Instrument-based detection methods for adulteration in spice and spice products – A review <s> Method <s> The identification of organic colorants is of high importance in the cultural heritage field, where they are found as paint components and textile dyes, and in forensic science, because of their use in inks and paints, food colorants and textile dyes. Surface-enhanced Raman spectroscopy (SERS) has emerged as a promising technique for the detection of these materials, yet concerns over the sensitivity of SERS spectra of dyes to chemical and instrumental variables (such as pH, choice of SERS substrates and/or aggregants, and excitation wavelength) have prevented its widespread use in analytical applications. Over the last few years, the development of several microanalytical approaches has considerably increased the chances of success in the identification of minute amounts of dyes by SERS. However, the need for searchable databases is still largely to be fulfilled. In this work, we have assembled the core of a comprehensive library which contains 100 Raman and SERS reference spectra of natural and synthetic organic colorants. Experiments to classify 20 query SERS spectra of dyes from a variety of museum objects were conducted using principal component analysis (PCA) and the correlation coefficient (CC) algorithm. The effect of spectral transformations such as baseline correction, selecting a standard frequency range, normalization, smoothing, as well as carrying out the search on the second derivative of the spectra, was systematically evaluated. With this study we demonstrate that SERS spectra of organic colorants can be reliably matched against a well constructed spectral library regardless of the instrumentation and the colloids used, and of the pH conditions at which the measurements were carried out. <s> BIB001 </s> Instrument-based detection methods for adulteration in spice and spice products – A review <s> Method <s> A substrate for Surface-Enhanced Raman Scattering spectroscopy (SERS), electropolished Al, is proposed as a tool for a rapid and low cost determination of Sudan I. This dye has been used as an additive in some foodstuffs but it is now banned because of the health risk associated with its carcinogenic and mutagenic properties. Despite the presence of fluorescence, Raman spectra of Sudan I can be obtained using excitation lasers at 633 and 785 nm. To get rid of the spectral noise and fluorescence background, Savitzky-Golay smoothing and polynomial corrections were applied, respectively. The Raman signal was proved to be enhanced. A linear dependence was found between the logarithmic intensity at 1598 cm(-1) peak versus the logarithmic concentration. The figures of merit were studied obtaining high sensitivity and low detection limits (10(-7) M). A multivariate exploratory analysis (PCA) was used to study the ability of SERS to distinguish Sudan I from other similar compounds. Therefore, results show that SERS is a potential tool to determine Sudan I quickly and effectively. <s> BIB002 </s> Instrument-based detection methods for adulteration in spice and spice products – A review <s> Method <s> The human nose is the primary instrument used to assess the smell or flavor of various natural and industrial products. The use of human nose has various limitations. Basically to overcome these limitations a concept of Electronic-nose is applied. It comprises an array of chemical sensors with partial specificity and an appropriate pattern recognition system, capable of recognizing simple or complex odor. Electronic nose has been used in a number of applications ranging from quality control of cosmetics, beverages freshness of food, detection of hazardous chemicals. This paper presents development of E-nose to identify Cardamom, Nutmeg or Clove oil using TGS 2610, TGS 2611 and TGS 2620 gas sensors. The gas sensor array was exposed to the Cardamom / Nutmeg / Clove oil alternatively. The data was acquired for two minutes for each spice oil using National Instruments Data Acquisition card, which was interfaced to the PCI slot of computer. The data acquisition was repeated sixteen times for each of the said spice oils and was saved in the separate excel files. A feed forward neural network of 3 input neurons, 5 hidden neurons and 3 output neurons was implemented using nprtool (Pattern Recognition Tool) with 34 training samples, 7 validation samples and 7 test samples with 1000 epochs in MATLAB software. This MATLAB tool leads through solving a pattern recognition classification problem with sigmoid function. The graphs showed different responses to the different spice oils and more than ninety seven percent correct classification was done by the neural network. <s> BIB003 </s> Instrument-based detection methods for adulteration in spice and spice products – A review <s> Method <s> An attempt was made to understand the variation in the curcuminoid profile of turmeric varieties. Eight cultivars namely, Local (check), IISR-Alleppey Supreme, IISR-Kedaram, IISR-Prabha, IISRPrathibha, Suvarna, Suguna and Sudharshana were utilized for the study. Curcuminoid content of fresh, dried and cured varieties after methanolic extraction and quantification through HPLC revealed that Local (check) turmeric in fresh form was significantly superior in curcuminoid content. IISR-Prabha and IISR-Prathibha in dry and cured form respectively, also exhibited high curcuminoid content. <s> BIB004 </s> Instrument-based detection methods for adulteration in spice and spice products – A review <s> Method <s> p-Aminobenzenethiol (ABT) is a popular molecule for surface-enhanced Raman scattering experiments (SERS), providing large signal enhancements on a range of metal surfaces. However, SERS intensities vary very much according to experimental conditions, and the interplay between ABT protonation, polymer state, and electronic structure/Raman cross section is still not completely clear. To