Current Strategies for Studying the Natural and Synthetic Bioactive Compounds in Food by Chromatographic Separation Techniques

: The present study summarizes the new strategies including advanced equipment and validation parameters of liquid and gas chromatography methods i.e., thin-layer chromatography (TLC), column liquid chromatography (CLC), and gas chromatography (GC) suitable for the identiﬁcation and quantitative determination of different natural and synthetic bioactive compounds present in food and food products, which play an important role in human health, within the period of 2019–2021 (January). Full characteristic of some of these procedures with their validation parameters is discussed in this work. The present review conﬁrms the vital role of HPLC methodology in combination with different detection modes i.e., HPLC-UV, HPLC-DAD, HPLC-MS, and HPLC-MS/MS for the determination of natural and synthetic bioactive molecules for different purposes i.e., to characterize the chemical composition of food as well as in the multi-residue analysis of pesticides, NSAIDs, antibiotics, steroids, and others in food and food products.


Introduction
Bioactive compounds are natural or synthetic (partially or totally) compounds that show biological activity i.e., have the ability to interact with living tissues and indicate an effect on human body including the promotion of good health, thus they are important as new ingredients of the current functional food (e.g., antioxidants) [1]. Food samples are very complex mixtures consisting not only of naturally occurring bioactive compounds with beneficial role on human health like for example vitamins, minerals, antioxidants but other substances coming from agrochemical treatments i.e., pesticides as well as promotors animals growth or veterinary drugs. Therefore monitoring the level of different veterinary drugs or organic pesticides coming from agrochemical treatments in food and food products could ensure the safety of potential consumers. Natural and synthetic bioactive compounds occur in foods in small quantities and represent a wide group of chemical compounds. Because of the complexity of food matrices, the separation and next accurate determination of their bioactive constituents with different chemical structure requires an universal analytical methodology like liquid and gas chromatography or combination of both chromatographic techniques.
For this fact, this article reviews new strategies including advanced equipment and validation parameters of liquid and gas chromatography methods dedicated for the identification and quantitative analysis of natural and synthetic bioactive compounds occurring in food and food products within the period of 2019-2021 (January). Special attention is given to optimization including the validation process of chromatographic analysis performed by using thin-layer chromatography (TLC), high-performance liquid chromatography

TLC Analysis of Selected Bioactive Compounds in Food Samples
The recently published papers indicate that thin-layer chromatography was successfully used for the quantification of selected antibiotics, alkaloids, aromatic amines, and gallic acid in food [2][3][4][5]. Both i.e., contact and immersion TLC-bioautography with the use of silica gel F 254 plates, 7.5% of KH 2 PO 4 , and Escherichia coli ATCC 8739 as a test bacterium were employed for the sensitive determination of streptomycin in the presence of kanamycin sulfate in frozen shrimp, thus to control the antibiotic abuse in frozen food [2]. The work of Foudah et al. [3] shows a rapid and sensitive HPTLC method with densitometry for the quantification of trigonelline content as important bioactive constituent of Arabic coffees at the level of ng/spot [3]. Another study [4] indicates the use of HPTLC-DPPH (high-performance thin-layer chromatography coupled with the use of 2,2-diphenyl-1-picrylhydrazyl) method for rapid and simple screening of antioxidant constituents i.e., gallic acid in honey, in natural food products. Similarly, the study of Piszcz and coworkers [6] demonstrated the ability of TLC method to separate two different forms of DPPH (i.e., DPPH and DPPH-H) and also for the measurement of total antioxidant potential in the meat samples. Another study describes a novel and fully validated HPTLC-MS method for the rapid identification and determination of toxic aryl azo amines in food matrices. The achieved level of detection and quantification of these compounds was in ppm [5].
Another authors; Turkmen and Kurada [7] confirmed the utility of HPTLC on silica gel 60F 254 plates with densitometric measurements to asses next toxic compound, namely patulin as contamination of fruit-based baby foods in Turkey.
In the vast majority of analyzes, fatty acids are investigated using the GC technique as fatty acid methyl esters. However, Dąbrowska et al. [8] developed a TLC method in combination with densitometry for the determination of omega-3 fatty acids: linolenic (ALA), docosahexaenoic (DHA), and eicosapentaenoic acids (EPA) in 15 dietary supplements and 5 cooking products.
Some studies indicate the important role of TLC and HPTLC methods as comprehensive techniques for the detection and identification of pesticides and the toxicity caused by these compounds [9][10][11][12][13][14][15]. Several new chromogenic reagents have been reported in the literature such as diphenylamine reagent for detection of organochloro insecticide endosulfan [9], stannous chloride and hydrochloric acid (reducing reagent) followed by a sodium nitrite in hydrochloric acid (coupling reagent) and β-napthol in sodium hydroxide for the detection of herbicide oxyfluorten [10], chloranil reagent with nitric acid for detection of organophosporus insecticide monocrotophos [11], 4-amminoantipyrene reagent with potassium ferricyanide for detection and identification of 2,4-dichlorophenol, an intermediate of 2,4-D (2,4-dichlorophenoxyacetic acid) herbicide [12], cupric acetate reagent for detection of organophosphate insecticide profenofos [13], and cobalt thiocyanate reagent for detection of organophosporus herbicide glyphosate [14]. Hussain et al. [15] developed an HPTLC method for the determination of residues of various pesticides in brinjal samples from a market of Pakistan. The authors showed that HPTLC can be an alternative method to HPLC for the detection of pesticide residues.
The scientific literature cited and discussed above indicates that TLC/HPTLC can be successfully used to detect and quantify a wide variety of synthetic and natural classes of bioactive compounds occurring in food and food products. There are many reports in the scientific literature combining TLC with a densitometry. However, there is an increase of works linking TLC with MS. Therefore, it seems that in the next few years there should be more scientific papers using TLC/MS.

