Accepted Manuscript

Current trends in the analysis and quality control of food supplements based on plant
extracts

Jakub Fibigr, Dalibor Šatínský, Petr Solich

PII: S0003-2670(18)30954-1
DOI: 10.1016/j.aca.2018.08.017
Reference: ACA 236191

To appear in: Analytica Chimica Acta

Received Date: 27 March 2018

Revised Date: 31 July 2018
Accepted Date: 4 August 2018

Please cite this article as: J. Fibigr, D. Šatínský, P. Solich, Current trends in the analysis and quality
control of food supplements based on plant extracts, Analytica Chimica Acta (2018), doi: 10.1016/
j.aca.2018.08.017.

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 ACCEPTED MANUSCRIPT

GC-MS

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FT-IR
LC-MS
LC-MS
TLC

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Endocrine
Pharmaceuticals disruptors

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LC-UV
ICP-MS
SFC-PDA
GC-FID

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Bioactive Toxic
substances metals

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NMR LC-MS
LC-MS GC-MS
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Adulterants Mycotoxins
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Current trends in the analysis and quality control of

food supplements based on plant extracts

Jakub Fibigr, Dalibor Šatínský*, Petr Solich

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The Department of Analytical Chemistry, Faculty of Pharmacy, Charles University,

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Akademika Heyrovského 1203, Hradec Králové 500 05, Czech Republic

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∗Corresponding author:

Dalibor Šatínský

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The Department of Analytical Chemistry

Faculty of Pharmacy, Charles University

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Akademika Heyrovského 1203

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Hradec Králové 500 05

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Czech Republic

Tel.: +420495067228; fax: +420495067164

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E-mail address: satinsky@faf.cuni.cz

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Abstract

Food (dietary) supplements include a wide range of products that are designed to be taken because of their
added nutrients and presumed health benefits. Global food supplement sales are experiencing rapid
growth and supplements that based on botanicals are among the most popular. The meteoric rise in sales
coupled with the general lack of a commitment to pass effective regulation make this market more
vulnerable to dishonest producers, increase the likelihood that supplements containing adulterants are sold

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on the market, and a greater prevalence of safety and quality issues (contamination by pesticides and
mycotoxins). In this paper, we present an overview of various sample preparation and analytical techniques

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that can be used for the determination of bioactive substances in food supplements based on plant extracts
and for making purity assessments of plant extracts in these preparations. The analysis looks at data

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collected from 2012 to 2017. The work is divided according to the different approaches taken when
analysing food supplements and groups of bioactive substances found in plant extracts (purity assessments
and the determination of bioactive substances).

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Keywords
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Food/dietary supplements; analytical methods; sample preparation; bioactive substances, plant extracts;
contaminants; adulterants
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1. Introduction

Food supplements, which are synonymous with the term "dietary supplements", are concentrated
sources of nutrients or other substances that have a nutritional or physiological effect and whose purpose
is to supplement one’s normal diet due to their presumed health benefits. Food supplements are marketed
in dosage forms, for example pills, tablets, capsules, or liquids in measured doses (EU regulation (Directive
2002/46/EC)) [1]. Global food supplement sales are experiencing rapid growth and those based on

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botanicals and plant extracts are among the most popular because they are believed to be safer and
healthier than synthetic drugs. They are also believed to be free of side effects. These increasing sales,

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which indicate the great popularity of food supplements, and the general lack of commitment to pass
effective legislation (e.g. a lack of guarantees concerning efficiency and safety, no documentation of the

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purity or stability of used substances, etc.) make this market more vulnerable to dishonest producers, a
greater likelihood that supplements contaminated by adulterants will be sold on the market, and to other
problems associated with the quality and safety of food supplements, not to mention the plant extracts

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themselves. In the EU, food supplements are regulated the same as food and the current legislation is
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focused only on the vitamins and minerals that are contained in food supplements [1]. In the US, "dietary
supplements" is the official term defined by FDA. According the FDA definition, dietary supplements are
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products taken by mouth that contain a "dietary ingredient." Dietary ingredients include vitamins, minerals,
amino acids, and herbs or botanicals, as well as other substances that can be used to supplement the diet
[2]. Generally, food supplements differ from nutraceuticals, which are, according to the European
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Nutraceutical Association, naturally derived bioactive compounds that are found in foods, dietary
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supplements and herbal products, and are presumed to aid in the prevention or treatment of disease.
However, the definition of the term food/dietary supplements and the widely used term nutraceuticals is
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relatively problematic because there is no unified legislation among different countries. In some countries,
the term nutraceuticals is almost equivalent to food/dietary supplements/products, on the other hand,
different food, food additives, and even drugs are marked as nutraceuticals. Therefore, the terms
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food/dietary supplements defined in EU and US legislation, respectively, should be used [2].

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Producers declare their preparations contain a certain amount of plant extract, but this information
says nothing about the content of bioactive substances. The composition and contents of bioactive
substances in natural plants can vary depending on climate, temperature, season, soil, and other factors. A
contradiction between the amount of used extract and the content of active substances in one dose can
have a substantial effect on dosing and lead to an overdose by consumers or to an inefficient preparation.
Although ensuring the health safety and harmlessness of food supplements is essential, no specific
assessments are demanded from their producers. In EU, no manufacturing standard controls that are at
least similar to the pharmaceutical level (GMP) exist. The most serious issues associated with the purity of

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food supplements concern undeclared chemical substances, such as PDE-5 inhibitors, slimming agents or
anabolic steroids. In most cases, these are illegally added to the supplements to enhance the claims stated
on their labels. This practice can lead to potentially serious health consequences [3-8]. Contaminants such
as pesticides, mycotoxins, toxic substances, and heavy metals present in plant extracts are another problem
with food supplement purity [9-15].
Although bioactive substances have low potency compared to pharmaceutical drugs, there are
numerous biological mechanisms by which food supplements based on plant extracts may influence

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pathophysiological processes. On the other hand, properly interpreting the clinical studies that have been
conducted thus far is made more difficult by inconsistent results, poorly designed experiments, or by the

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choices of food supplements investigated without analysing them before they were administered to
humans. Considering all these facts, there is no presumption that food supplements based on plant extracts

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may work or even contain any bioactive substances.
Unlike the EFSA (European Food Safety Authority) regulatory framework, the US FDA (Food and Drug

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Administration) requires that all food supplements produced in the USA be evaluated for identity, purity,
and potency as well as for contaminants and adulterants [2]. For US market, Current Good Manufacturing
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Practice (cGMP) in manufacturing, packing, labelling, or holding operations for dietary supplements is
obligatory. Still, analysts are challenged to develop more and more methods capable of providing the
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resolution, selectivity, and sensitivity to detect required bioactive substances. Thorough knowledge of the
composition and contents of these compounds is essential information for reasonable consumption of food
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supplements.
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To maintain the consumers’ trust in the safety and efficacy of products containing plant extracts, new
modern validated analytical methods are needed for the identification, quantification, and standardization
of bioactive substances in plant extracts to ensure quality control and dosage uniformity in food
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supplement preparations. Taking quality measurements is also important when establishing efficacy and
bioavailability after ingesting these bioactive substances in clinical trials (first quantifying the bioactive
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substances before intake and subsequently their metabolites). Complex natural matrices demand the use
of advanced analytical techniques, such as High- and Ultra-high-performance Liquid Chromatography (HPLC
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and UHPLC) in connection with modern photodiode array detectors (PDA) [16-22], fluorescence detectors
(FLD) [23,24], mass spectrometry (MS) [6,11,24-28], or a combination of more detection techniques [29-
42]. High-resolution mass spectrometry (HRMS) [3,4,12,39,43] is used to perform the non-target analyses
of plant extracts. Evaporative detectors, such as charged aerosol detector (CAD) [33] and evaporative light
scattering detector (ELSD) [31,35], are also used. The choice of analytical technique and sample preparation
procedure also depends on the target compounds and their physico-chemical properties. Therefore, gas
chromatography (GC) coupled with a flame ionization detector (FID) [44-45] and MS [9,10,46] is a
commonly used instrument when analysing plant extracts. Other perspective techniques that can be used

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to analyse plant extracts include super-critical fluid chromatography (SFC) [47-49] and, following the trend
of liquid chromatography, ultra-high performance supercritical fluid chromatography (UHPSFC) [50]. To test
the purity of food supplement preparations, inductively coupled plasma mass spectrometry (ICP-MS) [13-
15], nuclear magnetic resonance (NMR) [51-53], Fourier-transform infrared spectroscopy (FT-IR) [54-55],
and High performance thin layer chromatography (HPTLC) [56-57] have also been employed.
In this paper, we present the current status and the most recent advances in sample preparation and
analytical techniques used for the determination of various bioactive substances in food supplements

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based on plant extracts. We also identify those used for testing the purity of plant extracts in these
preparations from 2012 to 2017. The work is divided according to the different approaches taken when

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making the analyses (identification, assays, and purity tests) of food supplements and groups of bioactive
substances found in plant extracts.

