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Chapter 5
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY –
AN EFFECTIVE TOOL FOR QUALITY CONTROL OF
NATURAL CHOLESTEROL-LOWERING AGENTS
Ana Mornar*, Miranda Sertić and Biljana Nigović
Faculty of Pharmacy and Biochemistry University of Zagreb A.
Kovačića 1, 10 000 Zagreb, Croatia
ABSTRACT
Hyperlipidemia is a heterogeneous group of disorders characterized by an excess of lipids
in the bloodstream. Regardless of the cause, it is a major modifiable risk factor for
artherosclerosis or coronary artery disease. The most commonly used medication for the
treatment of high cholesterol levels are statins such as atorvastatin, simvastatin and
lovastatin. Although safe and effective, statins can cause muscle problems, lung and liver
disorders as well as kidney damage. Therefore, many patients seek alternative therapies
to control their cholesterol levels. Many natural cholesterol-lowering agents are widely
available to the public as dietary supplements. A dietary supplement, also known as a
food supplement, is a preparation intended to supplement the diet and provide nutrients.
Dietary supplements are usually used by patients at their own discretion, in an
unmonitored setting and without the input of their physicians. Dietary supplements are
readily available, not classified as over-the-counter medications, and not regulated as
such. Health practitioners and patients often consider these products safe and probably
effective. Unfortunately, information on dietary supplements in nonmedical literature and
even in scientific literature is usually unreliable. Moreover, numerous studies have used
products that were not well characterized. There is no uniform legislation in the dietary
supplements area despite their ever growing popularity and presence on the market.
Health safety, nutritional value and laboratory control of declared content is very rare.
Therefore, greater attention has recently been given to quality control of dietary
supplements. Today advance analytical techniques can be employed for identification and
quantification of active compounds as well as toxic ingredients in dietary supplements.
Due to its superior precision, high resolution and capacity to analyze thermally labile and
non-volatile compounds, high performance liquid chromatography is applied for the
* * Corresponding author: Email: amornar@pharma.hr.
Ana Mornar, Miranda Sertić and Biljana Nigović
quality control of dietary supplements. In this chapter a critical review on
chromatographic methods for quality control of most frequently used natural cholesterol-
lowering dietary supplements such as red fermented rice, artichoke, phytosterols, omega-
3 fatty acids, green tea, soybean, gugulipid, coenzyme Q10, taurine, flax seeds and
policosanol is given. Special attention is paid to determination of active ingredients as
well as toxic compounds. Additionally, sample preparation procedures and
chromatographic methods used for determination of active ingredients in biological fluids
are discussed.
Keywords: high performance liquid chromatography, hyperlipidemia, natural cholesterol-
lowering agents
INTRODUCTION
Hyperlipidemia is a metabolic syndrome characterized by diverse lipid profiles (e.g.
hypercholesterolemia, hypertriglyceridemia and familial combined hyperlipidemia) and may
have significant adverse effects on health such as atherosclerosis, cardiovascular diseases,
diabetes and obesity. Generally, hyperlipidemias are divided in two groups, primary and
secondary subtypes. Primary hyprelipidemias are probably genetically based, while secondary
hyperlipidemias may result from diseases such as diabetes, thyroid disease, renal and liver
disorders, Cusing’s syndrome as well as obesity, alcohol consumption, estrogen
administration and other drug-associated changes in lipid metabolism. The positive influence
of cholesterol-lowering drugs on decreased progression of cardiovascular diseases is clearly
established. Therefore, cholesterol-lowering therapy is essential for the prevention of the
progression of cholesterol-loaded plaques in vessel linings. Medications used to treat high
cholesterol levels are statins, fibrates, nicotinic acid and its derivatives. The most popular and
commonly prescribed cholesterol-lowering agents are statins, the 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA) reductase inhibitors. These drugs are generally well tolerated.
However, adverse events associated with their use such as myalgia, myopathy, myositis and
elevation of serum liver enzyme levels are well known [Mahamuni et al., 2012; Raza et al.,
2004]. Therefore, many patients seek alternative therapies to control cholesterol levels. There
is a growing number of commercially available dietary supplements that claim to be
beneficial for management of hyperlipidemia. These products are especially suited for
patients whose blood cholesterol levels are marginally high, but not high enough to warrant
the prescription of cholesterol-lowering medication and for treating patients who are unable to
tolerate statin therapy due to statin-associated myalgias. Furthermore, they are convenient for
patients who refuse to take synthetic cholesterol-lowering drugs because of philosophical
reasons [Chen et al., 2011].
A dietary supplement is a product taken by mouth that contains a ˝dietary ingredient˝
intended to supplement the diet. These products may contain vitamins, minerals, fiber, fatty
acids, amino acids, herbs, herbal extracts or substances such as enzymes and metabolites.
Since 1990s there is a sharp upward trend in the use of dietary supplements. The reasons for
their popularity are numerous. Both health practitioners and patients consider them as a
traditional mode of healing, safe and effective. They are easy accessible, relatively
inexpensive, ˝natural˝ products that are purported to improve or prevent a number of
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High Performance Liquid Chromatography – An Effective Tool for Quality Control …
conditions. Generally, dietary supplements, especially the ones that contain herbs and
vitamins, are assumed to be milder and safer than human-derived medications. Moreover,
they are usually used by patients at their own discretion, without the need for physician visit,
lifestyle changes or unpleasant treatments and procedures. They are readily available in
pharmacies, grocery stores and health food stores as well as by mail and via the Internet. The
manufacturers of dietary supplements aggressively promote their products to the public in
journals, magazines, on television and radio as well as over the Internet [Ashar et al., 2008].
Unfortunately, some of the products are promoted with unsubstantiated claims of benefit and
with any mention of potential toxicity, influence on pregnancy and breastfeeding, interactions
with drugs or quality control [Petroczi et al., 2011]. An additional problem related to
production and quality of botanical dietary supplements is that the composition and contents
of active as well as toxic ingredients in plants vary depending on the variety of plant and
environmental conditions such as season, climate, temperature, light conditioning, humidity,
soil, pest attack and several other factors. There is no uniform legislation in the dietary
supplements area despite their ever growing popularity and presence on the market. The
Dietary Supplement Health and Education Act of 1994 permits manufactures of dietary
supplements to sell their products without pre-market evidence of safety or efficacy. Hence,
they are not required to get approval by US Food and Drug Administration (FDA) or to
register their products with the FDA before producing or selling dietary supplements. The
manufacturers of dietary supplements or dietary ingredients are alone responsible for ensuring
that their product is safe before it is marketed. They just have to make sure that the product
label information is truthful and not misleading [Fontanarosa et al., 2003]. Taking into
consideration this increment in the use of dietary supplements worldwide and dramatic
variation from labeled values found in many products, it is obvious that the quality control of
dietary supplements has become an important concern for both health authorities and the
public.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
High performance liquid chromatography (HPLC) has proved to be an extremely
versatile technique for analysis of various active and toxic ingredients in dietary supplements
due to its superior precision and high resolution. It is especially suited for the analysis of
thermally labile and non-volatile ingredients of dietary supplements. Furthermore, it is
comprehensive in its qualitative and quantitative assessment of quality consistency as it can
be successively combined with a wild variety of detectors providing additional analytical
information about samples. The most commonly used detectors include diode array detector
(DAD), flourescence (FLD), electrochemical (ECD) and mass spectrometric (MS) detectors.
Except availability of more selective and sensible detectors for HPLC, a various
chromatographic columns, providing a different combination of hydrophobicity, silanol
activity, hydrolytic stability and interaction with analytes, are commercially available.
Furthermore, the appearance of ultra performance liquid chromatography (UPLC), a variant
of HPLC with chromatographic columns with particle size lower than 2 μm, has increased the
analysis efficiency, so that shorter analytical time and better separation have been achieved.
Taking into consideration all above mentioned advantages of HPLC technique, it is not
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Ana Mornar, Miranda Sertić and Biljana Nigović
surprising that the HPLC has become a common technique in the analytical laboratories
worldwide dealing with quality control of dietary supplements. Numerous HPLC methods
have been developed to determine active and toxic ingredients of dietary supplements.
Although several reviews of analytical methods for the determination of statins in
pharmaceutical formulations and biological samples have been given, a review of
chromatographic techniques for the quality control of natural cholesterol-lowering agents has
not been published yet [Nigović et al., 2012; Novakova et al., 2008]. Therefore, the purpose
of this chapter was to summarize the relevant literature on chromatographic methods for
quality control of most frequently used natural cholesterol-lowering dietary supplements such
as red fermented rice, artichoke, phytosterols, omega-3 fatty acids, green tea, soybean,
gugulipid, coenzyme Q10, taurine, flax seeds and policosanol.
Red Yeast Rice
Red fermented rice is one of the most popular natural hypolipidemic agents which has
found its way from Traditional Chinese medicine to modern, commercially available drugs,
called statins. Red fermented rice is obtained by the fermentation process of rice (Oryza
sativa, L.) substrates with a mold Monascus purpures. During the fermentation process a
group of substances, called monacolins, is produced. One of the 14 known monacolins is
monacolin K [Endo, 1980], which is structurally identical to commercially available drug
lovastatin, registered by the FDA in 1987 as Mevacor®. Monacolin K (lovastatin) acts as an
inhibitor of HMG-CoA reductase, the rate-limiting enyzme in the biosynthesis of cholesterol.
Besides monacolins, red fermented rice can contain other lipid lowering substances such as
saturated fatty acids, phytosterols, vitamin B, isoflavons etc.
Many research and clinical trials were conducted to assess efficacy of red fermented rice
[Heber et al., 1999]. A meta analysis of previous controlled trials summarized all given data
and it was concluded that red fermented rice produced significant reduction of serum total
cholesterol, triglyceride levels, and low-density lipoprotein cholesterol (LDL-cholesterol), as
well as a significant increase in high-density lipoprotein cholesterol (HDL-cholesterol) [Liu et
al., 2006]. The lipid modification was reported to be similar when compared with 10-20
mg/day of simvastatin, 10 mg/day of atorvastatina and 20 mg/day of lovastatin.
Today red fermented rice comes to the market registered as a dietary supplement, and it is
used by millions of people seeking an alternative to standard statin therapy. Its huge increase
in use is mostly contributed to the perception of red fermented rice being a natural cure for
hyperlipidemia, and probably incorrectly perceived absence of side-effects and drug
interactions [Childress et al., 2012].
However, since red fermented rice contains monacolin K (lovastatin) and its acid form,
which contribute to more then 90% of total monacolins content, FDA issued a statement in
1998 that red fermented rice does not comply with the definition of a dietary supplement.
Therefore, FDA determined that red fermented rice contained an unapproved drug and dietary
supplements containing 0.4% or more of monacolins were taken of the market in the United
States [Journoud et al., 2004]. However, if the producers of red fermented rice dietary
supplements do not make a label claim to containing lovastatin, then the products are not
subjected to FDA control. On the other hand, red fermented rice extracts containg monacolin
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High Performance Liquid Chromatography – An Effective Tool for Quality Control …
K (lovastatin) are available as dietary supplements in Canada, European countries, Asia and
through Internet sale.
Although the efficacy of red fermented rice dietary supplements is somewhat certain,
unfortunately safety and quality of these products is questionable. Important considerations
are raised involving the purity and standardization of red fermented rice products. Numerous
reports were published stating poor inconsistency in monacolins' content and significant
discrepancies between labeled and found amounts of monacolin K and total monacolins. In
addition, a toxic substance called citrinin can be found in red fermented rice products. Citrinin
is a nefrotoxin, produced as a secondary metabolite of the fermentation process, which causes
functional and structural kidney damage, as well as alterations in liver metabolism. The
maximum allowed level of citrinin in red fermented rice is 200 ppb in Japan, while the
European Union has a recommended limit of a 100 ppb.
Analysts have developed selective and precise methods to determine the content of
lovastatin lactone and its acid form, total monacolins and/or citrinin in red fermented rice
dietary supplements. Although several HPLC methods have been developed for the
determination of monacolins' content in red fermented rice powder, which was obtained from
different areas in Asia, or by different fermentation and production techniques, only a few
methods have been developed for the determination of lovastatin and other monacolins in red
fermented rice dietary supplements.
Heber and co-workers [Hebber et al., 2001] have published one of the first papers in
which they emphasised the need for standardization of the production and the labelling of red
fermented rice, in order to fulfil the potential of these products’ hypolipemic activity.
Equivalence of active ingredients content must be achieved among different red fermented
rice found on the market. According to the HPLC analysis that they have conducted, the same
levels and even the same profile of the monacolins present in the different dietary supllements
were not achieved. Total monacolins content vaired from 0-0.58% w/w and only one out of
nine tested preparations had ten monacolin compounds present. Citrinin was also found in
seven out of nine analysed samples.
