HPLC-MS(n) Applications in the Analysis of Anthocyanins in Fruits

*Seyit Yuzuak, Qing Ma, Yin Lu and De-Yu Xie*

## **Abstract**

Anthocyanins are water-soluble pink/red/blue/purple pigments found abundantly in the flesh and skin of fruits, flowers, and roots of different varieties of plants. Compared to vegetative tissues in many plants, fruits have much higher contents of anthocyanins. In general, anthocyanins have antioxidant, anti-inflammatory, antimutagenic, and antiapoptotic activities that benefit human health. To date, anthocyanins in many different fruits have gained intensive studies in structures, biosynthesis, genetics, and genomics. Despite this, difficulties exist in identifying anthocyanins with similar structures and precisely estimating contents within fruit matrices. To improve this challenge, high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) based metabolomics has been shown a powerful technology to distinguish structure-similar anthocyanins. This chapter reviews, summarizes, and discusses the application of HPLC-MS/MS in the annotation or identification of anthocyanins in fruits.

**Keywords:** anthocyanins, chromatography, HPLC, mass spectrometry, fruits

## **1. Introduction**

Anthocyanins are a class of plant flavonoids belonging to polyphenolics. Anthocyanins are water-soluble pigments that give pink/red/purple/blue color to plant tissues. Anthocyanins are found in the majority of higher plant species except in plant species of Caryophyllales. Moreover, anthocyanins have been found in some lower plants, such as mosses and ferns [1]. Certainly, anthocyanins are important agronomical traits in many crops, particularly ornamental ones for flowers and fruits [2]. Anthocyanins are synthesized in the cytosol and mainly transported to the central vacuoles. Plant cells such as epidermal cells in the peel of fruits and flower petals are the main locations with the active biosynthesis of anthocyanins. Generally, anthocyanins are stored as the colored flavylium ion form due to the acidic conditions of the vacuoles [3]. The color changes of plant tissues are normally associated with pH value variations in the central vacuoles.

#### **1.1 Structure**

All anthocyanins are derived from a specific chromophore core, namely 2-phenylbenzopyrylium or flavylium, which consists of two aromatic rings (A and B) and one heterocyclic pyran ring including three carbons (C) (**Figure 1**), thus is featured by C6-C3-C6. Seven positions (R3, R5, R6, R7, R3′, R4′, R5′) are commonly subjected to modification of monosaccharides, methyl, or other groups *via* –OH or carbon [4, 5]. Past structural studies have shown that anthocyanins predominantly found in nature are glycosylated or galloylated 2-phenyl-benzopyrylium or flavylium salts in acidic conditions. The flavylium structures also are subjected to modification by the vacuolar pH values to give different hue features of plant tissues.

To date, more than 700 distinct anthocyanins have been identified in the plant kingdom. Structurally, anthocyanins are composed of an aglycone also called anthocyanidin and carbohydrate residue. All anthocyanins are derivatives of anthocyanidin aglycones. Although different reports have listed 19 anthocyanidins (6 major and 13 minor groups) (**Table 1**), based on the biosynthesis from phenylalanine and three malonic acids, pelargonidin, cyanidin, and delphinidin form the three basic ones. Other types such as peonidin, petunidin, and malvidin result from methylation of the B-ring of the three basic forms. Actually, methylated anthocyanidins have different physical and chemical features from their precursors. To date, pelargonidin, cyanidin, delphinidin, peonidin, petunidin, and malvidin are commonly accepted to represent six major groups of anthocyanidins [6–8]. The glycosylation or methylation, acylation, galloylation, and other modifications diversify anthocyanin structures. Based on the structures reported, glycosides of anthocyanidin form the most predominant structures in nature. Past investigations have reported that glycosylation mainly occurs at the –OH group of C3, C5, or C7 of the core chromophore. The main monosaccharides involved in glycosylation include glucose, xylose, arabinose, rhamnose, rutinose, fructose, and galactose moieties. In addition, disaccharides are involved in glycosylation. The acylation, acetylation, and malonylation of anthocyanidins or anthocyanins lead to more diversity of structures. Based on structural and biosynthetic reports, the common acylation result from the addition of a coumaric, caffeic, ferulic, *p*-hydroxy benzoic, synaptic, malonic, acetic, succinic, oxalic, or malic acid to sugar moiety or moieties [7, 9]. The acylation can alter the physical or chemical features of anthocyanins, such as water solubility and color such as blue color. In addition, the hydroxyl and methoxyl groups as well as other modifications also lead to different colors and stability of anthocyanins [6, 8]. Furthermore, secondary, ternary, and more complicated modifications on sugars or acylate groups diversify

#### **Figure 1.**

*The core chemical structure of anthocyanidins. A; aromatic ring, B; phenyl ring, C; Benzopyran ring. R; -H, -OH, or -CH3.*


*HPLC-MS(n) Applications in the Analysis of Anthocyanins in Fruits DOI: http://dx.doi.org/10.5772/intechopen.110466*

#### **Table 1.**

*Basic and methylated or hydroxylated anthocyanidins (OMe: Methoxy group; OH: Hydroxyl group; H: Hydrogen) modified from ref. [6].*

anthocyanin structures. Most of those modifications result from enzymatic reactions catalyzed by glycosyltransferases, methyltransferases, acyltransferases, malonyltransferases, and others [4, 10]. These modifications are associated with the color, chemical stability, bioavailability, biological activity, and diverse molecular structures of anthocyanins in plants.

#### **1.2 Functions in plants and health benefits**

Anthocyanins play important physiological functions associated with plant reproduction and defenses. Anthocyanins act as visual signals to attract pollinators for the pollination of flowers and dispersers to spread seeds or fruits [4]. Anthocyanins can play as warning signals to repulse birds and insects for protection of plant tissues from being consumed [11]. They serve as filters to absorb UV-B light and visible light for protecting plant tissues from being damaged by severe irradiation [12]. The accumulation of anthocyanins protects leaves from radiation-caused damage of photosynthesis by absorbing extra light [13]. Past studies have reported that many anthocyanin structures defend plants against diseases infected by various pathogens and damage caused by abiotic stresses, including cold shock, drought, osmotic and wounding, and biotic stresses [5, 14, 15]. Furthermore, anthocyanidins are essential precursors of proanthocyanidins, which are powerful antioxidative, anti-pathogen, anti-radiative, and anti-pest flavonoids in most of the plant species [16].

Anthocyanins are of growing interest in beneficial values for human and animal health because of their antioxidative, antibacterial, and anticancer activities reported by *in vitro* experiments [17]. Some structures have been shown to scavenge free radicals such as reactive oxygen species generated in human cells [18]. More studies have shown that the antioxidative and anti-inflammation activities of anthocyanins help to improve visual acuity [19] and to protect from a heart attack [20, 21]. Metabolic studies have shown that certain anthocyanin structures can prevent obesity and diabetes by interfering the body weight gain and adipose tissue increase [22, 23]. Especially, different structures have been observed to associate with specific activities [24]. Accordingly, a general health fact is that regular consumption of high levels of dietary anthocyanin-rich plant sources, such as red, purple, and dark-colored berries, grapes, and vegetables, is considered to benefit human health [25].

## **2. Analysis of anthocyanins in fruits**

Anthocyanins are the main pigments responsible for red, magenta, violet, blue, and dark blue colors of many fruits and berries. Therefore, fruit or seed anthocyanins have gained a large number of studies, which include the formation and stability of color, and color changes during ripening, processing and storage, isolation, and identification from fruits (**Table 2**). Many have been developed as colorants for food, pharmaceutical, and nutritional industries.

Fruits are one of the main sources of novel anthocyanins with valuable health benefits [8, 26, 27]. A large number of anthocyanins have been isolated from fruits (**Table 2**).

To date, methods for the identification of fruit anthocyanins have been appropriately established by numerous studies. In generation, three main sequential steps are sample preparation, separation and purification, and identification.

Sample preparation, as an initial step for anthocyanin determination, is highly variable depending on the fruit samples and the objectives of the analysis. Sample preparation mainly consists of collection, drying, powdering, and extraction of samples. Liquid samples of fruit, such as juices, wines, and syrups, need very little preparation before the analysis. However, solid or dried fruit materials require to be fractionated, homogenized, crushed, or pulverized. The most commonly used solvents for the extraction of anthocyanins from fruits are the mixtures of ethanol, methanol, acetonitrile, and acetone compositions of water. Depending on the aim of the research, various types and different compositions of solvents can be implemented. For example, aqueous acetone solvents can be mostly preferred for a higher yield, an efficient and more reproducible extraction of anthocyanin [57, 58]. There is no universal and simplified sample preparation method to extract anthocyanins from fruit samples. However, a variety of modern techniques, including solid-phase extraction (SPE), accelerated solvent extraction (ASE), microwave-assisted extraction (MWE), ultrasound-assisted extraction (UAE), pressurized hot water extraction, and supercritical fluid extraction (SFC), have been developed based on maximizing the highest recovery, minimizing the amount of non-anthocyanins and degradation or alteration of the native anthocyanins [59].










*Fruit anthocyanin examples annotated or identified by HPLC-MS/MS-based profiling. Cy, cyanidine; Dp, delphinidin; Pg, pelargonidin; mv, malvidin; Pn, peonidin; Pt, petunidin.*

Because of the structural diversity of anthocyanins and their instability in light, high temperature and other conditions, it is necessary to avoid degradation during the sample preparation. In aqueous solutions, anthocyanins exist in four major forms, including the red flavylium cation, the blue quinonoidal base, the colorless carbinol pseudobase, and the colorless chalcone depending on pH. At pH below 2, anthocyanins are found primarily in the form of the red flavylium cation. Hydration of the flavylium cation gives the colorless carbinol pseudobase at pH values from 3 to 6, and the colorless chalcone pseudobase at pH values higher than 6. Since the flavylium cation form of anthocyanins is stable in a highly acidic medium, extraction solvents are required to be acidified by acetic, formic, hydrochloric, or sulfuric acids to prevent the degradation of the non-acylated anthocyanin pigments. However, the excessive acidic condition may have various effects during the extractions such as degradation or partial hydrolysis of the acylated anthocyanins as well. Solvents acidified with 0.1% hydrochloric acid (HCl, v/v) is the most commonly used for the extraction of anthocyanins from fruit samples [57, 58]. Anthocyanins can undergo a structural transformation during the sample treatment under a high temperature. When the temperature is increased, anthocyanins can turn into unstable chalcone formation, and even further degrade to brown products, giving a reduction in the concentration of the major anthocyanins. Therefore, using a lower temperature can improve the stability of anthocyanins during the sample treatment [58]. Temperature values between 30 and 50°C have been mostly used for the extraction of anthocyanins from fruit samples. Light is also destructive to anthocyanins. To minimize the degradation of anthocyanins, it is advisable to perform sample treatment and storage of extracts in darkness [58, 60]. An investigation provided parameters of the sample preparation based on the objectives of the study [61].

Due to the diversity and complexity of plant secondary metabolites, extractions methods for anthocyanins are nonselective and result in solutions with a lot of undesirable substances, such as sugars, proteins, fats, acids, and other water-soluble compounds. Based on this, a further efficient purification and separation method is normally required to remove other substances and elute anthocyanins from the extracts. These processes can normally lead to the loss of minor anthocyanin components, which result from heat, pH, metal complexes, and copigmentation [8, 62]. Undesired components, such as sugars, acids, and other water-soluble compounds, in the crude fruit extracts have been removed with C18 solid-phase extraction (SPE) cartridges containing octadecyl silica and Sephadex LH-20 containing cross-linked dextran resin. In order to purify anthocyanins by adsorption, silicone gels, such as Amberlite IRC 80, Amberlite IR 120, DOWEX50WX8, Amberlite XAD-4, and Amberlite XAD-7HP, have been used. Among the silicone gels, the Amberlite XAD-7HP has been proven to be the most effective resin for anthocyanin purification. Furthermore, other less polar polyphenolics or nonpolar compounds can be removed from the extracts by washing with ethyl acetate, chloroform, butanol, or acetonitrile in acidic conditions. After removal of other undesirable substances, anthocyanin fraction can be eluted with organic solvents, acidified with formic or hydrochloric acids (0.1%, v/v), containing water, ethanol, methanol or their composition in different ratios [57, 59, 63].

Separation of anthocyanins can be carried out by different chromatography techniques that have been developed in the past. Common methods include column chromatography, (CC), counter-current chromatography (CCC), paper chromatography (PC), thin-layer chromatography (TLC), capillary electrophoresis (CE), and high-performance liquid chromatography (HPLC) [61, 62].

A series of methods have also been developed for the characterization of anthocyanin structures. In brief, these methods include UV-visible spectrophotometry (UV-vis), HPLC facilitated with diode array detection or UV-detection, mass spectrometry (MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) [64, 65]. Since anthocyanins have a specific absorption in the visible wavelengths from 515 to 540 nm, spectrometry has been the main approach for quantification. Unfortunately, this is a measurement of total anthocyanins because it cannot measure specific components in crude extracts [26]. MS and MS/MS technologies are powerful in fragmenting anthocyanin molecules to generate featured fingerprints, which allow the annotation of unknown or known structures. NMR is a powerful technology for assigning hydrogen and carbon. There are different NMR tests such as homo and heteronuclear 2D and 3D techniques [66, 67].

#### **2.1 Chromatographic separation of anthocyanins**

Chromatography is a separation technique for the isolation of different compounds in a particular matrix. This type of technique is composed of a stationary phase formed from different materials, and a mobile phase (solvent). The separation of compounds of interest is based on their affinities for the stationary phase. The stationary phase retains the desired compounds, while the mobile phase elutes or migrates undesired substances. Based on the stationary phases, common chromatography techniques include column, paper, and thin-layer chromatography. Based on the mobile phase, the most used technique is liquid chromatography utilizing the physical and chemical features of analytes [68].