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2. Extraction procedures and sample preparation methods

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When describing sample preparation methods for food supplement samples, it must be taken in the
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consideration that the vast majority of the extracts are in a solid state. From this point of view,
homogenizing and pulverizing several dose forms (usually about 10) is the first step in all sample-handling
procedures. For the determination of bioactive substances in food supplements, ultrasound-assisted solid-
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liquid extraction (SLE) followed by filtration, centrifugation, or both are the most commonly selected
methods. The frequency of using of each technique is depicted (Fig. 1)
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2.1. Solid-liquid extraction

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As previously stated, ultrasound-assisted SLE is the most commonly used technique for the
determination of bioactive substances or adulterants in plant extracts. This is probably based on the fact
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that extracted compounds are easily soluble in the solvents used for extraction. In addition, these solvents
are compatible with the instrumentation methods used, so no further reconstitution step is needed. Due to
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the ease of the homogenization and pulverization of solid samples providing solvents with large exchange
surfaces and short diffusion paths, the fastest and most complete solid extraction possible can be achieved.
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After sonification, the undissolved material is typically removed by filtration [6,16-19,24,28,30,34,44,49]

and centrifugation [20,32,35,36,43,47,56]. Some works report the use of stirring [23], vortex mixing
[3,29,37], and shaking [37,39,57] of the samples in extraction solvents before performing sonification,
centrifugation, and filtration to facilitate extraction and to achieve the highest yields of the analysed
substances. Pure methanol is the most commonly used extraction solvent, followed by different aqueous
and acidified mixtures of methanol. All the extraction solvents used are specified in Table 1 and 2,
respectively.

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2.2. Solid-phase extraction

In case of MS, MS/MS, and HRMS analyses demanding a high purity of analysed samples, several forms
of solid-phase extraction (SPE) as a cleaning step are used. E.g., to minimize the presence of non-polar
components co-isolated from the oily matrix in the final sample, dispersive solid phase extraction (dSPE)
purification of the extracts obtained from the softgels was performed prior to their analysis [12]. For the
determination of phenolic compounds in dietary supplements by UHPLC-Q-TOF/MS, graphene oxide was

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used as the sorbent for dispersive micro solid-phase extraction (DMSPE) [38]. A recent work reported the
use of SPE based on the molecularly imprinted polymers (MISPE) technique to eliminate the matrix effects

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observed in MS. The suitability of the MISPE-UHPLC–MS/MS procedure for analysing real samples was
verified by the determination of lovastatin in dietary supplements based on red yeast rice [40]. MIPs

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provide high selectivity in the extraction step; therefore, they are advantageous for preparing samples for a
complex plant matrix. However, the number of available commercial MIP sorbents for the extraction of
biologically active compounds from plant extracts is limited.

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2.3. Liquid-liquid extraction and micro-extraction techniques
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Liquid-liquid extraction (LLE) was employed in a work dealing with the LC–HRMS determination of
stimulants, anorectic drugs, and phosphodiesterase 5 inhibitors in food supplements. A mixture of
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pentane/ethyl ether (9:1) was used as the acceptor liquid for compounds dissolved in a 1M solution of
sodium hydroxide [4]. Before analysing the plant sterols and stanols, saponification of their esterified forms
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had to be done. Whenever LC or GC instrumentation was employed, the LLE of free sterols and stanols was
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performed using an ethanolic solution of 2 M KOH after saponification at 80 °C. In a method employing LC-
CAD, the LLE was performed using a vortex mixing of the sample solution with diluted aqueous HCl and n-
hexane. Then, the organic part was filtrated and directly injected into the chromatographic system [33].
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This sample preparation method is quicker compared to the one used in the GC-FID/MS analysis, where
reconstitution and derivatization steps were performed before the sample was injected into the GC system
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[45]. LLE was used in all mentioned works as a cleaning step after extraction of desired analytes. Internal
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standards had to be used in all mentioned works to correct for the loss of analyte throughout the whole
sample preparation process, including LLE, which by its very nature can introduce a margin of error when
accurately measuring the target analytes. In addition, the LLE method is not “eco-friendly” in its standard
design. Therefore, the classic LLE approach is being replaced by new miniaturized techniques such as liquid-
liquid micro-extraction (LLME), dispersive liquid–liquid micro-extraction (DLLME), and single drop
microextraction (SDME) [68-70]. However, these micro-extraction approaches are rarely used to analyse
food supplements due to sufficient sample amounts and no necessity to reach low detection limits.

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2.4. Other procedures

For the ICP-MS method, ground and dried samples of dietary supplement were quenched with
concentrated ultrapure nitric acid (65%) and were subjected to mineralization by microwave digestion.
Then, clear solutions were quantitatively transferred into volumetric flasks and made up with deionized
water [13]. In two other works employing ICP-MS, a simple and rapid microwave-assisted extraction (MAE)
procedure using a microwave digester equipped with temperature and pressure sensors was performed

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[14,15].

3. Analytical methods

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3.1. Liquid chromatography methods

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Liquid chromatography in reversed phase mode (RP-LC) coupled with an ultraviolet-visible (UV/vis) or
photodiode array (PDA) is the most commonly used technique for analysing bioactive substances in food

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supplements based on plant extracts [16-22,30-34,36,54,57]. To achieve better selectivity and sensitivity, a
fluorescence detector was employed too [23,24,29]. In case of a low response of analytes in the UV
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spectrum, other types of detectors such as charged aerosol detectors (CAD, Corona detector) [33] and
evaporative light scattering detectors (ELSD) [31,35] had to be used. Despite the universal response of
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these detectors for all non-volatile or semi-volatile analytes without a chromophore or a fluorophore in its
molecule, no need for derivatization, and rather good sensitivity, they are not so widely used, probably due
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to the limitations in their linearity range and lack of spectral data.

One limitation of PDA-based methods for analysing compounds with similar spectral properties could
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be the misidentification of analytes, especially in the absence of authentic standards. This limitation can be
avoided using liquid chromatography (tandem) mass spectrometry (LC-MS/MS). With this instrumentation,
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low detection limits are achieved. In addition to sensitivity and selectivity, the emergence of new ionization
techniques allowing for the soft ionization of a wide range of compounds has encouraged the use of LC-
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MS/MS. The vast majority of employed ionization techniques is performed by (heated) electrospray (ESI)
[3,4,6,11,12,24-28,36-43].
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In works dealing with the analyses of metals such as bioactive compounds and toxic adulterants in
plant extracts, inductively coupled plasma mass spectrometry (ICP-MS) has been employed. ICP-MS
determination of trace elements in plants enables a rapid analysis with good precision and accuracy. The
results obtained in the reported works reveal that the studied medicinal plants and dietary supplements
contained elements in the µg/g range and that elemental concentrations varied widely. Toxic element
contents in the studied samples differed significantly from one another. This variability, according to the
authors of the study, is explained by environmental and agronomic conditions, variable exposure to
pollution, growing in contaminated areas, poor storage conditions, and bad purchasing sources [13]. ICP-

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MS coupled with HPLC was used for the determination of selenium [14] and cobalt [15] in food
supplements.
Advances in equipment and accessories have improved their quality and reliability in the recent years,
therefore alternative separation and determination techniques are currently being used to analyse plant
extracts and assays of bioactive substances. Columns with smaller particles and with core-shell technology
[17,19-21,29,32-34,41,57] have been employed to increase separation efficiency and elution speed with
satisfactory resolution and sensitivity. Another approach increasing elution speed is the use of monolithic

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columns [16,30]. For different selectivity, alternative stationary phases such as cyanopropyl [19],
pentafluorophenyl [29,32,34], and phenyl-hexyl [33] are used instead of conventional columns with a C18

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stationary phase. These alternative phases can offer better selectivity, shorter retention times, and higher
separation efficiency (e.g. C-30 for the separation of carotenoids, F5 for the separation of tocopherols [29]

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and anthocyanins [34], or a phenyl-hexyl stationary phase for the separation of phytosterols [33]).

3.2. Gas chromatography methods

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To determine bioactive compounds and adulterants in plant extracts, which due to their physico-
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chemical properties and way of detection are uneasily analysed with LC instrumentation, gas
chromatography was employed. A flame ionization detector (FID) was used for the assay of
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methylsulfonylmethane (MSM) in multicomponent dietary supplements [44] and for the determination of
plant sterols and stanols [45]. The analyses of low concentrations of adulterants in plant extracts demand a
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sensitive and selective detection technique, such as mass spectrometry with a triple quadrupole coupled
with gas chromatography (GC-MS/MS) [9,10]. Also, due to the unavailability of reference standards or in
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the case of peak co-elution, the identification of analytes could be based on MS spectra [45,46]. Regarding
the separation column, (5%-Phenyl)-methyl polysiloxane capillary columns with a length of 30 meters are
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the most commonly employed [9,10,45,46].