Column Liquid Chromatography
Extensive review of literature published in the two last years indicates that highperformance liquid chromatography (HPLC) with different detection systems such as ultraviolet detector (HPLC-UV), photodiode array detector (HPLC-PDA), or coupled to mass spectrometry or tandem mass spectrometry called as HPLC-MS and HPLC-MS/MS respectively is a powerful analytical tool with many applications including food analysis .

Column Liqiud Chromatography in Analysis of Selected Bioactive Compounds in Food Samples
Due the widespread use of agricultural chemicals in food production, people are exposed to low levels of pesticide residues through their diets. Because the organic pesticides usually exist in very small amounts in food samples and have different chemical structure containing, for example, triazine, imidazolinone, phenyluracyl, or macrocyclic lactone structure, thus there is a need to develop efficient and sensitive CLC systems for the simultaneous determination of compounds that are dangerous to human health, present in food and food products which belong to one of the presented groups as well as to various groups (i.e., multiclass pesticides) [31,32]. Table 1 shows the utility of selected CLC procedures with validation parameters that have been applied in analysis of food samples . The current literature review indicates that validated high performance liquid chromatography is a powerful analytical technique used to determine many single or multi-class pesticides present in different food matrices. Most developed methods were validated according to the European SANTE guidelines (SANTE/11945/2015, SANTE/11813/2017, SANTE/12682/2019) in terms of linearity, LOD, LOQ, accuracy, recovery, and precision, as shown in Table 1. As it can be observed, liquid chromatography is particularly appropriate for the analysis of polar, non-volatile, and/or thermally labile pesticides. Because of its high selectivity and sensitivity, HPLC and UHPLC in combination with MS/MS have mostly been used in this field especially to determine the insecticides and herbicides belonging to organophosphorus compounds, imidazolinone and pyridine carboxylic acid derivatives, and in study of samples containing multiclass pesticides [16][17][18][19][20][21][22][23][24]27,[30][31][32][33][34]. However, in a few cases, i.e., triazine and phenylurea herbicides, the HPLC coupled with spectrophotometric detection HPLC-UV or DAD has also been applied [25,26,28]. Various kinds of stationary phases (columns) have been used in the HPLC determination of pesticides, mainly C18 [16][17][18]20,[25][26][27][28][29][30][31], and also chiral [19,33], BEH HILIC [21], Hypercarb [22], Obelisc N HILIC [24,34], Acquity UPLC HSS T3 [23,32]. In general, water or water with formic acid or acetic acid or ammonium formate, acetonitrile, and methanol have been applied as mobile phases with gradient or isocratic elution, respectively.