#### *2.1.1 Column chromatography (CC) and counter-current chromatography (CCC)*

Column chromatography (CC) is an effective method to fractionate and purify anthocyanins. This separation method is based on the different distribution coefficients of anthocyanins in solid and mobile phases. Common materials used for the solid phase packed in the column include macroporous, polyamide, and sephadex resins, which do not contain ion exchange groups. Macroporous resins (MRs) have multiple advantages, such as fast, stronger, and large capacity of adsorption and desorption potential for anthocyanin purification. MRs are useful for the first step of isolation to obtain fractions. Polyamide and sephadex resins are normally used to separate anthocyanins. To date, although the CC is a favorable method for anthocyanin purification in the laboratory, it is a challenge for scale-up purification [62, 69].

Counter-current chromatography (CCC) has an industry-scale technology for the separation and purification of bioactive anthocyanins from a large amount of plant materials. CC is a support-free liquid-liquid chromatography. Its development is based on the fractionation of compounds between immiscible stationary and mobile liquid phases of a biphasic solvent system. In the separation of active anthocyanins, important factors that need to be considered are acidic solvents in the absence of oxygen, linear elution or gradient elution, pH zone refining, and strong ion exchange. The main drawback is that certain organic solvents used are toxic to human health [63, 70].

#### *2.1.2 Paper chromatography (PC) and thin layer chromatography (TLC)*

Paper and thin layer chromatography are two simple techniques used for the separation of anthocyanins. PC was one of the earliest methods. It depends on specific

samples, different mobile phases, and papers used. The advantage of PC is simple and fast to examine anthocyanins. The disadvantage of PC is the limited capacity to separate scale-up samples and the separation of metabolites is relatively poor. TLC uses silica or cellulose gel or both. The separation capacity depends on silica and cellulose size as well as developing solvents. In comparison, TLC can overcome poor separation problems occurred in PC. To date, these two methods are used in common for anthocyanin research in the laboratory because of simple, fast, and low-cost advantages [6, 65].

#### *2.1.3 Capillary electrophoresis (CE)*

Analysis of anthocyanins is difficult because they can undergo structural degradation under alkaline pH, light, and high temperature. Therefore, it is required to perform analytical methods as rapidly as possible for preventing their degradation and to avoid using more solvents during analysis. For this reason, CE is a promising separation technique due to being more rapid than other techniques and using only minor amounts of solvents [71]. CE is a separation method based on the electrophoretic motility of metabolites. This technique has excellent mass sensitivity, high resolution, low sample requirement, and decreased solvent waste. When a sample is introduced into the capillary tube at the anode, the basic or acidic mobile phase migrates some components of the sample toward the cathode while others are stuck at the anode. Because anthocyanins are not stable in basic solvents, acidic solvents are used to maintain protonated the flavylium cation form. In addition, CE is configured from cathode to anode. Based on its charge-to-size ratio, particular anthocyanins or other compounds are migrated in the CE system. The migration time of compounds with higher charge-to-size ratio takes a longer time. Detection of compounds is achieved by the UV-vis spectrophotometry coupled to CE, which records the spectra from 200 to 599 nm for each peak [72–74].

The separation of anthocyanins by the CE method includes the use of fused-silica capillaries non-coated or coated with Bare or poly-LA 313 (polycationic aminecontaining polymer). Non-coated capillaries are rarely used because anthocyanins cannot be excluded due to interactions between negatively charged silica surfaces and positively charged anthocyanins. Therefore, the coated capillaries are the most suitable for anthocyanin separation. Several background electrolyte (BGE) buffers, such as ammonium formate, ammonium acetate, borate, acetic acid, formic acid, and mixtures of formic acid and hydrochloric acid, are applied both basic and acidic. In order to get highly efficient separations, MS-compatible volatile BGE buffers are used. Non-volatile borate or phosphate buffers can be also applied but these buffers are not compatible enough with MS. Furthermore, the alkaline pH of borate buffers could cause the degradation of anthocyanins. However, an acidic BGE buffer helps to prevent anthocyanin degradation [71, 75].

#### *2.1.4 High-performance liquid chromatography (HPLC)*

High-Performance Liquid Chromatography (HPLC) is a separation technique where the mobile phase is pressurized so that it can flow through the column much more efficiently. HPLC is the most convenient for components that cannot withstand high temperatures. Thus, HPLC is widely used for the qualitative and quantitative analysis of anthocyanins. There are two main types of columns of HPLC depending on the aim of the study, including analytical columns for analysis and preparative

columns for isolating and refining specific compounds from samples. HPLC columns are packed with inert materials to form the stationary phase and vary in length and internal diameter. Analytical and preparative columns are normally designed for microgram-scale and milligram-to-gram-scale separation of compounds, and adjusted to the characteristics of each analyte. HPLC utilizes different separation modes depending on the primary characteristics of compounds such as polarity and electrical charge [6, 68, 76].

Depending on polarity, there are two types of HPLC separation modes, normalphase mode (NP) and reverse-phase mode (RP). The basic principle of these separation modes is that compounds with similar polarity will show much more attraction to each other. Furthermore, the separation result and accuracy will be depending on the retention time and the speed flow rate, respectively. The normal phase system consists of the non-polar mobile phase and polar stationary phase. When the sample enters the column, the metabolites (polar) with similar polarity to the stationary phase are retained, resulting in longer retention time while other metabolites (non-polar) with similar polarity to the mobile phase move along the column with shorter retention time. The retention time differences allow appropriate separation of anthocyanins and other metabolites. Past studies have reported that the NP system is effective only for the separation of proanthocyanidins but not for anthocyanins due to retaining in the NP's polar stationary phase. By contrast, the reverse-phase system uses a polar mobile phase and nonpolar stationary phase. Therefore, RP chromatography is effective for the separation of anthocyanins due to having a similar polarity with the mobile phase. In RP chromatography, compounds with higher polarity elutes earlier than non-polar compounds. For flavonoid analysis, there are different RP separation phases using C8, C12, C18, phenyl or phenyl-hexyl, pentafluorophenyl (PFP), and polar and polymeric embedded RP columns. In general, C8 and C18 columns, which are filled with particles of silica bonded with alkyl chains, have been used to separate anthocyanins in RP chromatography. The majority of anthocyanin separations in fruits have been performed using C18 columns with the column particle size mainly ranging from 1.7 μm to 5.0 μm [68, 76].

Multiple organic solvents have been used as mobile phases to elute anthocyanins from the columns. Commonly used ones include methanol, acetonitrile, isopropanol, or ethanol, which are mixed with water supplemented with acetic acid, formic acid, ammonium acetate, or trifluoroacetic acid to form aqueous/organic elution solvents. In many separation experiments, organic solvents and acidic water are used to develop a gradient binary solvent system for the ideal separation of different structures. Furthermore, the acidic water or solvents allow for maintaining the flavylium cationic species, thus increasing chromatographic performances [61, 76].

RP chromatography is effective for the separation of anthocyanins and anthocyanidin aglycones. This is associated with the solubility of the compounds in the mobile phase solvents. In general, an optimized gradient solvent system such as acetonitrile-water or methanol-water solutions supplemented with 0.11% acetic acid or formic acid are appropriate to elute anthocyanins. The elution order of anthocyanins through RP chromatography is normally a function of the number of hydroxyl groups and their degree of methoxylation. A general rule is that diglycosylated or more glycosylated anthocyanins are eluted earlier in the column, followed by monoglycosylated anthocyanins (in an order of galactosides, glucosides, arabinosides, xylosides, and rhamnosides) and aglycones [76]. For example, the elution order of anthocyanins in grapes follows a trend; delphinidin < cyanidin < pelargonidin < peonidin < malvidin, along with the number of glucosides and their acylation pattern following the order; diglucosylated < monoglucosylated < monoglucosylated–acetic acid < diglucosylated-coumaric acid < monoglucosylated-caffeic acid < monoglucosylated-coumaric acid [57].

Another separation mode being applied in the HPLC is hydrophilic interaction in liquid chromatography (HILIC). The development of this technique is based on polarity and hydrophilicity. HILIC uses a combination of NP's polar stationary phase, RP's aqueous-organic mobile phase, and the net surface charge of compounds (ion exchange), thus is a better separating method for small polar compounds. In HILIC, stationary phases usually consist of polymer-based, bare silica, or modified silica gels (Accucore HILIC and Acclaim HILIC 10). For the mobile phase, high organic water-miscible polar organic solvents and acetonitrile are used, giving a better polar component separation and an optimal retention time. When samples enter the column and move along the stationary phase, an interaction between water and silica will occur while acetonitrile will form layers above, giving a gradient of mobile phase, and retention caused by partitioning. More hydrophilic molecules retain more in the stationary phase. The preference of HILIC for polar metabolites has allowed its application to separate highly polar anthocyanins that cannot be separated by RP. To date, a column packed with ethylene bridged hybrid (BEH) amide (2.5 μm particle size) has been shown to provide efficient separation of diverse structures of glycosylated and acylated anthocyanins in fruits. However, HILIC may not be appropriate for the separation of isomeric anthocyanidin-hexosides and cis/trans acylated anthocyanin isomers [61, 68, 77, 78].

The polarity of anthocyanins can be reduced by acylation occurring at –OH or carbon on A, B, and C rings. Common acylation groups include malic, acetic, malonic, succinic, gallic, protocatechuic, hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, and synaptic acids. Acylated anthocyanins are usually eluted later than glycosylated ones. In addition, the anthocyanidin elution order of RP mode follows this trend; delphinidin < cyanidin < petunidin < pelargonidin < peonidin < malvidin. This is in contrast with the elution order reported from HILIC (hydrophilic interaction in liquid chromatography); malvidin, followed by peonidin, petunidin, cyanidin, and lastly delphinidin. Therefore, a combination of HILIC and RP-LC separation modes is useful for the comprehensive 2-dimensional liquid chromatographic (LC × LC) analysis of anthocyanins [76, 78, 79].

Because of the structural complexity of anthocyanin content, the polarities of different anthocyanin subgroups may yield peaks overlapped, causing unresolved chromatographic peaks. For instance, for a given aglycone base, the molecular masses for the 3,5-diglucoside and the caffeoyl glucoside are identical, resulting in a limitation for precise identification. Furthermore, anthocyanins are not easy to effectively separate from copigments such as phenolic acid and flavanols due to their similar structure as well. To alleviate these limitations associated with conventional C18 reversed-phase methods, an ion-exchange mode has been applied for anthocyanin separation. The stationary phase of ion-exchange columns consists of anion-exchange (AV-17-8, AV-17-2P, and EDE-10P) and cation-exchange (KU-2-8, Primesep B2, SCX, and 001X7) resins, which are having polar fixed groups. While the anion-exchange resins are positively charged, the cation-exchange resins have a negative charge. Depending on the net surface charge of analyte's, the cation-exchange and anionexchange stationary phases bind with negatively and positively charged compounds, respectively. In the case of the anion-exchange resins, the adsorption capacity increases, while pH of external solutions raises from acidic to neutral and alkaline values. However, anthocyanins can undergo partial degradation and lose their

biological functions in alkaline solutions. Therefore, to ensure maximum adsorption and separation, cation-exchange resins are mostly preferred for anthocyanin separation. Depending on the pH of environment, anthocyanins alternate between the cationic flavylium ion and the neutrally charged carbinol or quinoidal forms. In highly acidic conditions, anthocyanins would convert to positively charged flavylium cations because of the hydroxyl group in the 3-position, resulting in retarding on the negatively charged cation-exchange resins through the ionic interaction, and flushing other phenolic compounds that are not likely adsorbed by the resin. The mobile phase of ion-exchange mode is a solution that has counterions in general [56, 80, 81].

The separation mechanism for the ion-exchange is a mode based on the net charge contained in samples and their pH. It starts with an application of the counterion mobile phase including ion charges (Na+ and Cl− ). After loading of the sample, molecules with different net charges from the stationary phase bind to the resin while other unbound molecules are washed out by increasing the concentration of counterions or pH value of the mobile phase. For example, a cation exchange chromatography with 001X7 resin has been developed for copigments-free anthocyanins isolation both on a small and large scale from mulberry extracts. In this study, acidifed anthocyanin fractions were eluted with a mixture of methanol/NaCl solution. Cation-exchange 001X7 resin has been reported to be more advantageous with more than 95% purity compared to the macroporous adsorbent and strong cation exchange resins for the purification of anthocyanins [56, 81].

A novel separation mode combining both ion-exchange and reversed-phase separation mechanisms has been also developed, called mixed-mode ion-exchange reversed-phase chromatography. For example, for the mixed-mode separation method, Primesep B2 columns with embedded basic ion-pairing groups have been used for grapes, giving a significant improvement for chromatographic separation, purification, and detection of anthocyanidin diglucosides and acylated anthocyanins. However, the identification of anthocyanins is hard to predict by comparing with previously published data, because the ion-exchange elution mechanism significantly affects the retention orders of anthocyanins [56, 80].

The results from HPLC not only depend on column and separation mode but also on instruments and conditions used. Although instruments from different companies are comparable, the separation efficiency is always different among instruments. Once a new instrument is set up, it is better to optimize conditions and protocols for anthocyanins or other compounds. As such, results from the same instrument are highly reproducible. The detector can also provide informative characteristics of metabolites [82].

