Evaluation of Turmeric Powder Adulterated with Metanil Yellow Using FT-Raman and FT-IR Spectroscopy

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Article Evaluation of Turmeric Powder Adulterated with Metanil Yellow Using FT-Raman and FT-IR Spectroscopy

Sagar Dhakal, Kuanglin Chao *, Walter Schmidt, Jianwei Qin, Moon Kim and Diane Chan United States Department of Agriculture/Agricultural Research Service, Environmental Microbial and Food Safety Laboratory, Bldg. 303, Beltsville Agricultural Research Center East, 10300 Baltimore Ave., Beltsville, MD 20705-2350, USA; [email protected] (S.D.); [email protected] (W.S.); [email protected] (J.Q.); [email protected] (M.K.); [email protected] (D.C.) * Correspondence: [email protected]; Tel.: +1-301-504-8450 (ext. 260); Fax: +1-301-504-9466

Academic Editor: Malik Altaf Hussain Received: 14 April 2016; Accepted: 11 May 2016; Published: 17 May 2016 Abstract: Turmeric powder (Curcuma longa L.) is valued both for its medicinal properties and for its popular culinary use, such as being a component in curry powder. Due to its high demand in international trade, turmeric powder has been subject to economically driven, hazardous chemical adulteration. This study utilized Fourier Transform-Raman (FT-Raman) and Fourier Transform-Infra Red (FT-IR) spectroscopy as separate but complementary methods for detecting metanil yellow adulteration of turmeric powder. Sample mixtures of turmeric powder and metanil yellow were prepared at concentrations of 30%, 25%, 20%, 15%, 10%, 5%, 1%, and 0.01% (w/w). FT-Raman and FT-IR spectra were acquired for these mixture samples as well as for pure samples of turmeric powder and metanil yellow. Spectral analysis showed that the FT-IR method in this study could detect the metanil yellow at the 5% concentration, while the FT-Raman method appeared to be more sensitive and could detect the metanil yellow at the 1% concentration. Relationships between metanil yellow spectral peak intensities and metanil yellow concentration were established using representative peaks at FT-Raman 1406 cm´1 and FT-IR 1140 cm´1 with correlation coefﬁcients of 0.93 and 0.95, respectively.

Keywords: FT-Raman; FT-IR; turmeric powder; metanil yellow; quantitative analysis

1. Introduction Turmeric (Curuma long L.) is a herbaceous root commonly used for food seasoning as well as for medicinal purposes. Turmeric has a long history of medicinal use in Asian countries [1,2] and is used in root, oil, and powder forms. Its medicinal value is mainly due to its content of curcumin (diferuloyol methane) [3–7], with attributed medical properties including anti-inﬂammatory [8–12], anticarcinogenic [13–16], antioxidant [17–19], and wound-healing [20–22] effects. Curcumin has also been reported to have promise for development of therapies for Alzheimer’s disease [23]. The curcumin content in turmeric varies. Studies have shown that factors such as nutrient and acidity content in soil [24,25], fertilizer, soil type and cultivar [26–28] affects the curcumin content in turmeric. Reported curcumin concentrations in turmeric range from 0.3% to 8.6% [3,8,29–31]. Curcumin is isolated from turmeric for medicinal and cosmetic purposes. Although whole, dried, or fresh turmeric are typically free of contamination, turmeric powder can be adulterated with different chemical powders used as substitutes for curcumin [32]. Studies have reported the mixing of Curcuma zedoaria, a wild relative of turmeric, into turmeric powder due to its close resemblance with turmeric [32,33]. Similarly, metanil yellow (C18H14N3NaO3S) is a toxic azo dye that has been added to turmeric powder to mimic the appearance of curcumin [34–36] when the actual curcumin