Imidazolinone derivatives
Soybean, peanut, wheat, maize, rice  Recovery: 96.5 ÷ 100.9% [48] It is commonly known that the HPLC-UV (DAD) technique has a lower sensitivity compared to the LC-MS/MS. However, owing to the new SPE (solid phase extraction) systems consisting novel polymers as adsorbents e.g., porous organic polymer Car-DMB, Py-DMB HCP (heterocyclic hypercrosslinked polymer), HPLC analysis further allows the quantification of some pesticides in food samples at concentrations of ng/g [25,28]. As is shown in Table 1, many HPLC-MS/MS techniques with triple quadrupole (TQ), electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode or selected reaction monitoring (SRM) mode have been mainly used for the determination of different kind of pesticides [16,[18][19][20][21]27,31,33]. In addition, the HPLC-MS/MS methods with electrospray ionization (ESI) and quadrupole trap (QTRAP) in multiple reaction monitoring (MRM) mode have been also employed in the analysis of various pesticides [23,24,30,34]. Whereas UHPLC-Q Orbitrap-ESI-MS/MS has been applied for the determination of highly polar pesticides and contaminants (glyphosate, aminomethyl phosphonic acid (AMPA), phosphonic acid, fosetyl-Al, chlorate, and perchlorate) in processed fruits, vegetables, and infant foods [22].
Studies [19,33]  Several papers created during the last two years [35][36][37][38][39][40][41] demonstrate the importance of different CLC procedures to determine selected veterinary drugs in animal food and food products belonging to various groups including non-steroidal anti-inflammatory agents (NSAIDS), some antibiotics, and others according to EU Commission Decision 2002/657/EC requirements [35] to guarantee food safety.
Whereas, LC-MS/MS methods with triple quadrupole (TQ), electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode have been used for the determination of multiclass NSAIDs in meat of swine, chicken, eggs, and bovine [37,38,40]. Developed chiral UHPLC-TQ-ESI-MS/MS in MRM mode have been successfully applied to the simultaneous determination of four profens enantiomers including naproxen, carprofen, indoprofen, and flurbiprofen in fish tissues [39]. The obtained LODs and LOQs for each enantiomer ranged from 1 to 8 ng/g and 2 to 10 ng/g, respectively [39].
Kurjogi et al. [49] applied an HPLC-UV for the detection of antibiotics in milk samples originating from the dairy herds located in India. Similarly, Dinh et al. [50] elaborated QuEChERS-LC-MS/MS clean up method with UHPLC-MS/MS for the analysis of sulfonamides and potentiators, macrolides, lincosamides, quinolones and fluoro-quinolones, nitrofurans, nitroimidazoles, chloramphenicol, triphenyl-methane dyes, teracyclines, and metabolites in cultured and wild seafood sold (in red-meat fish, white-meat fish, and shrimp).
Studies confirm the vital role of HPLC with diode array detection method and mass spectrometry for the analysis of some steroids in current residual food analysis of meat products and eggs coming from farmed animals, thus to control steroids in meat [42,43]. A reliable and sensitive UHPLC-MS method was also constructed by Han and Liu to detect 17 endogenous and exogenous steroid hormones including estrogens, androgens, glucocorticosteroids, and mineralocorticosteroids in Antarctic krill (Euphausia superba Dana) [44].
Another study shows the utility of HPLC with MS/MS based on the operation of a triple quadrupole (LC-ESI-MS/MS) for quality control of the species of meat or products by determining the presence of thermostable dipeptides (e.g., anserine, carnosine and balenin) [51].
Several authors have also described the analytical methodologies based on HPLC to characterize the food composition i.e., to detect especially a new bioactive compounds with nutritional value and a proper biological activity, for example, antioxidant properties that are present in vegetables and fruits consumed in various countries. Developed methods are necessary to control the quality/authenticity of food and have been carried out by researchers during the last two years.
Numerous studies indicate that HPLC is the method of choice due to its precision and sensitivity for the determination and quantification of natural as well as synthetic antioxidants in various food/food products [45][46][47][59][60][61][62][63][64]. The main group of antioxidants investigated were phenolic compounds, especially phenolic acids, catechins, and flavonoids. Therefore the identification and assessment of antioxidant activity of different edible plant samples containing these bioactive compounds and their derivatives using highperformance liquid chromatography have been extensively investigated in the two last years. For example Yue et al. [45] developed and validated an HPLC-DAD method for the identification of selected synthetic phenolic antioxidants (SPAs) in chewing gum, noodle, snacks, nut, chocolate, fruit juices, coffee, oat, and biscuits. An interesting study performed by Cheung et al. [59] shows the utility of this technique for the determination of phenolic acids (16) and flavonoids (14) profiles in honey samples, thus for quality control of honey.