### **2.2 Detection, annotation, and identification**

Ultraviolent (UV) and visible (Vis) detectors are commonly used in HPLC. The detectors measure the absorbance intensity of UV and Vis spectra between the 190 and 900 nanometer (nm) wavelengths. There are two types of UV-vis detectors, including tunable and photodiode array (PDA), also known as diode array (DAD) detectors. The tunable UV-vis detectors can measure the maximum absorption of each analyte of a sample at one or more discrete wavelengths during the analysis. PDA detectors can measure the absorbance of each analyte at the entire wavelength range or a fixed wavelength in real time (at intervals of 1 second or less) during separation by HPLC with continuous eluate delivery [83]. The use of a detector is dependent on the metabolites analyzed. To detect and/or identify anthocyanins, detectors mostly

coupled with HPLC include UV-vis, /DAD, and PDA. These detector systems allow a few to full spectrophotometric scans on each peak as it elutes and provides a unique chromatogram for each anthocyanin that is used to compare with others for identification aims [6, 57]. In an acidic condition, the flavylium cation can maintain its red color and have absorption at a maximum between 510 and 545 nm (depending on the number of hydroxylated carbon atoms on the B-ring). The unique and maximum absorption wavelengths allow for distinguishing anthocyanins from other flavonoids for identification and quantification [6, 72]. PDA detector provides a spectral profile that can assist in detecting unknown peaks in the chromatograms, and provides characteristic spectra that give information about acylation and glycosylation patterns of anthocyanins [82, 84–86]. However, these detectors cannot distinguish anthocyanins with similar retention times and similar spectral characteristics. Also, the identification of anthocyanins with UV-vis dectors requires authentic standards, many of which are not commercially available [31, 82, 84, 87, 88].

Mass spectrometry (MS), an analytical technique, is used to measure the mass-tocharge ratio of ions. Mass spectrometry (MS) is completed on the mass spectrometer, which has MS detector. A mass spectrometer ionizes molecules and ionized molecules are sent to the mass analyzer, which is an electromagnetic field sorting and separating ions according to their mass and charge. Then, the mass detector detects and measures separated ions, and the results are displayed on a chart. HPLC coupled with a mass spectrometer has been used to effectively analyze anthocyanins (**Table 2**) and other plant secondary metabolites. Multiple accomplishments have been made to understand anthocyanins during fruit development [61, 89, 90]. In particular, HPLC-MS/MS or ultra-performance liquid chromatography (UPLC)-MS/MS is powerful to annotate unknown anthocyanins or identify known anthocyanins in fruits [61, 91]. These successes enhanced the understanding of anthocyanin biosynthesis and structures in fruits and other plant samples.

#### *2.2.1 Electrospray ionization (ESI) mass spectrometry (MS): ESI-MS*

The analysis of specific types of individual compounds by HPLC-MS requires an appropriate ionization interface between the physical coupling of LC and MS. Detection and quantification of an individual compound in MS-based measurements is determined by the level of ionization that generates intact molecular ions and/or a few fragments in MS1. Soft ionization techniques by desorption have been developed for nongaseous or thermally unstable natural compounds, for example, anthocyanins. These techniques cause a direct formation of gaseous ions by supplying power to solid or liquid sample, giving a little fragmentation and a simple mass spectrum for accurate molecular weight determination of the molecules. The most suitable ionization techniques for the chemical structure of anthocyanins are continuous-flow fast-atom bombardment (CF-FAB), desorption electrospray ionization (DESI), atmospheric pressure chemical ionization (APCI), matrix-assisted laser desorption ionization (MALDI), and electrospray ionization (ESI) [61, 82, 92–94].

In general, as an ionization mode, ESI has been mainly used, but some studies have reported the use of MALDI coupled with a time-of-flight (TOF) mass analyzer (MALDI-TOF) as an alternative. The main advantage of MALDI-TOF is the speed of analysis (a few minutes per sample). Also, MALDI-TOF mass spectrometry prevents the unwanted fragmentation of the molecules, giving a fingerprint mass spectrum for the desired molecules. Furthermore, this technique provides direct use of complex

sample mixture without prior separation. For example, anthocyanin profiling of the crude aqueous-methanolic extract of the pulp of Jamun fruit was performed by MALDI-TOF mass spectrometry operating in positive ion mode and using sodium chloride and 2, 5-dihydroxybenzoic acid as the matrix. However, MALDI-TOF is not capable of generating MS/MS data compared to ESI-MS/MS systems [95, 96]. MALDI-TOF mass spectrometer was applied for analysis of anthocyanins from blueberries, and found to be quicker and to give nontargeted quantitative estimates compared to HPLC-PDA-MS method, but unable to distinguish between anthocyanins and other flavonoids, which generate ions of the same m/z value, giving an inherent limitation of the method [95, 97].

For MALDI mode, 2,4,6-trihydroxyacetophenon (THAP) and cyano-4-hydroxycinnamic acid (CHCA) are used as a potential matrix for the flavonoids, but these matrixes tend to be fragmented and decomposed under the most instrumental conditions, resulting in a complicated mass spectra and difficulties to analyze flavonoids with a small molecular mass. However, the surfactant cetyltrimethylammonium bromide (CTAB) has been introduced as a MALDI matrix-ion suppressor and reported to yield a higher resolution and greater reproducibility than those without surfactant for qualitatively identifying anthocyanins from multiple berry extracts in a few minutes. Because of the specificity of the matrix-ion suppression, the method is called "surfactant-mediated" MALDI, and demonstrated as a complementary rapid screening technique for anthocyanins [98].

ESI is an appropriate method to generate ions in a positive or negative mode. The only prerequisite that ESI ionization needs are that the sample of interest must be soluble in appropriate solvents, and introduced to a mass spectrometer in the form of a solution. In addition, ESI is a common interface between LC/MS because of avoiding many problems seen with other LC/MS ionization interfaces. ESI-MS is a powerful versatile ionization for thermally labile, nonvolatile, and polar compounds because this soft ionization technique can produce intact ions from large and complex compounds. Past studies have shown that ESI is effective for anthocyanin ionization that produces gaseous ions from highly charged evaporating liquid droplets. To date, ESI-MS has been described and used as a powerful technology to identify the molecular structure and contents of anthocyanins in fruits. Although both positive and negative ionization modes have been generally reported for analyses of fruit anthocyanins, the positive ionization mode has been more commonly preferred by researchers [61, 86, 99–101].

Atmospheric pressure chemical ionization (APCI) is another ionization interface that has been used particularly for the broad class of flavonoids such as aglycons and glycosides. Because APCI produces a single charged product, the molecular mass spectrum of the product can be directly observed. In contrast to ESI mode, LC coupled to positive or negative ion mode APCI is more suitable for the analysis of weakly polar or nonpolar compounds due to the sample vaporization [102, 103]. For identification of anthocyanins with the same molecular mass, either sample treatment such as acid hydrolysis must be performed to release the anthocyanidin aglycons or MS fragmentation data must be obtained. The mass spectrometric data of LC-APCI-MS method provides information on the fragmentation of the anthocyanins, allowing the identification of the conjugate and the aglycone moiety. Therefore, LC-APCI-MS method allows the characterization of anthocyanins in samples without the need for sample preparation. For example, anthocyanins from red raspberries have been identified from the methanolic extract by reversed-phase HPLC through an atmospheric pressure chemical ionization probe operating in positive ion mode [30]. The combination

of both LC-APCI-MS and LC-ESI-MS methods was also reported to overcome the disadvantages of each ion source when applied individually, as well [103].

#### *2.2.2 Tandem mass spectrometry (MS/MS)*

Tandem mass spectrometry (MS/MS or MS<sup>n</sup> ) is powerful to characterize individual compounds and identify the structure of compounds by separate ionization and fragmentation steps. MS/MS allows for the formation of the fragments of each individual molecule by collision-induced dissociation (CID). Individual compounds are detected by the first quadrupole mass detector and then fragmented in the collision cell *via* a suitable gas, usually argon or nitrogen, and their fragments are detected by the second quadrupole mass analyzer [82]. In the last decade, the improvements in resolving power, selectivity, and sensitivity have accelerated the use of HPLC-MS/MS to identify known or annotate unknown anthocyanins [91].

## **2.3 HPLC coupled with ESI-quantitative time-of-flight MS/MS: HPLC/ESI-qTOF-MS/MS**

For LC-MS interfaces, there are different types of mass analyzers available, such as magnetic sectors, time-of-flight (TOF) analyzers, quadrupole mass filters, quadrupole ion traps, and ion cyclotron resonance. Mass analyzers can broadly be divided into two main groups including high- and low-resolution analyzers depending on their ability to distinguish ions with small mass-to-charge (m/z) differences. The high-resolution analyzers are useful in the structural annotation of anthocyanins. They can provide accurate m/z values of fragments, which allows to predict the location of structural fissions in MS/MS fragmentations. The resulting fragments can be useful to annotate anthocyanin structures.

Time-of-flight mass analyzers work on the principle that lighter ions travel faster than heavier ions following an initial acceleration by an electric field. All ions acquire the same kinetic energy during this initial acceleration period and are separated in the field-free flight tube, according to their different velocities. The physical property that is measured is flight time, which is directly related to the mass-to-charge ratio of the ion. Due to this mode of operation, TOF instruments offer very high mass ranges, very high acquisition rates, relatively high resolving power, and good sensitivity [6, 76, 104].

Ion trap analyzers enable the true MSn operation by allowing selective trapping and fragmentation of parent and/or daughter ions as a function of time. In general, ion trap analyzers are for qualitative analysis. The quadrupole or triple quadrupole ion traps are for quantitation purposes. For structure elucidation or annotation of an individual compound, a triple quadrupole ion trap has high specificity and sensitivity, thus it is an effective mass analyzer to filter the ion of choice. In the triple quadrupoles, the first quadrupole (Q1) and the third quadrupole (Q3) function as mass filters to isolate parent ions (precursor-ion) and to monitor selected daughter ions (fragment or production), respectively. The second quadrupole (Q2) serves as a collision cell, where parent ions are fragmented by ionization [6, 76, 105–107]. For example, anthocyanins from northern highbush blueberry extracts were identified by performing purification of solid-phase extraction, elution with acidified water and methanol, separation with the gradient mix of acidified water and acetonitrile through Zorbax SB-C18 column, and detected by reversed-phase HPLC coupled with electrospray ionization probe operating in positive ion mode, and time-of-flight tandem mass spectrometer [55].

## **3. Conclusion**

Anthocyanins are water-soluble glycosides acquiring different colors, from red to blue or violet. They are naturally the most occurring flavonoids containing sugar moiety and are synthesized abundantly in many fruits, particularly berries. Anthocyanins from fruits have high significance for food, cosmetic, and pharmaceutical industries. To obtain high-quality fruit anthocyanins, many different approaches, methods, and techniques have been created for extraction, structural characterization, and profiling. In this chapter, we introduced multiple methods for sample treatments, including extraction, chromatographic separation and purification, and for detection, annotation, and identification of fruit anthocyanins. We also discussed the use of HPLC-DAD in combination with mass spectrometry (MS) as an outstanding tool providing the chromatographic and spectral characteristics of the LC system and the resolution and separation by mass fragmentation. Especially, HPLC and MS/MS technologies were highlighted as powerful to understand anthocyanin profiles in fruits. As a result, the combination of the liquid chromatography (LC) method with electrospray ionization (ESI) and mass spectrometry (MS) or quadrupole time-of-flight (QTOF) with mass spectrometry (MS) was evaluated as the most popular and reliable methods for analyzing these compounds in fruit samples.

## **Acknowledgements**

The authors acknowledge the Foreign Specialized Project Program of the Ministry of Science and Technology of the People's Republic of China (2021) for financial support through the granted project (Grant Number: QN2021016002L).

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Seyit Yuzuak1 \*, Qing Ma2 , Yin Lu2 and De-Yu Xie3

1 Department of Molecular Biology and Genetic, Burdur Mehmet Akif Ersoy University, Burdur, Turkey

2 College of Biology and Environmental Engineering, Zhejiang Shuren University, Zhejiang, Hangzhou, China

3 Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, USA

\*Address all correspondence to: syuzuak@mehmetakif.edu.tr

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Fossen T et al. Anthocyanins of grasses. Biochemical Systematics and Ecology. 2002;**30**(9):855-864

[2] Fabroni S et al. Screening of the anthocyanin profile and in vitro pancreatic lipase inhibition by anthocyanin-containing extracts of fruits, vegetables, legumes and cereals. Journal of the Science of Food and Agriculture. 2016;**96**(14):4713-4723

[3] Passeri V, Koes R, Quattrocchio FM. New challenges for the Design of High Value Plant Products: Stabilization of anthocyanins in plant vacuoles. Front. Plant Science. 2016;**7**:153

[4] Kong JM et al. Analysis and biological activities of anthocyanins. Phytochemistry. 2003;**64**(5):923-933

[5] Harborne JB, Williams CA. Advances in flavonoid research since 1992. Phytochemistry. 2000;**55**(6):481-504

[6] Welch CR, Wu QL, Simon JE. Recent advances in anthocyanin analysis and characterization. Current Analytical Chemistry. 2008;**4**(2):75-101

[7] Delgado-Vargas F, Jimenez AR, Paredes-Lopez O. Natural pigments: Carotenoids, anthocyanins, and betalains - characteristics, biosynthesis, processing, and stability. Critical Reviews in Food Science and Nutrition. 2000;**40**(3):173-289

[8] He J, Giusti MM. Anthocyanins: Natural colorants with health-promoting properties. Annual Review of Food Science and Technology. 2010;**1**:163-187

[9] Wrolstad RE. Symposium 12: Interaction of natural colors with other ingredients - anthocyanin

pigments - bioactivity and coloring properties. Journal of Food Science. 2004;**69**(5):C419-C421

[10] Winefield C. The final steps in anthocyanin formation: A story of modification and sequestration. Advances in Botanical Research;**372002**:55-74

[11] Furuta K. Host preference and population-dynamics in an autumnal population of the maple aphid, Periphyllus-Californiensis Shinji (Homoptera, Aphididae). Journal of Applied Entomology-Zeitschrift Fur Angewandte Entomologie. 1986;**102**(1):93-100

[12] Page JE, Towers GHN. Anthocyanins protect light-sensitive thiarubrine phototoxins. Planta. 2002;**215**(3):478-484

[13] Ferreyra MLF, Rius SP, Casati P. Flavonoids: biosynthesis, biological functions, and biotechnological applications. Front. Plant Science. 2012;**3**:222

[14] Sun Y, Li H, Huang JR. Arabidopsis TT19 functions as a carrier to transport anthocyanin from the cytosol to tonoplasts. Molecular Plant. 2012;**5**(2):387-400

[15] Winkel-Shirley B, Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiology. 2001;**126**(2):485-493

