Principle of Chiral Chromatography and Its Presence in Food Analysis

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Food analysis is a very important branch of analytical chemistry which provides data about chemical composition of food stuffs, contaminants and ensures safety of foods during import and exports. Many compounds which are found in nature, can be chiral which may have one or more chiral center and can exist as enantiomers. Enantiomers exhibits similar physico-chemical properties with assymetric molecular configuration and they are not superimposable mirror images of each other. They rotate the plane polarized light in opposite directions and are referred as levo (-) and dextro (+) rotatory forms (Rocco et al., 2013).

In food analysis, it is important to know the enantiomeric composition of food to determine its quality and genuineness. Chiral compounds have been separated utilizing analytical techniques that have also been used for preparative purposes e.g., gas chromatography (GC), supercritical fluid chromatography and high-performance liquid chromatography (HPLC). Recently, miniaturized techniques, such as capillary electrophoresis and capillary liquid chromatography or nano- liquid chromatography, were also applied for enantiomeric analysis in food chemistry (Asensio‐Ramos et al., 2009)

Chiral chromatography could be used to check adulteration and authenticity of foods, determination of aroma components and development of flavors and pesticides biotransformation during food processing (Armstrong et al., 1990; Simó et al., 2003). Modern and ever more sophisticated analytical methods are needed for appropriate chiral analysis in food. These methods could be optimized to achieve fast separation at high separation efficiency and enantio-resolution, and enhanced sensitivity, and to give the possibility of correctly identifying analyzed compounds e.g., using mass spectrometric, polarimetric and/or circular dichroism detectors (Cifuentes, 2016)

Principle of Chiral Chromatography

A chiral environment is introduced to separate the enantiomers. Chiral separation can be obtained by applying two methods, indirect resolution and direct resolution method. In the indirect approach, the enantiomers react with a chiral selector on forming stable diastereoisomers that can be separated using conventional stationary phases. Many CHIRAL SELECTORS can be used based on the compound to be extracted. Mostly, chiral selectors are bonded or adsorbed to the stationary phases. When choosing the type of Chiral stationary phase and the chromatographic technique, it is important to know the chemical structure and the physical and chemical properties of the enantiomers to be separated (D'Orazio et al., 2017). In liquid chromatography, reversed phase, polar organic and normal phase modes are the most applied. In the direct resolution method, labile diastereoisomeric complexes are formed during the process of separation and is the most widely used method in analytical chemistry. Strong Coulomb bonds, hydrogen, pep, ion-dipole, dipole-dipole, van der Waals attractive interactions and steric hindrance as repulsion are reported between the enantiomers and the chiral separator (Berthod, 2006). The chiral separator has 3 sites and the two enantiomers bind differently to these sites. Thus, having different stability and can be easily separated. One of the two enantiomers will bind to the chiral separator with three of the adducts, while the mirror image will join with only two of its substituents (Berthod, 2006 and Davankov, 1997).

Chiral Separations in Food Analysis by Gas Chromatography

Gas chromatography is mostly employed to separate the volatile compounds having low boiling points. It also determines the constituents of foods such as fatty acids, contaminants etc (Ibañez and Cifuentes, 2001). Several applications of chiral GC relate to the study of toxicity, the processes of absorption, distribution, and degradation of chiral agrochemicals (e.g., pesticides, fungicides, and hormones) in organisms, vegetables and environment, which are often enantioselective ( sekhon 2009). Ali et al., (2010) determined the quantity of amino acid enantiomers in 26 wines and documented that D-amino acids, in particular, D-proline was present. A fused silica capillary column Chirasil®-L-Val was used as a chiral separator. In another study, α- and β-ionone concentrations were determined using multidimensional gas chromatography coupled to tandem mass spectrometric detection, and they serve as indicators for a potential adulteration (Langen et al., 2016). Also, flavor adulterations in wines has been reported using multi dimentional gas chromatography. The concentrations of (R)-(_)-1,2-Propanediol, (S)-(þ)-1,2-propanediol enantiomers were studied in wines which are industrial solvents. It has been demonstrated that a high enantiomeric ratio in favor of the (R)-enantiomer acts as a marker of flavor adulteration. The enantiomeric separation of the derivative was achieved with heptakis-(6-O-tert. butyl dimethylsilyl-2,3-di-O-acetyl)-β-cyclodextrin as the chiral selector. (Langen et al., 2016). Research has shown that, fifteen chiral monoterpenes in white wine were quantified from various wine matrices using head-space solid phase micro-extraction-MDGC-MS (Song et al., 2015). Another analysis of red wine documented that, p-menth-1-en-3-one was identified. Chiral multidimensional GC–MS was used to show that piperitone was present mainly in the (R) form in red wines (Pons et al., 2016)

