
Molecules 2019, 24, 1447 6 of 13
Molecules 2019, 24, x FOR PEER REVIEW 6 of 13
Molecules 2019, 24, x FOR PEER REVIEW 6 of 13

Figure
Figure 4. Acetate
4. Acetate celluloseelectrophoresis
cellulose electrophoresis able
able to
to detect
detect the
the presence
presenceofof(A)
(A)keratan
keratansulfate (KS)
sulfate or or
(KS)
(B) Figure 4. Acetate
(B) dermatan
dermatan sulfate cellulose
sulfate
(DS)(DS) electrophoresis
and/orand/or heparan
heparan able
sulfate to detect
sulfate
(HS) (HS)
in the
in presence of (A)
chondroitin
chondroitin sulfate keratan
sulfate
(CS) (CS)sulfate (KS) or
preparations
preparations (material
(B)
owned dermatan
(material
by theownedsulfate
same by the(DS)
same
author). and/or heparan sulfate (HS) in chondroitin sulfate (CS) preparations
author).
(material owned by the same author).
TheThe presence
presence of of thesenatural
these naturalpolymers
polymers in in CS
CS products,
products, also
also ininhigh
highpercentages,
percentages, alerts thethe
alerts
The presence
manufacturers
manufacturers for for of theseisolation
improved
improved natural polymers
isolation in as
procedures
procedures CSwell
products,
as well also in high agencies
as supervisory
as the the supervisory percentages,
agencies alerts the
for better
for better audits.
manufacturers
audits. Inthis
In addition, for improved
addition,
findingthisalso
finding isolation procedures
also compromises
compromises theasdesired
the desired well as
amounts the
amounts supervisory
of the of agencies
theingredient
real real ingredient for better
specified
specified on the
audits.
onclaims
label In addition,
the label
andclaims this
forewarns finding
and forewarns also compromises
the pharmacopeias
the pharmacopeias the desired amounts
to update
to update their of the real
their monographs.
monographs. ingredient
In fact,
In fact, general specified
general
non-specific
on the label
non-specific
analytical claims
methods andmethods
analytical
such asforewarns suchtheaspharmacopeias to update
CPC (cetylpyridinium
CPC (cetylpyridinium chloride) theirand
chloride)
titration monographs.
titration and In
carbazole fact, are
carbazole
assay general
assay
unable
non-specific
are unable toanalytical
detect methods
the presence such
of as CPC
other (cetylpyridinium
polysaccharides in chloride)
CS productstitration
due
to detect the presence of other polysaccharides in CS products due to their quite similar properties and
to carbazole
their quite assay
similar
andare unable to
properties
chemical anddetect
structures the presence
chemical (see below of other
structures (see
for polysaccharides
below
further for furtherin
analytical CS products
analytical due to their quite
considerations).
considerations). Finally, the similar
Finally,
presencethe of
properties
presence ofand chemical
variable amountsstructures
of the (see below forKSfurther
immunogenic poses analytical
serious considerations).
problems related to Finally,
the the
possible
variable amounts of the immunogenic KS poses serious problems related to the possible development
presence
developmentof variable amounts ofadverse
of immunogenic the immunogenic KS poses
reactions (Figure 3). serious problems related to the possible
of immunogenic adverse reactions (Figure 3).
development of immunogenic adverse reactions (Figure 3).
3.1.2. Other Biomolecules
3.1.2. Other Biomolecules
3.1.2. Other Biomolecules
Other (macro)molecules possessing similar properties of CS are copurified during CS extraction
Other (macro)molecules possessing similar properties of CS are copurified during CS extraction
fromOther (macro)molecules
tissues, possessing
such as nucleic acids similar
[31] and properties
proteins (Figureof5).
CSInare copurified
fact, duringand
animal tissues CS organs
extraction
are
from
from
tissues, such asasnucleic
tissues,
acids [31]and
andproteins
proteins (Figure 5). In fact, animal tissues and organs
rich in DNA, such
RNA and nucleic acids
proteins, [31]
and variable content(Figure 5).biomolecules
of these In fact, animal
aretissues
presentand organs
in CS are
extracts
arerich
richininDNA,
DNA, RNA andand proteins, variable
and variable content ofbiomolecules
these biomolecules are
in present in CS
depending onRNA
the sourceproteins,
and on and content
the purification of these
protocols adopted. are some
Moreover, present CS proteins
of these extracts
extracts
may depending
depending on theon the
source
have allergenic source
and on
and/or and on the purification
the purification
intolerance able toprotocols
protocols
capacity adopted. adopted.reactions.
Moreover,
develop immune Moreover,
some of thesesome of
proteins
Finally, these
these
may
proteins have allergenic
may
macromoleculeshave alsoand/or
allergenic intolerance
and/or
give positive capacity
intolerance
response able to develop
capacity
to aspecific analyticalimmune
able to controlsreactions.
develop immune Finally,
reactions.
such as CPC these
assayFinally,
[6]
macromolecules
these macromolecules
(Figure 5). alsoalso
give positive
give response
positive to aspecific
response analytical
to aspecific controls
analytical such
controls as CPC
such assay
as CPC [6] [6]
assay
(Figure
(Figure 5). 5).