[16] Xie DY, Dixon RA. Proanthocyanidin biosynthesis - still more questions than answers? Phytochemistry. 2005;**66**(18):2127-2144

[17] Poracova J et al. The importance of anthocyanins for human and animal health. Planta Medica. 2011;**77**(12):1447-1447

[18] Galvano F et al. Cyanidins: Metabolism and biological properties. Journal of Nutritional Biochemistry. 2004;**15**(1):2-11

[19] Matsumoto H et al. Stimulatory effect of cyanidin 3-glycosides on the regeneration of rhodopsin. Journal of Agricultural and Food Chemistry. 2003;**51**(12):3560-3563

[20] Folts JD. Antithrombotic potential of grape juice and red wine for preventing heart attacks. Pharmaceutical Biology. 1998;**36**:21-27

[21] Keevil JG et al. Grape juice inhibits human *ex vivo* platelet aggregation while orange and grapefruit juices do not. Journal of the American College of Cardiology. 1998;**31**(2):172a-172a

[22] Tsuda T et al. Dietary cyanidin 3-O-beta-D-glucoside-rich purple corn color prevents obesity and ameliorates hyperglycemia in mice. Journal of Nutrition. 2003;**133**(7):2125-2130

[23] Xie LH et al. Recent advances in understanding the anti-obesity activity of anthocyanins and their biosynthesis in microorganisms. Trends in Food Science & Technology. 2018;**72**:13-24

[24] Hou DX et al. Molecular mechanisms behind the chemopreventive effects of anthocyanidins. Journal of Biomedicine and Biotechnology. 2004;**5**:321-325

[25] Francisco RM et al. ABCC1, an ATP binding cassette protein from grape berry, transports Anthocyanidin 3-O-glucosides. Plant Cell. 2013;**25**(5):1840-1854

[26] Martín J et al. Anthocyanin Pigments: Importance, Sample Preparation and Extraction. Phenolic Compounds - Natural Sources, Importance and Applications, InTech, Crossref. Mar 2017. DOI: 10.5772/66892 [27] Yuzuak S, Xie DY. Anthocyanins from muscadine (Vitis rotundifolia) grape fruit. Current. Plant Biology. 2022;**30**:100243

[28] Prior RL et al. Identification of procyanidins and anthocyanins in blueberries and cranberries (Vaccinium spp.) using high-performance liquid chromatography/mass spectrometry. Journal of Agricultural and Food Chemistry. 2001;**49**(3):1270-1276

[29] Stintzing FC et al. A novel zwitterionic anthocyanin from evergreen blackberry (Rubus laciniatus Willd.). Journal of Agricultural and Food Chemistry. 2002;**50**(2):396-399

[30] Mullen W, Lean MEJ, Crozier A. Rapid characterization of anthocyanins in red raspberry fruit by high-performance liquid chromatography coupled to single quadrupole mass spectrometry. Journal of Chromatography. A. 2002;**966**(1-2):63-70

[31] Kim M-Y et al. Identification and antiradical properties of anthocyanins in fruits of Viburnum dilatatum thunb. Journal of Agricultural and Food Chemistry. 2003;**51**(21):6173-6177

[32] Määttä K, Kamal-Eldin A, Törrönen AR. High-performance liquid chromatography (HPLC) analysis of phenolic compounds in berries with diode array and electrospray ionization mass spectrometric (MS) detection: Ribes species. Journal of Agricultural and Food Chemistry. 2003;**51**(23):6736-6744

[33] Nakajima J-I et al. LC/PDA/ESI-MS profiling and radical scavenging activity of anthocyanins in various berries. Journal of Biomedicine and Biotechnology. 2004;**2004**:241-247

[34] Kammerer D et al. Polyphenol screening of pomace from red and white

grape varieties (Vitis vinifera L.) by HPLC-DAD-MS/MS. Journal of Agricultural and Food Chemistry. 2004;**52**(14):4360-4367

[35] Määttä-Riihinen KR, Kamal-Eldin A, Törrönen AR. Identification and quantification of phenolic compounds in berries of Fragaria and Rubus species (family Rosaceae). Journal of Agricultural and Food Chemistry. 2004;**52**(20):6178-6187

[36] Longo L, Vasapollo G. Determination of anthocyanins in Ruscus aculeatus L. berries. Journal of Agricultural and Food Chemistry. 2005;**53**(2):475-479

[37] Longo L, Vasapollo G, Rescio L. Identification of anthocyanins in Rhamnus alaternus L. berries. Journal of Agricultural and Food Chemistry. 2005;**53**(5):1723-1727

[38] Montefiori M et al. Pigments in the fruit of red-fleshed kiwifruit (Actinidia chinensis and Actinidia deliciosa). Journal of Agricultural and Food Chemistry. 2005;**53**(24):9526-9530

[39] Zanatta CF et al. Determination of anthocyanins from camu-camu (*Myrciaria dubia*) by HPLC-PDA, HPLC-MS, and NMR. Journal of Agricultural and Food Chemistry. 2005;**53**(24):9531-9535

[40] Wu X, Prior RL. Systematic identification and characterization of anthocyanins by HPLC-ESI-MS/ MS in common foods in the United States: Fruits and berries. Journal of Agricultural and Food Chemistry. 2005;**53**(7):2589-2599

[41] Kahle K et al. Studies on apple and blueberry fruit constituents: Do the polyphenols reach the colon after ingestion? Molecular Nutrition & Food Research. 2006;**50**(4-5):418-423

[42] Nicoué EE, Savard S, Belkacemi K. Anthocyanins in wild blueberries of Quebec: Extraction and identification. Journal of Agricultural and Food Chemistry. 2007;**55**(14):5626-5635

[43] Jin H et al. Characterization of anthocyanins in wild Lycium ruthenicum Murray by HPLC-DAD/QTOF-MS/MS. Analytical Methods. 2015;**7**:4947-4956

[44] Sharma RJ et al. Metabolite fingerprinting of Eugenia jambolana fruit pulp extracts using NMR, HPLC-PDA-MS, GC-MS, MALDI-TOF-MS and ESI-MS/ MS spectrometry. Natural Product Communications. 2015:**10**(6):969-976

[45] Ramirez JE et al. Anthocyanins and antioxidant capacities of six Chilean berries by HPLC-HR-ESI-ToF-MS. Food Chemistry. 2015;**176**:106-114

[46] Legua P et al. Polyphenolic compounds, anthocyanins and antioxidant activity of nineteen pomegranate fruits: A rich source of bioactive compounds. Journal of Functional Foods. 2016;**23**:628-636

[47] Oszmiański J, Lachowicz S. Effect of the production of dried fruits and juice from chokeberry (Aronia melanocarpa L.) on the content and Antioxidative activity of bioactive compounds. Molecules. 2016;**21**(8):1098

[48] Scorrano S et al. Anthocyanins profile by Q-TOF LC/MS in Myrtus communis berries from Salento area. Food Analytical Methods. 2017;**10**:2404-2411

[49] Li F et al. The novel contributors of anti-diabetic potential in mulberry polyphenols revealed by UHPLC-HR-ESI-TOF-MS/MS. Food Research International. 2017;**100**(Pt 1):873-884

[50] Sang J et al. Extraction and characterization of anthocyanins from Nitraria tangutorun bobr. Dry fruit and evaluation of their stability in aqueous solution and taurine-contained beverage. Journal of Food Measurement and Characterization. 2017;**12**:937-948

[51] Pastrana-Bonilla E, Akoh CC, Cerquera NE. Identification and quantification of anthocyanins in muscadine grapes by HPLC and HPLC-MS. ARPN Journal of Engineering and Applied Sciences. 2017;**12**(2):626-631

[52] Wu Q et al. Analysis of polyphenols composition and antioxidant activity assessment of Chinese dwarf cherry (Cerasus humilis (Bge.) Sok.). Natural Product Communications. 2019;**14**(6)

[53] Li CX et al. Phytochemical profiles, antioxidant, and antiproliferative activities of red-fleshed apple as affected by in vitro digestion. Journal of Food Science. 2020;**85**(9):2952-2959

[54] Stanoev JP, Stefova M, Bogdanov JB. Systematic HPLC/DAD/MSn study on the extraction efficiency of polyphenols from black goji: Citric and ascorbic acid as alternative acid components in the extraction mixture. Journal of Berry Research. 2021;**11**(269):1-20

[55] Pico J et al. Determination of free and bound phenolics in northern highbush blueberries by a validated HPLC/QTOF methodology. Journal of Food Composition and Analysis. 2022;**108**:104412

[56] Liao Z et al. Recovery of value-added anthocyanins from mulberry by a cation exchange chromatography. Current Research in Food Science. 2022;**5**:1445-1451

[57] Mazza G, Cacace J, Kay C. Methods of analysis for anthocyanins in plants and biological fluids. Journal of AOAC International. 2004;**87**:129-145

[58] Horbowicz M et al. Anthocyanins of fruits and vegetables-their occurrence,

analysis and role in human nutrition. Vegetable Crops Research Bulletin. 2008;**68**:5-22

[59] Avula B et al. Advances in the chemistry, analysis and adulteration of anthocyanin rich-berries and fruits: 2000-2022. Molecules. 2023;**28**(2):560. DOI: 10.3390/molecules28020560

[60] Lopez E et al. Effect of light on stability of anthocyanins in Ethanolic extracts of Rubus fruticosus. Food and Nutrition Sciences. 2014;**05**:488-494

[61] da Silva Oliveira JP et al. Metabolomic studies of anthocyanins in fruits by means of a liquid chromatography coupled to mass spectrometry workflow. Current. Plant Biology. 2022;**32**:100260

[62] Tan J et al. Extraction and purification of anthocyanins: A review. Journal of Agriculture and Food Research. 2022;**8**:100306

[63] Nunes AN et al. Alternative extraction and downstream purification processes for anthocyanins. Molecules. 2022;**27**(2):368

[64] Gonzalez-Paramas AM et al. Analysis of flavonoids in foods and biological samples. Mini-Reviews in Medicinal Chemistry. 2011;**11**(14):1239-1255

[65] de Rijke E et al. Analytical separation and detection methods for flavonoids. Journal of Chromatography A. 2006;**1112**(1-2):31-63

[66] Flamini R. Recent applications of mass spectrometry in the study of grape and wine polyphenols. International Scholarly Research Notices. 2013;**2013**:1-45

[67] Gardana C et al. Bilberry adulteration: Identification and

chemical profiling of anthocyanins by different analytical methods. Journal of Agricultural and Food Chemistry. 2014;**62**(45):10998-11004

[68] Fiorelia NE et al. Types of highperformance liquid chromatography (HPLC) columns: A review. FoodTech: Jurnal Teknologi Pangan. 2022;**5**(1):1-16

[69] Chen Y et al. Adsorption properties of macroporous adsorbent resins for separation of anthocyanins from mulberry. Food Chemistry. 2016;**194**:712-722

[70] Berthod A. Countercurrent chromatography: The support-free liquid stationary phase. Elsevier Science; 2002;**38**:1-397

[71] Petersson EV et al. Analysis of anthocyanins in red onion using capillary electrophoresis-time of flightmass spectrometry. Electrophoresis. 2008;**29**(12):2723-2730

[72] da Costa CT, Horton D, Margolis SA. Analysis of anthocyanins in foods by liquid chromatography, liquid chromatography-mass spectrometry and capillary electrophoresis. Journal of Chromatography A. 2000;**881**(1-2):403-410

[73] Calvo D et al. Migration order of wine anthocyanins in capillary zone electrophoresis. Analytica Chimica Acta. 2004;**524**(1-2):207-213

[74] Saenz-Lopez R, Fernandez-Zurbano P, Tena MT. Development and validation of a capillary zone electrophoresis method for the quantitative determination of anthocyanins in wine. Journal of Chromatography A. 2003;**990**(1-2):247-258

[75] Bednář P et al. Utilization of capillary electrophoresis/mass spectrometry

(CE/MSn) for the study of anthocyanin dyes. Journal of Separation Science. 2005;**28**(12):1291-1299

[76] de Villiers A, Venter P, Pasch H. Recent advances and trends in the liquidchromatography-mass spectrometry analysis of flavonoids. Journal of Chromatography A. 2016;**1430**:16-78

[77] Buszewski B, Noga S. Hydrophilic interaction liquid chromatography (HILIC)—A powerful separation technique. Analytical and Bioanalytical Chemistry. 2011;**402**:231-247

[78] Willemse CM, Stander MA, de Villiers A. Hydrophilic interaction chromatographic analysis of anthocyanins. Journal of Chromatography. A. 2013;**1319**:127-140

[79] Markham KA, Kohen A. Analytical procedures for the preparation, isolation, analysis and preservation of reduced nicotinamides. Current Analytical Chemistry. 2006;**2**(4):379-388

[80] McCallum J et al. Improved high performance liquid chromatographic separation of anthocyanin compounds from grapes using a novel mixedmode ion-exchange reversed-phase column. Journal of Chromatography. A. 2007;**1148**:38-45

[81] Pismenskaya N et al. Adsorption of anthocyanins by cation and anion exchange resins with aromatic and aliphatic polymer matrices. International Journal of Molecular Sciences. 2020;**21**:1-26

[82] Giusti MM et al. Electrospray and tandem mass spectroscopy as tools for anthocyanin characterization. Journal of Agricultural and Food Chemistry. 1999;**47**(11):4657-4664

[83] Swartz ME. HPLC detectors: A brief review. Journal of Liquid

Chromatography & Related Technologies. 2010;**33**:1130-1150

[84] Hong V, Wrolstad RE. Use of Hplc separation photodiode Array detection for characterization of anthocyanins. Journal of Agricultural and Food Chemistry. 1990;**38**(3):708-715

[85] Bakker J, Timberlake CF. The distribution of anthocyanins in grape skin extracts of port wine cultivars as determined by high-performance liquid-chromatography. Journal of the Science of Food and Agriculture. 1985;**36**(12):1315-1324