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Similarly, α-pinene, β-pinene, limonene, p-cymene were separated from cardamom essential oil using Chiraldex β-DM column. Two‐dimensional gas chromatography coupled to quadrupole–accurate mass time‐of‐flight mass spectrometry was used to separate chiral components of mono terpenes (Chin et al., 2014). Different chiral mono terpenoids were separated from Thymus vulgaris essential oil using GC ( Satyal et al., 2016). Enantiomeric purity screening and quantification of L- and D-carnitine was done using chiral gas chromatographic method. L-carnitine is mainly found in functional foods and food supplements and a cyclodextrin based stationary phase was used (Albreht et al., 2014)

The enantiomer ratios of chiral volatile organic compounds in rapeseed, chestnut, orange, acacia, sunflower and linden honeys were determined by multi-dimensional gas chromatography using solid phase microextraction (SPME). enantiomer distribution of some chiral organic compounds in honey was found to be dependent on their botanical origin. more than 270 compounds were detected in studied honeys that belong to various chemical classes and many of them were chiral ( Špánik et al., 2014).

Chiral Separations in Food Analysis by HPLC

High Performance Liquid Chromatography is the widely employed separative technique for both preparative and analytical purposes, due to the several advantages it offers, such as high selectivity, separation efficiency, large variety of stationary phases and columns commercially available, and appropriateness for non-volatile, polar, nonpolar, and thermally labile compounds. Furthermore, HPLC can be used as a multidimensional technique, so increasing its selectivity and allowing on-line clean-up of samples. Finally, coupling with a highly sensitive detector, such as MS, makes HPLC one of the most powerful analytical tools for solving complex analytical tasks. Many agrochemicals are composed of enantiomers and they may remain after processing of foods. These compounds may be toxic to humans and environment so their quantification and detection is focused using HPLC, SFC etc. Several HPLC methods have been developed for chiral agrochemicals analysis. determination of chiral pesticide flufiprole enantiomers using high-performance liquid chromatography has been established. The separation and determination were performed using reversed-phase chromatography on a carbamoyl–cellulose-type chiral stationary phase, a Lux Cellulose-2 column. An Alumina-N solid-phase extraction (SPE) column was used in the cleanup of the vegetables, fruits, and soil samples. The results confirmed that this method was convenient and accurate for the simultaneous determination of flufiprole enantiomers in food and environmental samples (Tian et al.,2015).

The separation of enantiomers of nutraceuticals-flavanone and 5 chiral flavanone derivatives was studied on polysaccharide-based chiral columns by HPLC with methanol, ethanol, acetonitrile, n-hexane/ethanol and n-hexane/2-propanol as mobile phases. The various column chemistry and mobile phases are quite complementary to each other from the viewpoint of enantiomer separation of flavanone and its chiral derivatives. Chiral columns based on cellulose 3,5-dichlorophenylcarbamate and amylose 3,5-dimethylphenylcarbamate were the most useful stationary phases (Fanali et al., 2016). Three stereoisomers of zeazanthin along with lutein were isolated from fish flesh using chiral HPLC-DAD. This method is suitable to detect and quantify meso-zeaxanthin and (3S,3′S)-zeaxanthin in fish flesh ( Prado-Cabrero et al., 2016). HPLC is used for the evaluation of antibiotics and veterinary drugs residues in animals. Clenbuterol is a steroid drug which is used in animals for veterinary purposes. The enantiomers were determined in beef and lamb meat by Wang et al., (2016). R-(−)-Clenbuterol and S-(+)-clenbuterol were completely baseline separated and detected by HPLC-MS/MS.