Foods 2016, 5, 36; doi:10.3390/foods5020036 www.mdpi.com/journal/foods Foods 2016, 5, 36 2 of 15 content is low [37]. Toxicologically, metanil yellow is classiﬁed as a CII category substance by the Joint FAO/WHO Expert committee on Food Additives, and it implies that it is a compound for which virtually no information on long-term toxicity is available [38]. Toxicity is categorized into four classes according to the toxicity of chemicals, where Category I chemicals are the most toxic and poisonous, and Category IV chemicals are least toxic and poisonous. Studies on rats show that long term consumption of metanil yellow causes neurotoxicity [39], hepatocellular carcinoma [40–42], tumor development [43], deleterious effect on gastric mucin [44], and lymphocytic leukemia [45]. A variety of conventional methods have been effectively used for detection of metanil yellow in food stuffs. Ion-pair liquid chromatography detected with 99% linearity the presence of azo dyes, such as metanil yellow, in the range of 0.05 ppm to 10 ppm in food [46]. Similarly other methods such as high performance liquid chromatography-electrospray ionization tandem mass spectrometry [47], high performance capillary electrophoresis [48], and micellar chromatographic method [49] have been used for detection of metanil yellow and other dyes in food and beverages. Despite their high accuracies and satisfactory detection limits, these conventional methods are limited in practicality due to their operational complexity and sample-destructive nature. In contrast, the relative simplicity of optical methods has driven their increasing use for safety and quality detection of foods and food products [50–54]. This study made use of FT-Raman and FT-IR spectroscopy for evaluation of metanil yellow in turmeric powder. Although FT-IR and FT-Raman spectroscopy have not been previously reported for detection of metanil yellow adulteration in food, these spectroscopy methods have been widely used for detection of other food adulterants. Lohumi et al. (2014) used FT-NIR and FT-IR spectroscopy to detect starch adulteration in onion powder [55]. Similarly, studies have reported analysis of starch-adulterated garlic powder [56] and detection of palm oil in extra virgin olive oil [57] using FT-IR spectroscopy, detection of hazelnut oil in olive oil using FT-Raman and FT-MIR spectroscopy [58], and adulteration of virgin olive oil using FT-Raman spectroscopy [59]. FT-IR and FT-Raman spectroscopy are also used for detection of chemical contamination in spices. Schulz et al. (2003) applied FT-IR and FT-Raman spectroscopy for detection of carcinogenic compounds (methyleugenol and estragole) in basil [60]. Similarly, FT-IR was applied for detection and quantiﬁcation of food colorants such as Tartrazine, Sunset yellow, Szorubine, Quinoline-yellow, Allura red, and Sudan II in saffron [61]. This study presents a comprehensive study of FT-Raman and FT-IR spectra of metanil yellow, turmeric powder, and turmeric adulterated with metanil yellow at different concentrations. The primary objectives of this study are to:

1 Study the FT-Raman and FT-IR spectra of metanil yellow and turmeric powder 2 Identify the FT-Raman and FT-IR spectral ﬁngerprint of metanil yellow to distinguish it from the spectral signal of turmeric powder 3 Evaluate turmeric adulterated with metanil yellow at different concentrations and determine the minimum concentrations detectable by the FT-Raman and FT-IR methods 4 Quantitatively evaluate the concentration of metanil yellow mixed with turmeric using FT-Raman and FT-IR methods

2. Materials and Methods

2.1. Sample Preparation Metanil yellow (70% dye, Aldrich, Carson City, NV, USA), organic turmeric powder (Norway, IA, USA), and de-ionized water (Thermo Scientiﬁc, St. Louis, MO, USA) were used to prepare sample mixtures. The high solubility of metanil yellow in water was used to obtain nicely mixed samples by ﬁrst adding 0.5 g of de-ionized water at room temperature to 0.01 g of metanil yellow, followed by 3 min of vortex mixing. An appropriate amount of turmeric powder was then added to the metanil yellow to achieve the targeted sample concentration. Samples were prepared in this way for eight concentrations of metanil yellow: 30%, 25%, 20%, 15%, 10%, 5%, 1%, and 0.01% (w/w). Three replicates of each Foods 2016, 5, 36 3 of 15 concentration were prepared for FT-Raman analysis, and three replicates were similarly prepared for FT-IR analysis.