[86] Garcia-Beneytez E, Cabello F, Revilla E. Analysis of grape and wine anthocyanins by HPLC-MS. Journal of Agricultural and Food Chemistry. 2003;**51**(19):5622-5629

[87] Gao L, Mazza G. Rapid method for complete chemical characterization of simple and Acylated anthocyanins by high-performance liquidchromatography and capillary gas-liquidchromatography. Journal of Agricultural and Food Chemistry. 1994;**42**(1):118-125

[88] Strack D, Akavia N, Reznik H. Highperformance liquid-chromatographic identification of anthocyanins. Zeitschrift Fur Naturforschung C-a Journal of Biosciences. 1980;**35**(7-8):533-538

[89] Baldi A et al. Hplc/Ms application to anthocyanins of Vitis-Vinifera L. Journal of Agricultural and Food Chemistry. 1995;**43**(8):2104-2109

[90] Wang J, Sporns P. Analysis of anthocyanins in red wine and fruit juice using MALDI-MS. Journal of Agricultural and Food Chemistry. 1999;**47**(5):2009-2015

[91] Saha S et al. Anthocyanin profiling using UV-Vis spectroscopy and liquid

chromatography mass spectrometry. Journal of AOAC International. 2019;**103**(1):23-39

[92] Sagesser M, Deinzer M. HPLC-ion spray tandem mass spectrometry of flavonol glycosides in hops. Journal of the American Society of Brewing Chemists. 1996;**54**(3):129-134

[93] Aramendia MA et al. Determination of Isoflavones using capillary electrophoresis in combination with electrospray mass-spectrometry. Journal of Chromatography A. 1995;**707**(2):327-333

[94] Favretto D, Flamini R. Application of electrospray ionization mass spectrometry to the study of grape anthocyanins. American Journal of Enology and Viticulture. 2000;**51**(1):55-64

[95] Wang J, Kalt W, Sporns P. Comparison between HPLC and MALDI-TOF MS analysis of anthocyanins in highbush blueberries. Journal of Agricultural and Food Chemistry. 2000;**48**:3330-3335

[96] Castañeda A et al. Identification of anthocyanins in red grape, plum and capulin by MALDI-ToF MS. Journal of the Mexican Chemical Society. 2012;**2012**:378-383

[97] Mullen W et al. Use of accurate mass full scan mass spectrometry for the analysis of anthocyanins in berries and berry-fed tissues. Journal of Agricultural and Food Chemistry. 2010;**58**(7):3910-3915

[98] Grant D, Helleur R. Rapid screening of anthocyanins in berry samples by surfactant-mediated matrix-assisted laser desorption/ionization timeof-flight mass spectrometry. Rapid communications in mass spectrometry: RCM. 2008;**22**:156-164

[99] Fenn JB et al. Electrospray ionization for mass-spectrometry

of large biomolecules. Science. 1989;**246**(4926):64-71

[100] Hutton T, Major HJ. Characterizing biomolecules by electrospray ionization mass spectrometry coupled to liquid chromatography and capillary electrophoresis. Biochemical Society Transactions. 1995;**23**(4):924-927

[101] Dugo P et al. Identification of anthocyanins in berries by narrowbore high-performance liquid chromatography with electrospray ionization detection. Journal of Agricultural and Food Chemistry. 2001;**49**(8):3987-3992

[102] de Rijke E et al. Liquid chromatography with atmospheric pressure chemical ionization and electrospray ionization mass spectrometry of flavonoids with triplequadrupole and ion-trap instruments. Journal of Chromatography A. 2003;**984**(1):45-58

[103] Commisso M et al. Performance comparison of electrospray ionization and atmospheric pressure chemical ionization in untargeted and targeted liquid chromatography/mass spectrometry based metabolomics analysis of grapeberry metabolites. Rapid communications in mass spectrometry: RCM. 2017;**31**(3):292-300

[104] Ignat I, Volf I, Popa VI. A critical review of methods for characterisation of polyphenolic compounds in fruits and vegetables. Food Chemistry. 2011;**126**(4):1821-1835

[105] Flamini R. Mass spectrometry in grape and wine chemistry. Part I: Polyphenols. Mass Spectrometry Reviews. 2003;**22**(4):218-250

[106] Huang ZL et al. Identification of anthocyanins in muscadine grapes with HPLC-ESI-MS. Lwt-Food Science and Technology. 2009;**42**(4):819-824

[107] Ganzera M, Sturm S. Recent advances on HPLC/MS in medicinal plant analysis-an update covering 2011- 2016. Journal of Pharmaceutical and Biomedical Analysis. 2018;**147**:211-233

## **Chapter 8**

## Current Trends in HPLC for Quality Control of Spices

*Prafulla Kumar Sahu, Sukumar Purohit, Swarnajeet Tripathy, Durga Prasad Mishra and Biswajeet Acharya*

## **Abstract**

India, the land of spices and condiments, is endowed with a plethora of herbs, spices, and unusual plants. Spices have been used as flavoring and coloring agents in Indian society since time immemorial. Spices have also been shown to have antioxidant, antibacterial, anticancer, and anti-inflammatory properties. Assessing spices' taste, nutritional, and bioactive qualities during postharvest processing is critical for quality control and preventing adulteration. Various illegal colors are frequently used to adulterate spices for fraudulent trading operations. For instance, Sudan dyes are widely substituted with hot chili, red pepper, or tomato products; metanil yellow in turmeric; tartrazine, amaranth, and sunset yellow FCF in ginger and chili powder; and magenta III and rhodamine B in saffron. These adulterants degrade the flavoring, fragrance, cosmetics, medicinal, and preservative value of spices, their authentication is critical in quality control. Apart from these adulterants, various aflatoxins secreted after fungal contamination also cause quality degradation of spices. According to the literature evaluation, HPLC is a rapid and adaptable technique for efficiently identifying these compounds in spices. The proposed chapter summarizes application of HPLC for detection, quantification, and quality assessment of various spices. Some of the recently published work on the said topic from various search engines (Google scholar, Scopus, science direct, etc.) is mentioned in the chapter.

**Keywords:** analytical HPLC, spice and condiments, adulteration, aflatoxin, quality control

## **1. Introduction**

Spice consumption has been a long-standing habit due to the great value of its color, flavor, pungency, and aroma properties. Spices are rich in lipids, proteins, minerals, and vitamins, in addition to their organoleptic qualities [1]. In addition, they are effective against microorganisms, oxidative stress, inflammation, diabetes, immunosuppressant, and mutation [2–5]. They are excellent for food preservation. As a result of its many advantageous effects, including the ability to purify blood and condition the skin, these spices have been mentioned in the ancient system of medicine such as Ayurveda, Unani, and Homeopathy. Besides the health benefits, global food habits include utilization of high spice levels, which makes the food more palatable and create an eye-catching garnish.

Global herb and spice market is valued at four billion USD and is believed to further grow up to 6–6.5 billion USD in next decade [6]. The increasing demand for spices is because of their flavor, aroma, taste, and color. Many of these spices and herbs include turmeric, ginger, garlic, chili, pepper, etc. used in every household worldwide. Apart from these whole spices, their powders (chili powder, turmeric powder, ginger powder, pepper powder, etc.) are also used for seasoning. Unfortunately, these powders are often contaminated with chalk powder, dyes, and many other chemicals to enhance the bulk and colored texture of the spice [7]. This food fraud practice can be detrimental to health condition of consumers. For instance, various reports suggest that nut protein mixed with cumin can cause anaphylaxis [8]. Yellow chalk powder is often mixed with turmeric powder, which may cause severe nausea, vomiting, and loss of appetite to the consumers [9]. Mixing olive leaves with oregano is another example of indirect type of food fraud, which can cost toxicity and mutagenicity to the customer [10].

Intentional addition or substitution of a substance with a structurally similar substance to enhance its quantity and decrease its production and processing cost for economic gain is called economically motivated adulteration [11]. This practice encourages fraudsters to mix various harmful illegal dyes with spices to make them more appealing to the customer. For instance, Sudan one and Sudan four are mixed with turmeric, chili, curry, pepper, etc. [12]. Rhodamine B is mixed with paprika, sumac, chili, etc. [13]. Para red is an illegal dye often mixed with cayenne pepper, chili, and paprika to enhance the color of the spice [13]. These above-mentioned banned dyes are genotoxic and carcinogenic in nature. Therefore, identifying these adulterants from herbs and spices has become an important step for their quality control.

Owing to the huge economic potential of the spice market, herbs and spices are heavily adulterated. Very often they are adulterated with low-quality and substandard products. This practice further enhances the possibilities of contamination. Improper storage and handling have a significant role in quality degradation of spice, which includes microbial growth [14]. Besides bacterial contamination, spices are more susceptible to fungal contamination. Mycotoxins are toxic materials released to the fungus-contaminated spices and herbs. Aflatoxins are a type of mycotoxins that are produced by certain fungi causing severe contamination in many agricultural crops including spices [15]. Aflatoxin contamination in spices occurs during their harvesting, drying, and storage [16]. These toxins must be identified and approaches should be considered to minimize such contamination for the quality enhancement of spices.

To verify the authenticity of herbs and spices due to the surging trends in spice contamination, many analytical techniques are taken into consideration. Most relevant techniques include spectroscopy and chromatography [17]. Visual inspection and microscopic methods are also considered, but they require huge manpower, trained professionals, and more analysis time. Many DNA-based methods, such as random amplified polymorphic DNA (RAPD), are also used for the detection of adulteration in spices. However, the authenticity of the result reproducibility is a matter of discussion [18]. High-performance liquid chromatography (HPLC) is a rapid, reliable, accurate, sensitive, and highly specific technique considered the gold standard for the detection of adulterants [19]. This chapter gives a complete overview of the ancient use of spices in India and their trading and fraudulent activities followed over the years to adulterate spices. Further, the report summarizes various recent findings based on identification of different adulterants including synthetic dyes, herbicides and pesticides, starch and fillers, etc. through HPLC methods that are mentioned. In addition to this, role of HPLC in quality assessment of drugs bearing essential oil and mycotoxins has been discussed elaborately. A comparison study between HPLC

analysis and other spectroscopic and chromatographic techniques is also discussed in this manuscript. This chapter will be the first documentation of its kind to cover the role of HPLC in quality boosting of different herbs and spices.

### **1.1 History, trivia, and background about spices of India**

Spices have various purposes, including cooking, aroma, personal care, medicine, and long-term food preservation. Most countries that import goods find spices important. There are many aromatic spices and flavors in Indian history. Spices have been pivotal in many realms, from mythology and the Middle Ages to modern politics and economics. Spices were used as exchange and gifts for marriages, war treaties, political tradings, etc. [20–22].

Spices have long been a globally traded commodity. Spices were one of the most important components of trade from the Indian subcontinent to the Roman Empire throughout the first to third centuries CE [23]. The Middle East initially used spices circa 5000 BC, which further moved through Egyptians exchanged between 3000 to 200 BC. They were cultivating garlic to use as a fragrance in the process of mummification. The Romans dominated the trade of spices including nutmeg, cinnamon, pepper, cloves, and ginger from 200 BC until 1200 AD. According to Indian history, in 1497, Portuguese explorer Vasco de Gama lured princes of India with spices such as ginger, pepper, and cinnamon. In 1663 AD, the Dutch people acquired exclusive permission for pepper trading with India and controlled Asian spices by the 17th century. In the late 17th century, the French became a superpower and stole cloves, cinnamon, and nutmeg from the Dutch. In 1780–1799, British took seized spice trading centers. By the year 1672 America joined the spice race through its geopolitical and economic eminence.

India is the world's greatest spice grower, accounting for 75% of worldwide spice production. The overseas trading of spices by India in 2018–2019 was 0.85 million tonnes (\$2.25 billion). The spice market's growth in recent years is a testament to the popularity of Indian spices abroad. Flavor, color, aroma, preservation, and therapeutic characteristics give spices considerable economic worth. Important seasonings, tree spices, seed spices, and miscellaneous spices all make up the spice family [24]. It is estimated that between 6.8 and 12.5 percent of spices are lost after harvest [25]. Spice businesses also experience pollution and adulteration. Adulteration causes health dangers, dangerous products, and poor quality.

The spice sector is constantly challenged by adulteration, which can take the form of introducing low-grade, harmful, or low-quality commodities as well as extraneous chemicals. Adulteration not only reduces the overall quality and authenticity of a product but it can also have a negative impact on consumer health, including the development of long-term conditions such as paralysis, cancer, and a compromised immune system because of the presence of poisonous products inside the human body [26]. Spice adulteration business stands at around \$30–\$40 billion per year as per the estimate given by Global Food Safety Forum. Adulteration is done to profit from cheaper raw materials and meets overpopulation's food need. Lack of knowledge about adulterants in spices lowers their marketability and safety.

## **1.2 Importance of spices for the Indian economy**

Spices are popular everywhere. India is the largest exporter and producer of herbs and spices worldwide. Spices are a highly traded commodity. Spice imports from developing countries like India dominate the worldwide market [27].

In India, the best quality spices are cultivated in the western ghat, Coorg, and Malabar region of southern states. Apart from that, other Indian states such as Madhya Pradesh, Kashmir, and Uttar Pradesh depend on their geo-climatic circumstances. In Uttar Pradesh, cumin, coriander, fennel, and black seed are commercially grown [28]. Spices are an ideal crop for small-scale farming in India. This firm may provide the family with more career options and emergency cash. Spices are a good business for women since they may be cultivated in home gardens, and they contribute to the local economy.

India's spices are the world's best. Black pepper has driven trade policy since time immemorial [29]. Spice trade fortunes affect agriculture exports. Spices accounted for 8.4% of all agricultural exports in 2017–2018, and their total worth was \$4.69 billion in 2016 [30].