Li and co-workers [Li et al., 2004] have developed a chromatographic chemical profiling
method for the determination of bioactive monacolins in red fermented rice products. The
chemical profiling was conducted by a HPLC/DAD method with separation performed on
reversed-phase Waters Symmetry C18 column (150 x 3.9 mm, particle size 5 μm). A gradient
elution was used with a mobile phase consisting of acetonitrile (eluent A) and 0.1%
trifluoroacetic acid (eluent B) at a flow rate of 1 mL/min. The total analysis time was 35 min.
To verify identification process, an additional LC/MS analysis was conducted in which the
separation was achieved using a reversed-phase Zorbax C18 column (100 x 2.1 mm, particle
size 5 μm) with a mobile phase consisting of acetonitrile (eluent A) and 0.2% acetic acid
(eluent B) at a flow rate of 0.3 mL/min, and gradient elution performed in the same way as
described in the HPLC/DAD method. 12 different monacolins were identified and chemical
profiling was performed in ten commercial red fermented rice products. Quantitation of all
monacolins was conducted by the proposed HPLC/DAD method and the content of other
monacolins were calculated with monacolin K used as the reference standard. Results
indicated that the contents of monacolins in tested red fermented rice tablets and capsules is
considerably different, both in total quantity of monacolins, as well as in individual
components amount.
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Ana Mornar, Miranda Sertić and Biljana Nigović
Recently, a paper has been published using flow injection tandem mass spectrometry for
fast screening of lovastatin in red fermented rice products [Song et al., 2012]. The most
important feature of the proposed method is speed, since it takes less then one minute per
analysis. The flow from the autosampler was connected to the electrospray ionization (ESI)
source of the mass detector by using a guard column Intersil ODS-3. An isocratic
chromatographic elution was used with 10% of eluent A, consisting of 4 mM ammonium
formate with 0.05% formic acid in water, while eluent B was comprised of 4 mM ammonium
formate with 0.05% formic acid in methanol. Flow rate was set to 0.4 mL/min with a stop
time of 0.65 min. Lovastatin was determined by monitoring five product ion transitions in ten
dietary supplements and two red fermented rice raw material samples. It should be taken into
account that the proposed flow injection tandem mass spectrometry method is only
semiquantitative, possible co-eluting interferences could cause over- and under-estimation of
quantities of the analytes. However, the presented method offers a cost effective way for fast
screening of lovastatin in red fermented rice products. The consumption of solvents is
significantly reduced and the HPLC column is not required.
A number of methods have been published for the determination of citrinin in red
fermented rice. However, in most of the proposed methods lovastatin and other monacolins
were determined by one method, while citrinin was determined by another detector, a
different method and in some cases even a diverse analytical technique. Several HPLC
method have been published for the determination of lovastatin in red fermented rice dietary
supplements in which lovastatin analysis was performed by a UV detector, while citrinin was
determined by an indirect competitive Enzyme-linked Immunosorbent Assay (ELISA)
method [Chen, F. et al., 2005], a separately developed HPLC method with the use of an MS
detector [Pattanagul et al., 2008] or by a thin-layer chromatography (TLC) method [Becker,
2009].
Two very similar methods for the simultaneous determination of lovastatin in both its
lactone and β-hydroxy acid form were published by the same group [Lee et al., 2006; Wu et
al., 2011]. For the separation a C18 Discovery column, 250 x 4.6 mm, particle size 5 μm, was
used. The authors have tested two HPLC methods using an isocratic and gradient elution, in
order to investigate the effect on the separation between the analytes and the peak to noise
ratio. Both approaches yielded good peak separation but gradient elution led to an unstable
baseline and high interference occurred in the integration of the citrinin peak area in the
chromatogram obtained by fluorescence detector. Therefore isocratic elution was chosen. In
HPLC method development the influence of pH of the mobile phase plays a crucial role on
the separation effectiveness, analysis time and peak shape of ionized molecules. Citrinin,
whose low expecting concentration in red fermented rice products was a huge challenge for
the method sensitivity, gave maximum fluorescence at an emission wavelength of 500 nm
when the pH of the mobile phase was set to 2.5. At this pH value citrinin is close to
hydrophobic character. Using a mobile phase pH above 4.6 would reveal citrinin at its
hydrophilic character, but the fluorescence would be lower. Hence the authors tested three
different acidifiers of the mobile phase, acetic, phosphoric and trifluoroacetic acid. The
maximum peak area was used as the discriminative factor. Acetic acid resulted in the lowest
peak area of citrinin in the fluorescence chromatogram. Phosphoric acid and trifluoroacetic
acid added as the acidifiers in the mobile phase gave sharp, high peaks of citrinin. However,
trifuloroacetic acid was selected since higher peaks with grater peak area values for lovastatin
in its lactone and β-hydroxy acid form were obtained. Since the ratio of organic solvent and
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High Performance Liquid Chromatography – An Effective Tool for Quality Control …
water in the mobile phase has direct influence on the analysis time and separation between
analytes in HPLC methods, 50-60% acetonitrile in water (v/v) mobile phases were tested. The
optimal conditions for the HPLC analysis were obtained using a mobile phase consisting of
acetonitrile, water and trifluoroacetic acid (55:45:0.05, v/v), at a flow rate of 1 mL/min.
Different extraction procedures were also studied in order to find conditions at which the
highest recoveries for citrinin and lovastatin in lactone and β-hydroxy acid form were
achieved. The authors wanted to compare their proposed HPLC method for simultaneous
determination of citrinin and lovastatin in lactone and β-hydroxy acid form with analytical
methods for individual determination of lovastatin and citrinin. Simultaneous methods surely
save time, but in analytical procedures accuracy of the method is far more important then
speed. According to their results, the proposed simultaneous method showed better accuracy
then individual methods found in the literature. The authors used an additional LC/MS
method in order to identify unknown peaks in the obtained chromatograms. They applied the
newly developed HPLC method for the analysis of several commercially available Monascus
capsules and foods. A high amount of total lovastatin was found, ranging from 1 161 to 31
250 mg/kg in the dietary supplements. Micotoxin citrinin was found in most of the tested
products, and in three examined samples in high concentrations, exceeding 10 000 μg/kg.
As mentioned above, same authors published another paper using a very similar HPLC
method for the analysis of citrinin, lovastatin in lactone and acid forms, as well as monascin
and ankaflavin, two yellow pigments of Monascus strain [Wu et al., 2011]. These two
pigments have been reported to exhibit anti-inflammatory potential, cytotoxic/cytostatic
activites, namely antimetastatic and antiangiogenic effects. For the separation of the analytes
they used a C18 Luna 250 x 4.6 mm, 5 μm particle size column. An isocratic elution was used
and the mobile phase consisted of acetonitrile, water and trifluoroacetic acid (62.5:37.5:0.05,
v/v) at flow rate of 1 mL/min, very similar to the above mentioned method.
Gordon and co-workers [Gordon et al., 2010] have also published a paper in which they
emphasized striking variability in monacolin content and the need for improved
standardization of red fermented rice products and their labeling. They have performed an
analysis of 12 commercial red fermented rice formulations by an HPLC method with a UV
detector. Separation was performed with a 250 x 4.6 mm Prodigy column. Confirmation of
analytes' identities was done by an LC/MS method. The authors analyzed twelve commercial
red fermented rice products for monacolin content and the presence of citrinin. Among the
tested samples the content of total monacolins, which was taken as the sum of lovastatin in
both its lactone and acid forms, ranged from 0.31-11.15 mg/capsule. Lovastatin lactone, the
most important monacolin in the red fermented rice, ranged from very low, 0.10 mg/capsule,
to relatively high value of 10.09 mg/capsule, which is considered to be the therapeutic level
of lovastatin. Citrinin was unfortunately also found in over one third of the analyzed samples,
ranging from 24-189 ppm. The authors emphasize that they did not test the variability of
monacolin levels within different product lots, which should also be taken into account.
Our group has recently developed an HPLC/DAD/FLD/MSn method for simultaneous
determination of lovastatin lactone, lovastatin β-hydroxy acid and citrinin, as well as other
monacolins present in the red fermented rice products [Mornar et al., 2013]. To achieve the
best separation of the analytes in the shortest possible analysis time, various columns and
mobile phases were tested. Different reversed phase columns were examine: Zorbax SB-C18,
250 x 4.6 mm, 5 μm column, Hypersil C18, 150 x 4.6 mm, 5 μm column, Symmetry C18, 150
x 4.6 mm, 3.5 μm column, and XBridge C18, 50 x 3.0 mm, 2.5 μm column. The shortest
7
Ana Mornar, Miranda Sertić and Biljana Nigović
analysis time with good resolution and peak shapes without tailing was observed using the
XBridge C18 column. A problem of interconversion between lovastatin lactone and β-
hydroxy acid can occur while analyzing these products [Yang et al., 2006]. Since this can lead
to severe variance in accuracy of the method, and yield false positive and/or negative results
in the lovastatin content, several factors had to be attended during the method development
and the analysis of the samples. The pH of the mobile phase has the most important influence
on the interconversion process. Since the minimum interconversion rate is at pH 4.5 this value
was applied for the chromatographic analysis. The proposed mobile phase consisted of
acetonitrile, water and formic acid (10:90:0.1, v/v) as the eluent A, and acetonitrile, water and
formic acid (90:10:0.05, v/v) as the eluent B. Both isocratic and gradient elutions were tested,
but too long analysis time was obtained with isocratic solvent system. Therefore, the gradient
elution was used for the analysis of the red fermented rice products and the binary gradient
program was set as follows: 40-70% B (0-7 minutes) and 70-90% B (7-10 minutes) at a flow
rate of 1 mL/min and constant temperature of 25 °C. Detection was performed using three
different detectors. The UV detection and quantitation of monacolins was performed at 237
nm. The specific and sensitive fluorescence detection was used for the quantitation of citrinin,
expected in trace amounts at ppb levels. The excitation and emission wavelenths were set at
331 and 500 nm, respectively. The mass detection was carried out on an Agilent MSD Trap
system equipped with an ESI source and an ion trap analyzer system. Various samples,
including red fermented rice grains, capsules, tablets, sofgel capsules and liquid capsules,
containing different matrixes were investigated; therefore the extraction procedure was
carefully optimized. Different organic solvents (methanol, ethanol and acetonitrile) used as
the extraction solvents were tested. The best recoveries for all analytes were achieved using
methanol, so further investigations were performed using methanol with different proportions
of ultrapure water (50, 40, 30, 20, and 10%). The use of 80% methanol yielded the maximum
amount of all target compounds. It is known from literature that during extraction with
methanol lovastatin β-hydroxy acid can react with methanol and form an ester [Huang et al.,
2010]. Therefore, MS detector was used to identify extracted analytes from red fermented rice
products using 80% methanol as the extraction solvent and no ester products were found. The
influence of extraction time in the range from 15-90 minutes on the extraction efficacy was
also investigated. The maximum amount of each analyte was obtained at 60 minutes
extraction time, while further increase of the extraction time did not increase extraction
efficacy. Extraction temperature was also examined but high temperatures showed no
improvement in the extraction of monacolins. On the other hand, citrinin is known to undergo
thermal degradation at high temperatures, so in order to avoid it, the extraction procedure was
performed at room temperature. The proposed method was used for the analysis of two
different red fermented rice food products, as well as six commercially available dietary
supplements form different manufacturers. The results obtained by the newly developed
HPLC/DAD/FLD/MSn method revealed a dramatic variation from labeled values in
monacolin content in the tested products (2-155%). This demonstrates that the red fermented
rice dietary supplements are poorly standardized, thus resulting in inconsistent
pharmacological activity. In most samples lovastatin lactone is the dominant monacolin
found, while in two tested products lovastatin β-hydroxy acid was found in the highest
amount. In most investigated dietary supplements other monacolin were found, e.g.
monacolin L, monakolin M and dehydromonacolin K. However, levels of other monacolins
were quite low, except in one sample were 3 277 μg/g of other monacolins was found. When
8
High Performance Liquid Chromatography – An Effective Tool for Quality Control …
daily serving recommendation was taken into account, quite different amounts of monacolins
were taken using different products of red fermented rice, ranging from 0.05-11.85 mg.
Therefore, significantly diverse hypolipemic effects would be manifested using different red
fermented rice products. It should be noted that 10 mg is already considered as the therapeutic
level for lovastatin. We have also performed batch-to-batch variability evaluation. For most
of the tested products a relatively good uniformity was obtained, but for one tested dietary
supplements a large bath-to-batch variations was exhibited (15%) (Figure 1).
All these results emphasize the importance of quality control of red fermented rice dietary
supplements. Since some manufactures standardize their products to lovastatin lactone, while
others standardize them to total monacolins content or do not mention the presence of these
hypolipemic agents at all, the standardization as well as detailed and precise labeling of these
products is of great importance. Hence careful and particular consideration should be paid to
red fermented rice dietary supplements. Developing new, faster, more selective and more
sensitive HPLC methods are the way to ensure effectiveness, safety and high quality of red
fermented rice products.