Gbylik-Sikorska et al. [46] developed for the first time an UHPLC-MS/MS method for the estimation of the pharmacokinetic parameters of quercetin in milk samples of dairy cows.
A few papers indicate the HPLC studies of different phenolic compounds in green coffee and the fruits of the three European plum cultivators [47,60].
Pepe et al. [61] undertook the study of the composition of polyphenols (26) and anthocyanins (12) found in Citrus sinensis and Vitis vinifera. RP-UHPLC-PDA combined with LCMS-IT-TOF (ion trap-time of flight mass spectrometer) was used in analysis of polyphenols and anthocyanins. HPLC with UV-Vis detection was also used for the determination of anthocyanin in skins and seeds of five Greek red grape varieties [62].
Similar study by means of HPLC-MS/MS method was performed to estimate the contents of some antioxidant components in grapevine seeds Vitis vinifera L cultivated in Italy [63]. The results of chromatographic analysis confirmed the presence of nine major flavonoids (apigenin, astragalin, hyperoside, isorhamnetin, kaempferol, myricetin, quercetin, quercitrin, and rutin) and two procyanidins (procyanidin A 2 and procyanidin B) in the studied extracts.
Carotenoids and polyphenols were evaluated and quantified by HPLC-DAD and UHPLC-Q-Orbitrap HRMS, respectively, in two-pigmented Lactuca sativa L. var. [64]. Separation and quantification of carotenoids were performed by HPLC-DAD on C18 column. Polyphenols analysis was performed by UHPLC-Q-Orbitrap HRMS on biphenyl column. LODs and LOQs of analyzed compounds were in the range of 0.03-0.05 and 0.10-0.16 ng/g, respectively.
Another author Cirilli et al. [65] investigated iberin (an isothiocynate with chemoprevention of different tumors) in natural products and in different food supplements. Analysis was performed by UHPLC-PDA-ESI/MS. Three degradation products of iberin were identified, namely: thiourea, methyl thiocarbamate, and ethyl thiocarbamate. Similar study refers to 6-methoxymellein as the main ingredient responsible for the bitterness of carrot (Daucus carota L.) [66].
Summarizing, it can be stated that the studies described above confirm that validated high-performance liquid chromatography methods coupled with DAD, UV-Vis, MS/MS, and HPLC-TQ-ESI-MS/MS are the powerful tools in analysis i.e., separation, identification, and quantification of different natural and synthetic bioactive compounds occurring in food and food products for different purposes, i.e., authenticity and safety of food and food products.
It was stated that examined by column liquid chromatography bioactive compounds in food samples belonged to different chemical classes e.g., steroids, phenolic compounds, variety antibiotics (fluoroquinolones, tetracyclines, β-lactams), organophosphorus, phenyluracyl or triazines pesticides, and others. Therefore, both the factors, chemical diversity and the complexity of investigated mixtures, i.e., the kind of studied matrix were the biggest challenges in the case of HPLC technique and were accurately described in this review paper. A broad variety of packing material of column including a new one such as molecularly imprinted magnetic polymers as well as modern extraction systems like solid-phase extraction and salting-out extraction combined with switchable-hydrophilicity solvent liquid-liquid microextraction to sample preparation allow separation and quantification of new bioactive compounds like synthetic antioxidants or trace levels of different chemical groups of pesticides simultaneously (i.e., multiclass pesticides) in food. The use of chiral stationary phases improves the separation and determination of the selected stereoisomers (S-and R-form) of some imidazolinonen herbicides in food samples (e.g., soybean, peanut, wheat, maize, rice) and some NSAIDs belonging to profens i.e., ibuprofen, indoprofren, pranoprofen, flurbiprofen, ketoprofen, caprofen, naproxen and loxoprofen in fish tissues simultaneously at the level of ng/g.
Properly validated for optimal conditions HPLC method by means of DAD (PDA) and UV-Vis detector with gradient elution program makes this technique enough sensitive for the quantitative determination of different bioactive compounds including the selected pesticides and drugs in food samples in µg/mL or ng/g, respectively.