#### **1.3 Scope of adulteration in the spice quality**

Adulteration uses forbidden substances such as sand, pebbles, woodchip, colors, oil, floral stalks, and so on to improve the physical appearance of the spices. Lead and arsenic contamination can be developed during food preparation and handling [26]. Ground spices are more likely adulterated because of their shape and texture, which can be easily admixed during grinding or milling. Spice adulteration is done during processing of the spices or transpiration for the benefit of the trader. Common adulteration practices for different spices are presented in **Figure 1**.

Authenticity of food products, herbs, and spices has become a significant step of their quality control. Today, the consumers rely more on 100% safer and natural products. Therefore, identification of adulterants mixed with spices has become very important for checking the authenticity of the spices. Pure and authentic product assures the high quality of the product to the purchaser, dealer, and exporter. To authenticate the originality on the scale of quality and standard index, the analytical

#### **Figure 1.**

*Example of some household spices with their adulterants. Red brick dust (a) is mixed with red chili powder (b), metanil yellow (c) is mixed with turmeric powder (d), papaya seeds (e) is mixed with black pepper (f), grass seed (g) is used as adulterant for cumin seed (h), cassia (i) is used as a substitution for cinnamon (j), corn threads (k) are mixed with saffron (l), de-oiled cardamom (m) is mixed with good quality cardamom for bulk (n), agremon seed (0) is often mixed with mustard seed (p), and soap stone (q) is mixed with asafoetida (r).*

approaches are classified into three major basic strategies: physical, chemical, and instrumental analysis. Though physical methods are simple but claim limited applications due to time consumption. Chemical and instrumental techniques have been widely used. Although these techniques involve complex instrumentation and data processing, their routine applications cannot be restricted owing to their powerful benefits of rapidity, sensitivity, accuracy, and cost-effectiveness.

## **1.4 Role of HPLC in detecting adulterants in spices**

Chromatography-based methods successfully separate a mixture of components. It has been used for identifying secondary plant metabolites for ages [31, 32]. However, it is a popular approach for detecting and identifying food adulterants also. Based on its principle, HPLC separates chemical entities from a sample mixture based on their affinity for the column adsorbent or mobile phase, causing constituents to flow at various speeds and separate. It was once called as high-pressure liquid chromatography because it used high-pressure pumps [33]. There are many toxic materials and banned dyes; adulterant compositions have been identified through HPLC analysis. For instance, Bhooma et al. [34] reported presence of magenta III and rhodamine B in pink saffron. They collected 104 commercial saffron samples from 16 different countries and confirmed presence of magenta III and rhodamine B in 20 samples for the first time. Both the toxic dyes were identified by HPLC and ESI MS analysis. Further, Sahu et al. [35] used reversed-phase HPLC for simultaneous determination and separation of curcumin, metanil yellow, demethoxy curcumin, and bismethoxy curcumin. Metanil yellow is a carcinogenic and genotoxic banned dye often used as an adulterant for turmeric powder. The authors reported that the RP-HPLC was very accurate and precise to detect turmeric adulterants with a detection limit of 0.37–2.48 mg/ml concentration. Another report by Vickers et al. suggested that a minimum limit of quantification (0.1 mg/kg) was achieved using HPLC for the detection of genotoxic Sudan dyes (I, II, III, and IV) through HPLC analysis from different spices from Egypt [36]. Adulteration in spices alone cannot deteriorate their quality aspects. Improper harvesting, processing, drying, and storage lead to poor quality of spices [37]. Inadequate measures followed for drying spices promote higher moisture content and further encourage microbial contamination. Spices are prone to get contaminated with toxic fungi and molds. Fungal species such as *Aspergillus* and *Penicillium* release toxic secondary metabolites such as mycotoxin. Aflatoxins are a group of mycotoxins that are potentially dangerous when released into spices and further degrade the food material. However, recent chromatographic techniques such as HPLC have become a go-to tool for detection of such toxic material in spices. Mixing of starch and other harmful powder in powdered drugs has also become a concern these days. However, some recent reports suggest HPLC plays a vital role in detecting such type of misconduct. This chapter summarizes some of the recent literature where HPLC has been employed for the detection of adulterants such as dyes, mycotoxins, pesticides, and powder fillers in spices.

## **2. Identification of synthetic dyes using HPLC**

Synthetic dyes are commonly used to enhance the color of spices such as chili powder, paprika, and turmeric. While some of these dyes are approved for use in food, others may be unsafe and pose a risk to human health. High-performance liquid



*wavelength detection, HR-Q-TOF: high-resolution quadrupole time-of-flight mass spectrometry, GPC: gel permeation chromatography, FLD: fluorescence detection.*

## **Table 1.**

*List of adulterant dyes detected from spices by using HPLC analysis.*

## *Current Trends in HPLC for Quality Control of Spices DOI: http://dx.doi.org/10.5772/intechopen.110897*

chromatography (HPLC) is a highly accurate and reliable method for identifying the presence of synthetic dyes in spices. To identify these dyes in spices using HPLC, a sample of the spice is first extracted with a suitable solvent, preferably methanol or ACN. The extract is then filtered and injected into the HPLC column. The column is typically equipped with a UV-VIS detector that can detect the characteristic absorption spectra of the synthetic dyes. Different synthetic dyes have distinct retention times and spectral properties, making them easy to identify using HPLC. For example, Sudan I, II, III, and IV are commonly used to enhance the color of chili powder, and their presence can be detected using HPLC with UV-VIS detection. These dyes have characteristic absorption spectra with peaks at 480 nm, 503 nm, 528 nm, and 440 nm, respectively. By comparing the retention times and spectral properties of the synthetic dyes in the sample to those of known standards, it is possible to identify and quantify the levels of synthetic dyes in the spice sample.

A study by Yun et al. developed an HPLC method for the simultaneous identification and quantification of six synthetic dyes in spice samples, including chili powder and paprika. The authors found that some of the samples were contaminated with Sudan I and II [38]. Another study by Duan et al. used HPLC-MS/MS to analyze 15 synthetic dyes in chili powder samples from China. They discovered seven of the samples were contaminated with illegal dyes such as Sudan I and Rhodamine B [39]. Adulteration of Sudan I and II and Rhodamine B was further confirmed by Maria et al. in chili powder and chili powder using an HPLC-MS/MS method [40].

The immense usability and popularity of HPLC method for detection of adulterants in spices are because of its sensitivity, accuracy, and low detection limits. For instance, Zhang et al. developed an HPLC method for the determination of 14 synthetic dyes in 16 spice and seasoning samples. The method was found to be reliable and sensitive, with detection limits ranging from 0.005 to 0.05 mg/kg. Similarly, Wang et al. used HPLC-MS/MS to determine the synthetic dyes and succeed in determining eight synthetic dyes in 30 spice samples [41]. The limit of detection was ranging from 0.005 to 0.05 mg/kg. Zhang et al. used HPLC-PDA (photodiode array) detector and successfully identified 16 synthetic dyes from 30 spices with a detection limit ranging from 0.003 to 0.02 mg/kg [42]. Hu et al. developed an HPLC-MS/ MS method for the determination of seven synthetic dyes in various spices [43]. The method was found to be sensitive and accurate, with detection limits ranging from 0.003 to 0.05 mg/kg. Apart from this literature, **Table 1** summarizes various other dyes used as adulterants for various spices and their detection method. The extraction and detection methods of synthetic dyes are also listed in **Table 1**.

From the above illustration, it can be understood that HPLC is a highly effective method for identifying synthetic dyes in spices. The UV-VIS detection system or mass spectroscopy coupled with HPLC can further assure the accuracy of the experiment. Comparing the retention time of test sample with the standard sample in UV-VIS method or standard library searching for MS analysis is more helpful for identifying and quantifying synthetic dyes in the spices. This can further help to ensure that food products are safe and free from harmful additives.

## **3. Quantification of pesticides and herbicides in spices by HPLC**

Pesticides and herbicides are often used in the cultivation of spices to protect the crops from pests and weeds [54]. However, these chemicals can pose a risk to human health if consumed in excessive amounts. HPLC is a powerful analytical technique

#### *Current Trends in HPLC for Quality Control of Spices DOI: http://dx.doi.org/10.5772/intechopen.110897*

that can be used to quantify the levels of pesticides and herbicides in spices [22, 55]. To quantify pesticides and herbicides in spices by HPLC, a sample of the spice is first extracted, filtered, and then injected into the HPLC column. The column is typically equipped with a UV-VIS detector, and the elute is monitored at a specific wavelength that corresponds to the absorption spectra of the target pesticides or herbicides. Different pesticides and herbicides have different retention times and spectral properties, making them easy to identify and quantify using HPLC. The retention time is influenced by the chemical and physical properties of the compound, such as its molecular weight, polarity, and solubility. Once the retention time of the target pesticide or herbicide is determined, a calibration curve is constructed by analyzing a series of standard solutions containing known amounts of the target compound. The calibration curve allows for the quantification of the levels of the target compound in the sample, based on the peak area or height of the eluate corresponding to the target compound. In some cases, it may be necessary to use a mass spectrometer in combination with HPLC to identify and quantify trace levels of pesticides and herbicides in spices. Mass spectrometry can also provide additional information on the molecular mass of the target compound, allowing for more accurate identification and quantification. In this section, extensive literature review of published articles in the last 5 years focused on the quantification of pesticides and herbicides in spices by HPLC is furnished.

A study by Tesemma et al. used HPLC to analyze the presence of different pesticides in lemon, black pepper, and fenugreek seed samples and the most commonly detected pesticide was chlorpyrifos present in the level of 1.6 to 1.9 μg/kg [56]. In a different study by Jiao et al., HPLC was used to quantify the levels of pesticides in black cumin samples [57]. The authors found that the most commonly detected pesticides were pyrethroids and that the levels of pesticides were higher in samples that had been stored for longer periods of time. Wei et al. also used QuEChERS coupled with HPLC-MS/MS and quantified clothianidin and acetamiprid in black pepper samples [58]. Ultra-HPLC-quadrupole-orbitrap mass spectrometry was used by Arnab et al. for the detection of pesticide content in various spices such as chili, coriander, black pepper, cardamom, turmeric, etc. and the detection limit was 2 to 5 ng/ml [59]. Xuan et al. developed aLC-Q-TOF/M method for the simultaneous detection of various pesticide residues in chili and Sichuan peppesamplesand the LOQ of ≤5 μg kg was detected [60]. They reported that some of the samples were contaminated with imidacloprid. An illegal pesticide (chlorpyrifos) was detected by Yep et al. using HPLC method to quantify the pesticide in black pepper samples [61]. The method was found to be accurate and reliable.

From the above discussion, it can be noted that the presence of pesticide residues in spices is a significant problem and HPLC is an effective method for the determination of pesticide residues in spices. This study also demonstrates the continuous and effective application of HPLC for the quantification of pesticides and herbicides in spices.

## **4. Detection of mycotoxins in spices by HPLC**

Mycotoxins are toxic secondary metabolites produced by certain molds that can contaminate various food products, including spices [62, 63]. The presence of mycotoxins in spices can pose a significant risk to human health, as some mycotoxins are carcinogenic or can cause other adverse health effects [64]. HPLC has become an analytical tool for the detection and quantification of mycotoxins in spices [65].



*Current Trends in HPLC for Quality Control of Spices DOI: http://dx.doi.org/10.5772/intechopen.110897*


## **Table 2.**

*Recent reporting on aflatoxin detection in spices through HPLC method.*

#### *Current Trends in HPLC for Quality Control of Spices DOI: http://dx.doi.org/10.5772/intechopen.110897*

This section summarizes some of the recent findings where HPLC is used for the detection of mycotoxins in various spices.

A study reported by Iqbal et al. showed simultaneous detection of aflatoxins and ochratoxin A in spices by HPLC method. The developed HPLC method was found to be reliable and sensitive and was able to detect mycotoxins at concentrations as low as 0.5 μg/kg [66]. Da Silva et al. also used HPLC to analyze the presence of Ochratoxin A in black pepper (29 powder and 31 grains) and found the range of this mycotoxin between 0.05 and 13.15 μg/kg [67]. Zareshahrabadi et al. reported that out of 80 spice samples, 40 were contaminated with aflatoxins and 48 were ochratoxin A (some were common spices where both the mycotoxins were identified) through HPLC [68]. Ainiza et al. checked aflatoxins in fennel, coriander, turmeric, cumin, and chili and found and reported aflatoxins B1, B2, G1, and G2 through HPLC. The authors further stated that the optimized HPLC method can be utilized for the detection of various other mycotoxins from adulterated spices [69]. Aflatoxins (AFs) and ochratoxin A (OTA) contamination in *C. annuum* was reported by Costa et al. [70]. In a different report from Ali et al., aflatoxins and ochratoxin A detection were carried out by HPLC coupled with fluorescent detector on dried chili, black and white pepper, coriander, turmeric, fennel, cumin, poppy seed, etc. The limit of detection for aflatoxin was found to be 0.01 ng/g and 0.10 ng/g for ochratoxin A [71]. Palma et al. optimized and validated the quantification of various aflatoxin in Marken using HPLC-FLD (fluorescence detection). The optimized method furnished LOD with a minimum range of 0.6–20 ng/g [72]. Aflatoxin adulteration in coriander seed was checked by Ouakhssase et al. using UPLC MS/MS. The limit of quantification was found to be between 0.12 to 0.5 μg/kg [73]. This method validated aflatoxin contamination in coriander seeds successfully. Koutsias et al. confirmed presence of aflatoxins B1 in various spices of Greece [74]. HPLC with fluorescence detector was used for the detection of aflatoxins and the quantification limits were ascertained at 0.1ng/g and 0.45ng/g.

Mycotoxins are the unwanted toxic toxin released by the pathogens formed in spices, which are poorly stored or processed. Their detection is very important for the quality control of the spices. Therefore, employment of HPLC for the detection of mycotoxins in spices is considered vastly. The aforementioned literature review suggested that HPLC coupled with UV-VIS detector or mass spectroscopy has successfully identified many mycotoxins in wised varieties of spice samples. Some of the recent reporting on different aflatoxin-contaminated spices and their detection through HPLC is presented in **Table 2**.