Abbreviations: MKA – monacolin K acid, ML – monacolin L, MK – monacolin K, MM – monacolin M
and DMK – dehydromonacolin K.
Figure 1. UV chromatogram of red fermented rice dietary supplement (A) and MS2 spectrum of
monacolin K (B).
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Ana Mornar, Miranda Sertić and Biljana Nigović
Artichoke
Artichoke (Cynara scolymus, Asteraceae) is an ancient herbaceous perennial plant,
originating from the Mediterranean area. Artichoke leaves were used in ancient herbal
medicine for a variety of diseases, mostly for the treatment of dyspepsia. In recent years,
artichoke leaf extracts have been tested for various pharmacological activities:
hepatoprotective, anticarcinogenic, antioxidative, antibacterial and anti HIV activities as well
as the ability to inhibit cholesterol biosynthesis and LDL oxidation [Lattanzio et al., 2009;
Lupattelli et al., 2004]. The chemical components of artichoke have been studied extensively
and it was found to be a rich source of polyphenols, flavoniods and mono- and
dicaffeoylquinic acids. Several studies have shown that cynarin (1,5-dicaffeoylquinic acid)
was mainly responsible for the inhibition of HMG-CoA reductase and phosphatidate
phosphohydrolase enzymes [Gebhardt, 1998; Heidarian et al., 2011].
The main phenolic compounds, monocaffeoylquinic acids, dicaffeoylquinic acids and
flavonoides, in different batches of dried artichoke leaf extracts have been separated and
identified by LC/MS technique [Häusler et al., 2002]. Using a gradient elution of 35 minutes
and a slow flow rate (0.2 mL/min) a baseline separation of nine compounds was obtained.
Unfortunately, two dicaffeoylquinic acids coeluted as one peak. Neither addition of modifiers
nor use of a buffer as mobile phase improved the selectivity of the proposed method. In all
samples the highest levels were observed for monocaffeoylquinic acids, while the amounts of
flavonoids were quite low. High variability of poliphenolic content between different batches
was reported. Therefore, it can be concluded that polyphenol composition and content in
artichoke leaf extracts highly depends on the plant material used.
Fritsche and co-workers [Fritsche et al., 2002] have reported a novel method for
isolation, characterization and determination of several polyphenolic compounds in
commercially available artichoke products (powders and dietary supplements prepared in
form of juice as well as capsules). Preparative liquid chromatography (Sephadex LH-20) was
successfully employed to isolate minor artichoke compounds. Structural characterization of
isolated compounds was performed using LC/MS technique. In contrast to above described
method [Häusler et al., 2002] better sensitivity of MS detector was obtained using positive
ESI technique. Furthermore, quantitation of polyphenolic constituents of investigated
artichoke products was performed using LC/DAD technique and rosmarinic acid as internal
standard. The content of cynaropicrin, the key ingredient of artichoke, responsible for its
effective treatment for acute gastritis, showed a considerable variation. Moreover, in some
products it was not found at all. This can be easily explained by the seasonal influence as the
highest content of cynaropicrin in artichoke can be found in early summer. Wide variation in
the content of cynarin was also found. Contrarily to expected, this cholesterol-lowering
compound was not found in two dietary supplements. It is well known that aqueous extraction
of artichoke leaves increases the formation of cynarine by intraesterification of its isomer 1,3-
dicaffeoylquinic acid, while alcoholic extraction decreases the cynarin formation.
Furthermore, the cynarin content highly depends on the physiological state of the vegetal
tissues, decreasing intensivly as the plant ages. It should be pointed out that the highest
content of cynarin was found in dietary supplement prepared as juice.
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High Performance Liquid Chromatography – An Effective Tool for Quality Control …
The phenolic profile of cultivated and wild artichoke extracts as well as artichoke-based
dietary supplements was investigated using LC/DAD/MSn technique [Gouveia et al., 2012].
The analysis time was a little bit longer comparing to above described methods, but 29
different polyphenolic compounds were separated and structurally characterized by tandem
mass spectrometry. MSn analyses were performed using ESI in negative mode and ion trap
analyzer. The fragmentation pattern of all analytes was explained in detail. The phenolic
profile of cultivated artichoke was found to be much more diversified than that for wild
artichoke. Free luteolin aglycone, hexoside of rosmarinic acid and dimmer of rosmarinic acid
were only found in the dragées sample, while the only sample in which free quercetin
aglicone was found is juice sample. A slightly modified HPLC method using only UV
detection was used for quantitation of biological active ingredients. Cynarin was found in
fresh leaves of both cultivated and wild artichoke as well as in liquid dietary supplement.
This, one of the most important ingredients of artichoke, was not found in dragées sample.
This can be a result of the use of old leaves instead of young ones or of poor manufacture.
As many artichoke based dietary supplements are poorly manufactured, we have
proposed a new LC/DAD/MSn method for identification of characteristic compounds in
artichoke based dietary supplements (Figure 2) [Mornar et al., 2012].
Abbreviations: CN – cynarin.
11
Ana Mornar, Miranda Sertić and Biljana Nigović
Figure 2. UV chromatogram of artichoke dietary supplement (A) and MS2 spectrum of cynarin (B).
Phytosterols
Plant sterols or phytosterols are compounds that may be found in a greate variety of
different plants as they serve to stabilize phospholipid bilayer in plant cell membrane just as
cholesterol does in animal cell membranes. More than 250 different types of phytosterols and
related compounds have been reported in plant species. In all plant tissues, they occur in five
common forms: free sterols, fatty-acid esters, steryl glycosides, acylated steryl glycosides,
and hydroxycinnamic acid steryl esters [García-Llatas et al., 2012]. There are several
proposed mechanisms of hypocholesterolemic activity of phytosterols. Firstly, phytosterols
compete with cholesterol molecules for incorporation into mixed micelles in the intestinal
tract [Rozner et al., 2006]. Phytosterols have also been described to interfere in cholesterol
absorption by competing with cholesterol transporters. Both cholesterol and phytosterol
require the Niemann-Pick C1–like 1 protein in the small intestine for their absorption. In
addition, phytosterols may affect cholesterol absorption also through the upregulation of the
ATP-binding cassette transporters ABCG-5 and ABCG-8, located at the apical surface of
enterocyte [Brufau et al., 2008]. Although cholesterol-lowering effects of phytosterols are
known since 1950s, due to poor solubility and bioavailability of the free phytosterols, the
hypolipidemic effect was not consistent and very high doses were required for efficacy. More
recently, there has been considerable interest and commercial marketing of phytosterols
dietary supplements as it was found that their solubility can be easily increased by esterifying
phytosterols with fatty acids, while their hypocholesterolemic activity remains unchanged.
Furthermore, they can be easily incorporated in the micelles or prepared as suspensions or
emulsions in lecithin [Rozner et al., 2006].
The high lipophilicity of sterols makes sample processing and HPLC analysis of dietary
supplements containing phytosterols a quite challenging task. Still, two methods for quality
control of phytosterol based dietary supplements and dietary supplement row materials have
been published. A simple, rapid and validated HPLC method with UV and evaporative light
scattering detections (ELSD) was developed for the analysis of two sterols (stigmasterol and
ß-sitosterol) and a stanol (stigmastanol), as marker compounds of commercial dietary
supplements [Nair et al., 2006]. The separation of all three marker compounds and internal
standard, cholesterol, was accomplished within 12 minutes on a C8 column using 95%
methanol (v/v) as the mobile phase. After the UV detection at 210 nm, the chromatographic
column effluent was subjected to detection by ELSD. The most important parameters
affecting the ELSD response are the nebuliser gas flow rate and the drift tube temperature.
The gas flow rate influences the droplet size of the column effluent before evaporation
occurs. Higher flow rates result in the formation of smaller aerosol droplets and less
scattering of light with subsequent lower sensitivity but an increased stability. On the other
hand, lower gas flow rates are associated with larger droplet formation, augmented light
scattering and therefore a higher response, but baseline stability is decreased. It is necessary
to optimize this parameter to ensure that the optimal signal to noise ratio is obtained.
Therefore, the gas flow rate was carefully investigated and optimal sensitivity and baseline
stability was obtained using flow of 0.7 L/min. A relatively high drift tube temperature (100
°C) was required for the adequate evaporation since the sterols are not highly volatile and the
mobile phase consisted of polar solvents. Comparing UV and ELSD detections, it should be
12
High Performance Liquid Chromatography – An Effective Tool for Quality Control …
pointed out that ELSD detection provided advantages over UV detection in terms of
sensitivity as investigated phytosterols gave weak UV signals due to their poor
chromophores. Moreover, stigmastanol could only be analysed using ELSD. The HPLC
technique using atmospheric pressure chemical ionization/mass spectrometric (APCI-MS)
detection was used for the separation of the phytosterols campesterol, cycloartenol, lupenone,
lupeol, ß-sitosterol, and stigmasterol, while brassicasterol and cholesterol were also included
in the study as internal standards [Bedner et al., 2008]. A wide variety of analytical columns
with different selectivities was investigated during method development and the best
separation was provided by two columns: ACE C18 column (150\×\3.0\mm, particle size
3\μm) and XTerra phenyl column (150\×\3.9\mm, particle size 3.5\μm). Although none of the
columns allowed complete separation of all analytes, the different mass transitions in the
tandem mass spectrometric detection provided satisfying selectivity.
Omega-3 Fatty Acids
Fatty acids are organic compounds formed by a hydrocarbonated chain and a carboxylic
group that is normally bounded with glycerol forming acylglycerides (mono-, di- or
triglycerides). Depending on the nature of the hydrocarbonated chain, fatty acids can be
saturated or unsaturated, which in turn can be mono- or polyunsaturated fatty acids. The fatty
acids can be synthesized by humans, but some of polyunsaturated fatty acids are the essential
fatty acids and the human body cannot produce them. Polyunsaturated fatty acids can be
divided in two categories: omega-3 (n-3) and omega-6 (n-6) fatty acids, depending on the
location of the first double bond. The most important natural sources of omega-3 fatty acids
are marine organisms, such as fish, seafood and algaes, which are fed, directly or indirectly,
from marine phytoplankton, the primary producer of omega-3 acids in the trophic chain.
Other rich sources of omega-3 fatty acids include walnuts, flaxseed and canola oil [Lee et al.,
2003]. A number of studies have been published showing the relation between omega-3
enriched diet and prevention or treatment of some diseases such as myocardial infarction,
bronchial asthma, rheumatoid arthritis, cystic fibrosis and Crohn’s disease. Furthermore,
omega-3 fatty acids have been shown to lower plasma cholesterol and triglyceride levels
through the inhibition of LDL and triglyceride synthesis in the liver without reducing
production of beneficial HDL molecules [Fetterman et al., 2009].
Although gas chromatography (GC) is the predominant technique used for omega-3 fatty
acid analysis, HPLC technique could have an important role in their analysis as it operates at
ambient temperature so there is relatively little risk to sensitive functional groups, especially
if unusual and complex samples are analyzed. Still, very few analytical data are available on
the quantitative determination of omega-3 fatty acids in dietary supplements by HPLC
technique. A chromatographic method has been developed for the determination of
eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) in fish oil dietary supplements
containing triglycerides rich in polyunsaturated fatty acids [Teng et al., 1993]. Prior the
analysis the samples were hydrolized by methanolic solution of potassium hydroxide.
Afterwards, the samples were acidified with chloric acid and extracted with mixture of
hexane and isopropyl alcohol. Following the ester saponification, free fatty acids were
resolved by TLC into two fractions: a more mobile group of saturated and monounsaturated
fatty acids and a less mobile group containing polyunsaturated fatty acids. The second
13
Ana Mornar, Miranda Sertić and Biljana Nigović
fraction was subjected to HPLC analysis. Good separation of investigated omega-3 fatty acids
and other constituents of dietary supplements was achieved using isocratic elution with a
mobile phase composed of tetrahydrofuran, acetonitrile, water and acetic acid (25:35:75:0.4,
v/v/v/v). The detection of analytes was performed using UV and MS detectors. Yoo and
McGuffin [Yoo et al., 1992] have developed a new method for determination of 14 saturated
and unsaturated fatty acids, including the omega-3 fatty acids, in fish oil dietary supplements
using reversed-phase liquid chromatography and a slightly unusual laser-induced
fluorescence detector. The major advantage of the proposed method is that the
physiologically important fatty acids with 12 - 22 carbon atoms and 0 - 6 double bonds were
determined at the femtomole level.