2.2. FT-Raman and FT-IR Spectral Acquisition A Fourier Transform spectroscopy (Thermo Scientific, Madison, WI, USA) that consisted of FT-IR module (Nicolet 6700, Thermo Scientific, Madison, WI, USA) and FT-Raman module (NXR-FT Raman module, Thermo Scientific, Madison, WI, USA) was utilized for collection of spectral data from samples. Each spectroscopy was designed with a separate operation chamber. The FT-IR module consisted of a triglycine sulfate (DTGS) detector with KBr beam splitter for collection of sample spectra in the spectral range of 650 cm´1 to 4000 cm´1. The attenuated total reflection (ATR) technique was utilized for FT-IR spectral collection. A small amount of the sample was placed on the Germanium crystal of the ATR device pressurized by pointed tip to ensure uniformity in surface area of contact between the Germanium crystal and sample. A background spectra was first acquired with the empty Germanium crystal prior to spectral collection of the sample. The crystal plate and pointed pressure tip of the ATR device was cleaned thoroughly using cotton soaked with methanol after spectral acquisition of each sample. An average of 32 successive scans at 4 cm´1 intervals were acquired and saved (“comma separated values” format) for further analysis. The FT-IR spectral signal of three replicate samples were acquired at each concentration level. FT-Raman spectroscopy utilized a Germanium detector and CaF2 beam splitter for spectral acquisition in the spectral range of 100 cm´1 to 3700 cm´1. The Germanium detector was cooled by liquid nitrogen to reduce dark current noise and enhance signal-to-noise ratio. A Nuclear Magnetic Resonance (NMR) tube sampling accessory was used to hold the NMR tube in the FT-Raman compartment. Samples were put in NMR tubes (5 mm diameter, 50 mm length) to fill approximately one fourth of each tube’s length. Each sample tube was clamped in the NMR sampling accessory inside the FT-Raman compartment one at a time. The accessory was designed to move in horizontal and vertical directions using two stepper motors. Horizontal movement ensured the alignment of the NMR tube with the laser, and vertical movement was used to achieve the best laser focus. A laser source of 1064 nm (laser power 1 W) was used for sample excitation. An average of 32 successive scans at 8 cm´1 intervals were acquired and saved (“comma separated values” format) for further analysis. The FT-Raman spectral signal of three replicate samples were acquired at each concentration level. The overall system was controlled by OMNIC software provided by Thermo Scientific (Madison, WI, USA). Prior to collection of the spectral signal, OMNIC software was adjusted to operate in FT-IR module (for FT-IR spectral collection) and FT-Raman module (for FT-Raman spectral collection).

2.3. Processing of Spectra Signal Pre-treatments for the FT-Raman and FT-IR spectra were performed using Matlab (MathWorks, Natick, MA, USA). The raw FT-Raman spectral data were first treated for noise removal by applying a Savitzky-Golay filter, fitting a 5th order polynomial with 3-point interval by linear least squares method. Polynomial curve fitting was then used for removal of the fluorescence background, which is important even though the FT-Raman technique utilizing 1064 cm´1 is reportedly affected less by fluorescence background compared to other Raman techniques. Although other methods are available, the polynomial fitting method was utilized in this study because of its simplicity [50,51]: a polynomial curve is generated to fit the background fluorescence signal, and this curve is subtracted from the Raman spectra to remove the fluorescence background from the Raman spectral data. A 6th order polynomial curve was generated to fit the background fluorescence signal in the range of 850 cm´1 to 2800 cm´1. Since all the spectral peaks of metanil yellow, turmeric, and mixture samples were inside this spectral range, all further analysis used only the corrected FT-Raman data from this region. Similarly, the raw FT-IR spectra were first pre-treated for noise removal by a Savitzky-Golay filter using a 5th order polynomial with 3-point interval to fit the spectral signal by linear least squares Foods 2016, 5, 36 4 of 15 method. Multiplicative Scatter Correction (MSC) was utilized to remove the baseline drift from the FoodsFT-IR 2016 spectra, 5, 36 of mixture samples. 4 of 15 Two spectral analysis methods were compared for evaluating metanil yellow concentration in correlatedthe mixture with samples. the ac First,tual the concentration spectral peak of intensity metanil of yellow metanil in yellow the mixture after data-pretreatment sample. The second was approachcorrelated was with athe band actual ratio concentration method, whereby of metanil a yellow ratio inwas the obtained mixture sample. of the Theintensity second at approach a peak representativewas a band ratio of metanil method, yellow whereby against a ratio the was intensity obtained of a of neutral the intensity spectral at band, a peak and representative the ratio was of correlatedmetanil yellow with metanil against yellow the intensity concentration of a neutral in the spectral sample band,mixtures. and The the neutral ratio was band correlated was selected with asmetanil being yellowone not concentration exhibiting any in thesharp sample peaks mixtures. due to metanil The neutral yellow, band turmeric was selected powder as, beingor any one other not chemicalexhibiting constituents. any sharp peaks Each due of to these metanil two yellow, methods turmeric was powder, applied or using any other the chemical FT-Raman constituents. spectral measurementsEach of these two and, methods separately, was using applied the usingFT-IR thespectral FT-Raman measurements. spectral measurements and, separately, using the FT-IR spectral measurements. 3. Results and Discussion 3. Results and Discussion 3.1. Spectral Interpretation 3.1. Spectral Interpretation The comparable degree of extended conjugation in both metanil yellow and curcumin/turmeric is responsibleThe comparable for the similar degree yellow of extended color present conjugation in each in bothchemical metanil substance. yellow andHowever, curcumin/turmeric the chemical compositionis responsible in for each the is similar very different. yellow color Metanil present yellow in each contains chemical three substance. nitrogen atoms However, (N=N the and chemical –NH) andcomposition one sulfate in (SO each3−) is group; verydifferent. it contains Metanil no methylene yellow (CH contains2) or methyl three nitrogen (CH3) groups, atoms (N=Nand no and oxygens –NH) ´ exceptand one for sulfate those (SOon 3the) group;sulfate itsite contains (Figure no 1a). methylene Curcumin/Turmeric (CH2) or methyl contains (CH3) groups,six oxygen and atoms, no oxygens two ofexcept which for are those in carbonyl on the sulfate groups site (C=O), (Figure no1a). nitrogen Curcumin/Turmeric or sulfur atoms, contains two sixmethyl oxygen groups, atoms, and two one of singlewhich methylene are in carbonyl site at groups the center (C=O), of the no nitrogenmolecule orbetween sulfur atoms,the two two carbonyl methyl sites groups, (Figure and 1b). one Either single onemethylene of the ketones site at the in centerthe O=C of– theCH molecule2–C=O site between can also the be two in the carbonyl enol form, sites (FigureO=C–CH1b).–C Either–OH, onewhich of extendsthe ketones the in degree the O=C–CH of conjugation2–C=O site can but also does be in notthe enol change form, the O=C–CH–C–OH, chemical composition which extends of curcumin/turmeric.the degree of conjugation but does not change the chemical composition of curcumin/turmeric.