The main disadvantage of GC is the derivatization step needed when moderate polar or nonpolar
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substances are analysed to obtain symmetric peaks, ensure satisfactory precision, and to improve the
separation and detection of these compounds [45,46]. This is probably one of the reasons why LC
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instrumentation has been used much more frequently (by a factor of several times) than GC to analyse
bioactive substances and the determination of adulterants in plant extracts over the past five years.

3.3. Super-critical fluid chromatography methods

Supercritical fluid chromatography (SFC) is a suitable alternative method used for the determination of
bioactive compounds, especially for the separation of isomers (L/D-amino acids) and substances with very
similar structures [47-49]. SFC instrumentation has become a replacement for normal phase liquid
chromatography (NP-LC), which is now hardly ever used to analyse plant extracts. Carbone dioxide is the

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most commonly used mobile phase in SFC and moreover, it is considered environmental friendly. Above its
critical pressure (7.3 MPa) and temperature (31 °C), the supercritical fluid has lower viscosity and higher
diffusivity relative to conventional liquids, and higher density and dissolving capacity compared to
conventional gases. These unique properties make SFC an effective analytical tool for analysing thermally
unstable and non-volatile compounds. Relative to the corresponding HPLC separation, SFC allows for higher
flow rates and shorter run times. Owing to the low polarity of CO2, SFC is efficient at separating
hydrophobic compounds, such as carotenoids, which are difficult to separate using conventional LC or with

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long runtimes [50]. In addition, organic co-solvents such as methanol, can be added to change the polarity.
Recent technological advancements that employ sub-2 µm particles in UHPLC have been transferred to

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ultra-high performance supercritical fluid chromatography (UHPSFC) [50].

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3.4. Other techniques

Among the previously-mentioned analytical methods, some works have used other techniques for

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assay determination, and especially for testing the purity of plant extracts. These include Fourier transform
infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), and thin layer chromatography (TLC).
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It has been reported that an application of near-infrared spectral imaging technology has been used
for the detection of illegally added drugs in food supplements [5], the quantitative determination of
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coenzyme Q10 from dietary supplements [54], and the detection of regulated herbs and plants in plant
food supplements [55]. Due to the characteristics of infrared spectroscopy, its detection limit cannot reach
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the level of the HPLC and HPLC-MS methods. However, HPLC runtimes for complex plant materials take
hours, while near-infrared spectral imaging takes only minutes. Spectral imaging can provide the initial
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screening for illegally added drugs and adulterants in food supplements before employing methods that are
more precise and time consuming.
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The routine application of NMR is a useful addition to the standard analytical methods used for
bioactive substance quantification in food supplements. 1H NMR spectroscopy has proven to be a robust
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analytical tool yielding highly reliable quantitative results in a very short time. The approach is
advantageous because it minimizes sample preparation and allows for the analysis of a large number of
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samples without human intervention. A simple and direct 1H NMR assay was developed to determine the
total statin content in red yeast rice [58] and coenzyme Q10 [51] in dietary supplements. The same
instrumentation was employed for the detection, identification, and quantification of adulterants in herbal
dietary supplements marketed for improving sexual performance [52] and for weight loss [53].
Recently, a number of enhancements have been made to the basic method of thin layer
chromatography (TLC) to automate different steps, to increase resolution, and to allow for more accurate
quantitative measurements. This enhanced form, high performance thin layer chromatography (HPTLC),
can be a very powerful technique for making a qualitative and quantitative analysis of problematic

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compounds, for example, due to their instability. A method was established for making a rapid on-site
detection of anti-diabetes chemicals used to adulterate botanical dietary supplements for diabetes.
Surface-enhanced Raman spectroscopy (SERS) was employed for making a qualitative identification of trace
substances on the HPTLC plate after the separation of analytes and pharmaceutical matrix components by
HPTLC [8]. Additionally, a new Camag TLC–MS interface has proven to be a useful tool to directly confirm
the identity or structure elucidation of carotenoids resolved on a TLC plate. [56]. HPTLC has also been
utilized to make a qualitative comparison of barley juice extracts from different regions of origin. The

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HPTLC fingerprint analysis showed chemical homogeneity with small qualitative differences [57]. From this
point of view, HPTLC offers a simple and fast solution for analysing plant extracts.

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4. Purity assessments and the safety of plant extracts and food supplements

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When dealing with purity assessments of food supplements based on plant extracts, several aspects
must be taken into consideration. Plant extracts are a concentrated form of botanical products. The plant

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itself can be exposed to different toxic substances involved in agricultural processes, such as pesticides,
mycotoxins, and toxic metals, which can be detected in the final dietary supplement preparation. Another
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problem with the purity of food supplements is the presence of adulterants and pharmaceuticals that were
intentionally added by their producers to enhance their potency. The presence of these drugs represents a
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serious health-risk for consumers, especially considering the fact that preparations are not just being taken
by healthy people, but mostly by ill patients to improve their health. A list of analytical techniques and
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sample preparation methods used for the determination of adulterants and contaminants present in food
supplements based on plant extracts is shown in Table 1 and Table 2, respectively. From both tables, it is
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obvious that the safety control and analysis of contaminants in food supplements based on plant extracts is
a widely discussed and current research topic.
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4.1. Analysis of mycotoxins present in plant extracts

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Common extraction procedures used for the production of extracts from plant material do not avoid
the co-isolation of various contaminants that may be present in the raw material. Improper handling,
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storage, and distribution of medicinal plants can be source of fungi contamination that may result in the
production of wide spectrum of mycotoxins. Therefore, the increasing consumption of contaminated plant
extracts can be cause of health problems due to the lack of effective surveillance of their use. Food
supplements based on botanicals contaminated with mycotoxins can contribute to adverse human health
problems and therefore represent a special hazard [59]. A UHPLC-MS/MS method for the determination of
34 mycotoxins in dietary supplements containing green coffee bean extracts was developed, evaluated, and
used in the analysis of 50 commercial products containing these extracts. In this study, the presence of
ochratoxin A, ochratoxin B, fumonisin B1, and mycophenolic acid was confirmed and number of

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contaminated preparations have ranged from 10 to 36% [11]. LC-MS/MS instrumentation was also
employed in two other works. The first one deals with the determination of 57 mycotoxins in dietary
supplements used in the treatment of liver diseases (milk thistle), the reduction of menopausal effects (flax
seed, soy, and red clover), and preparations containing green barley, goji berries, yucca, and nettle. The
main contaminating mycotoxins were Fusarium trichothecenes, zearalenone, enniatins, and Alternaria
mycotoxins. The co-occurrence of all mentioned mycotoxins has been observed in many cases. The highest
concentrations of mycotoxin were found in milk thistle-based supplements (up to 37 mg/kg in the sum)

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[60]. In the second study, a multi-class method for determination of more than 250 contaminants (including
pesticides and mycotoxins) in ginkgo biloba products was developed. There were found 17 different

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contaminants (14 pesticides and 3 mycotoxins) in most of Ginkgo biloba products [61]. A further study
based on immunoaffinity clean-up step for sample preparation and HPLC-FD determination reported that

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75% of tested preparations were contaminated with OTA at levels ranging from 1.16−20.23 μg/kg [62]. In a
multi-class method employing HRMS developed to identify and quantify 260 toxic substances in soy

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nutraceutical products, green tea, and royal jelly supplements, 6 pesticides and 2 mycotoxins in some of
the studied samples were found [71,72].
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In case of contaminated preparations, the possible health risks associated with mycotoxin occurrence
should be considered because there is a huge risk associated with the fact that consumers may take several
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food supplements with different contaminated plant extracts simultaneously. Moreover, considering the
fact that milk thistle preparations are used by people who suffer from liver disease, a high intake of the
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hepatotoxic mycotoxins from food supplement preparations, which claim to be beneficial, is of concern.
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4.2. Analysis of endocrine disruptors in the plant extracts of food supplements

Endocrine disruptors (EDs) are mostly synthetic compounds that interact with the human or animal
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endocrine system and disturb its function. EDs in food supplements based on plant extracts may have
different origins, e.g. from the contamination of plants through industrial pollution or bioaccumulation in
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the food chain. Persistent and bio-accumulative compounds include alkylphenols (nonylphenol,
bisphenols), polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT), and dioxins, which
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pose serious risks in plant and livestock production. Herbal-based preparations on the market are mostly
formulated as capsules containing a dried ethanolic extract of active compounds [60] or dried enriched
plant material. Unfortunately, the common practices employed when producing food supplements based
on plant extracts do not avoid the co-isolation of potential contaminants. These may be present in the raw
plant materials and owing to their similar physico-chemical properties as targeted biologically active
compounds, they are also co-extracted. In this context, their enrichment from trace levels to levels
exceeding permitted limits can pose a health hazard to consumers.