Green Tea
Tea is produced from the leaves of Camilla sinensis (Theaceae), which grows mainly in
China and southeast of Asia. Tea is the most popular hot beverage in the world. The
consumption of tea is a very ancient habit and legends from China and India indicate that it
was initiated about five thousand years ago. The most important active compounds in green
tea are the polyphenols, more specifically the catechins. The most abundant catechin species
in green tea include (+)-catechin, (-)-epicatechin, (+)-catechin gallate, (-)-epicatechin gallate,
(+)-gallocatechin, (-)-epigallocatechin, (+)-gallocatechin gallate and (-)-epigallocatechin
gallate. These compounds are widely distributed in the leaves of green tea and constitute up to
one third of the dry leaf weight. Much interest has been focused on catechins, not only for
their antioxidant activity, but also because of their known antimutagenic and antitumorigenic
properties. Recent studies have suggested that green tea and its components can play a
protective role in development of hyperlipidemias [Dufresne et al., 2001]. One of the
underlying mechanisms by which tea catechines reduce plasma cholesterol level is up-
regulation of LDL receptor activity [Bursill et al., 2007]. Furthermore, it was found that
catechins stimulate CYP7A1 enzyme in human hepatoma cells at mRNA level [Lee et al.,
2008]. Regardless that most of the people prefer to take green tea as hot beverage in relaxing
atmosphere, it should be pointed out that green tea dietary supplements are the fourth best
selling dietary supplement in the USA.
Although number papers have focused on analysis of catechins in green tea leaves or
brewed teas [Zuo et al., 2002], few reports have been published on the determination of
catechins in dietary supplements. As green tea-based dietary supplements have gained
popularity in the world in recent years, Sun and co-workers [Sun et al., 2011] have evaluated
the difference in phytochemical composition of green tea dietary supplements and green tea
leaves using a LC/MS fingerprinting technique coupled with chemometric analysis. Five
components that are most responsible for class separation among samples were identified as
epicatechin gallate, strictinin, trigalloylglucose, quercetin-3-O-glucosyl-rhamnosylglucoside
and kaempferol-3-O-galactosyl-rhamnosylglucoside. Chemometric analysis has shown a
significant variance in the chemical composition across the investigated dietary supplements.
Furthermore, the concentrations of flavonol aglycones were higher in dietary supplements
than in tea leaves, indicating the degradation of flavonol glycosides during the manufacturing
and storage of dietary supplements.
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High Performance Liquid Chromatography – An Effective Tool for Quality Control …
Catechins have also been determined in green tea extract using HPLC technique [Bonoli
et al., 2003]. The authors have used a C18 chromatographic column with a 3 mm i.d.
Detection limits of all anaytes obtained with narrow column were lower than values obtained
using classical 4.6 mm column. Moreover, the 3 mm column required lower eluent flows than
the 4.6 mm column to obtain a similar separation, maintaining a relatively low pressure.
Using a gradient elution with total analysis time of 40 minutes, including the additional 6
minutes for column conditioning after each run, all catechins were separated. Applying the
optimized method for determination of catechins in the samples, it was found that two peaks,
corresponding to gallocatechin and gallic acid, one of the most abundant phenolic acids in
green tea, have not been resolved well. Therefore, the same group of authors has developed
an another HPLC method for determination of catechins in green tea extract using the same
column and the same gradient elution program, but detection of analytes was performed using
both detectors DAD and MS with ESI source in positive mode [Pelillo et al., 2004].
Calculated amounts of gallocatechin were significantly different between UV and MS
detection, indicating a possible interference of a UV detectable molecule coeluting with the
gallocatechin. Using the extracting ion chromatogram tool by MS detector it was confirmed
that gallocatechin and gallic acid coelute. Therefore, the quantitative analysis of tea
catechines was performed using MS detector and selected ion monitoring mode. Although
MS detection was found to be more selective and sensitive than UV detection, both detectors
showed comparable precision for quantitation of catechins. Since repeatability conditions
were maintained for a longer time using UV quantitation, regular calibration of MS detector
was recommended, in order to compensate fall of robustness.
Another HPLC method for determination of catechins and gallic acid in green tea dietary
supplements was proposed by Bedner and Duewer [Bedner et al., 2011]. The most commonly
used a C18 chromatographic column with 4.6 mm i.d., a gradient elution with formic acid and
acetonitrile and both UV and ESI-MS detection were employed. The results from both
detection techniques were evaluated with 14 quantitation models, all of which were based on
the analyte response relative to an internal standard, proxyphylline. For all analytes, response
factors obtained with UV detection were stable. Moreover, comparable results with low
variability were observed regardless of the quantitation model used. On contrary, the highly
selective MS responses were found to vary both with time and as a function of the calibrant
concentration. The variability in the quantitative results obtained by MS detection was
reduced by a dynamic quantitation model based on polynomial data-fitting.
Green tea has a unique composition, which includes already mentioned catechins,
flavonols and glycosides, phenolic acids, xanthines and proanthocyanidins. Therefore, HPLC
analysis of catechins, theaflavins and alkaloids in commercial teas and green tea dietary
supplements (capsules, tubes, powders and chewing gums) was proposed [Friedman et al.,
2006]. As extraction is one of the most important factors affecting the analytical results,
extraction efficacy of boiling water and different ethanol – water mixtures was investigated.
According to obtained results, the best extraction efficacy of all analytes was obtained using
80% ethanol at 60 °C as extraction solvent. The chromatographic separation was achieved
using a C18 column and a gradient system consisted of a mixture of acetonitrile and 20 mM
potassium hydrogen phosphate. Although the method was quite long (90 minutes including
the time necessary for column conditioning after each run) 14 catechins, theaflavins and
alkaloids were separated in a single run. All seven catechins, included in the study, were
found in green tea dietary supplements, but the amounts of tea catechins found in dietary
15
Ana Mornar, Miranda Sertić and Biljana Nigović
supplements were significantly lower than the amounts listed on the labels. All capsules
contained caffeine, theobromine and theophylline, with caffeine levels approximately 20
times more than levels of other two tea alkaloids. None of the investigated supplements
contained beneficial theaflavins.
There are several other HPLC methods developed for determination of catechins in
green tea dietary supplements [Seeram et al., 2006; Manning et al., 2003]. All authors
reported wide variability in catechin content between green tea products. Most of obtained
values were significantly lower than label claims. These results demonstrate the urgency of
quality control for green tea dietary supplements regarding to catechin content.
Caffeine is an alkaloid that occurs naturally in the leaves, seeds and fruit of tea.
Therefore, green tea dietary supplements may contain caffeine, even if caffeine is not listed as
an ingredient on the label. HPLC technique was used for determination of caffeine content in
53 dietary supplements containing different botanicals such guarana, yerba mate, kola nut and
green tea as well as vitamins and minerals [Andrews et al., 2007]. The proposed method was
quite simple. Caffeine was extracted from complex matrices with boiling water in five
minutes followed by chromatographic separation using a C18 column and a gradient elution.
Total analysis time per sample was around 30 minutes. In all samples, containing green tea
extract, caffeine was found from 0.60 to 828.71 mg/day, although 18 from 41 samples had no
specific label information about caffeine content. For most of the green tea supplements that
listed a level of caffeine on the label, the mean analyzed level calculated on a per day basis
was similar to the labeled amount.
The HPLC method for determination of three catechins (epicatechin, epigallocatechin
and epigallocatechin gallate), procyanidin B2 and caffeine in green tea and grape seed dietary
supplements was developed by Hadad and co-workers [Hadad et al., 2012]. Initial efforts to
develop a separation method using a C18 column and an isocratic elution system with a
methanol-based mobile phase were unsuccessful. Therefore, acetonitrile was used as organic
modifier in the mobile phase. A satisfactory separation of all analytes in only 12 minutes was
obtained using a mobile phase containing water with 0.05% orthophosphoric acid and
acetonitrile (85:15, v/v). Increasing acetonitrile concentration in the mobile phase led to
inadequate separation, while at lower acetonitrile concentrations, separation was achieved but
with excessive tailing and long analysis time. The method was fully validated and the
developed method was found to be linear, sensitive, accurate and precise; therefore it is suited
for rapid, routine analysis of principal components in green tea and grape seed extract, dietary
supplements with antioxidant activity.
Soybean
Soy (Glycine max, Fabaceae) is a subtropical plant, native to southeastern Asia. It has
been a dietary staple in Asian countries for at least 5000 years. During the Chou dynasty in
China (1134 – 246 B.C.), fermentation techniques were discovered that allowed soy to be
prepared in more easily digestible forms such as miso and tamari soy sauce. Soy was
introduced to Europe in the 1700s and to the United States in the 1800s. In addition to its
culinary uses, soy has also been investigated for benefit in terms of decreasing menopausal
symptoms, risk of breast cancer in women, prostate cancer in men, cardiovascular disease and
osteoporosis. Furthermore, the relationship between the consumption of soybean and the
16
High Performance Liquid Chromatography – An Effective Tool for Quality Control …
improvements in lipid levels is well known [Ricketts et al., 2005; Taku et al., 2007]. The
hypocholesterolemic effect of soybean is attributed to the proteins, fiber and phytochemicals
including the soy isoflavonoids. A significant reduction of total and LDL-cholesterol as well
as of ischemic and celebrovascular events with a daily soy protein intake was reported [Sirtori
et al., 2009]. Soy isoflavonoids, genistein and daidzein, have demonstrated to inhibit ACAT
activity in hepatocyte [Borradaile et al., 2002].
As the main ingredients of soy dietary supplements are isoflavonoides, strongly depend
on the soy cultivar, geographical area, clime and extraction procedure, a number of HPLC
methods are developed to evaluate the labeling accuracy and product uniformity of available
soy isoflavone products. Griffith and Collison [Griffith et al., 2001] have performed a
comprehensive study on optimization of extraction procedure of isoflavonoids from soy based
dietary supplements. Acetonitrile (50%) was found to be more efficient in extraction of
malonyl isoflavones, acetyl isoflavones and aglucones than commonly used mixtures of
methanol or ethanol and water [Chua et al., 2004; Prabhakaran et al., 2006]. The authors have
proposed two similar HPLC methods for quantitation of 12 isoflavones. The proposed
methods use a C18 column (250 x 3 mm, 5 μm particle size) and a gradient elution with 0.1%
acetic acid in water and 0.1% acetic acid in acetonitrile. Both methods are quite long 57 and
73 minutes, respectively. Although these two long methods are suitable for analysis of 12
isoflavones in complex matrices due to high peak capacity, the authors have proposed an
another rapid HPLC method using a short chromatographic column (53 x 7 mm, 3 μm particle
size) and fast flow of 3 mL/min. This type of method was shown to be suitable for rapid
analysis of isoflavones and may be particularly useful for analysis of purified dietary
supplements. Third HPLC method using MS detection with ESI in positive mode and ion trap
analyzer was developed to elucidate the structures of degradation products in standard and
sample solutions. In all above described methods apigenin was used as internal standard.
According to authors, structural similarity to isoflavone aglucones and its hydrophobicity
make apigenin an ideal internal standard for isoflavone determination. The same method was
used to analyze soy based dietary supplements by 13 collaborating laboratories in 6 countries
[Collison, 2008].
LC/MS/MS analysis using APCI in negative mode and ion trap analyzer was used for
the identification and quantitation of daidzein, genistein, glycitein, daidzin, glycitin, 6˝-O-
acetyldaidzin, 6˝-O-acetylglycitin and 6˝-O-acetylgenistin in soy based dietary supplements
[Chen, L.J. et al., 2005]. The mixture of acetonitrile and water was also used as extraction
solvent. Supersonic bath at room temperature was used for extraction of analytes from
samples. It was found that the concentration of analytes maintained constant after 20 minutes
of supersonic time. Moreover, too long time of extraction resulted in deesterification of acetyl
isoflavones. To investigate the isoflavone profile, the hydrolysis of samples was not
performed. The proposed method with gradient elution was quite long but good separation
between analytes and internal standard, biochanin A, was obtained. The method enabled
structural characterization of unknown components in investigated dietary supplements and
fragmentation pathways of nine isoflavones were systematically suggested for the first time.
In the investigated samples acetyl isoflavones were prominent compounds, while malonyl
isoflavones were not detected. Glycoside and aglycone isoflavones only contained small
portion of whole isoflavones.
Boniglia and co-workers [Boniglia et al., 2009] have proposed a simple isocratic HPLC
method with UV detection for determination of isoflavone aglycones in soy based dietary
17
Ana Mornar, Miranda Sertić and Biljana Nigović
supplements. The authors have performed the hydrolysis of the samples as aglycones are
considered to be the bioactive part of the isoflavone molecule. The amounts of isoflavones
expressed as aglycones in analysed samples did not coincide with the values stated on the
labels. Three samples had higher levels of total isoflavones than stated by the manufacturer,
while the others contained much less than the amounts stated on the labels.
Yanaka and co-workers [Yanaka et al., 2012] have used HPLC technique to determine
the content of 13 isoflavones, including 3 succinyl glucosides, in soy food and dietary
supplements. The obtained results suggest that the total isoflavone contents maybe
underestimated in the previous studies that have not included succinyl glucosides.