## **5. HPLC analysis of essential oils in spices**

Essential oils are natural products that are extracted from various parts of plants, including leaves, stems, flowers, and fruits [87, 88]. They are widely used in the food and beverage, cosmetics, and pharmaceutical industries for their flavor, aroma, and medicinal properties [89]. However, substandard drugs are used for adulterating the genuine crude drugs. For instance, de fat clove, funnel, and coriander are mixed with their genuine counterparts. HPLC finds its way to the analysis of essential oil composition in substandard spices [90]. There are reports suggesting coupling of HPLC with UV-VIS detector or MS offers more accuracy for the detection of essential oil [91].

Bendif et al. used HPLC to analyze the essential oil content and chemical composition of thyme. The study found that the major component of the essential oils in thyme was monoterpenes and sesquiterpenes and oxygenated monoterpenes [92].

The authors suggested that HPLC can be an effective tool for the identification and quantification of the major components of essential oils in spices. Chen et al. also used HPLC to analyze the essential oil content and composition of black pepper and white pepper to report higher concentrations of monoterpenes and sesquiterpenes [93]. A study by Ling et al. reported the use of HPLC-MS to identify and quantify essential oil including cinnamaldehyde in cinnamon bark [94]. In a study by Ji et al., HPLC was used to analyze the essential oil components in Sichuan pepper and confirmed the presence of monoterpenes [95]. HPLC is a method of choice for analyzing less volatile or nonvolatile constituents in essential oils. HPLC detects nonvolatile adulteration, such as synthetic compounds or vegetable oils [96, 97]. Ding et al. used HPLC-based fingerprint analysis to evaluate quality of 24 cinnamon bark and 32 cinnamon twig samples sourced from various countries. The study separated and determined seven major marker compounds: cinnamaldehyde, eugenol, coumarin, cinnamyl alcohol, cinnamic acid, 2-hydroxyl cinnamaldehyde, and 2-methoxy cinnamaldehyde [98]. Lee determined the total volatile material contents of black pepper and white pepper via the SDE (Linkens-Nikens type simultaneous steam distillation and extraction apparatus) aided HPLC method [99]. In addition, Yeh et al. determined the essential oil content in two different varieties of ginger root using HPLC analysis [100]. A study by Yang et al. reported use of HPLC-MS/MS to identify 101 small molecules including flavonols and flavones, phenolic acids, lactones, terpenoids, phenylpropanoids, and flavanols from waste cinnamon leaves [94].

This section summarizes the importance of HPLC in determining various essential oil in spices. By the application of HPLC method, substandard varieties of spices can be avoided for commercial marketing. HPLC analysis for essential oil determination can also limit chances of spice adulteration.

## **6. HPLC quantification of starch and other fillers in spices**

The addition of fillers to spices is a common form of adulteration, which can lead to reduced quality, taste, and nutritional value of the spice [101]. One of the most common fillers used in spices is starch, which can be derived from various sources, such as corn, wheat, or rice [102]. Starch quantification is carried out by first extracting the sample with water or ethanol followed by filtration and subjection into the HPLC column. The column is typically equipped with a refractive index detector (RID), which detects changes in the refractive index of the eluent as the sample components pass through the column [103, 104]. Excessive fillers destroy the authenticity of the spices, and to detect the fillers in the powdered spices, HPLC is an alternate solution. In this section, some of the recent findings on detection of starch and fillers in spices have been mentioned.

Ordoudi et al. [105] controlled saffron shelf life using FT-MIR spectra. They confirmed presence of glucose molecules and glycoside linkage damage in spice samples at 1028 cm−1 and in the range 1175–1157 cm−1 band intensity, respectively. The obtained FT-MIR spectroscopic data were analyzed using PCA. To verify the outcomes of the FT-MIR-PCA procedure, HPLC-DAD analysis was carried out [106]. HPLC-DAD method was used to check the authenticity of the results obtained from FT-MIR-PCA analysis. This signifies that HPLC method for detection of unwanted and harmful adulterants is the most reliable method of choice.

Starch and other fillers can be added to spices to increase their weight and bulk, making them more profitable for manufacturers. However, the presence of fillers in spices can reduce their quality and potentially cause health hazards. Therefore, it is important to determine the amount of fillers in spices to ensure that they meet the standards for purity and quality.

## **7. HPLC in comparison to other analytical techniques in quality control of spices**

This section is based on the comparison of HPLC with other physical, chromatographical, and spectroscopical methods of adulteration detection in spices. Several analytical techniques are in use for checking the authenticity of spices. These methods include physical authentication techniques and spectroscopic and chromatographic analysis. Macroscopic and microscopic standardization of spices are done under the physical authentication method. Color, flavor, shape, size, etc. are examined personally by workers to identify the adulteration in the macroscopic analysis method. On the other hand, arrangement of tissue, tissue layering, fiber structure, and root and rhizome structure is checked through microscopic lens in the microscopic method. These methods have become outdated because they are nonreliable, lack repeatability, and time-consuming. These methods also require many skilled people for the microscopic studies. Moreover, different spectroscopic methods such as FTIR, NMR, Raman, XRD, and mass spectroscopy have garnered popularity for detection of illegal substances in spices. They analyze the ingredients and structure of spices efficiently and are very sensitive and accurate techniques. However, they are very expensive and also come up with very complex software algorithm to analyze the results.

Other chromatographic techniques such as gas chromatography (GC) and thinlayer chromatography (TLC) are the commonly used separation methods for the isolation and detection of adulterants in spices. However, each method has its advantages and limitations. GC is widely used for the detection of mycotoxins in spices, but it requires derivatization and has limited selectivity for nonvolatile compounds. TLC is a cost-effective and rapid method, but it lacks sensitivity and requires extensive sample preparation.

Spectrophotometry is a simple and rapid method, but it lacks specificity and is limited to certain compounds. Infrared spectroscopy is a nondestructive method, but it is less sensitive and requires skilled operators. In contrast, HPLC offers high sensitivity, specificity, and selectivity for a wide range of compounds, including mycotoxins, synthetic dyes, pesticides, herbicides, and starch. Moore et al. [17] found that when comparing different methods for identifying compounds and adulterants, IR spectroscopy had the second-highest number of references after HPLC paired with a specific type of detection equipment. Nevertheless, mass spectrometry [107] and IR spectroscopy techniques were found to have the most reports of adulterants in the literature.

HPLC has several advantages over other analytical methods for the detection of adulterants in spices. Firstly, it can separate and identify multiple compounds in a single run, which saves time and reduces the cost of analysis. Secondly, HPLC is highly sensitive and can detect adulterants at low levels, ensuring the safety and quality of spices. Thirdly, HPLC is highly specific and can distinguish between different compounds, ensuring the accuracy and reliability of results. However, there are some limitations associated with HPLC that require expensive equipment and skilled operators, which may limit its use in some settings. Further HPLC may require extensive sample preparation, which may increase the cost and time of analysis.

Finally, HPLC may not be able to detect certain compounds that are not amenable to its separation and detection methods.

## **8. Conclusions**

Now days food quality and safety have become one of major concerns because of the serious health hazards observed due to a range of food intake. However, various fraudulent activities that include adulteration of toxic material into spices have become a serious concern. To address this serious issue, different physical, microscopical, and analytical methods have been carried out. HPLC is one of the chromatographic techniques relied on mostly for the detection of adulteration in spices. The combination of HPLC with other analytical instruments such as mass spectrometry, NMR, etc. is found to be efficient in identifying adulterants in trace amounts also. This method has become most trustworthy because of its sensitivity, accuracy, and reproducibility. The current chapter explains the recent application of HPLC for the quality control of spices. HPLC coupled with UV-VIS or MS/MS detectors have been proven effective in determination of mycotoxins, starch, fillers, and pesticides in verity of spices. Different carcinogenic and synthetic dyes are also successfully identified through HPLC analysis. This present study furnishes extensive scientific information related to utilization of HPLC for the detection of various harmful adulterants in spices. However, more literature on detection of powder fillers and spoiled volatile oils in spices (clove, coriander, and fennel) by HPLC analysis is due in this study.

## **Conflict of interest**

The authors declare no conflict of interest for this publication.

## **Author details**

Prafulla Kumar Sahu\*, Sukumar Purohit, Swarnajeet Tripathy, Durga Prasad Mishra and Biswajeet Acharya School of Pharmacy, Centurion University of Technology and Management, Odisha, India

\*Address all correspondence to: prafulla.sahu@cutm.ac.in

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Sasikumar B et al. Advances in adulteration and authenticity testing of herbs and spices. In: Advances in Food Authenticity Testing. Woodhead Publishing: Elsevier; 2016. pp. 585-624

[2] Jessica Elizabeth DLT et al. Spice use in food: Properties and benefits. Critical Reviews in Food Science and Nutrition. 2017;**57**:1078-1088

[3] Martínez-Graciá C et al. Use of herbs and spices for food preservation: Advantages and limitations. Current Opinion in Food Science. 2015;**6**:38-43

[4] Nair KP, The Agronomy and Economy of Turmeric and Ginger: The Invaluable Medicinal Spice Crops: Newnes. London: Elsevier; 2013

[5] Sanlier N, Gencer F. Role of spices in the treatment of diabetes mellitus: A minireview. Trends in Food Science & Technology. 2020;**99**:441-449

[6] M. Manual, Web Directory for Organic Spices, Herbs and Essential Oils, 2006.

[7] Sudhabindu K, Samal L. Common adulteration in spices and Do-at-home tests to ensure the purity of spices. Food Science Repoets. 2020;**1**:66-68

[8] Sicherer SH, Sampson HA. 9. Food allergy. Journal of Allergy and Clinical Immunology. 2006;**117**:S470-S475

[9] Nallappan K et al. Identification of adulterants in turmeric powder using terahertz spectroscopy. In: 2013 38th International Conference on Infrared, Millimeter, and Terahertz Waves (IRMMW-THz). Germany; 2013. pp. 1-2

[10] WHO. Exposure to highly hazardous pesticides: A major public health concern. 2010

[11] Spink J, Moyer DC. Defining the public health threat of food fraud. Journal of Food Science. 2011;**76**:R157-R163

[12] Tarantelli T, Sheridan R. Toxic Industrial Colorants Found in Imported Foods. New York: State Department of Agriculture & Markets Food Laboratory; 2011

[13] Galvin-King P et al. Herb and spice fraud; the drivers, challenges and detection. Food Control. 2018;**88**:85-97

[14] Moses J et al. Climate change and its implications on stored food grains. Agricultural Research. 2015;**4**:21-30

[15] Ismaiel AA, Papenbrock J. Mycotoxins: Producing fungi and mechanisms of phytotoxicity. Agriculture. 2015;**5**:492-537

[16] Kabak B, Dobson AD. Mycotoxins in spices and herbs–an update. Critical Reviews in Food Science and Nutrition. 2017;**57**:18-34

[17] Moore JC et al. Development and application of a database of food ingredient fraud and economically motivated adulteration from 1980 to 2010. Journal of Food Science. 2012;**77**:R118-R126

[18] Babaei S et al. Developing an SCAR and ITS reliable multiplex PCR-based assay for safflower adulterant detection in saffron samples. Food Control. 2014;**35**:323-328

[19] Reemtsma T. Liquid chromatography– mass spectrometry and strategies for trace-level analysis of polar organic pollutants. Journal of Chromatography A. 2003;**1000**:477-501

[20] Dalby A. Dangerous Tastes, The Story of Spices. Berkeley: University of California Press; 2000

[21] Freedman P. Out of the East: Spices and the Medieval Imagination. USA: Yale University Press, Chicago press; 2008

[22] Rodinson M, Arberry AJ, "Medieval Arab Cookery". USA: Prospect books; 2001

[23] Galli M. Beyond frontiers: Ancient Rome and the Eurasian trade networks. Journal of Eurasian Studies. 2017;**8**:3-9

[24] Bharathi S et al. Instrument-based detection methods for adulteration in spice and spice products–A review. Journal of Spices and Aromatic Crops. 2018;**27**:106-118

[25] Kar M. Towards sustainable indian Agri-commodities' markets: Experiences, innovative model and future agenda. Pragati: Journal of Indian Economy. 2020;**7**:48-63

[26] Bansal S et al. Food adulteration: Sources, health risks, and detection methods. Critical Reviews in Food Science and Nutrition. 2017;**57**: 1174-1189

[27] Jaffee S. Delivering and Taking the Heat: Indian Spices and Evolving Product and Process Standards. USA: The World Bank group; 2005

[28] Kumari C, Singh SG. Mycoflora of spices, A review. Plant Archives. 2021;**21**:99-102

[29] DeWaal CS, Brito GRG. Safe food international: A blueprint for better global food safety. Food and Drug Law Journal. 2005;**60**:393-405

[30] Thomas L, Sanil P. Competitiveness in spice export trade from India: A

review. Food Science and Biotechnology. 2019;**28**

[31] Moges A et al. Dietary and bioactive properties of the berries and leaves from the underutilized Hippophae salicifolia D. Don grown in Northeast India. Food Science and Biotechnology. 2021;**30**:1555-1569

[32] Purohit S et al. Exploration of nutritional, antioxidant and antibacterial properties of unutilized rind and seed of passion fruit from Northeast India. Journal of Food Measurement and Characterization. 2021;**15**:3153-3167

[33] Sahu PK et al. An overview of experimental designs in HPLC method development and validation. Journal of Pharmaceutical and Biomedical Analysis. 2018;**147**:590-611

[34] Bhooma V et al. Identification of synthetic dyes magenta III (new fuchsin) and rhodamine B as common adulterants in commercial saffron. Food Chemistry. 2020;**309**:125793

[35] Sahu PK et al. A robust RP-HPLC method for determination of turmeric adulteration. Journal of Liquid Chromatography & Related Technologies. 2020;**43**:247-254

[36] Vickers NJ. Animal communication: When i'm calling you, will you answer too? Current Biology. 2017;**27**:R713-R715