Figure 5. Natural biomolecules possibly present in chondroitin sulfate preparations and their related
Figure
5. 5. Natural
problems.
Figure Natural biomolecules
biomoleculespossibly present
possibly in chondroitin
present sulfate sulfate
in chondroitin preparations and their and
preparations related
their
problems.
related problems.
Molecules 2019, 24, 1447 7 of 13

Molecules 2019, 24, x FOR PEER REVIEW 7 of 13

3.1.3. Infective Agents
3.1.3. Infective Agents
The animal origin of CS may pose safety problems due to the possible presence of pathogen
The animal
contamination andorigin of CS mayinfective
transmissible pose safety problems
agents such due to the possible
as bacteria residues,presence of pathogen
viruses and prions
contamination and transmissible infective agents such as bacteria residues, viruses
potentially causing spongiform encephalopathy in bovines (BSE), foot-and-mouth disease, influenza and prions
potentially causing spongiform encephalopathy in bovines (BSE), foot-and-mouth disease, influenza
spread in birds and other animal diseases (Figure 5). The possible presence of infective agents in
spread in birds and other animal diseases (Figure 5). The possible presence of infective agents in the
the organs and tissues used for CS production requires the application of specific chemical steps
organs and tissues used for CS production requires the application of specific chemical steps able to
able to destroy or inactivate and eliminate bacteria, viruses and prions [36,37] possibly introducing
destroy or inactivate and eliminate bacteria, viruses and prions [36,37] possibly introducing chemical
chemical modifications in the CS structure such as degradation, desulfation, oxidation of carbohydrate
modifications in the CS structure such as degradation, desulfation, oxidation of carbohydrate chains,
chains, introduction
introduction of chemical
of chemical groups,
groups, etc. etc.
In fact, In fact,
prions prions
adhere adhere very
very tenaciously tenaciously
to surfaces, to surfaces,
making them
making
hard them hard toand
to remove, remove,
their and their resistance
resistance to proteaseto protease treatment,
treatment, certain chemical
certain chemical agents andagents
heatand
denaturation represents a major concern for pharmaceutical products derived from terrestrial
heat denaturation represents a major concern for pharmaceutical products derived from terrestrial
animals
animals [36].
[36]. Moreover,
Moreover, thepurification
the purificationsteps
steps useful
useful to
to remove
removethetheinfective
infectiveagents
agentscan
canalso affect
also thethe
affect
biological
biological capacities
capacities ofof extractedCS
extracted CS(Figure
(Figure6).6).

Figure
Figure 6. Common
6. Common possiblepathogen
possible pathogencontamination
contamination and
andtransmissible
transmissibleinfective agents
infective in in
agents chondroitin
chondroitin
sulfate
sulfate preparations
preparations and
and theireffects.
their effects.

Another
Another keykey point
point is isthe
theadoption
adoptionofofspecific
specific and selective
selectiveanalytical
analyticalassays
assaystoto
detect thethe
detect possible
possible
presence
presence of of
thethe differentinfective
different infective agents
agents in
in the
thefinal
finalproducts.
products.In fact, the the
In fact, presence of prions
presence may be
of prions may
easily demonstrated
be easily demonstrated with withaa diagnostic
diagnostictest.
test.However,
However,the thecurrent
currentavailable
availableanalytical assays
analytical areare
assays
characterized
characterized byby high
high costsand
costs andlimited
limitedto
tospecialized
specialized laboratories
laboratories[38].
[38].Finally, other
Finally, pathogens
other pathogens show
show
similar attributes as prions for the potential risk of carry-over into final purified products.
similar attributes as prions for the potential risk of carry-over into final purified products.