LC/MS technique with ESI in both positive and negative modes and quadrupole
analyzator was employed to determine content of six isoflavones in soy dietary supplements,
soy based health products and infant formulas [Prabhakaran et al., 2006]. To facilitate
quantitation and accurately evaluate the total isoflavones in samples, extraction of analytes
was carried out in 80% aqueous methanol followed by mild saponification. The profile of
isoflavones found in different dietary supplements varied among one other. Eight dietary
supplements contained genistein as the major isoflavone while five products had daidzin in
higher amounts. The obtained results suggest that the majority of supplements were derived
from American or Japanese soybeans having genistein as the major isoflavone, while the
others may have been derived from Korean soybeans which had slightly higher levels of
daidzin then genistin. Soy based infant formulas are used primarily to replace milk or milk
based formulas in the diets of children who have galactosemia or are intolerant to milk. The
isoflavone content in these products ranged from 59.5 to 226.8 μg/g of the formula.
Therefore, the authors have pointed out that the possible chronic effects, which might occur
after high levels of intake of isoflavones in early age, need to be further studied to confirm the
safety of these products.
Delmonte and co-workers [Delmonte et al., 2006a] have developed a HPLC method with
UV detection capable of quantifying isoflavones in dietary supplements regardless of the
botanical composition. Six samples of dietary supplements containing soy, red clover and
kudzu were investigated. The samples were analyzed directly after extraction and after acid or
base hydrolysis. The 90 minutes gradient elution with acetonitril, water and acetic acid
allowed separation and simultaneous quantitation of 19 isoflavone present in investigated
dietary supplements. The choice of internal standard was a quite challenging task as
investigated samples are complex matrices with a number of unknown compounds. Several
compounds were tested with respect to their retention times and stability during extraction or
acid and basic hydrolysis. 2´-methoxy-flavone and 6-metoxy-flavone were considered
suitable internal standards as they elute from column after all analytes and were quantitatively
recovered after sample preparation. More recently, HPLC method with both UV and MS
detection for analysis of isoflavones in dietary supplements containing soy and red clover was
developed [Romani et al., 2010]. Using MS detector high selectivity and sensitivity was
obtained. In addition, antiradical activity of samples was investigated. Hence, HPLC coupled
with UV and/or MS detectors is analytical technique of choice for determining isoflavones in
soy based dietary supplements [Delmonte et al., 2006b; Krenn et al., 2006].
Nevertheless, liquid chromatography coupled with uncommonly used coulometric
electrode array detector has also been shown to provide impressive results in soy analysis
[Nurmi et al., 2002]. Good separation of all analytes was obtained using an endcapped C18
column with a gradient elution with mixtures of 50 mM sodium acetate buffer (pH = 5),
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High Performance Liquid Chromatography – An Effective Tool for Quality Control …
methanol and acetonitrile as eluents. Furthermore, a simple gradient reversed phase HPLC
method using a multi-channel electrochemical detector for the determination of four
isoflavones in dietary supplements has been developed [Tian et al., 2002]. Peak assignment at
various retention times was confirmed by comparison of the simultaneous response of sample
peaks to standards, at glassy carbon electrodes held at different oxidation potentials.
An UPLC technique, which provides significantly better separation than the traditional
HPLC and enables much faster analysis, was used for the simultaneous determination of soy
isoflavones in dietary supplements [Fiechter et al., 2010]. Reversed phase separations were
performed at 35 °C using an optimized gradient elution and a C8 chromatographic column
with 1.7 μm particle size. The proposed method enabled adequate separation of all 12 soy
isoflavones within 19 minutes, hence demonstrating significant improvements especially
regarding the reduced analysis time compared to above described HPLC methods.
A through literature search has revealed that only a few analytical methods are available
for determination of process related impurities and forced-degradation products in dietary
supplements. Thus, the papers referring to this subject are very interesting and valuable. The
HPLC/DAD method for determination of soy isoflavonoids in dietary supplements was
published [Chua et al., 2004]. Moreover, the same method was used for determination of
product impurities. Only 4 of the 13 products contained at least 90% of the isoflavone content
claimed on the label and two of products contained impurities in the chromatogram that
exceeded 40%.
Guggulipid
Gum guggul is obtained from the thorny plant Cammiphora whighitii which belongs to
the family Burseraceae and is found in the dry regions of Indian subcontinent, mainly India,
Bangladesh and Pakistan. About 400 g of dry guggul is obtained from a typical plant in one
season. The recognition of the therapeutic and medicinal value of guggul in Indian Ayuvedic
medical system dates from 600 BC. Guggul is regarded as the most important resin in the
authoritative monograph Charaka Samhitra for the treatment of a variety of diseases and
disorders including obesity, atherosclerosis, osteoarthritis, infectious diseases and skin
disorders. Guggulipid, the herbal extract from this oleo gum resin, was approved in India as a
cholesterol-lowering dietary supplement in 1986. Afterwards, guggulipid was introduced to
Western medicine and its popularity as effective natural hypolipidemic is increasing. In 1994
FDA approved guggulipid as a dietary supplement [Shen et al., 2012]. More than 300
molecules have been identified in the guggul including terpenoids, minerals, proteins, sugars,
flavonoids, sterols and ferrulates. Two C21 steroid isomers Z- and E-guggulsterones have
attracted lots of interest for their hypolipidemic properties. The biological effects of
guggulsterones on plasma lipoprotein levels have been studied and at least four mechanisms
have been proposed to explain their activity. It has been well established that guggulsterones
are an effective antagonists of farnesoid X receptor, a key transcriptional regulator for
maintenance of cholesterol and bile acid homeostasis [Sinal et al., 2002]. More recently, it
was demonstrated that guggulsterones upregulate the bile acid salt export pump, an efflux
transporter responsible for removal of cholesterol metabolites, bile acids from liver [Deng,
2007]. Moreover, guggulsterones have been shown to enhance the uptake of LDLs by the
liver through stimulation of LDL receptor-binding activity in the membranes of hepatic cells.
19
Ana Mornar, Miranda Sertić and Biljana Nigović
Finally, guggulsterones have been shown to increase fecal excretion of bile acids and
cholesterol by decreasing the absorption of cholesterol in the intestine [Shishodia et al.,
2008].
Mesrob and co-workers [Mesrob et al., 1998] have developed two HPLC methods for
quality control of guggul resin extracts and formulated dietary supplements (tablets and
capsules). Both of the methods used C18 chromatographic columns and mobile phases
consisted of acetonitrile and water. Also, the gradient elution was employed in both methods.
The first method was developed for qualitative identification of the resinous components. It is
suitable for screening both row materials and formulated products for the authenticity of the
used resin. In order to obtain better insight in the sample composition, the chromatograms
were monitored at two different wavelengths, 245 and 327 nm. The second method was
developed for quantitative determination of the bioactive components Z- and E-
guggulsterones. Therefore, the chromatograms were recorded only at 245 nm, the wavelength
of maximum absorbance of guggulsterones. Although the method is slightly shorter than the
first one, the better separation of guggulsterones from other components of samples was
achieved. Both methods were validated for linearity, accuracy and ruggedness. In addition,
the samples were analyzed using LC/MS technique. The MS detectors were used to evaluate
the selectivity of the proposed methods and to confirm the purity of the peaks. Two different
ionization modes were investigated: electron impact and APCI. The second method was
successfully applied for determination of Z- and E-guggulsterones in six commercially
available guggul resin extracts as well as in various dietary supplements, tablets and capsules,
containing formulated guggulipid. The obtained results showed that the content of both
guggulsterones in these materials varied widely and was always significantly lower than
claimed on the label of the products. Moreover, in one formulated product none of
guggulsterones were found.
More recently, a significantly shorter method for simultaneous determination of Z- and E-
guggulsterones in dietary supplements containing guggulipid was developed [Nagarajan et al.,
2001]. Although, a simple isocratic eluation was employed, the good separation of
guggulsterones from other components of samples was achieved. The authors have also found
relatively low amounts of both guggulsterones in commercial formulated products in
comparison with the manufactures’ claim.
An another HPLC method for analysis of guggulipid based dietary supplements was
developed by Gottumukkala and co-workers [Gottumukkala et al., 2005]. As in the previously
described methods, E-guggulsterone was eluted from the column prior Z-guggulsterone using
a reversed phase chromatography. The authors have also used a simple isocratic elution; still
analysis time was quite longer than in the method proposed by Nagarajan and co-workers
[Nagarajan et al., 2001].
Musharraf and co-workers [Musharraf et al., 2011b] have used a short C18
chromatographic column with small particle size (1.8 µm) and a gradient elution for
separation of Z- and E-guggulsterones in guggul resin, guggulipid and marked dietary
supplement. The satisfactory resolution between investigated guggulsterones was obtained.
Moreover, unknown compound, present in all investigated samples, was eluted between two
isomers. The unknown compound was isolated using preparative chromatography. Firstly,
guggulipid was dissolved in ethyl acetate and spotted on a TLC plate as the wide band. After
development of the plate, bands between Z- and E-guggulsterones were scratched from the
plate and the scratched silica was extracted by ethyl acetate. Afterwards, the extract was
20
High Performance Liquid Chromatography – An Effective Tool for Quality Control …
filtrated and unknown compound was isolated and purified using a column chromatography.
In order to clarify the structure of unknown compound tandem mass spectrometric analysis
was performed. The sample was introduced into MS detector consisted of ESI mode and
tandem mass spectrometric analayzer quadrupole-time-of-flight by direct infusion. The mass
spectrum of isolated compound showed the protonated pseudomolecular ion at m/z 315. The
MS/MS spectra of isolated compound was also recorded and found to be very similar to E-
and Z-isomers of guggulsterone with characteristic fragments at m/z 109 and 97. According to
the literature and mass spectrometric data, the structure of isolated compound was identified
as 17,20-dihydroguggulsterone. The same group of authors has also published another
validated HPLC method for screening of E- and Z-guggulsterones in row materials and
dietary supplements containing Commiphora mukul extract [Musharraf et al., 2011a].
Kamal and co-workers [Kamal et al., 2012] have developed and validated a new HPLC
method for simultaneous determination of piperine, Z- and E-guggulsterones, the main
ingredients of Habb-e-Khardal Unani tablets. Several mobile phases containing methanol,
acetonitrile and water in different ratios on a C18 chromatographic column were tested.
Although water and acetonitrile in the ratio of 60:40 (v/v) showed well-defined peaks of all
analytes in standard solutions, the separation of analytes in sample was not well resolved.
Therefore, a gradient elution system consisting of water and acetonitrile at flow rate of 1
mL/min was used. The satisfactory separation of all three analytes was achieved in 15
minutes. Furthermore, a nanoemulsion of piperine and guggulsterones using sefsol-218 as an
oily phase, cremophor-EL as a surfactant, transcutol as a co-surfacant and distelled water as
an aqueous phase was prepared. This formulation was optimized on the basis of optimum
globule size, lower viscosity, lower surfactant concentration, higher solubilization of drug in a
minimum amount of oil as well as greater drug release. In vitro drug release pattern for a
novel approach of fast drug release delivery system with conventional tablet formulation has
also been compared. A dissolution study was carried out in simulated gastric and intestinal
fluids. According to obtained results, the nanoemulsion approach was considered as a
promising drug delivery system for poorly soluble drugs. The nanosize and higher area
permitted faster rate of drug release and improved absorption and bioavailability of active
ingredients. The proposed HPLC method was successfully applied for simultaneous
determination of piperine and guggulsterones in a nanoemulsion for the first time.
Coenzyme Q10
Coenzime Q10 (ubiquinone) is a component of the mitochondrial electron transfer chain
and in its reduced form (ubiquinol) can function as an oxidant that protects membrane
phospholipids, mitochondrial membrane protein and LDL from free radical-induced oxidative
damage. The Q10 can be synthesized in tissue from farnesyl diphosphate and tyrosine. Much
like cholesterol, Q10 is a downstream product of the mevalonate pathway. Therefore, a
biosynthesis of Q10 is a complex process requiring enzyme HMG-CoA reductase [Pacanowski
et al., 2008]. It appears that levels of Q10 are decreasing with increasing age, which may
account in part, for age-related increase observed in oxidative damage to protein and DNA.
Several studies have shown that coenzyme Q10 has potential for use in prevention and
treatment of cardiovascular disease, particularly hypertension, hyperlipidemia, coronary
artery disease and heart failure [Sarter, 2002]. As a small amount of Q 10 can be obtained from
21
Ana Mornar, Miranda Sertić and Biljana Nigović
food such as meat, fish and vegetables, the administration of Q10 dietary supplements may be
beneficial for treatment of hyperlipidemias alone or as adjuvant therapy for patients on
statins, the inhibitors of the biosynthesis of both cholesterol and Q10.
An HPLC method with UV detection for determination of Q10 in row materials and
dietary supplements was proposed by Orozco and co-workers [Orozco et al., 2007]. Since Q10
can exist also as the reduced form, the application of an oxidizing agent, ferric chloride, drove
the equilibrium mechanics to the fully oxidized state and allowed for quantification of total
Q10, sum of oxidized and reduced forms, in the samples. The proposed method is simple and
does not require unusual or specialized equipment not commonly found in the typical
analytical chemistry laboratory. Using a C18 chromatographic column and a isocratic elution
with acetonitrile, tetrahydrofuran and water (55:40:5, v/v/v), the good separation of Q10 and
structurally similar Q9 and idebenone was achieved in only 5 minutes. A detailed validation of
method was performed and the assay has proven to be linear, accurate, repeatable, selective,
and rugged over the intended dynamic range. Afterwards, the same method was used for
interlaboratory study to analyze Q10 dietary supplements by 11 collaborating laboratories in 4
different countries [Lunetta et al., 2008].