Matrix/Compound
Chromatographic Conditions Other Parameters Refs.

Matrix/Compound Chromatographic Conditions Other Parameters
Refs.

Non-steroidal anti-inflammatory compounds (profens) and Steroids
Mussels Mytilus edulis trossulus NSAID (5) Crude fat, total saturated acids, and total trans fatty acids in home meal replacements, and restaurant foods were analyzed using GC-FID (gas chromatography-flame ionization detector). Total crude fat contents were 0.61 ÷ 6.75 g/100 g, and 0.22 ÷ 5.69 g/100 g for home meal replacements and restaurant foods, respectively. Total saturated fatty acids contents were 0.08 ÷ 1.42 g/100 g, and 0.07 ÷ 1.44 g/100 g for home meal replacements and restaurant foods, respectively. Total trans fatty acids contents were 0.0 ÷ 0.11 g/100 g, and 0.0 ÷ 0.07 g/100 g for home meal replacements and restaurant foods, respectively [77]. Fatty acids in the form of methyl esters were also determined using the GC-FID technique in four bee products. The authors of the study compared the total fatty acid concentration (saturated, unsaturated, omega-3, omega-6, the ratio of saturated and unsaturated, omega-3/omega-6 fatty acids and trans fatty acids) [78]. Fruehwirth et al. [79] investigated the lipid oxidation in stored margarine using GC-FID method. Volatile components and fatty acids present in margarines were tested. Acetone and hexanal increased in all types of margarine during storage.
Study [80] shows the applicability of GC-MS analysis for identification of chemical components with different activity including antioxidant properties of varieties, not well described in literature, of edible plants and fruits cultivated in different countries. GC-MS was successfully applied for the separation and identification of chemical components with antioxidant activity such as different phenolic acids from citrus fruits cultivated in India i.e., grapefruits. The major components found were: limonene, methyl-cyclohexane, hexane-3one, 3-hexanol, 2-hexanol, myrcene, sabinene, nonanal, neral, geranyl acetate, ostole. These compounds might contribute to the antioxidant activity of the juice and oil [80].
The reviewed papers confirm that gas chromatography has recently been used to study food and edible plants (the contents of pesticides, endocrine disrupting chemicals, NSAIDs, natural estrogens, glyoxal, methylglyoxal, fatty acids, compounds with antioxidant properties, such as e.g., flavonoids, phenolic compounds). The most commonly used gas chromatography was combined with a mass spectrometer or a dual mass spectrometer with electrospray ionization (GC-EI-MS, GC-EI-MS/MS). The presented papers show the utility of this technique for both, i.e., residue analysis of multiclass pesticides and NSAIDs simultaneously in food and food products as well as for the determination of new antibacterial and antitumor agents in edible plants.