(a) (b)

FigureFigure 1. 1. ChemicalChemical structure structure of: of: Metanil Metanil yellow yellow ( (aa),), and and Curcumin/Turmeric Curcumin/Turmeric ( (bb).).

BothBoth chemicals chemicals contain contain substituted substituted aromatic aromatic compounds. compounds. In In metanil metanil yellow, yellow, the the three three a aromaticromatic ringsrings (I, (I, II, II, and and III) III) are are not identical: I I is is mon monosubstituted;osubstituted; II II is is di di (1,4 (1,4-)-substituted,-)-substituted, and and III III is is di di (1,3(1,3-)-substituted.-)-substituted. In Curcumin/Turmeric, the twotwo ringsrings (A(A andand B) B) are are structurally structurally identical identical and and are are tri tri(1,3,4-)-substituted. (1,3,4-)-substituted. BecauseBecause the the chemical chemical structure structure of of the the two two compounds compounds are are so so dissimilar, dissimilar, the the vibrational vibrational modes modes specificspeciﬁc to to precisely precisely their their discretely discretely different different chemical chemical structures structures will will be be different. different. Figures Figures 2 and3 3 showshow the FT-Raman FT-Raman and and FT-IR FT-IR spectra, spectra, respectively, respectively, of metanil of metanil yellow, yellow, turmeric, turmeric, and curcumin and curcumin powder powder(Aldrich, (Aldrich, Carson Carson City, NV, City, USA). NV, TableUSA).1 Tablepresents 1 presents the vibrational the vibrational modes modes of metanil of metanil yellow yellow and andturmeric turmeric in the in Infrared the Infrared (IR) and (IR Raman) and frequency Raman frequency range. The range. assignment The assignment of structural of components structural componentsto vibrational to frequencies vibrational is frequencies critical in this is critical study because in this structurally study because different structurally components different can componentscoincidentally can have coincidentally vibrational have modes vibrational that appear modes in close that or appear even thein close same or frequency even the ranges. same frequencyExperimental ranges. evidence Experimental is required evidence to assign is required the spectral to assign regions the and spectral their relativeregions and intensities, their relative and to intensities,determine whichand tointensities determine co-vary which intensities with concentration. co-vary with The experimentalconcentration spectral. The experimental regions most spectral unique regionsto each most component, unique andto each preferably component, with theand highest preferably relative with intensity, the highest can berelative used tointensity, ﬁngerprint can thebe used to fingerprint the presence of metanil yellow in curcumin/turmeric. Because of the abundance of spectral information collected in this experiment, further analysis of the data already collected can be used as confirmatory evidence in the fingerprint data. In metanil yellow, sharp vibrational modes specific to the N=N site (IR 1597 cm−1, 1140 cm−1; Raman 1606 cm−1, 1452 cm−1, 1437 cm−1, and 1147 cm−1) [62–64] are most definitive to its identification