The Q10 is a lipophilic compound which bioavailiability from food and dietary
supplements is significantly improved when it is applied incorporated in lipid matrix. In
addition, the presence of emulsifiers or surfactants may also increase its absorption from
supplements. An HPLC method for determination of Q10 in dietary supplements in different
formulations (tablet, hard capsule, soft capsule, granule, liquid, jelly and inclusion complex
with γ-dextrin) was proposed [Kettawan et al., 2007]. After extraction with ethanol and n-
hexane, Q10 was separated on a reversed phase column from other constituents and detected
using an electrochemical detector. The major advantage of the proposed method is possibility
to measure simultaneously both forms of Q10, reduced and oxidized, respectively. In order to
determine the total amount of Q10 in samples, the Q10 was converted into the corresponding
reduced form by treatment with sodium borohydride solution. Most of investigated
supplements were well manufactured and the content of Q10 was in accordance with labeled
value. However, a few products exhibited insufficient quality. In several products besides the
oxidized form, the reduced form of Q10 was also found. Moreover, in one product Q10 was
recovered entirely as the reduced form. Furthermore, it was pointed out that all samples in
which reduced form of Q10 was detected contained vitamins E and/or C.
Another rapid, sensitive and validated method for the determination of coenzyme Q10 in
dietary supplements by automated HPLC with coulometric detection was published [Tang,
2006]. The Q10 and its internal standard, Q9, were monitored at an analytical cell that
contained 2 coulometric electrodes, where Q9 and Q10 were reduced to the corresponding
ubiquinol-9 and ubiquinol -10 and then oxidized to produce currents.
A sensitive HPLC method for the simultaneous quantification of the fat-soluble vitamins
(vitamin A, vitamin A-acetate, vitamin A-palmitate, vitamin E and vitamin E-acetate)
together with coenzyme Q10 in multivitamin dietary supplements using a C30
chromatographic column and dual-wavelength detection was developed [Breithaupt et al.,
2006]. A good separation of all six analytes in 40 minutes was achieved using a gradient
elution. As expected, vitamin A-acetate and vitamin A-palmitate showed higher retention
times on C30 column than vitamin A in form of alcohol. The same behavior was observed for
vitamin E and its ester. The Q10 showed the highest retention time, which is in accordance
22
High Performance Liquid Chromatography – An Effective Tool for Quality Control …
with ten isoprene units forming its side chain. For additional identification of analytes, APCI-
MS detection in positive ionization mode was used.
As mentioned above, a few analytical methods for determination of process related
impurities and forced-degradation products in dietary supplements are published. One of them
is the method developed by Nageswara Rao and co-workers [Nageswara Rao et al., 2008].
The separation and determination of Q10 and its process related impurities was examined by
non-aqueous reversed-phase liquid chromatography using a C8 chromatographic column and
a photodiode array detector. As mentioned above, Q10 is highly lipophilic compound which
retention on chemically bonded reversed phases is large. Therefore, non-aqueous solvents
such as methanol, ethanol, acetonitrile, isopropoyl alcohol and tetrahydrofuran were tested to
accomplish the elution in an acceptable analysis time. The best separation of Q 10 and its four
related impurities were obtained in 20 minutes using mobile phase containing acetonitrile and
isopropyl alcohol (84:16, v/v). In order to identify the impurities with more than 0.1% area,
APCI-MS detection in positive ionization mode was used.
Taurine
Taurine, β-sulphonic amino acid, was first isolated more than 150 years ago. It is
consider being an essential nutrient for human and widely distributed in mammalian cells and
tissues. It is a common constituent of the human diet and most important sources of taurine
are seafood and meat. Many biological effects of taurine are well established. It is able to act
as a neurotransmiter, antioxidant and modulator of cellular calcium levels. Moreover, taurine
also has beneficial effects on the liver that include prevention of liver damaged due to toxic
chemicals. Although, taurine is well-known to humans for many years, just recently studies
regarding its beneficial effects on serum lipids in animals and humans have been performed
[Choi et al., 2006; Zhang et al., 2004]. In a 2008 study, taurine has been shown to reduce the
secretion of lipids and apolipoprotein B100, essential structural component of LDL in HepG2
cells [Yanagita et al., 2008].
A simple LC/MS method for simultaneous determination of caffeine, theobromine,
theophylline and taurine in different dietary supplements was reported [Marchei et al., 2005].
Chromatographic separation was achieved using a C18 column with water, methanol and
acetic acid (75:20:5, v/v/v) as eluent at a flow of 0.7\mL/min. Mass spectrometer was operated
in positive ESI mode with selected ion monitoring acquisition. Although it was not mentioned
on the labels of products, taurine was found in three dietary supplements.
SIMULTANEOUS ANALYSIS OF NATURAL
CHOLESTEROL-LOWERING AGENTS
In order to reduce lipid levels more efficiently, nowadays many cholesterol-lowering
dietary supplements are produced and sold as products containing multiple herbal
components. The development of the method for simultaneous determination of several
natural-cholesterol lowering agents is a quite challenging task as these compounds are
structurally diverse. An additional problem is that the composition of these multiple herbal
23
Ana Mornar, Miranda Sertić and Biljana Nigović
dietary supplements is extremely complex including several active and inactive ingredients of
each herb as well as various excipients.
Far to our knowledge, only one HPLC method coupled with MS detector equipped with
an ESI source and quadrupole – ion trap hybrid analyzer was developed for multi-target
screening of biomarker compounds in different herbal dietary supplements, including natural
cholesterol-lowering agents such as artichoke, soybean and flax seed [Mathon et al., 2013].
Different types of formulations were purchased; a majority of samples were powders, tablets
and capsules, while others were in liquid form as drinkable ampoule or oral suspensions. 98
analytes with quite different structures and physical-chemical properties were unselectively
extracted by sonification in a mixture of methanol and water. The aim of work was to keep
extraction procedure as simple as possible, even though this might lead to some matrix effects
due to limited sample purification. For the separation, a short reversed phase Synergi Polar
analytical column (50\×\2.0\mm, particle size 2.5\μm) was used. The mobile phase consisted
of water and methanol for the negative ionisation mode, while the mixture of methanol and
water with 5\mM ammonium formate buffer at pH 4 for the positive ionisation mode was
used. In both modes, the same solvent gradient elution was used. The proposed method was
fairly short (25 minutes). Still, based on mass spectrometric recognition, 98 selected
biomarker compounds were identified and quantified in 79 different dietary supplements.
BIOANALITICAL METHODS
The investigation of bioavailability, pharmacokinetic properties and metabolism of active
ingredients of cholesterol-lowering dietary supplements requires large-scale clinical trials that
involve rapid, validated assays for the identification and quantification of the active
ingredients and their metabolites in biological samples. HPLC has proved to be a fully
automatable instrumental technique of choice for this purpose due to its superior precision
and high resolution. One of the main advantages of HPLC is the possibility to couple the
technique to various detectors in order to obtain higher selectivity and sensitivity of methods.
However, biological samples are complex matrices and sample preparation, a delicate, time-
consuming and tedious step of bioanalytical methods, can not be avoided. The endogenous
proteins and other biological compounds can cause increased column backpressure and may
lead to the decrease in the performance of chromatographic columns.
So far little has been known about the metabolism of active ingredients of artichoke leaf
extract. Therefore, a validated method for the simultaneous determination of the
hydroxycinnamates caffeic, dihydrocaffeic, ferulic, dihydroferulic and isoferulic acid as well
as the flavonoid luteolin in human plasma as metabolites derived from artichoke leaf extract
was developed [Wittemer et al., 2003]. To obtain a nice peak shape of analytes, the sample
preparation procedure included the pH adjustment to 5.0 with acetic acid. Chromatographic
separation of analytes was performed on a reversed phase column with a polar endcapping,
while superior sensitivity (limits of quantitation were lower than 2.2 ng/mL) was achieved
using electrochemical array detection. The total analysis time may be too long for routine
analyses (97.5 minutes), most probably due to simple and unselective sample preparation
procedure. Afterwards, the developed method was used for the determination of plasma and
urine metabolites in a clinical study of the bioavailability and pharmacokinetics of artichoke
24
High Performance Liquid Chromatography – An Effective Tool for Quality Control …
leaf extract components after oral administration [Wittemer et al., 2005]. In order to assay the
free metabolites beside their conjugates each plasma sample was prepared with and without
enzymatic hydrolysis by sulfatase/glucuronidase of conjugated metabolites prior to HPLC
analysis. After administration of extract none of the genuine target extract constituents could
be detected in human plasma. However, after ß-glucuronidase treatment caffeic acid, its O-
methylated products ferulic and isoferulic acid, the hydrogenation products dihydrocaffeic
and dihydroferulic acid as well as luteolin could be identified. All of these compounds, except
of dihydroferulic acid, were present as phase-II-conjugates (sulfates or glucuronides).
However, the presence of nonconjugated caffeic, dihydrocaffeic and ferulic acid in plasma
cannot be completely excluded since they were detected in urine. It is believed that the
concentration of these compounds in plasma was below the limit of detection.
The development of a method for analysis of omega-3 fatty acids at physiological levels
represents a challenge based on the high endogenous levels of these fatty acids, as well as the
vast variability between subjects due to dietary intake and genetics. A method for
simultaneous determination of most abundant omega-3 fatty acids, EPA and DHA, as well as
arachidonic acid in human plasma after oral administration of a fish oil dietary supplement
using HPLC/MS/MS was developed [Salm et al., 2011]. Free and esterified forms of fatty
acids were hydrolyzed and analytes were extracted from biological samples using liquid-
liquid extraction with chloroform. The chromatographic analysis was performed using a small
reversed phase column (50 x 2 mm, particle size 3 μm) and total analysis time was only 3.5
minutes. To minimize sample carry-over effects, a programmed injector needle wash of three
consecutive cycles with 85% methanol (v/v) was used. The MS detection was performed on a
linear ion trap quadrupole tandem mass spectrometer. The proposed method was proven to be
particularly suitable for monitoring omega-3 fatty acids in clinical studies that may aid in
achieving optimal concentrations of these fatty acids in patients who could be at risk of
sudden cardiac death.
The clinical studies regarding pharmacokinetic behavior of tea catechins are extremely
valuable as most of bioavailability and tissue distribution studies have been performed mostly
using laboratory animals. The pharmacokinetic properties of tea polyphenols in plasma and
urine eight hours after a bolus consumption of green tea, black tea or green tea dietary
supplement were investigated [Henning et al., 2004]. The samples were purified and analytes
concentrated using reversed phase solid phase extraction. The columns were washed with
sodium acetate at pH 6.5 and the elution of analytes form cartridges was performed with ethyl
acetate. The polyphenol levels were measured using HPLC coupled to coulometric array
electrochemical detector and a small C18 chromatographic column with unusually large
internal diameter (53 x 7 mm, particle size 5 μm). Separation of all analytes was achieved
using a gradient elution and total analysis time was 35 minutes. The polyphenols
administrated in the form of green tea supplement showed enhanced bioavailability compared
with that of green and black tea, which led to a small but significant increase in antioxidant
capacity. The HPLC technique coupled to coulometric array electrochemical detector was
also used in pharmacokinetic study of green tea polyphenols after multiple-dose
administration of epigallocatechin gallate and Polyphenon E, a defined, decaffeinated green
tea polyphenol mixture [Chow et al., 2003]. A more common C18 chromatographic column
(150 x 4.6 mm, particle size 5 μm) and a gradient elution with phosphate buffer, acetonitrile
and tetrahydrofuran were employed. The total analysis time was a little bit longer (44
minutes) than in above mention method. A simple sample preparation procedure was
25
Ana Mornar, Miranda Sertić and Biljana Nigović
employed. The plasma samples were extracted with methylene chloride to remove lipid
components. Afterwards, the remaining aqueous phase was extracted with ethyl acetate. The
patients were instructed to take green tea polyphenol dietary supplements in amounts similar
to the epigallocatechin gallate content in 16 Japanese-style cups of green tea ones daily for
four weeks. It was found that the daily administration of high doses of green tea polyphenols
is safe and well tolerated in healthy human subjects. Repeated administration of high doses
resulted in a significant increase in the systematic exposure of epigallocatechin gallate, most
likely due to inhibition of presystematic elimination of this cahechin.