Combined Techniques
In many cases, not one but two or more analytical techniques are required for determining the active substances present in food matrices. Nowadays, these combined techniques are powerful analytical tools with many applications. Several papers reported their utility in food analysis [81][82][83][84][85][86].
Carotenoids, phenolic compounds, and fatty acids were determined in tomato seed oil derived from cold break and hot break processing lines [81]. HPLC-DAD-ESI-MS on C18 column and two mobile phases in the gradient elution were used in the investigation of phenolic compounds. HPLC-DAD on C18 column and two mobile phases were used for the quantitative and qualitative analysis of carotenoids. Fatty acid profile was determined by GC-MS. Higher levels of carotenoids (lutein, lycopene, β-carotene) and phenolic compounds ((caffeic acid-glucoside isomer (CG), caffeic acid (CA), syringic acid (SyA), di-caffeoylquinic acid (di-CQA), and tri-Caffeoylquinic acid (tri-CQA)) were found in the cold pressed oil. The following fatty acids were the most abundant in the oil: linoleic acid, oleic acid, and palmitic acid [81].
Migas et al. [82] determined lutein and lutein mixed with zeaxanthin in eight dietary supplements. BMD-TLC (bivariant multiple development thin layer chromatography) was used for the analysis of lutein, β-carotene in samples. HPLC-DAD-ESI-MS was used for the isolation and identification of mixture of lutein and zeaxanthin. The proposed method was linear in the range 90 ÷ 500 ng/point. Limits of detection and quantification were 50 ng/point and 90 ng/point, respectively. Method was precise, accurate, and robust. TLC was used for monitoring the formation of γ-aminobutyric acid (GABA) in traditional Indonesian foods fermented with thirty strains of lactic acid. For this purpose, silica gel 60F 254 plates and n-butanol-acetic acid-distilled water (5:2:2) mobile phase were used. On the other hand, for the quantitative determination of GABA, UPLC was used with the C18 column [83].
Aflatoxins are produced by fungi, including those on spoiled food. TLC on silica gel 60 plates using acetonitrile-methanol-trifluoroacetic acid (9:1:0.2, v/v/v) mobile phase and with the visualization using vanillin, p-anisaldehyde solutions, or iodine vapor was a simple, robust, and non-quantitative method for the detection of aflatoxins. HPLC-DAD (λ = 200 ÷ 410 nm) with C18 column and two eluents in gradient elution were used for the quantitative determination of aflatoxins. TOF/Q-TOF MS/MS was used for the detection of aflatoxin metabolites, and the sixteen possible metabolites were identified [84].
A novel and highly sensitive metastable state nanoparticle-enhanced Raman spectroscopy combined with thin layer chromatography (TLC-MSNERS) has been successfully used for the determination of pesticides such as thiabendazole, phosmet, and triazophos on fruit skin. An amphiphilic polymer polyurethane-Ag nanoparticle (AgNPs) has been employed as the MSNERS substrate [85]. Another work developed and validated a modified QuEChERS method to determine multiclass pesticides (207) in honey samples using both LC-MS/MS (154 compounds) and GC-MS/MS (53 compounds) [86].
In summary, the necessity to analyze samples with a complicated composition requires the use of combined techniques. Sometimes the matrix is so complex (it contains chemical compounds belonging to different chemical classes) that there is a need to use at least two analytical techniques to determine the composition of the analyzed sample. The reliability requirements of the analytical results often preclude the possibility of identifying the analytes solely on the basis of the retention time. Only the combination of the ability to separate complex mixtures using chromatographic methods with structural information (HPTLC/MS, LC/MS, GC/MS) enables reliable identification of food constituents.
Owing to the use of combined techniques, it is possible to significantly speed up and reduce the cost of analyzing due to less requirements for the stage of sample preparation for analysis.
The advantages of the combined techniques in food analysis are: the ability to identify unknown food constituents, information about their molecular weight and/or structure, easy detection of the overlap between peaks, and faster end results. In contrast, the disadvantages of the combined techniques are high investment costs.

Conclusions
The reviewed papers confirm that of all chromatographic techniques, liquid chromatography (LC) is the most universal technique that enables successful analysis of complex matrices including food products. The current high-performance liquid chromatography systems are crucial to assess the quality of food. HPLC method in combination with various detection modes i.e., HPLC-UV, HPLC-DAD(PDA) and HPLC-MS or HPLC-MS/MS, respectively is selective, sensitive, accurate, and robust for the simultaneous determination of natural and synthetic bioactive molecules belonging to different chemical classes in complex food samples as residue of food production such as multiclass pesticides, NSAIDs or steroids, as well as a new food constituents (e.g., antioxidants) in edible plants cultivated in different countries. The use of modern spectroscopic techniques such as MS as detection system allows the identification and accurate study of the structure of all components occurring in food matrices.
While thin-layer chromatography coupled to densitometry and mass spectrometry could be the most suitable technique for preliminary screening and determination the antioxidant properties (TLC-DPPH) of food components.
Gas chromatographic methods (GC-EI-MS, GC-EI-MS/MS) are also essential for the screening of different bioactive compounds including the pesticides and fatty acids in edible plants and in food products. Pesticides profiling in food samples done by HPLC and GC in combination with prior sample separation by means of modern microextraction systems can be valuable in rapid quality control of food and ensures food use safety.

Conflicts of Interest:
The authors declare that there is no conflict of interests regarding the publication of this paper.