3.2.3.2. Intentional
Intentional Adulterations
Adulterations
Besides
Besides thethe presence
presence ofofnatural
naturalcontamination,
contamination, finished
finished CS
CSpreparations
preparationsmay
may also bebe
also adulterated
adulterated
by the voluntary addition of cheaper polysaccharides and organic derivatives unable
by the voluntary addition of cheaper polysaccharides and organic derivatives unable to be determined to be
with usual analytical controls utilized in the nutraceutical industry (Figure 7). In fact, CS isCS
determined with usual analytical controls utilized in the nutraceutical industry (Figure 7). In fact, well
is well known to be one of the most adulterated supplements in the market, a widespread and
known to be one of the most adulterated supplements in the market, a widespread and probably
probably underestimated problem, abetted by the lack of industry-wide specific and sensitive quality
underestimated problem, abetted by the lack of industry-wide specific and sensitive quality control
control analytical methods with several of the adulterated agents going undetected [6,39].
analytical methods with several of the adulterated agents going undetected [6,39].
Molecules 2019, 24, 1447 8 of 13
Molecules 2019, 24, x FOR PEER REVIEW 8 of 13

Figure
Figure 7. Artificially
7. Artificially adulteratedchondroitin
adulterated chondroitinsulfate
sulfate by
by identified
identifiedsubstances
substancesand
andtheir consequences.
their consequences.

Approximately
Approximately 80–90%
80–90%ofofthe theCSCSsupplied
supplied to to the US and
the US andEurope
Europemarkets
marketsisisproduced
produced in in China
China
andandintentional
intentional adulteration is possibly practiced by nutraceutical manufacturers globally with theaim
adulteration is possibly practiced by nutraceutical manufacturers globally with the
aim to reduce
to reduce ingredientingredient
costs bycosts by using
using cheapercheaper substances
substances [40].[40].
TheThe price
price pressure
pressure is isalso
alsoresponsible
responsiblefor
poor for poor purification procedures with the production of CS preparations rich in salts and having
purification procedures with the production of CS preparations rich in salts and having lowlowtitle
buttitle
alsobut also to minimize
to minimize analytical
analytical controls controls
to onlytocommon
only common non-specific
non-specific and inexpensive
and inexpensive tests tests
[40].[40].
In this
In this some
scenario, scenario,
rawsome raw and
material material andsupplement
dietary dietary supplement
products products
containcontain
less thanlessthethan the claimed
claimed amount
amount
of CS, in someof CS, in some
cases cases
as little as little[41,42]
as 5–10% as 5–10% with[41,42]
a huge with a huge
fraud fraud to
to clients, clients,and
traders traders and
consumers
consumers (Figure 7). In fact, many studies available in scientific literature
(Figure 7). In fact, many studies available in scientific literature demonstrate the poor quality and a demonstrate the poor
titlequality and a titletonot
not conforming conforming
label declaration to of
label
many declaration
CS finishedof many
productsCS finished
in several products
countries in [6,41–45].
several
countries [6,41–45]. of specific analytical methods such as eHPLC (enzymatic HPLC), HPSEC
The application
The application
(high-performance size-exclusionof specific analytical methods such
chromatography) andaselectrophoresis
eHPLC (enzymatic HPLC),
(Figure 4 and HPSEC (high-for
see below
performance size-exclusion chromatography) and electrophoresis (Figure 4 and see below for
analytical methods) on commercially available nutraceuticals has demonstrated the poor quality of
analytical methods) on commercially available nutraceuticals has demonstrated the poor quality of
many of these products in the global market showing a lower concentration of CS than specified
many of these products in the global market showing a lower concentration of CS than specified on
on the label as well as their intentionally adulteration [39,41,42]. The adulteration is obtained with
the label as well as their intentionally adulteration [39,41,42]. The adulteration is obtained with
molecules that may interfere with aspecific widely-used analytical methods of CS quantitation, such as
molecules that may interfere with aspecific widely-used analytical methods of CS quantitation, such
the asCPC
the titration
CPC titrationand carbazole
and carbazole assay, to give
assay, an artificially
to give inflated
an artificially inflatedestimate
estimate ofofCS CSconcentration
concentration[6].
Many (macro)molecules have been identified for CS adulteration,
[6]. Many (macro)molecules have been identified for CS adulteration, such as carrageenan,such as carrageenan, proteins
proteinsand
and surfactants,
surfactants, cheaper cheaper polysaccharides,
polysaccharides, sodium
sodium alginate,propylene
alginate, propylene glycol
glycol alginate
alginatesulfatesulfatesodium,
sodium,
sodium
sodium hexametaphosphatecommonly
hexametaphosphate commonly known knownwith withthethename
name of of
Calgon
Calgon andand
usedused as a as
detergent or
a detergent
water-treatment additive [39], maltodextrin and lactose [42] (Figure 7). These substances can be be
or water-treatment additive [39], maltodextrin and lactose [42] (Figure 7). These substances can
separated
separated from from
realreal
CSCS
by by their
their difference
difference in in electrophoretic
electrophoretic mobility(Figure
mobility (Figure4)4)andandaacorrect
correcttitle
titleof
of CS
mayCSbemay be obtained
obtained by other
by other specificspecific analytical
analytical approaches
approaches suchsuch as eHPLC
as eHPLC andand HPSEC
HPSEC (see
(see below),and
below),
notandwithnot with commonly-used
commonly-used methods methods [6,39,41,42]
[6,39,41,42] (Figure
(Figure 7). 7).