Foods 2016, 5, x 5 of 15

sulfate (IR 1043 cm−1) [65] are confirmatory of metanil yellow presence. Because metanil yellow lacks any CH3 or CH2 sites, peaks near 3000 cm−1 (IR 3014 cm−1 and 2976 cm−1) would be CH sites, but

Foodsassigning2016, 5 ,the 36 peaks to any specific CH site would be far from definitive. The site that results in5 of the 15 unique CH bending peak (IR 1188 cm−1 and Raman 1190 cm−1), however, most likely correlates to one of these CH stretching frequencies. Peaks above 3100 cm−1 are too broad, and often moisture presencedependent of as metanil well, yellowto be analytically in curcumin/turmeric. useful. The CH Because bending of the vibrational abundance modes of spectral on aromatic information sites collectedbelow 950 in thiscm−1 experiment, will be different further between analysis ofmetanil the data yellow already and collected curcumin/turmeric can be used as and conﬁrmatory could be evidenceconfirmative in the evidence ﬁngerprint of its data. presence.

(a)

(b)

(c)

Figure 2. FT-Raman spectra of: (a) Metanil yellow; (b) Turmeric powder; (c) Curcumin. Foods 2016, 5, 36 6 of 15

(c) Foods 2016, 5, 36 6 of 15 Figure 2. FT-Raman spectra of: (a) Metanil yellow; (b) Turmeric powder; (c) Curcumin. 0 4 1 1 ) s t i n U

. b r a (

8 e 8 c 1 n 1 a b 7 r 4 o 0 1 s b A 7 2 9 4 6 3 5 2 1 7 2 1 1 5 9 4 1 4 4 4 1 2 3 1 1 0 5 5 0 0 3 3 4 4 1 0 3 1 9 8 9 1 4 8 1

-1 Wavelength (cm ) (a) 2 3 0 1 ) s t i n 2 1 U 5

. 3 1 5 b 1 r 8 1 a 2 2 ( 6 8

1 2 3 e 1 0 c 7 6 0 n 1 1 2 3 a 1 4 b 1 r 3 o 1 s 8 9 b 0 7 5 3 A 8 1 4 2 9 2

Wavelength (cm-1)

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2 9 2 7 e 1 3 1 c 0 1 n 8 1 1 3 2 a 3 1 6 0 b 8 1 6 r 1 6 o 5 s 8 b A 9 7 7 3 8 1 0 8 1 5 3

Wavelength (cm-1)

(c)

FigureFigure 3. 3.Fourier Fourier Transform-Infra Transform-Infra Red Red (FT-IR) (FT-IR) spectra spectra of: of: ((aa)) MetanilMetanil yellow; ( (bb)) Turmeric Turmeric powder; powder; (c) (c)Curcumin. Curcumin.

Foods 2016, 5, 36 7 of 15

Table 1. Assignment of FT-Raman and FT-IR spectral bands [62–65].

Metanil Yellow Turmeric Infra Red (IR) Raman (cm´1) Assignment IR (cm´1) Raman (cm´1) Assignment (cm´1) 3342 br O–H str intermolecular bonded 3064 ν (C–H) 3073 O–H str alcohol 3030 3043 ν(C–H) 3014 sharp 3017 2976 sharp 2974 2956 2959 2923 weak 2926 weak 2924 2874 2854 2855 1739 C=O stretching 1681 Conjugated C=O stretching 1628 1630 Disubstituted C=C stretching 1597 sharp 1606 sharp ν(N–N) stretching (III) 1603 1603 C=C stretching 1581 1591 ν(C–C) stretching (III) 1585 1534 δ(Ar–O + Ar–O–R) bending 1514 1524 weak ν(C–C) stretching (III) 1512 1505 1493 1496 weak ν(C–C) stretching (I) 1475 ν(N=N), δCH 1465 CH bending 1455 weak 1452 sharp ν(N=N), δCH 1451 1456 1431 1437 sharp ν(N=N) 1431 1429 1412 1417 ν(N=N) stretching (I) 1400 1402 S=O str C–H bending in 1379 1374 O=C–CH2–C=O 1371 S=O str 1343 S=O str 1325 νas(SO2) 1318 1304 1306 νC–C stretching (III) 1309 1282 νC–N stretching 1282 1264 weak ν(C–Nazo)δ(C–H) 1268 COH + CO(CH3) stretching 1232 1243 weak νC–X stretching (I) 1234 1244 CH bending 1223 1218 weak δ(N–H) 1207 1202 C–O–(CH3) stretching 1188 1190 δ(C–H) 1187 1186 CH3 deformation 1171 ν(C–Nazo)δ(C–H) 1173 CH bending 1156 1140 sharp 1147 sharp ν(C–Nazo)δ(C–H) 1115 sharp 1120 1125 1126 1082 1084 weak βCH bending (II) ip 1076 1084 1047 1052 weak 1045 1034 νs(SO3) 1032 1028 C–O stretching 1023 1026 weak 997 997 sharp Ring breathing (II) 969 966 =CH wag trans 945 936 935 weak 930 938 908 γCH wagging (I,II) op 901 882 weak γCH wagging (II) op 888 Ar CH bending 870 weak 866 weak γCH wagging (I) op 870 860 Ar CH bending 845 842 weak γCH wagging (II) op 856 Ar CH bending 830 γCH wagging (III) op 837 810 813 weak 1,4-Ar CH bending 813 812 Ar CH bending