understand main factors affecting Raman intensities, density functional theory (DFT) and matrix polarization theory (MPT) models were used to generate the spectra and compare to the experiment. The simulations showed that ABT protonation as well as its binding to the metal surface shift the absorption threshold, which invokes resonance or preresonance conditions favorable to the signal enhancement. The MPT approximation enabled modeling of the effect of the metal bulk and orientation of the dye on the metal surface on the enhancement and relative band intensities. The simulations can be done relatively easily and reveal chem... <s> BIB005 </s> Instrument-based detection methods for adulteration in spice and spice products – A review <s> Method <s> Cardamom (Elettaria cardamomum Maton.) is one of the most expensive spices in the world. Essential oil is the most functionally important component of cardamom and is described as sweet, spicy, warm, camphoraceous and citrusy. The quality of cardamom is assessed by the essential oil content and its composition. Design of hand-held E-nose and evaluation of quality of cardamom in terms of its essential oil content is reported here. <s> BIB006
Typically, analytical measurements in UV-Vis spectroscopy are carried out between 200 to 800 nm (Isengard & Breithaupt 2015) . The radiation source for UV-Vis spectroscopy could be a hollow cathode lamp, a tungsten filament lamp or a mercury vapour lamp. The sample can either be a liquid or a solid dissolved in suitable solvents. The sample is placed in a cuvette and enclosed in a compartment which prevents the loss of radiation and the possibility of interference from  SERS technique is highly specific as it identifies molecules based on their vibrational fingerprints and thereby permits detection with high sensitivity BIB001 . Essentially, SERS process involves three steps: preparation, measurement and detection. Preparation involves manufacturing of SERS-active substances and its adsorption to targeted molecules leading to enhanced Raman scattering. The measurement process relies on the sensitivity and specificity under energetic, temporal and spatial resolutions. Detection and data analysis involve acquisition of unique spectra of different molecules present in the sample (Ding et al. 2014 ). Several studies have been conducted for the application of SERS in detecting adulterants in spices; especially with Sudan dye, considering its carcinogenic and mutagenic effects (Cheung et al. 2010; Gao et al. 2015; BIB002 . Di Anibal et al. (2012) compared different Raman spectroscopic modalities (Normal Raman, FTRaman and SERS) and found that SERS is better suited for detection of Sudan I dye in culinary spices. A portable Raman spectrometer developed using SERS and multivariate chemometrics was used to provide quantitative data of Sudan I dye in spice products. The limit of detection was 48 µg kg -1 of chilli powder and the substrate used was gold citrate reduced sol (Cheung et al. 2010) . Similarly, Lopez et al. (2013) proposed a tool to detect Sudan I dye quantitatively, using electropolished aluminium as a substrate for SERS with low detection limits of 3x 10 -7 M. Recently, a biosensor functioning using molecularly imprinted polymers, thin layer chromatography and SERS to detect Sudan I in paprika powder at levels as low as 1 ppm with gold colloid as SERS substrate has been fabricated. This biosensor exhibited rapidity in both separation (30-40 s) and detection (0.1-1 s) and can be used as an effective screening tool for Sudan I adulteration (Gao et al. 2015) . To detect water insoluble dyes, in the presence of water soluble competitor, Jahn et al. (2015) developed a hydrophobic surface modified sensor layer on the SERS active substrate (enzymatically grown silver nanoparticles) and detected Sudan III adulterants upto concentration of 9 µmol/L in methanol extracted real food samples. In an FT-IR spectrometer, the monochromator is replaced with an interferometer which allows instantaneous passage of radiation of all wavelengths into the sample instead of filtering or dispersing them. Radiation from the source is allowed to strike a beam splitter, that reflects a portion of the radiation to a fixed mirror and transmits other portion to a movable mirror. (Fig. 2) shows peak at varying wavelengths because of variation in response of functional groups to IR radiation. For instance, the peak at 1140 cm -1 in metanil yellow is due to N=N site. After noise removal using a Savitzky-Golay filter the method can be used in authentication of spice oils. Importantly, there is reduced or near-zero use of hazardous solvents and reagents, making the approach 'green'. Nurrullhidayah et al. (2011) used FTIR to detect black cumin (Nigella sativa L.) seed oil adulterated with grape seed oil. In the spectra obtained, black cumin seed oil showed sharp peaks at 1117 and 1098 cm -1 , whilst grape seed oil showed peak at 1744 cm -1 only.  The major components of IRMS include a sample delivery system in which either the sample is combusted and transformed into gas using an elemental analyzer or a gas chromatography unit, an electron ionization source to ionize and accelerate the gas molecules, a magnetic sector to disperse mono-energetic ions of varying mass through the magnetic field, a faraday-collector to measure the current of each ion beam and a computer controlled data acquisition system to amplify and display values as digital output (Brand 2004; Kelly 2003; BIB005 ). In general, naturally occurring substances are enriched with lighter isotopes and the ratio of isotopes is considered to be unique to a substance and could indicate origin from a particular geographic location. Adulteration will alter this ratio. IRMS has been useful in authenticity control of spices and spice products such as essential oils (Frank et al. 1995; Greule et al. 2008) . Gruele et al. (2010) used an IRMS approach for rapid detection of