6-Prenylnaringenin (6PN) is a chiral prenylflavonoid found most prevalently in hops (Humulus lupulus) and present in hops and hop products. It is an isomer of the potent phytoestrogen, 8-prenylnaringenin. The method was found to be accurate and precise for enantiospecific quantification of 6PN. The method was successfully applied to the content analysis of 39 commercially available natural health products and dietary supplements reported to contain H. lupulus plant material, extracts and label claims of 6PN (Martinez, S. E., & Davies, 2015).

Using high-performance liquid chromatography (HPLC), flavanol enantiomers, (+)- and (-)-epicatechin and (+)- and (-)-catechin, are eluted isocratically using ammonium acetate and methanol mobile phase. The mobile phase is applied to a modified β-cyclodextrin chiral stationary phase and the flavanols detected by fluorescence (Machonis et al., 2014).

Chiral Separations in Food Analysis by Supercritical Fluid Extraction

For chiral SFC separations, most HPLC chiral stationary phases (CSPs) can be directly used. Similar to what observed in chiral HPLC, the most commonly used CSPs in SFC are the ones based on polysaccharides (Rocco, A., Aturki, Z., & Fanali, S. (2013).) Ergostanes are major bioactive constituents of the medicinal mushroom Antrodia camphorata. These tetracyclic triterpenoids usually occur as 25R/S epimeric pairs, which renders their chromatographic separation difficult. In this study, we used analytical supercritical-fluid chromatography (SFC) to separate seven pairs of 25R/S-ergostanes from A. camphorata. Chiralcel OJ-H column (4.6 × 250 mm, 5 μm, chiral), eluted by 10% MeOH in CO2 at 2 mL/min with a back pressure of 120 bar and a column temperature of 40 °C. Particularly, this chiral-SFC method could rapidly and efficiently separate low-polarity epimers like antcin A and antcin B, which were very difficult for RP-HPLC. Aside from high separation efficiency, SFC also showed advantage over HPLC in short analysis time and low consumption of organic solvents (Qiao et al., 2014).

A green and sensitive chiral analytical method was developed to determine flutriafol enantiomers in vegetables (tomato, cucumber), fruits (apple, grape), and soil by supercritical fluid chromatography–tandem mass spectrometry. The simple and fast QuEChERS pretreatment method was adopted. The enantioseparation was performed within 3.50 min using Chiralpak IA-3 column with CO2/methanol (88:12, v/v) as the mobile phase at a 2.2 mL/min flow rate (Tao et al., 2014). An efficient and sensitive chiral analytical method was established for the determination of propiconazole stereoisomers in wheat straw, grape, and soil samples by supercritical fluid chromatography–tandem mass spectrometry (SFC-MS/MS). Stereoisomeric separation was performed on a Chiralpak AD-3 column with CO2/ethanol (93:7) as the mobile phase. The four propiconazole stereoisomers were well separated in 4.7 min with resolutions above 2.0. (Cheng et al., 2016).

Conclusion and Future Trends

  1. For the detection of chiral agrochemicals QuEChERS is the most employed method. Li et al. 2012 combined the small-scale extraction of QuEChERS with a conventional acetonitrile extraction to enhance removing food colorings, such as chlorophyll, carotene, and water soluble materials, for the determination of difenoconazole stereoisomers and their hydroxylated metabolite, difenoconazole alcohol, in some vegetables and soil.
  2. Recent developments in LC answered the need for high separation efficiency and rapid analysis. As an alternative to HPLC, ultrahigh- pressure LC (UPLC), was proposed for working at higher pressure conditions to carry out fast analysis with columns packed with sub-2-lm porous particles or sub-3-lm core-shell particles. Eto et al. 2011 performed the analysis of D-AAs and L-AAs using UPLC coupled with a circular dichroism detector (CDD) in less than 6 min. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was selected as pre-column derivatization reagent in order to enhance the sensitivity and the selectivity of UV and CDD detection. Fig. 3 shows the chromatograms of the AA enantiomers (after derivatization) obtained by UPLC.
  3. Electromigration techniques and miniaturized liquid chromatography. Even though GC, HPLC and more recently SFC are chromatographic separation techniques widely used in chiral in food analysis, alternative miniaturized methodologies has gained importance due to their great potential in terms of selectivity, short analysis time and low analysis costs.
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