4. Analytical
4. Analytical Quality
Quality Controls
Controls
CS CSforfor pharmaceuticalapplications
pharmaceutical applications isis strictly
strictly evaluated
evaluated forfor quality,
quality, content,
content,structural
structural
characterization
characterization and
and parameters(such
parameters (suchas
ascharge
charge density
density and
andmolecular
molecularmass)
mass) byby
means
means of of
sensitive,
sensitive,
specific, validated and published analytical approaches [6]. These features are of
specific, validated and published analytical approaches [6]. These features are of paramount importance paramount
importance
to ensure to ensurereproducibility
therapeutic therapeutic reproducibility
and safe use,andand
safe use, and to protect
to protect patientspatients from ineffective
from ineffective and/or
and/or unsafe and potentially dangerous drugs. As discussed below, is it possible to affirm the same
unsafe and potentially dangerous drugs. As discussed below, is it possible to affirm the same for food
for food and nutraceutical CS preparations?
and nutraceutical CS preparations?
European and USA pharmacopoeias require some analytical tests for CS identification such as
European and USA pharmacopoeias require some analytical tests for CS identification such
infrared spectroscopy, specific optical rotation, intrinsic viscosity, electrophoresis and CPC titration
as infrared spectroscopy, specific optical rotation, intrinsic viscosity, electrophoresis and CPC
[46,47]. Some of these assays are very old and usually no longer used in modern quality control
titration [46,47]. Some
laboratories due to of these
their lackassays are very old
of specificity, lowand usuallyand
sensitivity no longer used in modernInquality
poor reproducibility. control
particular,
laboratories due to their lack of specificity, low sensitivity and poor reproducibility. In particular,
infrared spectroscopy and specific optical rotation cannot give specific information belonging to
Molecules 2019, 24, 1447 9 of 13
Molecules 2019, 24, x FOR PEER REVIEW 9 of 13