In metanil yellow, sharp vibrational modes specific to the N=N site (IR 1597 cm´1, 1140 cm´1; Raman 1606 cm´1, 1452 cm´1, 1437 cm´1, and 1147 cm´1)[62–64] are most definitive to its identification and quantitation. Additionally, peaks due to ring breathing in ring II (IR and Raman 997 cm´1), and sulfate (IR 1043 cm´1)[65] are confirmatory of metanil yellow presence. Because ´1 ´1 ´1 metanil yellow lacks any CH3 or CH2 sites, peaks near 3000 cm (IR 3014 cm and 2976 cm ) would be CH sites, but assigning the peaks to any specific CH site would be far from definitive. The site that results in the unique CH bending peak (IR 1188 cm´1 and Raman 1190 cm´1), however, most likely correlates to one of these CH stretching frequencies. Peaks above 3100 cm´1 are too broad, and often moisture dependent as well, to be analytically useful. The CH bending vibrational modes on aromatic Foods 2016, 5, 36 8 of 15 Foods 2016, 5, 36 8 of 15

´1 γCH wagging (I,II) sites below908 950 cm will be different between metanil yellow and901 curcumin/turmeric and could be confirmative evidence of its presence.op γCH wagging (II) 882 weak 888´ 1 ´1 Ar CH bending In turmeric, vibrational modes betweenop 1740 cm and 1628 cm cannot be present in metanil yellow870 weak because the866 carbonyl weak γ groupCH wagging and (I) conjugated op 870carbonyl groups860 are onlyAr present CH bending in turmeric γCH wagging (II) components.845 An example842 weak where structurally dissimilar856 sites can have similar vibrationalAr CH bending modes is methyl deformation (IR 1187 cm´1,op Raman 1186 cm´1) versus C–H bending in metanil yellow (IR γCH wagging (III) 830´ 1 ´1 837 1188 cm , Raman 1190 cm ). Twoop other vibrational modes fully discrete from metanil yellow ´1 are from810 aromatic813 (Ar) weak bending 1,4-Ar at CH Ar–O bending and Ar–O813 (CH 3) (Raman812 1534 cm )Ar and CH CHbending bending at ´1 ´1 O=C–CH2–C=O (IR 1379 cm and Raman 1374 cm ). Curcumin is is a a chemically chemically purified purified fraction fraction of of turmeric. turmeric. Vibrational Vibrational spectroscopy spectroscopy can can identify identify a structurala structural difference difference between between curcumin curcumin and and turmeric turmeric based based upon upon which which vibrational vibrational modes modes are areno longerno longer present present in the in purified the purified fraction. fraction. Curcumin Curcumin has only has one only vibrational one vibrational mode for modeC=O (IR for 1628 C=O cm (IR−1, Raman1628 cm 1630´1, Raman cm−1) whereas 1630 cm turmeric´1) whereas also turmerichas two other also hasC=O two peaks other (IR C=O 1739 peakscm−1 and (IR 1681 1739 cm−´1).1 Theand changes1681 cm ín1 ).the The Ar– changesCH vibrational in the Ar–CHmodes vibrationalbetween the modes two below between 950 thecm−1 two in both below IR 950and cm Raman´1 in spectraboth IR suggest and Raman turmeric spectra may suggest contain turmericstructural may analogs contain of curcumin structural as analogswell. The of Ar curcumin–OH and asAr well.–O– CHThe3 Ar–OHsite at 1,3,4 and- Ar–O–CH on both rings3 site A at 1,3,4-and B oncould both be rings a 1,2,4 A and- at BA could or B, beor athe 1,2,4- methyl at A group or B, or could the methyl be at thegroup 3- site could instead be at of the, or 3-even site in instead addition of, to or, the even 4- site. in addition Additional to, thechemical 4- site. and Additional spectroscopic chemical analys andes wouldspectroscopic be required analyses to confirm would the be required identity toof confirmthe other the components identity of in the turmeric. other components Quantitative in turmeric.analysis isQuantitative required to analysisdetermine is requiredthe mole toratio determine of components. the mole Although ratio of components. the chemical Although structure theof curcumin chemical canstructure be determined of curcumin precisely, can be determinedthe ratio of curcumin precisely, theto that ratio of of other curcumin components to that in of otherturmeric components depends onin turmericvariables depends including on botanical variables and including horticultural botanical factors and [3,8,24 horticultural–31]. factors [3,8,24–31].