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Recently, a number of studies have reported that food supplements are a high-risk foodstuff with the
potential to contain estrogenic endocrine disruptors as active constituents [63-65]. One study published the
determination of a wide spectrum of organophosphorus pesticide residues in traditional Chinese medicine
food supplements [66]. Eight percent of the tested samples were positive for contamination by
chlorpyriphos-ethyl and one sample by dichlorvos. Nevertheless, the contents of both pesticides were
lower than the limits set by the United States Environmental Protection Agency (EPA). No other pesticide
residues were found in this study [66]. Several studies have also focused on analysing pesticide EDs in soy-

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based nutraceutical products [10], in nutraceutical products with Camellia sinensis [74] or carbamate
residues in different food supplements containing herbal products, such as Garcinia cambogia, Cynaras

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colymus, Equisetum arvense, soy lecithin, Spirulina maxima, Fucus vesiculosus, olive leaf, and garlic extract
[42].

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Although food supplements containing herbal products or plant extracts can be contaminated by
pesticides used in crop production or other EDs from soil and water contamination, there is only a limited

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number of publications dealing with the problem of food supplement safety analyses. The difficulty in
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regulating and ensuring the safety of these products is caused by the incapacity to establish an inspection
agency that can effectively control the food supplement products on the market. Moreover, due to the
large complexity of food supplements and their dual nature (food vs. medicine), the maximum established
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residue levels of pesticides and other EDs are still not well defined, especially in the European Union.
Though pesticides may be largely present in most human food commodities, their screening in food
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supplements based on plant extracts and adequate methods for making such analyses are still scarce [42].
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It can be concluded that the quality control of plant materials is critical whenever a botanical product is to
be used as a plant food supplement. A systematic review that widely summarizes the problems of
contaminants, mycotoxins, and the intentional adulteration of plant food supplements was published by
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Sanzini et. al. in 2011 [67].

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4.3. Determination of pharmaceuticals, adulterants and contaminants occurring in food supplements

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The most serious issues associated with the purity of food supplements concern undeclared chemical
substances and pharmaceuticals, such as PDE-5 inhibitors, and anorectic and anti-diabetic drugs, which are
illegally added to the supplements in order to enhance the claims stated on their labels. To consumers, it
can lead to potentially serious health consequences and it poses a global challenge for analysts. One special
case is the detection of high levels of glucose, as an adulterant, in food supplements based on Stevia
rebaudiana extracts. This finding could be very alarming for consumers who might be trying to limit their
sugar intake and those who are already suffering from diabetes [31].

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Advanced detection techniques, especially high-resolution MS, has in the past few years enabled
complex and non-targeted analyses of tested food supplements. UHPLC-HRMS methods have been used for
the determination of PDE-5 inhibitors, anorectic drugs [3,4], secondary metabolites, and multiple
pharmaceuticals [12] in plant extracts. Mass spectrometry has been employed in almost all other studies
for analysing multiple pharmaceuticals, anti-hypertensive drugs, steroidal hormones, and some alkaloids
occurring in supplement preparations [6,9,10,26,28,36,39,41,42,61]. Only a minority of works have
reported the use of additional analytical techniques for assessing the purity of plant extracts and food

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supplements, such as FT-NIR for pharmaceutical drugs [5], TLC-SERS for anti-diabetics [8], and NMR for
PDE-5 inhibitors [52,53].

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The quality control results of the tested food supplements were alarming. Among the more than 100
batches of analysed preparations for sexual performance improvement, 67 contained one drug (Sildenafil,

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Tadalafil, Vardenafil, or analogues), while 33 contained more than one. The amount of Sildenafil found in 8
dietary supplements reached concentrations of about 100 mg/g [3,26,52]. Such high amounts are equal to

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those contained in the prescribed registered preparations. Sildenafil was also found in low concentrations
in preparations intended to support healthy blood pressure management [12]. Ephedrine and caffeine
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were found in dietary supplements that are sold in fitness centres and in unauthorized online markets [4].
Among the 187 analysed samples of food supplements for weight loss, only 44% were either truly natural
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or had a composition that conformed to the label. The others were tainted by six drugs, mainly Sibutramine
and Phenolphthalein, or a mixture of these two compounds [12,53]. Among the other pharmaceuticals that
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were detected in the tested samples include Nifedipine, Diclofenac, Metformin, and Phenformin in dietary
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supplements of different origin labelled for anti-diabetes and anti-inflammation [5,8].

Contaminants contained in plant extracts may exist in raw materials, as was described with regard to
mycotoxins in the previous chapter. Generally, these toxic compounds include plant toxins, secondary
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metabolites, pesticides, and heavy metals. Chronic exposure to high levels of these toxic metals can cause a
variety of adverse health effects, including skin allergies, internal cancers, and cardiovascular and
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neurological effects [13]. LC-ICP-MS is the technique of choice for the determination of toxic metals in plant
extracts [13-15].
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Regarding other contaminants, pesticides were found in eight samples with amounts above the
maximum residue limits for each toxic substance regulated by EU legislation [61]. Pesticides at
concentration lower than 21 mg/kg were also found in food supplements containing grape seed extracts
[75].
In general, these results point to the poor quality of manufacturing practices and to intentional
adulteration of food supplements by their producers. This is an alarming fact. Taking into account the
different classes of adulterants that were found, including both pharmaceuticals and toxic substances,

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methods that allow for multi-class studies (e.g. UHPLC-HRMS) are necessary in order to ensure better
quality control for food supplement products and, therefore, increased health safety for consumers.

5. Bioactive substances in food supplements based on plant extracts and their determination

A list of analytical techniques and sample preparation methods used for the determination of bioactive
substances in food supplements based on plant extracts is summarized in Table 3. A simple characterization
of biologically active compounds mostly used in plant extracts and the analytical techniques used for their

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determination are briefly mentioned in the following chapters.

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5.1. Terpenoids

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Today, HPLC coupled with UV–Vis detection or diode array detection (DAD) using a C30 column is the
technique of choice for analysing carotenoids in different matrices, although due to their chromophores,
carotenoids can be easily detected by TLC. Furthermore, there are some advantages of HPTLC over HPLC

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when making a qualitative and quantitative analysis, such as lower solvent consumption, minimal sample
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preparation, and concurrent high throughput analyses with minimal costs [50,56]. The developed HPTLC
method for the determination of carotenoids was also connected to MS detection using a Camag TLC–MS
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interface [56]. Another very suitable and alternative separation technique to LC is super-critical fluid
chromatography and its modification using sub-2 µm particle-size columns. The obtained results
demonstrated UHPSFC as a powerful tool for the separation of carotenoids, especially for their structural
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isomers α-carotene and β-carotene, lutein, and zeaxanthin [50].

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Triterpenoid saponins are triterpenes that belong to the group of glycosides saponins. Evaporative
light scattering detection (ELSD) with UHPLC is the method of choice for the quantification of steroidal
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saponins from dietary supplements because of their low UV absorption [35].

Pentacyclic triterpenes as a major group of ubiquitous secondary plant metabolites offer a wide range
of health promoting biological activities. One study using GC–MS for the determination of 38 different
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commercial plant extracts sold as ingredients for dietary supplements was published [46].
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Phytosterols, phytostanols, and their esters are natural steroids that are important structural
components of plant membranes. They are known for their ability to decrease the level of blood LDL-
cholesterol in humans. Phytosterols from the plant Serenoa repens have been also used as food
supplements for the auxiliary treatment of prostatic hyperplasia. Measuring phytosterols in plant extracts
requires problematic alkaline saponification and/or acid hydrolysis to convert all the conjugated
phytosterols into their free forms. The sample preparation steps are then usually followed by liquid-liquid
solvent extraction, sample pre-concentration, and possible solvent exchange prior to performing the
chromatographic analysis. A GC method employing FID and MS detection has been reported to analyse

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phytosterols and two phytostanols [45]. While GC methods are more common for the analysis of
phytosterols because of the lack of chromophor in their structure, they are not ideal because the
phytosterols usually must be derivatized prior to the analysis to render the target analytes volatile and
thermally stable. However, a new UHPLC method using a Corona-charged aerosol detector (CAD) was
presented for the first time to analyse phytosterols and phytostanols by employing a core-shell column
with phenyl-hexyl stationary phase, which provided a rapid and highly efficient separation of seven plant
sterols and stanols in complex matrices of plant extracts [33]. CAD enabled to reach sufficient detection

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limits for campesterol and campestanol that lack of chromophores. Though, the response of CAD was linear
only in a limited range. Therefore, calibration using a second order polynomial function was used to achieve

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correlation coefficients as good as those found with the linear response of UV detectors. Changes in mobile
phase composition during a slow gradient program did not show significant effect on the suppression of the

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CAD response. Despite these limitations, CAD can be ideal choice for compounds lacking of chromophores
[33].