Since the bioavailability of soy isoflavones is quite variable, measurement of plasma
levels could help dosage adjustment, above all in the patients who failed to response the
therapy. An analytical method based on use of an HPLC system with coulometric detector,
equipped with a high sensitivity analytical cell was developed [Saracino et al., 2010]. The
plasma samples needed careful clean-up before proceeding to the step involving the
measurement of free and total amounts of soy isoflavones (genistein, daidzein and glycitein).
Therefore, the solid-phase extraction was employed. Different cartridges that exhibit polar
and weak non-polar interactions with analytes were tested: diol, cyanopropyl, C2, C8 and
hydrophilic-lipophilic balance sorbents. Better results in term of extraction yield were
obtained using the hydrophilic-lipophilic balance cartridges (recoveries more than 90%) than
the lipophilic sorbents, C2 and C8 (recoveries less than 60%). Polar solvents have shown to
be inadequate to clean-up plasma samples because of much interference found in
chromatograms of samples. Two different chromatographic columns were tested: C18 and
pentafluorophenylpropyl reversed phase columns. As pentafluorophenylpropyl column did
not show good resolution, all experiments were performed using C18 column, characterized
by a lower retention of lipophilic soy isoflavones. An excellent separation of all analytes was
obtained in 21 minutes using isocratic elution with phosphate buffer, triethylamine and
acetonitrile. Good results in terms of precision (relative standard deviations lower than 5.4%),
sensitivity (limits of quantitation were 0.25 and 0.5 ng/mL) and accuracy (recoveries higher
than 89%) were obtained. The hypocholesterolemic effect of soybean is attributed mostly to
the soy isoflavonoids, which are also well known as phytoestrogens. Therefore, a new
chromatographic method for the simultaneous determination of 13 phytoestrogens, including
soy isoflavonoides, and their most important precursors and gut microbial metabolites in
human serum and urine was developed [Wyns et al., 2010]. The sample preparation procedure
consisted of enzymatic hydrolysis using Helix pomatia β-glucuronidase/sulfatase. Analytes
were extracted from urine samples using liquid-liquid extraction with diethyl ether, while
serum samples required more efficient and selective solid-phase extraction. Chromatographic
separation of all analytes in one single run was performed using a C18 column at temperature
of 55 °C. During the chromatographic run composition as well as flow rate of mobile phase
were changed. Although complete resolution of several analytes on UV detection was not
accomplished, subsequent mass selective detection allowed full separation of all compounds.
The proposed method was fully validated and was sensitive enough (limits of detection were
from 0.2 to 132.6 ng/mL) to be applied for the analysis of serum and urine samples collected
in a clinical trial with dietary supplements combining different classes of phytoestrogens,
including soybean.
As mentioned above, coenzyme Q10 is strongly lipophilic compound, practically insoluble
in aqueous solution and has poor bioavailability in humans. Its bioavailability is markedly
influenced by delivery system. In addition, emulsifiers and surfactants may affect the release
26
High Performance Liquid Chromatography – An Effective Tool for Quality Control …
of active ingredient from formulations. The importance of product formulation was
recognized early in the development of Q10 dietary supplements. Therefore, several studies
regarding to bioavailability of Q10 are published. A randomized cross-over study was
performed to investigate the bioavailability of dietary Q10 in humans by supplementation with
single doses of coenzyme Q10, administered either as a meal consisting of cooked pork heart
or as coenzyme Q10 dietary supplement [Weber et al., 1997]. After liquid-liquid extraction
with hexane, the quantification of Q10 in human serum was performed by means of HPLC
with UV detection. The isocratic elution with mixture of organic solvents (ethanol, hexane
and isopropanol) was employed. The concentration of coenzyme Q10 in serum was increased
after administration of both cooked meal as well as dietary supplement. Interestingly, the
maximum concentration for the two administrations did not differ significantly. The influence
of two dietary supplement formulations, oil- and granule-based preparations, on absorption of
coenzyme Q10 was investigated using HPLC technique [Kaikkonen et al., 1997]. Two methods
were proposed for determination of only reduced form of Q10 as well as for determination of
both reduced and oxidized forms. Furthermore, two sample preparation procedures were
described: liquid-liquid extraction with hexane and solid phase extraction with C18
cartridges. It was found that the granular preparation elevated better plasma Q10 at baseline,
but the increase did not differ significantly from the oil-based capsule, as assessed by area
under the curve. In summary, the bioavailability of coenzyme Q10 varied more between the
subjects than between preparations. Afterwards, the bioavailability of coenzyme Q10
formulated as an emulsion in a soft gelatin capsule was compared with a hard gelatin powder-
filled capsule [Wahlqvist et al., 1998]. Before the determination of Q10 plasma level, the
samples were oxidized with 0.1% ferric chloride solution in the presence of diluted
hydrochloric acid. The mixture was than shaken with n-propanolol and supernatant was
injected to the HPLC systems connected to UV detector. The new formulation, soft gelatin
capsule, which contained coenzyme Q10 as a complex micelle in an emulsion, had higher
bioavailability than the previously used hard gelatin powder-filled capsule. Most likely the
presence of surfactants in the new formulation contributed to the enhanced solubilization and
release of Q10. Tang and co-workers [Tang et al., 2001] have proposed a simple and rapid
HPLC procedure with coulometric detection for simultaneous determination of both forms of
coenzyme Q10. Afterwards the same method was used for bioequivalence study of coenzyme
Q10 dietary supplements [Miles et al., 2002]. It was found that the liquid formulation may be
useful for individuals who have difficulty or are unable to swallow solid formulations. The
similar method was used for the investigation of the bioavailability of coenzyme Q10
supplements as well as for determination of the most efficient dose of coenzyme Q10
[Molyneux et al., 2004; Molyneux et al., 2007]. The comparison of Q10 plasma levels
following multiple oral doses administered as sustained release or regular tablets was
performed [Lu et al., 2003]. The sample preparation procedure included only the precipitation
of proteins using methanol as organic solvent and the extraction of analytes with hexane. The
separation of Q10 and Q9, internal standard, from other constituents of biological sample was
obtained using a simple isocratic elution with non-aqueous mobile phase consisted of
methanol and ethanol (9:1, v/v). UV detector was used for quantitation of Q10, still good
sensitivity of method was achieved. The minimum concentration of coenzyme Q10 that could
be detected in plasma was 0.02 mg/L. The obtained pharmacokinetic parameters showed that
the sustained release preparation indeed provided longer systematic exposure of Q10.
However, the effect was not statistically significant compared to the regular preparation. To
27
Ana Mornar, Miranda Sertić and Biljana Nigović
investigate the safety of high doses of coenzyme Q10 a double-blind, randomized, placebo-
controlled study was performed and Q10 plasma levels were determined using HPLC coupled
to electrochemical detector [Ikematsu et al., 2006]. Recent studies have shown that the
determination of Q10 plasma levels provides only limited information on patient’s coenzyme
Q10 status. Therefore, an HPLC method coupled with electrochemical detector for analysis of
coenzyme Q10 in plasma, erythrocytes and platelets was proposed [Niklowitz et al., 2004].
More recently, an HPLC method coupled to tandem mass spectrometric detector for
determination of Q10 levels after oral supplementation in lymphocytes was developed [Arias et
al., 2012]. The method is particularly suited for the monitoring and dose adjustments in the
patients with coenzyme Q10 deficiency.
RELATED TECHNIQUES
Thin-Layer Chromatography
Now-a-days TLC is becoming a routine analytical technique due to its advantages of low
operating cost, high sample throughput and need for minimum sample preparation. The
unique feature of picture-like image of TLC supplies an intuitive visible profiling of samples.
It is worth noting that the technique of TLC is also being updated in progress. With
densitometric scanning, it is possible to get useful qualitative and quantitative information
from the developed TLC plate. The major advantage of TLC over HPLC is that several
samples can be run simultaneously using a small quantity of mobile phase.
In the work of Reich and co-workers [Reich et al., 2006] a high performance thin layer
chromatographic (HPTLC) method for identification of green tea and green tea extract is
presented. The flavonoid fingerprint of green tea was obtained in order to determine the
geographical origin of the material. The mobile phase consisted of toluene, acetone and
formic acid (9:9:2, v/v/v) allowed the discrimination of green tea from black and other
specialty teas, based on the polyphenol pattern. Additionally, two other chromatographic
systems were used for investigation of an alkaloid and an amino acid profile.
Agrawal and co-workers [Agrawal et al., 2004a] have proposed an HPTLC method for
quantitative determination of Z- and E-guggulsterones in herbal extracts and dietary
supplements. The silica gel aluminium plates 60F-254 (20 x 10 cm, thickness 250 μm) were
used. The mobile phase was consisted of toulene-acetone mixture (9:1, v/v) and densitometric
scanning of the plates was performed on Camag TLC Scanner III. The method was validated
and the statistical analysis proved that the method is an accurate, precise, robust and
reproducible. Comparing the proposed TLC method to above described HPLC methods, it
may be concluded that selectivity of all HPLC method was significantly better. The proposed
TLC method was applied for investigation of guggul herbal extract and dietary supplement.
The video densitometric image of chromatographic plate has clearly shown very low
concentration of Z- and E-guggulsterones in both extract and capsules. The same HPTLC
method was employed for stress degradation studies on E- and Z-guggulsterones [Agrawa et
al., 2004b]. The degradation of guggulsterones under different International Conference on
Harmonization (ICH) prescribed stress conditions (acid and base hydrolysis, oxidation, dry
and wet heat degradation and photodegradation) and establishment of a stability indicating
28
High Performance Liquid Chromatography – An Effective Tool for Quality Control …
HPTLC assay were investigated. The results of this study have shown that guggulsterones are
unstable in almost all forced degradation conditions. More recently, Lalla and co-workers
[Lalla et al., 2007] have published another HPTLC method for determination of E- and Z-
guggulsterones as biomarkers of dietary supplements containing guggul. Besides the above
described HPLC method, Musharraf and co-workers [Musharraf et al., 2011b] have also
proposed HPTLC and two-dimensional HPTLC methods for simultaneous determination of
E- and Z-guggulsterones in oleo resin guggul, herbal extract guggulipid and their
pharmaceutical products. HPTLC analysis was performed using the same chromatographic
plates as Agrawal and co-workers [Agrawal et al., 2004a] had used, still mobile phase
composition was slightly different (toulene-acetone mixture, 9.0:0.7, v/v). To improve
selectivity of the method two runs with the same mobile phase were carried out. Two-
dimensional HPTLC analysis was performed by spotting samples at the lower right corner on
smaller plates (10 x 10 cm) with the band width of 0 mm which resulted the spotting in
spherical shape. Well resolved spots for E- and Z-guggulsterones were obtained using both
methods. Moreover, the good separation of both guggulsterone isomers from 17,20-
dihydroguggulsterone was achieved. An HPTLC method for simultaneous estimation of E-
and Z-guggulsterone and tinosporaside in Jivitprada vati (an Ayurvedic formulation consisted
of Guggul, Shilajit and Galodhan) was developed [Jariwala et al., 2011]. The proposed
method was found to be rapid, simple and accurate of quantitative estimation of all analytes in
different formulation.
Gas Chromatography
Many pharmacologically active components in dietary supplements are volatile chemical
compounds. GC is a well established analytical technique commonly used for the
characterization and quantitation of volatile compounds. The powerful separation efficiency
of capillary columns and sensitive detection with two most frequently used detectors, flame
ionization (FID) or MS, make GC a useful tool for the analysis of active and toxic ingredients
in complex matrices such as dietary supplements. Despite its advantages, GC analysis of
these products is usually limited to the volatile oils. The possible degradation of thermo-
instable compounds and the pre-requisite of volatile compounds make GC unsuitable for
many active ingredients in dietary supplements for which derivatization is not always
possible.
Capillary GC is the technique of choice for the analysis of phytosterols and related
compounds. Therefore, Bedner and co-workers [Bedner et al., 2008] have used GC-FID
method for affirmation of newly developed HPLC method for determination of phytosterols
in dietary supplements. The dimethylpolysiloxane fused silica capillary column and a gradient
temperature program were used. The obtained results are in good agreement with those
obtained by HPLC method. Another GC-FID method using 5% phenyl-methyl siloxane
capillary column was developed for determination of three phytosterols in dietary
supplements [Sorenson et al., 2006]. Afterwards, the same method was used for collaborative
study between 10 laboratories [Sorenson et al., 2007]. The major advantage of the method
proposed by Bedner and co-workers [Bedner et al., 2008] was that no derivatization step was
required before GC analysis. On the other hand, the demanding sample preparation
29
Ana Mornar, Miranda Sertić and Biljana Nigović
procedure, including derivatization of phytosterols to trimethylsilyl ethers, was required in the
method developed by Sorenson and Sullivan [Sorenson et al., 2006; Sorenson et al., 2007].
Although several HPLC methods for determination of omega-3 fatty acids in fish oil
based dietary supplements are described above, there can be little doubt that GC is the only
technique that need be considered for routine analysis of most fatty acid samples. Still, it must
be taken into account that all GC methods, before the analysis, include time-consuming
derivatization of fatty acids to methyl esters derivatives. A capillary GC-FID method for
determination of 11 fatty acids in dietary supplements was proposed [Tatarczyk et al., 2007].