adulteration in vanillin by comparing the carbon and hydrogen stable isotope values of vanillin molecule and vanillin methoxyl groups of authentic and synthetic vanillin samples of different origin. This technique requires 1 mg and 4 mg of samples for stable carbon and stable hydrogen analysis, respectively. The approach can be used to control adulteration in vanilla; a serious concern, particularly to natural vanilla. A typical HSI system consists of an illumination source (usually tungsten halogen or LED source), an objective lens through which light waves get reflected from the sample that moves on a motorized stage, a spectrograph to separate the reflected light into its component wavelengths, a camera operated usually in conjunction with either charge coupled device or complementary metal oxide semiconductor sensors for two-dimensional image acquisition and a personal computer where two-dimensional images of the sample are stacked to form a threedimensional hypercube (Gowen et al. 2007 ). By using obtained physical and chemical characteristics from the HSI spectra, the presence of foreign matter or adulterants in food and agricultural products can be identified. Vermaak et al. (2014) suggested that short wave infrared (SWIR) hyperspectral imaging with chemometric data analysis can be used to detect adulteration in star anise (Illicium verum), a dried fruit used in the treatment of infant colic, with Japanese star anise (Illicium anisatum), an adulterant containing neurotoxic compounds. The technique has proven potential in quality analysis of bulk samples when conveyed through conveyor belt systems. In a High Performance/Pressure Liquid Chromatography (HPLC) system, the mobile phase carrying components of an analyte mixture (sample) flows through a stationary phase in a narrow and uniform column made of porous silica capillaries. Usually, the stationary phase is polar, whilst the mobile phase is non-polar. However, a reverse phase HPLC uses a non-polar stationary phase and a polar mobile phase. In food quality applications, reverse phase HPLC is preferred as polar components elute faster than non-polar components due to hydrophobic interactions. Presence of gas bubbles in the mobile phase may lead to distortion of detected signal. In order to remove gas bubbles prior to functioning, either a vacuum pump is used or the mobile phase sparged with an inert gas can be used (Kealey & Haines 2002) . HPLC instrumentation consists of a reciprocating pump to maintain a constant flow of a mobile phase, a loop injector to disperse the sample in the mobile phase, a separating column to separate the components of the sample, a detector for quantitative determination of different constituents separated from the sample and a computer to display the results (Harvey 2000). HPLC has been used not only in differentiation of varieties of spices BIB004 ) but also in detection of adulterants such as Sudan dyes, Para-red and Rhodamine-B (Ertaº et al. 2007; Fu et al. 2015) . Tateo & Bononi (2004) used combination of HPLC and APCI-MS (Atmospheric Pressure Chemical IonizationMass Spectrometry) to detect and quantify Sudan I present in chillies. The study found that extracts from Sudan I from chilli added food products can be efficiently detected and quantified using HPLC and APCI-MS at very low concentrations. From  Fig 3, it can be observed that sharper the peak, the more concentrated the component; whereas, wider peak indicates dispersed components. Though HPLC is an excellent choice for quantitative detection, time and cost requirements are high (Di Anibal et al. 2009 ). An e-nose consists of a sample delivery system which absorbs volatiles from the sample to be analysed, a detection system and a computing system. The detection system consists of sensors which can be broadly classified as hot sensors (eg: Metal oxide semiconductors and metal oxide semiconducting field effect transistor) and cold sensors (eg: Conducting polymers, oscillating sensors, optical or electrochemical sensors) based on operating temperature range (Haugen 2001 ). An important component of an e-nose system is the data processing unit, operated using principal component analysis, partial least squares, functional discriminant analysis, hierarchical cluster analysis, fuzzy logic or artificial neural network algorithms  . Neural networks operate on the basis of functional mapping between known and unknown samples. Information processing in an e-nose is done along a number of input and hidden layers that are grouped on the basis of similarities and differences to give a single digital output (Adak & Yumusak 2016 ). E-nose systems can be used in quality monitoring of food products; especially in spices, where aroma, flavor and color are of prime concern BIB006 . BIB003 successfully used e-nose to classify three spice oils (cardamom, nutmeg and clove oil) with classification accuracy of around 97.1%. Recently, Heidarbeigi et al. (2015) demonstrated detection of saffron adulteration with safflower and corn stigma. Enose, containing MOS (Metal Oxide Semiconductors) sensors, successfully yielded aroma fingerprints of the pure compound and the adulterated compounds. Obtained features were analysed with principal component analysis and artificial neural network and it was found that e-nose could provide classification accuracies of 100% and 86.87% for original saffron and other adulterants, respectively. The concern over safety and authenticity of spices has resulted in establishing higher standards for quality evaluation and improved methods to function at various stages of post-harvest, storage and shipment. Though development of various analytical tools employed, standardization and authentication of spice products have increased. Besides adulterants, the quality of spice is lost due to the presence of contaminants, filth matter or microbial toxic substances like aflatoxin, mycotoxins and heavy metals like lead, cobalt, 