3.2. Evaluation Evaluation of of Metanil Yellow in Mixture by FT-RamanFT-Raman ´1 ´1 Figure 44 shows the the original original FT FT-Raman-Raman spectra spectra in inthe the 250 250 cm− cm1 to 3500to cm 3500−1 region cm forregion all twenty for all- fourtwenty-four sample mixtures sample mixtures (three replicates (three replicates for each of for eight each concentrations). of eight concentrations). A majority Aof majority the FT-Raman of the peaksFT-Raman of turmeric peaks and of turmeric metanil andyellow metanil are exhibited yellow areby the exhibited mixture by sa themples. mixture However, samples. the FT However,-Raman ´1 ´1 ´1 peaksthe FT-Raman of turmeric peaks at 1244 of turmeric cm−1 and at of 1244 metanil cm yellowand of at metanil 1120 cm yellow−1, 1282 at 1120cm−1, cmand 1475, 1282 cm cm−1 are, andnot ´1 seen1475 in cm the mixtureare not seen sample in the spectra mixture at any sample concentration. spectra at anySimilarly, concentration. FT-Raman Similarly, peaks of FT-Raman metanil yellow peaks ´1 ´1 ´1 ´1 ´1 atof 1402 metanil cm− yellow1, 1437 cm at 1402−1, and cm 1452, 1437cm−1 cmhave ,shifted and 1452 to 1406 cm cmhave−1, 1433 shifted cm− to1, and 1406 1452 cm cm, 1433−1, respec cm tively,, and ´1 in1452 the cm mixture, respectively, spectra. in the mixture spectra. 3 0 6 1 0 3 6 1 y t i 0 7 s 9 4 1 1 n 6 1 1

e 0

t 4 6 1 n 8 I 1

1 2 e 3 5 v 3 2 i 4 1 t 1 3 a l 1 6 e 3 5 1 R 4 6 1 6 9 7 9 9

-1 Raman Shift (cm ) Figure 4. Original FT-Raman spectra of mixture samples at all concentration levels. Figure 4. Original FT-Raman spectra of mixture samples at all concentration levels.

The FT FT-Raman-Raman spectra were first first smoothed using a Savitzky Savitzky-Golay-Golay filter filter (5th order polynomial curve at 3-point3-point intervals) to remove noise. noise. Each Each spectrum spectrum was was then then corrected corrected to to remove the underlying fluorescence fluorescence background background by by subtracting subtracting a a 6th 6th order polynomial function function fitted fitted to the spectral fluorescence. During the spectral correction it was observed that the 6th order polynomial curve best fit the spectral region of 850 cm−1 to 2800 cm−1. Since all the spectral peaks of metanil yellow