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Tocopherols are known as Vitamin E homologues that exist naturally in four of their corresponding
homologues (α-, β-, γ-, δ-tocopherols and α-, β-, γ-, δ-tocotrienols). Each homologue shows distinct
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biological activity (anticancer, antioxidant, cholesterol-lowering properties, neuroprotective, etc.). Vitamin
E in food supplements is usually present as α-tocopheryl acetate. Monolithic C-18 column was used for the
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rapid separation (3.5 min) and determination of vitamin E acetate in dietary supplements [16]. In other
study, a tandem connection of PDA and a fluorescence detector was used for the simultaneous
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determination of tocopherols (α-, β-, γ-, δ-), tocotrienols (α-, β-, γ-, δ-), α-tocopherol acetate, and α-
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tocopherol nicotinate in food supplements using a pentafluorophenyl (PFP) column [29]. Generally, a PFP
stationary phase is very suitable for the separation of tocopherol isomers and other isomers and aromatic
compounds. PFP can offer a different selectivity compared to a C18 phase owing to multiple retention
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mechanisms, including π-π interaction, dipole-dipole interaction, hydrophobic and H-bonding.

Ginkgo biloba extracts containing ginkgolides, bilobalides, flavonol glycosides, and ginkgolic acids are
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commonly used to improve age-related loss of mental function. In a recent study, SFC was used to
determine both ginkgolic acid and terpene lactone in dietary supplement samples. The high diffusivity and
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low viscosity of supercritical CO2 enabled fast and efficient separation approach compared to reversed
phase LC methods. MS was proven as the more suitable detector for chromatographic separation, which
was verified by a tandem connection of PDA and MS detectors [49]. In a study employing HRMS to identify
and quantify relevant marker compounds in ginkgo biloba products, ginkgolic acids, which are considered
undesirable in ginkgo biloba extracts due to negative properties attributed to these compounds, were
identified. Furthermore, the results showed a great variability among the presence of isoflavonoids, which
can provoke negative effects in certain type of people [77].

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Steviol glycosides, a group of diterpene glycosides, are used as natural sweeteners (400 times sweeter
higher than sucrose). They are found in the leaves of the perennial herb Stevia rebaudiana Bertoni. Because
of their low absorption in the UV spectrum, UHPLC-UV coupled with ELSD was used for the determination
of rebaudioside A, stevioside, rebaudioside D, dulcoside A, and steviolbioside from Stevia rebaudiana
extracts. However, the results of the study report that ELSD and UV detections showed almost comparable
results [31].

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5.2. Polyphenols

Flavonoids and phenolic acids are known for their strong anti-oxidative activity [17,18]. A large family

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of flavonoids is divided into five subclasses, including flavonols, flavones, anthocyanins, catechins, and
flavonones. Some flavonoids, such as rutin, hesperidin, and diosmin are widely used in the auxiliary

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treatment of civilization diseases (high blood pressure, chronic venous insufficiency, haemorrhoids, and
phlebitis). The methods for their determination are usually based on fast reversed phase HPLC separations

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coupled with PDA detection [17,18,22,25,30]. Analytical method details are mentioned in Table 3.
A unique group of pharmacologically active natural substances present isoflavones. In contrast to
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regular flavonoids, isoflavones possess hormone-like activities that can explain their wide use in food
supplements as phytoestrogens. They are used for auxiliary reduction of menopausal and post-menopausal
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symptoms. The SFC technique was used for the rapid baseline separation and quantitative determination of
nine isoflavones (aglyca and glycosides) in dietary supplements containing soy (Glycine max), red glover
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(Trifolium pratense), and kudzu (Pueraria lobata) [47]. This study is not just the first one overall describing
the use of SFC for making an isoflavone analysis, it also serves as convincing evidence for the efficiency and
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benefits of this still rarely used analytical technique.

Anthocyanins are often used in food supplements obtained from blueberry and açaí berry extracts.
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They are found in plants as glycosides derived from anthocyanidins via the addition of a sugar group. The
most common anthocyanins are cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin [34].
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A UHPLC-PDA method with an F-5 stationary phase for the fast determination of six anthocyanins contained
in acai berry and blueberry extracts has recently been developed [34]. Our experience after a comparison
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of the separation efficiency of three stationary phases – Kinetex F5, Luna Omega Polar C18, and Luna
Omega PS C18 showed that the fluorinated stationary phase is the most suitable for the separation of
anthocyanins in respect of resolution and peak symmetry (Fig 2).
Among the best-known flavonolignans belong silychristin, silydianin, silybin A, silybin B, isosilybin A,
and isosilybin B. All are contained in silymarin, an extract of the purple flowering plant Silybum marianum,
which is known for its abilities to protect the liver against toxic substances and alcohol. The methods for
analysing silymarin extract are quite challenging due to the difficult separation of diastereoisomers silybin A
and B, which are the major components of silymarin. For this reason, the use of UHPLC and an alternative

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fluorinated stationary phase with high separation efficiency have been reported for six flavonolignans in
herbal extracts [32].
Resveratrol and its glycosylated form piceid (polydatin) are the most well-known stilbenoids.
Currently, resveratrol is typically extracted from red grapes and Japanese knotweed. Resveratrol exists in
cis- and trans- isomeric forms. The trans-form is the preferred steric form in nature and is relatively stable
[19]. HPLC-UV and HPLC-MS methods have been developed for the quality control of resveratrol and piceid
in multi-component food supplements [18,19], grape seed extract, and Reynoutria japonica extract used in

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food supplements [27].
Emodin belongs to a class of cyclic organic compounds with two carbonyl groups - quinones. Food

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supplements made from lower purity plant extracts may contain unacceptably high amounts of emodin. It
can cause stomach cramping, diarrhoea, or bloating However, moderate amounts of emodin have been

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used in traditional Chinese herbal medicine as a natural laxative. A RP-HPLC method has been reported for
making the simultaneous analysis of emodin in 28 food supplements with resveratrol [18]. Another

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representative of this group is ubiquinone Coenzyme Q10. The reason for the popularity of CoQ10 is in the
prevention and treatment of many disorders... A methodology utilizing 1H NMR spectroscopy has been
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developed to measure the concentration of coenzyme Q10 in dietary supplements. On the basis of the
labelled amounts, only two products contained substantially lower concentrations of CoQ10 (57 % and 51
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%). All other concentrations varied between 83 % and 190 % with respect to labelling [51]. A different
approach was used with the Fourier transform near-infrared spectroscopy (FT-NIR) method for the
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quantitative determination of CoQ10 from several dietary supplements. The combination of FT-NIR
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spectroscopy and multivariate calibration methods is a very fast and straightforward way to replace the
commonly used HPLC-UV method; because in contrast with the traditional techniques, sample pre-
treatment and reagents are not required, and no wastes are produced [54].
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5.3. Aromatic acids

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The three proteinogenic aromatic amino acids, tryptophan (Trp), phenylalanine (Phe), and tyrosine
(Tyr) are used as food supplements for their auxiliary purposes. Phe and Tyr are recommended for appetite
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control and as anti-depressive agents. Trp promotes the release of the neurotransmitter serotonin that
plays role in sleep regulation and pleasure. Due to the chiral nature of amino acids, for food supplements
containing Phe, Trp and Tyr, only the L-forms of acids are allowed in their natural configuration. Their D-
forms may have different biological properties and worsen metabolic efficacy. SFC was successfully used
for chiral analysis of three aromatic amino acids in a very short time (7 min) employing a Chirobiotic T2
column [48]. The results of several food supplements analysis did not show any enantiomeric impurities.
The other aromatic acids analysed in food supplements from green coffee extract are known as
chlorogenic acids. Green coffee extract has become popular over the past few years as a food supplement

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for quick weight reduction. A novel in-line sample preparation flow method coupled with LC analysis was
developed for the automated dissolution testing of chlorogenic acids from dietary supplements based on
green coffee extracts. The study simulated in-vitro dissolution conditions through the use of synthetic
gastric juice [21]. In another study, dispersive micro solid-phase extraction (DMSPE), together with UHPLC-
Q-TOF/MS was employed for the determination of protocatechuic aldehyde, caffeic acid, and rosmarinic
acid in dietary supplements [38].