[37] Cokosyler N. Farkh yontemlerle kurutulan kirmizi biberlerde Aspergillus flavus gelisimi ve aflatoksin olusumunun incelenmesi. Gida. 1999;**24**:297-306

[38] Zhu Y et al. A screening method of oil-soluble synthetic dyes in chilli products based on multi-wavelength chromatographic fingerprints comparison. Food Chemistry. 2016;**192**:441-451

*Current Trends in HPLC for Quality Control of Spices DOI: http://dx.doi.org/10.5772/intechopen.110897*

[39] Duan H-L et al. Magnetically modified porous β-cyclodextrin polymers for dispersive solid-phase extraction high-performance liquid chromatography analysis of Sudan dyes. Food Analytical Methods. 2019;**12**:1429-1438

[40] Khalikova MA et al. On-line SPE–UHPLC method using fused core columns for extraction and separation of nine illegal dyes in chilli-containing spices. Talanta. 2014;**130**:433-441

[41] He T et al. Dummy molecularly imprinted polymer based microplate chemiluminescence sensor for one-step detection of Sudan dyes in egg. Food Chemistry. 2019;**288**:347-353

[42] Zhang M et al. A composite polymer of polystyrene coated with poly (4-vinylpyridine) as a sorbent for the extraction of synthetic dyes from foodstuffs. Analytical Methods. 2020;**12**:3156-3163

[43] Hu Z et al. Simultaneous determination of multiclass illegal dyes with different acidic–basic properties in foodstuffs by LC-MS/MS via polarity switching mode. Food Chemistry. 2020;**309**:125745

[44] Cornet V et al. Journal of Agricultural and Food Chemistry. 2006;**54**:639-644

[45] Genualdi S et al. Method development and survey of Sudan I–IV in palm oil and chilli spices in the Washington, DC, area. Food Additives & Contaminants: Part A. 2016;**33**:583-591

[46] Sebaei AS et al. Determination of seven illegal dyes in Egyptian spices by HPLC with gel permeation chromatography clean up. Journal of Food Composition and Analysis. 2019;**84**:103304

[47] Bessaire T et al. A new highthroughput screening method to determine multiple dyes in herbs and spices. Food Additives & Contaminants: Part A. 2019;**36**:836-850

[48] Ullah A et al. Banned Sudan dyes in spices available at markets in Karachi, Pakistan. Food Additives & Contaminants: Part B. 2022;**2022**:1-8

[49] Amelin V et al. Simultaneous determination of dyes of different classes in aquaculture products and spices using HPLC–high-resolution quadrupole timeof-flight mass spectrometry. Journal of Analytical Chemistry. 2017;**72**:183-190

[50] Thalhamer B, Buchberger W. Adulteration of beetroot red and paprika extract based food colorant with Monascus red pigments and their detection by HPLC-QTof MS analyses. Food Control. 2019;**105**:58-63

[51] Zhu Y et al. Simultaneous determination of 14 oil-soluble synthetic dyes in chilli products by high performance liquid chromatography with a gel permeation chromatography clean-up procedure. Food Chemistry. 2014;**145**:956-962

[52] Qi P et al. Development of a rapid, simple and sensitive HPLC-FLD method for determination of rhodamine B in chili-containing products. Food Chemistry. 2014;**164**:98-103

[53] Botek P et al. Determination of banned dyes in spices by liquid chromatography-mass spectrometry. Czech Journal of Food Science. 2007;**25**:17-24

[54] Reinholds I et al. Mycotoxins, pesticides and toxic metals in commercial spices and herbs. Food Additives & Contaminants: Part B. 2017;**10**:5-14

[55] Wang Y et al. Analytical methods to analyze pesticides and herbicides.

Water Environment Research. 2019;**91**:1009-1024

[56] Mekonnen TF et al. Investigation of chlorpyrifos and its transformation products in fruits and spices by combining electrochemistry and liquid chromatography coupled to tandem mass spectrometry. Food Analytical Methods. 2018;**11**:2657-2665

[57] Ersoy N et al. Determination of pesticide residue present in cumin plant (Nigella orientalis L.) with LC-MS/MS and GC-MS. Asian Journal of Chemistry. 2016;**28**:1011

[58] Yao W et al. Multi-residue analysis of 34 pesticides in black pepper by QuEChERS with d-SPE vs. d-SLE cleanup. Food Analytical Methods. 2019;**12**:176-189

[59] Goon A et al. A simultaneous screening and quantitative method for the multiresidue analysis of pesticides in spices using ultra-high performance liquid chromatography-high resolution (Orbitrap) mass spectrometry. Journal of Chromatography A. 2018;**1532**:105-111

[60] Liu X et al. Determination of pesticide residues in chilli and Sichuan pepper by high performance liquid chromatography quadrupole timeof-flight mass spectrometry. Food Chemistry. 2022;**387**:132915

[61] Yap C, Jarroop Z. Residue levels and dissipation behaviors of chlorpyrifos in black pepper berries and soil. Food Research. 2018;**2**:587-593

[62] Milićević DR et al. Real and perceived risks for mycotoxin contamination in foods and feeds: Challenges for food safety control. Toxins. 2010;**2**:572-592

[63] Tahir NI et al. Nature of aflatoxins: Their extraction, analysis, and control. Journal of Food Safety. 2018;**38**:e12561

[64] Bryden WL. Mycotoxins in the food chain: Human health implications. Asia Pacific Journal of Clinical Nutrition. 2007;**16**:95-101

[65] Rahmani A et al. Qualitative and quantitative analysis of mycotoxins. Comprehensive Reviews in Food Science and Food Safety. 2009;**8**:202-251

[66] Iqbal SZ et al. Survey of aflatoxins and ochratoxin A in retail market chilies and chili sauce samples. Food Control. 2017;**81**:218-223

[67] da Silva ARP et al. Ochratoxin A and related fungi in Brazilian black pepper (Piper nigrum L.). Food Research International. 2021;**142**:110207

[68] Zareshahrabadi Z et al. Detection of aflatoxin and ochratoxin A in spices by high-performance liquid chromatography. Journal of Food Quality. 2020;**2020**:1-8

[69] Ainiza WW et al. Simultaneous determination of aflatoxins and ochratoxin A in single and mixed spices. Food Control. 2015;**50**:913-918

[70] Costa J et al. Occurrence of aflatoxins and Ochratoxin A during Merkén pepper powder production in Chile. Food. 2022;**11**:3843

[71] Ali N et al. Natural occurrence of aflatoxins and ochratoxin A in processed spices marketed in Malaysia. Food Additives & Contaminants: Part A. 2015;**32**:518-532

[72] Palma P et al. Adaptation, optimization, and validation of a sensitive and robust method for the quantification of total aflatoxins (B1, B2, G1, and G2) in the spice merkén by HPLC-FLD with post-column derivatization. Microchemical Journal. 2022;**178**:107342

*Current Trends in HPLC for Quality Control of Spices DOI: http://dx.doi.org/10.5772/intechopen.110897*

[73] Ouakhssase A et al. Modified-QuEChERS-LC/MS method for the analysis of aflatoxins and ochratoxin A in coriander seeds. Food Additives & Contaminants: Part A. 2023;**2023**:1-9

[74] Koutsias I et al. Occurrence and risk assessment of aflatoxin b1 in spices marketed in Greece. Analytical Letters. 2021;**54**:1995-2008

[75] Asghar MA et al. Aflatoxins in composite spices collected from local markets of Karachi, Pakistan. Food Additives & Contaminants: Part B. 2016;**9**:113-119

[76] Garcia MV et al. Aflatoxigenic and ochratoxigenic fungi and their mycotoxins in spices marketed in Brazil. Food Research International. 2018;**106**:136-140

[77] Kollia E et al. Aflatoxin B1 in sesame seeds and sesame products from the Greek market. Food Additives & Contaminants: Part B. 2016;**9**:217-222

[78] Asghar MA et al. Development and validation of a high-performance liquid chromatography method with post-column derivatization for the detection of aflatoxins in cereals and grains. Toxicology and Industrial Health. 2016;**32**:1122-1134

[79] Vandekerckhove M et al. Development of an LC-MS/MS method for the detection of traces of peanut allergens in chili pepper. Analytical and Bioanalytical Chemistry. 2017;**409**:5201-5207

[80] Wang Y-H et al. Cassia cinnamon as a source of coumarin in cinnamonflavored food and food supplements in the United States. Journal of Agricultural and Food Chemistry. 2013;**61**:4470-4476

[81] Santos L et al. Co-occurrence of aflatoxins, ochratoxin A and zearalenone in Capsicum powder samples available on the Spanish market. Food Chemistry. 2010;**122**:826-830

[82] Romagnoli B et al. Aflatoxins in spices, aromatic herbs, herb-teas and medicinal plants marketed in Italy. Food Control. 2007;**18**:697-701

[83] Akpo-Djènontin DOO et al. Mold infestation and aflatoxins production in traditionally processed spices and aromatic herbs powder mostly used in West Africa. Food Science & Nutrition. 2018;**6**:541-548

[84] Fundikira S et al. Awareness, handling and storage factors associated with aflatoxin contamination in spices marketed in Dar es Salaam, Tanzania. World Mycotoxin Journal. 2021;**14**:191-200

[85] Mair C et al. Assessment of Citrinin in spices and infant cereals using Immunoaffinity column clean-up with HPLC-fluorescence detection. Toxins. 2021;**13**:715

[86] Saito K et al. Residual analysis of aflatoxins in spice by HPLC coupled with solid-phase dispersive extraction and solid-phase fluorescence derivatization method. Journal of AOAC International. 2020;**103**:1521-1527

[87] Irshad M et al. "Biological importance of essential oils," Essential Oils-Oils of Nature. Vol. 1. London: IntechOpen; 2020

[88] Vankar PS. Essential oils and fragrances from natural sources. Resonance. 2004;**9**:30-41

[89] Irshad M et al. Biological importance of essential oils. Essential Oils-Oils of Nature. 2020;**1**

[90] Zhang J et al. Basic sensory properties of essential oils from aromatic plants and their applications: A critical

review. Critical Reviews in Food Science and Nutrition. 2023;**2023**:1-14

[91] Turek C, Stintzing FC. Application of high-performance liquid chromatography diode array detection and mass spectrometry to the analysis of characteristic compounds in various essential oils. Analytical and Bioanalytical Chemistry. 2011;**400**:3109-3123

[92] Bendif H et al. Total phytochemical analysis of Thymus munbyanus subsp. coloratus from Algeria by HS-SPME-GC-MS, NMR and HPLC-MSn studies. Journal of Pharmaceutical and Biomedical Analysis. 2020;**186**:113330

[93] Chen S-X et al. Comparison of chemical compositions of the Pepper EOs from different cultivars and their AChE inhibitory activity. Natural Product Communications. 2020;**15**:19345

[94] Yang Y-L et al. LC-MS-based identification and antioxidant evaluation of small molecules from the cinnamon oil extraction waste. Food Chemistry. 2022;**366**:130576

[95] Ji Y et al. Chemical composition, sensory properties and application of Sichuan pepper (Zanthoxylum genus). Food Science and Human Wellness. 2019;**8**:115-125

[96] Do TKT et al. Authenticity of essential oils. TrAC Trends in Analytical Chemistry. 2015;**66**:146-157

[97] Smelcerovic A et al. Recent advances in analysis of essential oils. Current Analytical Chemistry. 2013;**9**:61-70

[98] Ding Y et al. Discrimination of cinnamon bark and cinnamon twig samples sourced from various countries using HPLC-based fingerprint analysis. Food Chemistry. 2011;**127**:755-760

[99] Lee M-S. Simple rapid quality estimation method in black and white pepper grounds by determination of volatile oil content. The Korean Journal of Food and Nutrition. 2009;**22**:352-356

[100] Yeh H-y et al. Bioactive components analysis of two various gingers (Zingiber officinale Roscoe) and antioxidant effect of ginger extracts. LWT-Food Science and Technology. 2014;**55**:329-334

[101] Momtaz M et al. Mechanisms and health aspects of food adulteration: A comprehensive review. Food. 2023;**12**:199

[102] Adigwe OP et al. "Starch: A Veritable Natural Polymer for Economic Revolution," 2022.

[103] Murillo MMS, Granados-Chinchilla F. Total starch in animal feeds and silages based on the chromatographic determination of glucose. MethodsX. 2018;**5**:83-89

[104] Sluiter JB et al. Direct determination of cellulosic glucan content in starch-containing samples. Cellulose. 2021;**28**:1989-2002

[105] Ordoudi SA et al. On the quality control of traded saffron by means of transmission Fourier-transform mid-infrared (FT-MIR) spectroscopy and chemometrics. Food Chemistry. 2014;**150**:414-421

[106] Kucharska-Ambrożej K, Karpinska J. The application of spectroscopic techniques in combination with chemometrics for detection adulteration of some herbs and spices. Microchemical Journal. 2020;**153**:104278

[107] Osman AG et al. Overview of analytical tools for the identification of adulterants in commonly traded herbs and spices. Journal of AOAC International. 2019;**102**:376-385

## *Edited by Oscar Núñez, Sònia Sentellas, Mercè Granados and Javier Saurina*

Increasing interest in topics related to health and quality of life in recent years has led to a growing need in food, environmental and bioanalytical research for high-throughput separation techniques able to cope with the qualitative/quantitative determination of a large number of compounds in very complex matrices. High-performance liquid chromatography (HPLC) is a well-established separation technique widely employed in many fields. The versatility of chromatographic separation modes, coupled with lowresolution and high-resolution mass spectrometry, makes HPLC among the best options to solve emerging analytical problems. This book provides an overview of new advances in high-performance liquid chromatography and its applications in different fields.

Published in London, UK © 2023 IntechOpen © Septumia Jacobson / Unsplash

High Performance Liquid Chromatography - Recent Advances and Applications

High Performance Liquid

Chromatography

Recent Advances and Applications

*Edited by Oscar Núñez, Sònia Sentellas,* 

*Mercè Granados and Javier Saurina*