manyinfrared
other spectroscopy and specific
(macro)molecules, such asoptical rotation cannot
the absorption give specific
of chemical groups information
in the spectrum belonging to
of infrared
many other(for
wavelengths (macro)molecules, such as the[48]
infrared spectroscopy) absorption of chemical
or the capacity to groups in the
rotate the spectrum
plane of infrared of
of polarization
wavelengths (for
plane-polarized lightinfrared spectroscopy)
by chiral [48] or the such
chemical compounds capacity to rotate the plane
as carbohydrates of polarization
(for optical rotation)of[49].
plane-polarized
Intrinsic light by chiral is
viscosity measurement chemical
a very compounds
old technique suchforasthe
carbohydrates
determination (forofoptical rotation) [49].
the molecular weight
of aIntrinsic
polymer viscosity
replacedmeasurement
by the more is a modern
very old technique for thechromatography
size-exclusion determination of the molecular
coupled withweight
various
of a polymer
detectors replaced
[50] (Figure 8). by theindustry-wide
The more modern method
size-exclusion chromatography
for analysis of CS is CPC coupled with various
titration. However,
detectors [50] (Figure 8). The industry-wide method for analysis of CS is CPC titration. However,
several flaws in this old testing method show relevant limits, as it can be tricked by the use of natural
several flaws in this old testing method show relevant limits, as it can be tricked by the use of natural
macromolecules and/or artificial adulterants and cannot distinguish between CS and other material
macromolecules and/or artificial adulterants and cannot distinguish between CS and other material
having similar physicochemical properties, e.g., nucleic acids, anionic proteins, other polysaccharides,
having similar physicochemical properties, e.g., nucleic acids, anionic proteins, other
etc. [6]. Moreover, CPC assay is also influenced by the molecular weight of tested CS as well as for
polysaccharides, etc. [6]. Moreover, CPC assay is also influenced by the molecular weight of tested
theCScarbazole assay that is another non-specific quantitative test [6]. On the contrary, agarose-gel
as well as for the carbazole assay that is another non-specific quantitative test [6]. On the contrary,
electrophoresis according to European
agarose-gel electrophoresis accordingpharmacopoeia, electrophoresis
to European pharmacopoeia, on acetate of on
electrophoresis cellulose
acetate(ACE)
of
according
cellulose (ACE) according to US pharmacopoeia, and HPSEC and eHPLC according to the AOAC of
to US pharmacopoeia, and HPSEC and eHPLC according to the AOAC (Association
(Association
Official of Official
Analytical Chemists) Analytical
[51] are Chemists) [51] are
highly specific highly specific
analytical analytical
approaches for theapproaches
determinationfor the
of CS
determination of CS quality, quantity, chemical properties and structure.
quality, quantity, chemical properties and structure.

Figure
Figure 8. Specific
8. Specific analytical
analytical procedures
procedures able
able to distinguish
to distinguish between
between chondroitin
chondroitin sulfate
sulfate produced
produced from
from different
different origin
origin and and toCS
to assess assess CS quality
quality and
and real real quantity.
quantity.

Besides
Besides thethelow
lowtitle,
title, also
also origin
origin of ofCS
CShashasthe been
the reported
been reportedto betoabe
puzzle and inand
a puzzle general not
in general
notdeclared
declared onon
product
productlabels [6]. The
labels [6].origin of the CS
The origin ofutilized
the CSinutilized
a finishedinproduct
a finishedcan only be detected
product can only
by ratherby expensive
be detected rather expensiveand detailed
and detailedbiochemical/structural
biochemical/structural analyses by by
analyses determining
determining thethe
disaccharide/oligosaccharide pattern and molecular mass parameters
disaccharide/oligosaccharide pattern and molecular mass parameters [6]. According to this, [6]. According to this, a a
combination of specific analytical methods to define the final quality, origin and
combination of specific analytical methods to define the final quality, origin and properties of the product properties of the
product isSuch
is required. required.
methods Such methods
include include electrophoresis,
electrophoresis, HPSEC,enzymatic
HPSEC, selective selective enzymatic
treatmenttreatment
and further
and further separation of the constituent disaccharides/oligosaccharides
separation of the constituent disaccharides/oligosaccharides using sophisticated techniques including using sophisticated
techniques
capillary including capillary
electrophoresis [31,52,53], electrophoresis [31,52,53],
HPLC [5,6,54,55], FACE HPLC [5,6,54,55], FACE (Carbohydrate
(Fluorophore-Assisted Fluorophore-
Assisted Carbohydrate Electrophoresis) [42,56] and mass spectrometry [57]. CS profile is achieved using
Electrophoresis) [42,56] and mass spectrometry [57]. CS profile is achieved using well-established
well-established multi-analytical laboratory techniques, particularly electrophoresis, HPSEC and
multi-analytical laboratory techniques, particularly electrophoresis, HPSEC and eHPLC (Figure 8) [5,6]
eHPLC (Figure 8) [5,6] due to their high throughput, sensitivity and reproducibility. These
due to their high throughput, sensitivity and reproducibility. These techniques, applied before and
techniques, applied before and after enzymatic digestion of CS into smaller components, can
after enzymatic digestion of CS into smaller components, can distinguish between CS derived from
distinguish between CS derived from different animal or marine sources based on disaccharide
different animal
content, or marine
patterns sources
of sulfation and based on disaccharide
molecular size of thecontent,
polymerpatterns
(Figure of 8) sulfation
also usingand molecular
analytical
sizestandards
of the polymer
of CS of different origin (see below). However, a big problem remains for the below).
(Figure 8) also using analytical standards of CS of different origin (see sure
identification
However, of the origin
a big problem of CS when
remains for themixed
sure source materialof
identification is the
usedorigin
to its production
of CS whenormixedin the case
source
of possible cross-contamination.
material is used to its production or in the case of possible cross-contamination.
The use of animal-derived sources to produce commercial CS also represents a concern for
vegetarians and people with dietary restrictions related to religious and supply issues. In fact,
Molecules 2019, 24, 1447 10 of 13