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5.4. Glucosinolates

Glucosinolates, the main group of nutrients found in cruciferous vegetables, are sulphur-containing

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compounds. During food preparation, chewing, and in an acidic medium, they quickly convert into bioactive
compounds, such as isothiocyanates, nitriles, and indoles with health promoting properties. Rich in

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glucosinolates, food supplements based on cruciferous vegetables are popular health-promoting products
that deliver nutrients the immune system needs with anti-cancer and anti-aging properties [20,43]. An

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HPLC-UV method was developed for the rapid determination of indole-3-carbinol and its degradation
products. Degradation products that were found showed poor stability of these preparations [20].
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5.5. Chlorophylls
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Chlorophyll, whose basic structure is a porphyrin ring that is coordinated with a central atom, can be
found in the chloroplasts of green plants. The antioxidant and anti-mutagenic health benefits associated
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with food supplements containing chlorophyll have been discovered. An HPLC-UV method for ensuring the
quality control of barley grass juice extracts containing chlorophylls and a comparative HPTLC analysis was
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performed for making a comparison of barley juice extracts from different regions of origin. The HPTLC
fingerprint analysis showed chemical homogeneity with small qualitative differences [57].
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5.6. Amines

This chapter covers every other compound containing some class of amine in its molecule that was not
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mentioned in previous chapters or could not be classified in one of the previous groups. Amines of Citrus
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aurantium L., also known as bitter orange, are aimed at reducing body weight through their adrenergic
activity. The C. aurantium extract is permitted in food supplements, but several countries have established
limits for the content of active amines (octopamine, synephrine, tyramine, N-methyltyramine, and
hordenine). A problem associated with C. aurantium is that it can act as an alternative to Ephedra, with
possible risks for consumers. Therefore, an LC/UV/Fluorescence assay for making a quantitative analysis of
five active amines in raw C. aurantium and derivatives has been fully validated according to FDA Guidelines
[23].

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A sympathomimetic compound, 1,3-Dimethylamylamine (DMAA), is currently being incorporated into
some dietary supplements. Significant controversy exists regarding the natural origin of DMAA, as claimed
by the manufacturers of supplements. The aim of a study using UPLC-MS/MS instrumentation was to
determine whether DMAA is naturally present in plant species. The result was that no measurable levels of
DMAA in the plant species claimed to be used in the mentioned food supplements were present above a
detectable limit. This study indicates that the DMAA contained in food supplements is of a synthetic origin
and was not present in the analysed plant extracts [37]. This is a finding of much concern.

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Melatonin supplements are used widely in the EU and US to alleviate jetlag and other sleep-delay
disorders. A recent study was aimed at determining the dosage of melatonin in food supplements

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marketed in Europe (Spain) and the US by validating an LC-DAD. Additionally, the contaminants present in
melatonin supplements were analysed using UHPLC coupled with an orbitrap mass spectrometer [39].

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6. Quality assurance/quality control (QA/QC) AN
A quality assurance/quality control (QA/QC) protocol must be established to guarantee reliable and
precise results. Although authors commonly refer to different validation guidelines, such as US Food and
Drug Administration (FDA) guidelines on Bioanalytical Method Validation [3,23,45], guidelines established
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by the International Conference on Harmonisation (ICH)[6,11,16,17,28,31,33-36,47-49,57] European

Pharmacopoeia recommendations [16,17,19,20,32,44,57], the US Pharmacopeial Convention [21], and
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others [9,10,12,18,26,29,38-42,50,54,55,58,60,61], no complete validation method for assaying bioactive

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substances and contaminants in herbal dietary supplements include selectivity, sensitivity (LOD/LOQ),
linearity, accuracy and precision, recovery, matrix effect (in case of MS detection), stability of samples,
carryover, and robustness of the proposed methods in most of the reported works. The limits for all these
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parameters were adopted from the above-mentioned guidelines and recommendations. For LC, GC, and
SFC methods, a system suitability test is an essential part of the validation procedure. For the identification
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of bioactive substances, retention times and spectra are often used. But due to the complexity of plant
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extracts, verification of the authenticity of the analysed substances has to be performed when the method
itself cannot guarantee an exact match between the analysed substance in the reference and sample
solutions, respectively, or to detect peak coelution (for example, due to missing spectral data for the peak
purity test). This verification is usually performed using MS, MS/MS, and HRMS data
[8,24,30,31,35,36,39,52,53].
For adulterants (pharmaceuticals deliberately added in food supplements), these kinds of validation
parameters already exist. They are present in both FDA guidelines and the OMCL (Official Medicines
Control Laboratory) guidelines and are audited under the ISO 17025 standard [77].

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7. Conclusion and future perspectives

In this work, we presented an overview of various sample preparation and analytical techniques
employed for the determination of bioactive substances in food supplements based on plant extracts and
for assessing the purity of the plant extracts contained in these preparations. The analysis looked at data
collected from 2012 to 2017.

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It can be concluded that the preferred analytical technique for analysing bioactive substances in food
supplements is HPLC coupled with PDA/UV detectors. This is probably based on the fact that this technique

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is well established in most of the laboratories that perform routine analyses of different samples, not just
dietary supplements. Despite this, UHPLC instruments have become more common in recent years with

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alternative detection techniques instead of UV, such as FD, CAD, ELSD and mostly MS. The GC method has
also been used for the determination of bioactive substances, but a more promising technique in the future

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seems to be SFC (and its enhanced form UHPSFC). SFC is a powerful tool that offers high selectivity for
difficult separations of isomeric compounds not achievable with conventional LC in such short times. The
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suitability of various analytical techniques and their method of detection for the determination of specific
groups of bioactive substances is given in Fig 3.
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When dealing with testing the purity of plant extracts, UHPLC coupled with MS, MS/MS and the HRMS
detection technique are almost exclusively used. A minority of works covers GC-MS/MS methods and ICP-
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MS for analysing toxic metals. The use of such advanced instrumentation is necessitated by the complexity
of the sample matrices, low concentrations of the target compounds, and by the need to cover a wide
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range of analytes. There is a clear trend showing the increasing prevalence of MS detection over
conventional UV in LC and FID in GC methods, respectively. For complicated plant extracts, the HRMS
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technique is the gold standard used to reliably identify target bioactive substances. Ultrasound-assisted
solid-liquid extraction followed by filtration or centrifugation is still the main sample preparation technique
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used for the determination of bioactive substances in food supplements with plant extract content, as well
as for testing the purity of these extracts.
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The clear majority of the studied works report full or partial validation of the proposed methods. This
indicates that the results obtained by the analyses are not random and it is necessary to take them
seriously because they highlight the poor quality of food supplements. The low content of declared
bioactive substances is one problem, but the purity of the plant extracts used in these preparations (and
thus, their safety) is more alarming. The presence of contaminants, such as pesticides, toxins, or endocrine
disruptors, and illegally added drugs with the intention to enhance the potency of plant extracts, could
have especially serious consequences for consumers. Therefore, for the ultimate protection of consumers,
quality control tests should be performed throughout all processing stages, beginning with the raw plant

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extract to the finished product. Unfortunately, there is no universal analytical method that can assure the
quality control of all parameters of these supplements. That clearly shows the importance of employing
modern analytical methods for ensuring the quality control of food supplements based on plant extracts
and the need for more advanced techniques for making multi-class purity assessments of these extracts.
This will lead to improved health safety for consumers. Another problem associated with the quality control
of food supplements is the level of demand for regulatory requirements. This varies greatly in each region.
The global harmonization of legislation can help to produce better quality supplements with a defined

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dosage for consumers. Defining the optimal daily dose is another problem associated with plant extracts
because the preparations differ from producer to producer and their region of origin. Standardizing the

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potency of plant extracts according these new and globally harmonized guidelines is one way to make the
food supplement market more transparent and safer for consumers.

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Acknowledgments

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The authors would like to acknowledge the financial support of the specific research project, no. SVV
260 412 and grant GAUK no. 181216.
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This work was also supported by the EFSA-CDN project (No. CZ.02.1.01/0.0/0.0/16_019/0000841) co-
funded by the ERDF.
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Conflict of interest
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The authors report no conflicts of interest in this work.