Quantitative analysis was completed with the use of tridecanoic acid as internal standard. All
samples contained considerable amounts of long chain omega-3 fatty acids, with highest
percentage of EPA and DHA. Comparison with manufactures´ information showed that
amounts found in four dietary supplements did not differ significantly from the labeled value,
while four samples held significantly more fatty acids than stated and one manufacturer did
not declare the amount of omega-3 fatty acids contained in their product. Regarding
manufacturer’s recommendations for daily intake, one of supplements exceeded the amount
recommended by healthcare organizations. In the study of Opperman and co-workers
[Opperman et al., 2011] a large number of dietary supplements containing fish oil were
analyzed using GC-FID method. The separation of two most frequently present omega-3 fatty
acids, EPA and DHA, was achieved in 20 minutes using a BPX-70 fused silica capillary
column and a linear gradient temperature program. Interestingly, more than half of
investigated dietary supplements contained the amount of EPA and DHA more than 89%
lower than the claimed value on the labels of the products. A health concern related to fish
and fish oil supplements is that some species of fish may accumulate in tissue significant
amounts of lipid-soluble persistent organic pollutants, including polychlorinated biphenils.
These compounds can be immunotoxic, carcinogenic and can elicit adverse developmental,
reproductive and endocrine effects. Therefore, Bourdon and co-workers [Bourdon et al.,
2010] have proposed a new GC method with electron capture detector for determination of 36
polychlorinated biphenils in fish oil and fish oil alternatives dietary supplements. In all
samples, except flax seed oils, polychlorinated biphenils were found in the range from 0.8 to
high 793 ng/g. The obtained results indicate that some omega-3 dietary supplements may
contain substantial levels of polychlorinated biphenils. Therefore, the manufactures of omega-
3 dietary supplements should give detailed information on content of persistent organic
pollutants in their products to regulatory agencies in order to evaluate the quality of marked
products.
Policosanol is the common name that refers to a mixture of long chain (20 - 36 carbon)
aliphatic primary alcohols isolated from the waxes of the plants sugarcane and yams as well
as beeswax. Its use as a natural cholesterol-lowering agent is quite new. In the early 1990s,
researchers at Dalmer Laboratories in La Habana Cuba isolated and produced the first
policosanol formulated product from sugarcane wax. Although research studies have come to
conflicting conclusions regarding the efficacy of the policosanol in lowering LDL or raising
HDL levels, currently dietary supplements containing policosanol are sold in more than 40
countries all over the world [Marinangeli et al., 2010]. Identification and quantification of
long chain aliphatic primary alcohols in policosanol dietary supplement were performed using
GC coupled to MS detector [Singh et al., 2006]. The analytes were extracted from the tablets
by methylene chloride. Three major peaks corresponded to hexacosanol, octacosanol and
triacontanol, while heptacosanol and nonacosanol were present as minor constituents. More
30
High Performance Liquid Chromatography – An Effective Tool for Quality Control …
comprehensive analysis of policosanol content in beeswax, sugar cane and wheat extracts as
well as in three commercial dietary supplements was performed by Irmak and co-workers
[Irmak et al., 2006]. To obtain a complete extraction of long chain aliphatic primary alcohols
from different matrices, the ground samples were hydrolized by refluxing with 0.1 N sodium
hydroxide solution in methanol for 30 minutes. The further purification of samples was
performed by extraction with diethyl ether. Trimethylsilyl derivatives of alcohols were
analyzed by GC coupled to MS detector. Good separation of nine analytes on a fused silica
capillary column was obtained using gradient temperature program in 57.5 minutes and the
elution of compounds was molecular weight dependent. However, the chromatograms of
samples were quite complicated and displayed numerous peaks. The mass spectral library
search indicated that some of the phytosterols and fatty acids present in the samples had
similar chromatographic behavior as investigated long chain aliphatic primary alcohols.
Finally, it should be pointed out that all investigated dietary supplements contained lower
amounts of policosanol than was claimed on the labels.
Capillary Electrophoresis
Capillary electrophoresis (CE) meets the requirements for the quality control of dietary
supplements because of its versatility and high separation power. Moreover, it has become
popular in separation science because of their low solvent consumption, decreasing the
operational costs and causing less negative impact on the environment. Surprisingly, a few
CE methods for determination of active and toxic ingredients in cholesterol-lowering dietary
supplements have been published.
So far only one CE method was proposed for the determination of lovastatin in red
fermented rice products [Li et al., 2007]. They used a capillary zone electrophoresis method
for the qunatitation of lovastatin in Monascus capsules. The background electrolyte consisted
of 16% ethanol (v/v) in 60 mM glycine-sodium hydroxide buffer (pH 9.5). Separation was
performed at 16 kV applied voltage and 22 °C. Injection was performed by pressure set at 14
mbar for 5 s. Extraction of the 50.0 mg of the red fermented rice samples was performed with
ethanol, and supernatant after the centrifugation was collected and transferred to another tube.
The residue was extracted once more by ethanol and the two organic phases were combined
and dried by water bath at 80 °C. The residue after dryness was dissolved in 1 mL ethanol.
During method development influence of the buffer pH and concentration, organic modifier
and applied voltage was examined. The deficiency of the proposed method is that the
lovastatin in the lactone form was converted to the β-hydroxy acid form which was
subsequently determined in the analyzed samples.
Therefore, we have recently developed a CE method for simultaneous determination of
lovastatin in both its lactone and β-hydroxy acid form, and the possible nefrotoxic by-product
citrinin [Nigović et al., 2013]. First step in the development of a CE method is the selection of
the buffer pH since it plays the key role in the extent of the ionization and electrophoretic
mobilities of the analytes, as well as the electroosmotic flow rate. Special attention was given
to the possible interconversion problem between the lactone and β-hydroxy acid form of
lovastatin. To avoid possible conversion during the analysis, and yet to keep the analysis time
as short as possible, phosphate buffer was used and the pH was tested in the range between
5.5-7.5. Since lovastatin lactone is a neutral molecule, micellar electrokinetic chromatography
31
Ana Mornar, Miranda Sertić and Biljana Nigović
had to be employed. Sodium dodecyl sulphate (SDS) was used as the surfactant to act as the
pseudostacionary phase. Although phosphate buffer at pH 7.5 gave the shortest migration
times of all analytes studied, the best results were obtained at pH 7.0 because best peak shape,
symmetry, and good resolution was achieved in short analysis time (under 2 min). During
further method development phosphate buffer concentration, SDS concentration and applied
voltage was also tested. Optimized conditions were as follows: 20 mM phosphate buffer pH
7.0, 30 mM SDS, and 25 kV applied voltage and 25 °C temperature of the capillary. To
enhance the detection sensitivity, an extended light path capillary, possessing a bubble cell at
the detection point was used for the separation. The capillary with an optical path length of
150 μm increased the peak area citrinin, the critical analyte because of low expected
concentration in the investigated products, by 3.5-fold over standard capillary with 50 μm
internal diameter. After optimization, method was fully validated according to the ICH
guidelines, and selectivity, linearity range, limits of detection and quantification, precision,
accuracy, and the robustness of the method were examined. Due to the complexity of the
analyzed dietary supplements, different sample extraction procedures were tested. The
optimization of the extraction process was performed on tablet, capsule and gel cap product.
Among acetonitril, ethanol, and methanol, the highest extraction efficiency was achieved with
80% methanol. Extraction was performed at room temperature in an ultrasonic bath for 60
minutes. After the extraction, the mixture was centrifuged at 3000 rpm for 10 minutes and the
supernatant was collected and evaporated to dryness under a nitrogen stream. The residue was
redissolved in 1 mL of methanol thus enhancing the sensitivity of the method even more. As
with the HPLC method, were noticed. In two investigated products the levels of both
lovastatin forms were very low, thus the investigated products were under dosed. In three
samples lovastatin lactone was the main component, while one product contained the β-
hydroxy acid form as the major active ingredient. The obtained results in total monacolin
contents compared to declared values ranged from 11-178%, again showing severe
discrepancy between actual and labeled values. In two samples total monacolin values were
above 11 mg, which is in the lovastatin therapeutic range. Citrinin was found in two analyzed
products, under the European Union recommended limit. The proposed method is the first
capillary electrophoresis method for simultaneous determination of lovastatin lactone,
lovastatin β-hydroxy acid, as the key active ingredients in red fermented rice products, and
citrinin, a possible nefrotoxic contaminant. It represents a fast, environmental friendly, cost
effective alternative to usually employed HPLC methods and can be used for fast screening of
red fermented rice products by the manufacturers, as well as the regulatory agencies.
A micellar electrokinetic chromatography (MEKC) was developed for the analysis of
green tea dietary supplements [Weiss et al., 2006]. The authors developed a first MEKC
method for the determination of five catechins, and caffeine. Much consideration was put to
optimization of the extraction process for which they prepared dummy capsules containg
catechin and caffeine standards. Extraction with 80% methanol (v/v) has proven to be the
most efficient one. During method development, the choise of buffer type and pH, SDS and
buffer concentrations were examined. Best results were obtained using a background
electrolyte containg 5 mM borat buffer, 60 mM phosphate buffer, 50 mM SDS at pH 7.0.
Separation was conducted using 27 kV voltage in a 80.5 cm x 50 μm i.d. fused silica
capillary. The authors emphasise that the separation of the most common expected catehins
was not difficult, the challenge was to separate all other unknown compounds present in the
green tea extract from the analytes of interest. The method was then applied for the analysis
32
High Performance Liquid Chromatography – An Effective Tool for Quality Control …
of three commercially available green tea dietary supplements from different manufactures.
All labels claimed to contain catechins in the range between 60 and 90%, depending on the
product. Obtained results indicated a significant variation in caffeine and catechins amounts
among different brands. The amount of total catechins was greater than the manufacturer
claimed on the products in two tested samples. In one case under dose of catehins was found.
Considerable variability was found between capsules of the same batch (349 mg versus 167
mg), as well as between batches (179 mg versus 268 mg). The authors highlighted that the
manufacturers had not indicated whether their products had been prepared using Good
Manufacturing Practices and suggested a need for tightened regulation of dietary
supplements.
A microchip-micellar electrokinetic chromatography with pulsed amperometric detection
was used for the direct analysis of naturally occuring flavonoids, catechin, epigallocatechin
gallate, epicatechin, and epicatechin gallate in commercial green tea extract supplements
[Hompesch et al., 2005]. During method development the effect of buffer pH, surfactant
concentration, detection potential and signal stability were examined. The analysis time was
very short, 4.5 minutes, thus the proposed CE method is 10 times faster than the usually
employed HPLC methods.
A comparison between a borate-phosphate-SDS based MEKC and a reversed phase
HPLC method for the separation of seven tea catechins and gallic acid in green tea extracts
was performed [Bonoli et al., 2003]. The MEKC method showed significantly higher
sensitivity, resolution, efficiency and migration times repeatability than the proposed HPLC
method.
CONCLUSION
The sale of dietary supplements has increased considerably over the last 20 years in the
industrial countries. Although seemingly ˝natural˝ way of healing, safe from serious side
effects, they should be tested under strict conditions to ensure efficacy and safety. Therefore,
development of the analytical methods for identification and quantitation of active ingredients
in dietary supplements has received a great deal of attention in the field of quality control of
these products. This chapter includes current trends in HPLC and related techniques in quality
control of natural cholesterol-lowering agents.
The extraction of analytes of interest from this complex samples is a quite tedious
procedure and usually extraction with organic solvents with different polarities was
employed. HPLC was found to be the most popular analytical technique for analyzing
cholesterol-lowering dietary supplements. Most of developed methods have used a reversed
phase C18 chromatographic columns, except in several methods when C8, C30 and phenyl
columns were used. The gradient elution was usually employed for separation of active and
toxic ingredients. As HPLC requires rather expensive machinery and often uses large
volumes of environmental unfriendly solvents, this chapter surveys the application of other
separation techniques (TLC, GC and CE) for the investigation of dietary supplements.
The results obtained in the most studies revealed a great diversity in the amounts of
active and toxic components in dietary supplements produced by different manufactures.
Moreover, high batch-to-batch variability was found for several products. Unfortunately,
33
Ana Mornar, Miranda Sertić and Biljana Nigović
most of dietary supplements had lower amounts of active ingredients than the claimed value
on the product, which means lower pharmacological activity. Because of these poorly
manufactured products, most of health practitioners and patients consider all dietary
supplements as products with low efficacy and quality. Still, the use of cholesterol-lowering
dietary supplements has increased in recent years and is expected to enhence further in years
ahead because hyperlipidemias are being diagnosed more frequently. Thus, the development
of new chromatographic techniques for determination of active and toxic compounds,
degradation products of currently commercially available dietary supplements as well as
novel upcoming products will be future challenging task for analysts all over the world.
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