religious reasons and/or the practice of abstaining from the use of animal products have precluded the
introduction of CS dietary supplements both in key emerging markets, such as Middle East and Asia,
and in consolidated markets.
The application of the above-illustrated multi-analytical techniques, electrophoresis, HPSEC and
eHPLC requires highly qualified laboratories equipped with dedicated instrumentation and qualified
technicians with specific analytical know-how producing very expensive activity and elevated costs
for high quality CS management (Figure 8). Moreover, an accurate and reproducible quantitative assay
requires reference standards with high specificity, purity and well-known physicochemical properties
and structures [6]. This is not a simple task in the case of CS due to its high heterogeneity of structure
and properties mainly depending on the source and purification protocols, as previously discussed.
However, several reference standards are commercially available, such as the CS standard from bovine
cartilage manufactured by Bioiberica (www.bioiberica.com/) and possessing a purity greater than
98% and approved in 2004 as Chemical Reference Substance (CRS) by the European Pharmacopeia
Commission [6]. Furthermore, other standards are available such as the European pharmacopoeia
reference standard of CS sodium of marine origin (https://www.sigmaaldrich.com/) or the CS sodium
salt from shark cartilage (https://www.sigmaaldrich.com/).

5. Conclusions
In 2008, heparin manufactured in China was intentionally adulterated with an oversulfated CS
polymer having a very high charge density quite similar to natural heparin, which provoked hundreds
of deaths in Europe and USA after intravenous administration [58]. As for heparin, an important
natural drug derived from animals, intentional adulteration of CS is difficult to detect since the tissue
supply chain for CS in slaughterhouses generally lacks current good manufacturing processes (cGMP)
oversight assuring high-quality CS production along with specific analytical quality controls. Moreover,
as mentioned above, CS is derived from a variety of animal tissues potentially containing infectious
agents leading to the transmission of viral, bacterial and prion diseases and the intentional or accidental
mixing of CS from different animal species is highly suspected. In fact, some countries do not have a
strict biosecurity system able to reduce the risk of the introduction and spread of disease agents.
Even if specific and high-level analytical methods are generally able to distinguish from various
sources, the detection of the presence of small amounts of different raw material remains very difficult.
Additionally, CS structure can vary with environmental factors, animal subspecies, age and/or tissues
providing additional difficulties for its quality and properties standardization. Moreover, the missing
guidelines allow manufacturers of finished products to often change the sources of supply of the
animal tissues or CS raw materials for competitive advantages. Consequently, the urgent solution to
this scenario is the introduction and utilization in the current market of validated, specific, scientifically
sound and largely published analytical controls. Manufacturers should ensure traceability of raw
material right through to the finished product, with analytical evaluation made of each lot supplied to
guarantee same quality, reproducibility and safety, as well as avoid misleading label information on
finished products as reported in the literature.
In conclusion, specific and accurate analytical procedures should be enforced for the control of
high-quality products and applied by quality control laboratories to confirm the purity and label claims
of CS in raw materials and finished products for food and nutraceutical applications. Finally, there
are no definite regulations and common acceptance governing the origin of the ingredients in these
supplements and the origin of the ingredients in products of natural and extractive origin is one of the
most important factors ensuring quality, safety and efficacy.

Funding: This research received no external funding.

Conflicts of Interest: The author declares no conflict of interest.
Molecules 2019, 24, 1447 11 of 13

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