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Table 1 Analytical techniques and sample preparation methods used for determination of adulterants in food supplements based on plant extracts

Adulterants in food supplements Analytical technique Mobile Column Extraction method Runtime Reference
phase (min)
PDE-5 inhibitors UHPLC-HRMS (Q-Orbitrap) acetonitrile+0.1% FA/0.1% FA (g) Hypersil GOLD aQ C18 (100×2.1 mm, 1.9 µm) SLE (50% acetonitrile) 10.0 [3]
NMR - - SLE (CD3CN/D2O 80:20) - [52]

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Anorectic drugs, PDE-5 inhibitors UHPLC-HRMS (Orbitrap) methanol+0.1% FA/10% methanol+0.1% FA (g) Acclaim RSLC 120 C18 (100×2.1 mm, 2.2 µm) LLE (methanol, pentane/ethyl ether) 14.5 [4]
NMR - - SLE (CD3CN/D2O 80:20) - [53]
Antihypertensive drugs HPLC-MS/MS (QqQ) acetonitrile+0.5 mM AA/0.5 mM AA (g) Atlantis dC18 (150×2.1mm, 3 μm) SLE (methanol) 40.0 [6]

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Antidiabetic drugs TLC-SERS dichloromethane/methanol/water 8:2:0.2 silica gel 60-F254 plates SLE (methanol) - [8]
Multiple pharmaceuticals FT-NIR - - - - [5]
UHPLC-HRMS (Q-Orbitrap) methanol/5 mM ammonium formate+0.1% FA (g) Acquity UPLC HSS T3 (100×2.1 mm, 1.8 µm) SLE (acetonitrile+2% FA) 15.0 [12]

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HPLC-MS (IT-TOF) acetonitrile+0.1% acetic acid/0.1% acetic acid (g) Zorbax Eclipse AAA (150×4.5 mm, 5 μm) SLE (methanol/acetonitrile+1% FA 1:1) 45.0 [26]
UHPLC-MS/MS acetonitrile/0.1% FA (g) Kinetex C18 (150×2.6mm, 1.7 μm) SLE (acetonitrile) 11.0 [41]
(Iso)miroestrol HPLC-MS/MS (Q-Orbitrap) acetonitrile+0.1%FA/0.1% FA (g) Xbridge C18 (150×2.1 mm, 3.5 μm) SLE (methanol) 20.0 [28]
Glucose UHPLC-PDA acetonitrile+0.05% FA/0.05% FA (g) Acquity UPLC BEH C18 (100×2.1 mm, 1.7 µm) dissolution (10% methanol) 14.0 [31]

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PDE-5 - phosphodiesterase type 5; HRMS – High resolution mass spectrometry; (g) – elution gradient; FT-NIR - Fourier transform near infrared spectroscopy; IT – ion trap; TOF – time of flight; Q – quadrupole; QqQ – triple Q; SERS -
surface-enhanced Raman spectroscopy; SLE – solid-liquid extraction; LLE – liquid-liquid extraction; FA- formic acid; CD3CN/D2O - deuterated acetonitrile/deuterated water; AA – acetic acid

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Table 2 Analytical techniques and sample preparation methods used for determination of contaminants in food supplements based on plant extracts

Contaminants in food supplements Analytical technique Mobile Column Extraction method Runtime Reference
phase (min)
Pesticides GC-MS/MS (QqQ) helium (temperature gradient) HP-5MS fused silica (30m×0.25 mm) SPE (50% acetonitrile, toluene) 55.3 [9]
GC-MS/MS (QqQ) helium (temperature gradient) VF-5MS (30mx 0.25 mm, 0.25 µm film) SLE (ethyl acetate, acetonitrile) 23.0 [10]

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UHPLC-MS/MS (QqQ) methanol+0.01% formic acid/0.01% formic acid (g) Zorbax Eclipse Plus RRHD (50×2.1 mm, 1.8 μm) SLE (50% acetonitrile) 10.5 [42]
Pesticides, mycotoxins UHPLC-MS (Orbitrap) methanol/4 mM Ammonium formate+0.01% FA (g) Hypersil GOLD aQ C18 (100×2.1 mm, 1.9 µm) SLE (acetonitrile+1% acetic acid) 14.0 [61]
Mycotoxins UHPLC-MS/MS methanol/5 mM ammonium formate (g) Acquity UPLC HSS T3 (100×2.1 mm, 1.7 µm) SLE (acetonitrile) 13.0 [11]

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HPLC-MS (Orbitrap), methanol/5 mM ammonium formate and 0.2% FA Acquity UPLC HSS T3 (100×2.1 mm, 1.8 µm) SLE, DSPE (acetonitrile) 13.5 [60]
HPLC-FD acetonitrile/water/acetic acid 99:99:2 Synergi Hydro-RP (150×3 mm, 4 µm) SLE (60% acetonitrile) 10.0 [62]
Plant toxins, secondary metabolites UHPLC-HRMS (Q-Orbitrap) methanol/5 mM ammonium formate+0.1% FA (g) Acquity UPLC HSS T3 (100×2.1 mm, 1.8 µm) SLE (acetonitrile+2% FA) 15.0 [12]

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Metals, toxic metals ICP-MS - - MAE (65% nitric acid/water) - [13]
HPLC-ICP-MS SPS+citric acid+methanol/NaH2PO4+methanol (g) Econosphere C18 (250×4.1 mm, 5 μm) MAE (SPS/citric acid/methanol) 10.0 [14]
HPLC-ICP-MS 22% methanol/8 mM ammonium acetate Perkin Elmer C8 (33×3.0 mm, 3 μm) MAE (0.5% nitric acid) 10.0 [15]
Sesquiterpenes, pyrrolizidine alkaloids UHPLC-UV/MS (TOF) acetonitrile+0.05% FA/50 mM ammonium formate Acquity UPLC HSS T3 (100×2.1 mm, 1.8 µm) SLE (methanol) 24.5 [36]

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Tryptophan-related contaminants UHPLC-MS/MS (Orbitrap) methanol+0.1% formic acid/0.1% formic acid (g) SB-C18 (100×2.1 mm, 1.8 µm) SLE (50% methanol) 15.0 [39]
ICP - inductively coupled plasma; SPE – solid-phase extraction; DSPE – dispersive SPE, IA – immunoaffinity, MAE - microwave-assisted extraction; SPS - sodium 1-pentanesulfonate

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Table 3 Analytical techniques and sample preparation methods used for determination of bioactive substances in food supplements based on plant extracts

Bioactive substances Analytical technique Mobile Column Extraction method (solvent) Runtime Reference
phase (min)
Tocopherols HPLC-PDA methanol/water 98:2 Chromolith Performance RP-18e (100×4.6 mm) SLE (methanol) 3.5 [16]
UHPLC-PDA/FD methanol/water (gradient) Kinetex PFP column (150×2.1 mm, 2.6 µm) SLE (2-propanol) 10.0 [29]
Flavonoids HPLC-PDA acetonitrile/water solution of acetic acid 30:70 Ascentis Express RP-Amide (100×3.0 mm, 2.7 µm) SLE (DMSO) 6.0 [17]
HPLC-UV methanol/0.1% formic acid (gradient) Luna C18 (250×4.6 mm, 5 μm) SLE (methanol) 22.0 [18]
UHPLC-UV acetonitrile/0.05% trifluoroacetic acid Chromolith Fast Gradient RP-18e (50 mm×2.0 mm) SLE (methanol) 5.0 [30]
HPLC-MS (Q-TOF) 0.1% FA in methanol/0.1% FA in water (gradient) Zorbax Eclipse plus C18 (100×2.1 mm, 1.8 µm) SLE (methanol/0.1% HCl 70:30) 35.0 [25]

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Resveratrol HPLC-UV methanol/0.1% FA (gradient) Luna C18 (250×4.6 mm, 5 μm) SLE (methanol) 22.0 [18]
HPLC-PDA acetonitrile/water solution of acetic acid 20:80 Ascentis Express ES-Cyano (100×3.0 mm, 2.7 µm) SLE (methanol) 3.5 [19]
HPLC-PDA-FD-MS/MS acetonitrile/0.1% acetic acid (gradient) Kromasil 100 C18 (150 mm × 4.0 mm, 5 µm) SLE (methanol) 37.0 [24]

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Indole-3-carbinol HPLC-PDA acetonitrile/water (gradient) Kinetex XB-C18 (100×4.6 mm, 5 µm) SLE (methanol) 6.3 [20]
Caffeoylquinic acids HPLC-PDA acetonitrile/0.2% acetic acid (gradient) Kinetex C18 (250×4.6 mm, 5 μm) dissolution (0.1M HCl) 24.0 [21]
Polyphenols, secoiridoids HPLC-PDA acetonitrile/aqueous FA pH 3.2 (gradient) Lichrosorb RP18 (250×4.6 mm, 5 μm) dilution (water) 82.0 [22]
Amines HPLC-UV/FD acetonitrile/sodium dodecyl sulfate (gradient) LiChrospher RP-18 (250×4.0 mm, 5 μm) SLE (0.1 N HCl/water/methanol) 30.0 [23]

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Steviol glycosides UHPLC-UV/ELSD acetonitrile with 0.05% FA/0.05% FA (gradient) Acquity UPLC HSS T3 (100×2.1 mm, 1.8 μm) dissolution (80% methanol) 17.5 [31]
Flavonolignans UHPLC-PDA methanol/phosphate buffer pH 2.0 (gradient) Kinetex F5 (150×2.1 mm, 1.7 µm) SLE (methanol) 10.5 [32]
Phytosterols, phytostanols UHPLC-PDA/CAD acetonitrile/water (gradient) Kinetex Phenyl-hexyl (100×2.1 mm, 1.7 µm) LLE (ethanolic KOH, n-hexane) 8.5 [33]
GC-FID/MS hydrogen (temperature gradient) SE-54 (30 m × 0.32 mm, 0.25 µm film) LLE (ethanolic KOH, diethyl ether) 69.0 [45]

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Anthocyanins UHPLC-PDA acetonitrile/5% aqueous FA (gradient) Kinetex PFP (150×2.1 mm, 1.7 µm) SLE (methanol/5% aqueous FA 1:3) 5.0 [34]
Steroidal saponins, diosgenin UHPLC-ELSD acetonitrile with 0.05% FA/0.05% FA (gradient) Acquity UPLC BEH Shield RP18 (100×2.1mm,1.7 μm) SLE (methanol) 21.5 [35]

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1,3 dimethylamylamine UHPLC-MS/MS acetonitrile/ammonium formate + HFBA (gradient) Acquity UPLC BEH C18 (50x2.1 mm, 1.7 μm) SLE (50% methanol) 35.1 [37]
Phenolic compounds UHPLC-MS (Q-TOF) acetonitrile/0.01% FA (gradient) Zorbax StableBond-C18 (50x2.1 mm, 1.8 μm) DM-SPE (70% methanol) 7.0 [38]
Melatonin HPLC-PDA methanol/0.1% FA 40:60 Superspher 100 RP-18 (250 mmx4 mm, 5 μm) SLE (50% methanol) 15.0 [39]
Lovastatin UHPLC-MS/MS (QqQ) acetonitrile/0.5mM ammonium acetate (gradient) BEH C18 (50×2.1 mm, 1.7 μm) MI-SPE (acetonitrile) 5.0 [40]

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H NMR - - dissolution (ethanol) - [58]
Glucosinolates UHPLC-HRMS (Orbitrap) acetonitrile with 0.1% FA/0.1% FA (gradient) Hypersil Gold AQ RP-C18 (100×3.0 mm, 1.9 μm) SLE (50% methanol) 30.0 [43]
Methylsulfonylmethane GC-FID helium at 190 °C Alltech AT-624 (30 m×0.32 mm, 1.8-μm film) SLE (acetone) 5.0 [44]

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Triterpenic acids GC-MS helium (temperature gradient) DB-5 (30 m× 0.25 mm, 0.25-μm film) SLE (ethyl acetate) 60.0 [46]
Isofavones SFC-PDA 0.05% phosphoric acid in methanol/CO2 (gradient) Acquity UPC2 BEH (100×3.0 mm, 1.7 μm) SLE (80% methanol) 20.0 [47]

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Aromatic amino acids SFC-PDA 40% modifier (methanol/water 90:10) Chirobiotic T2 (250×2.1 mm,5 μm) SLE (water) 10.0 [48]
Ginkgolic acids, terpene lactones SFC-PDA/MS IPA/methanol with ammonium acetate/CO2 Acquity UPC2 BEH 2-EP (150×3.0 mm, 1.7 μm) SLE (methanol) 19.5 [49]
2
Carotenoids UHPSFC-PDA methanol/ethanol 1:2/ CO2 (gradient) Acquity UPC HSS C18 SB (150×3.0 mm, 1.8 μm) SLE (DMSO/water 3:1/0.1% BHT) 15.0 [50]
HPTLC methanol/acetone 1:1 with 0.1% TBHQ C18 RP HPTLC glass-backed plates (4x10 cm) SLE (ethyl acetate/0.1% BHT) 30.0 [56]
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Coenzyme Q-10 H NMR - - dissolution (CDCl3) 19 [51]
FT-IR - - homogenization - [54]
HPLC-UV acetonitrile/THF/water 65:30:5 Zorbax XDB C18 (50×2.1 mm, 3.5 μm) SLE (ACN/THF/water 40:55:5) - [54]
Chlorophylls HPLC-UV methanol/water/ethyl acetate 40:10:50 Ascentis Express C18 (150×4.6 mm, 2.7 μm) SLE (acetone/water 5:1) 6.0 [57]
C

HPTLC four solvent system silica gel 60 F254 glass plates 10×10 cm SLE (acetone/water 5:1) - [57]
FD – fluorescence detection; ELSD – evaporative light scattering detector; CAD – charged aerosol detector; FID – flame ionization detector, SFC – super-critical fluid chromatography, UHPSFC – ultra-high performance SFC; HTPLC –
AC

high performance TLC; DM – dispersive micro; MI – molecular imprinted; FA – formic acid; DMSO – dimethyl sulfoxide; HCl - hydrochloric acid; HFBA - heptafluorobutyric acid; IPA – isopropanol; CDCl3 - deuterated chloroform
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Fig. 1 Sample preparation techniques employed for determination of bioactive

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substances and purity tests of plant extracts

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Fig. 2 Comparison of six anthocyanins separation on three columns: Kinetex PFP 150 × 2.1 mm, 1.7 µm; Luna
Omega C18 PS 150×2.1 mm, 1.6 µm; and Luna Omega C18 Polar 150×2.1 mm, 1.6 µm. Peaks identification: 1

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Delphinidin-3-galaktoside; 2 Delphinidin-3-glucoside; 3 Delphinidin-3-rutenoside; 4 Cyanidin-3-glucoside; 5
Cyanidin-3-rutenoside; 6 Peonidin-3-glucoside [34]

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LC

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UV/PDA FD CAD/ELSD MS

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Anthocyanins
Amines

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Flavonoids Phytosterols Amines
Flavonolignans Stilbenoids Phytostanols Flavonoids
Hydroxycinnamic Tocopherols Steroidal saponins Glucosinolates

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acids Steviol glycosides Stilbenoids
Stilbenoids

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Tocopherols

SFC HPTLC GC

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PDA PDA FID MS

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Aromatic amino acids
Phytosterols
Carotenoids Carotenoids Phytosterols
Phytostanols
Isoflavones Chlorophylls Phytostanols
Triterpenic acids
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Terpene lactones
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Fig. 3 Suitability of analytical techniques for determination of specific group of bioactive substances in food

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supplements based on plant extracts

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Highlights

Analytical methods for quality control of food supplements based on plant extracts were
reviewed

Advantages and limitations of extraction techniques and separation methods were discussed

Critical evaluations of the poor quality of food supplements were highlighted

Adulterants, endocrine disruptors, pesticides and mycotoxins contamination were pointed out

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Perspectives in quality control of food supplements based on plant extracts were summarized

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Petr Solich is full professor at the Department of Analytical Chemistry, Faculty of Pharmacy in Hradec
Králové, Charles University in Prague. He obtained RNDr. and PhD. degrees in Pharmaceutical
Analysis in 1980 and 1986, respectively. Prof. Solich became head of the Department of Analytical
Chemistry in 2004 and faculty vice-dean in 2014. He is co-author of over 220 articles and holds 6
patents. His research interests are automation in analytical chemistry, focused on Sequential
Injection Analysis and Sequential Injection Chromatography and the development of novel sample
preparation techniques and frontend chromatographic separation approaches for pharmaceutical,
bioanalytical, and environmental analysis.

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Jakub Fibigr is currently a PhD student in Pharmaceutical Analysis at the Department of Analytical
Chemistry, Faculty of Pharmacy in Hradec Králové, Charles University under the supervision of assoc.
prof. Dalibor Šatínský. Jakub Fibigr received his master degree in Pharmacy in 2014. His research
interest is focused on chromatography, alternative stationary phases for separation of isomers, and
fast separation methods for quality control of nutraceuticals and food supplements based on plant
extracts. He has published 6 scientific articles.

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Dalibor Šatínský is associated professor at the Dept. of Analytical Chemistry, Faculty of Pharmacy in
Hradec Králové, Charles University. He obtained PhD degree in Pharmaceutical Analysis in 2003. His
research interests are automation in flow methods, focused on chromatography separations and the
development of novel sample extraction techniques including on-line SPE. He is involved in a wide
scope of research projects being focused on pharmaceutical analysis, food and food supplements
analysis, mycotoxins and contaminants monitoring in food and environmental, and bioanalytical
applications. Since 2016, his domain of expertise focuses on nano-fibrous extractions in
chromatography systems. He has published over 80 scientific articles with about 1400 citations.

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