Foods 2016, 5, 36 9 of 15 and turmeric were located in this range, only this region was utilized for spectral correction and further analysis. Figure 5 shows the corrected spectra of representative samples at each of the concentration levels. As discussed earlier, the FT-Raman spectral peaks due to N=N (at 1606 cm−1, 1452 cm−1, 1437 cm−1, and 1147 cm−1), at 997 cm−1 (ring breathing in ring II), and at 1402 cm−1 due to S=O stretching can be utilized for identification of metanil yellow. Although each of these spectral peaks are representative peaks of metanil yellow, a few of them happen to overlap with those of turmeric powder. For example, the metanil yellow peak at 1606 cm−1 (due to N=N) is very close to the turmeric peak at 1603 cm−1 (due to C=C stretching). Similarly, the metanil yellow peak at 1437 cm−1 shifted to a new position at 1433 cm−1 (Figure 5), which is also in close proximity to the turmeric peak at 1429 cm−1 (Figure 2b). Because the metanil peaks at 997 cm−1, 1406 cm−1, and 1147 cm−1 did not overlap with any turmeric peaks, these three peaks were initially considered as representative FT-Raman peaks for metanil yellow. In addition to uniqueness or non-overlap of each compound’s characteristic peaks, selection criteria for representative peaks also included the spectral intensity of the characteristic peaks. Although the metanil yellow peaks at 997 cm−1 and 1147 cm−1 did not overlap with those of Foodsturmeric2016, 5powder,, 36 the mixture samples only exhibited these key peaks for the higher concentrations9 of 15 of metanil yellow (Figure 5). Consequently, the FT-Raman peak at 1406 cm−1 was the final selection −1 spectralfor representi ﬂuorescence.ng metanil During yellow. the spectral The intensity correction at it 1406 was observed cm is high that the for 6th samples order polynomial with high curveconcentrations best ﬁt the of spectralmetanil yellow region and of 850 the cm intensity´1 to 2800 reduces cm´ 1gradually. Since all with the spectraldecreasing peaks concentration, of metanil yellowshowing and a relativeturmeric relationship were located between in this range, peak onlyintensity this region and metanil was utilized yellow for concen spectraltration correction in the −1 andsamples. further The analysis. minimum Figure concentration5 shows the of corrected metanil spectra yellow of for representative which the peak samples at 1406 at each cm of was the concentrationdetectable was levels. 1%. 3 0 6 1 7 6 0 4 0 3 1 y 4 6 1 t 1 1 i s n e t n I

3 0 e 3 9 4 v 1 i 1 1 t

a 6 l 8 e 1 1 R 2 5 2 1 6 3 6 1 9 3 1 7 9 9

-1 Raman Shift (cm ) Figure 5. Corrected FT-Raman spectra for representative samples at each concentration level. Figure 5. Corrected FT-Raman spectra for representative samples at each concentration level.

´ ´ AsThe discussedspectral peak earlier, position the FT-Ramanis utilized to spectral identify peaks and distinguish due to N=N chemicals (at 1606 in cma mixture1, 1452 sample, cm 1, ´ ´ ´ ´ 1437while cm the 1intensity, and 1147 of the cm spectral1), at 997 signal cm can1 (ring be utilized breathing to evaluate in ring II), the and chemical at 1402 concentration cm 1 due to in S=O the stretchingmixture. Two can bemodels, utilized one for based identification on spectral of metanilpeak intensity yellow. at Although 1406 cm each−1 and of the these other spectral on the peaks ratio are of representativethe peak intensity peaks against of metanil intensity yellow, at a a neutral few of them band, happen were developed to overlap withfor evaluating those of turmeric metanil powder. yellow ´ Forconcentration example, the in turmeric metanil yellow powder. peak The at first 1606 model cm 1 evaluated(due to N=N) the intensity is very close of the to metanil the turmeric yellow peak peak at ´1 ´1 1603at 1406 cm cm(due−1 after to C=Cfluorescence stretching). removal Similarly, against the the metanil metanil yellow yellow peak concentrations at 1437 cm shiftedof the samples. to a new ´ ´ positionFigure 6 atshows 1433 the cm relation1 (Figure between5), which the is metanil also in yellow’s close proximity peak intensity to the turmericand its actual peak concentration at 1429 cm 1 ´ ´ ´ (Figurefor samples2b). Becauseat seventhe different metanil concentrations, peaks at 997 cm with1 ,a 1406correlation cm 1, andcoefficient 1147 cm of 0.91 did3. not overlap with any turmeric peaks, these three peaks were initially considered as representative FT-Raman peaks for metanil yellow. In addition to uniqueness or non-overlap of each compound’s characteristic peaks, selection criteria for representative peaks also included the spectral intensity of the characteristic peaks. Although the metanil yellow peaks at 997 cm´1 and 1147 cm´1 did not overlap with those of turmeric powder, the mixture samples only exhibited these key peaks for the higher concentrations of metanil yellow (Figure5). Consequently, the FT-Raman peak at 1406 cm ´1 was the final selection for representing metanil yellow. The intensity at 1406 cm´1 is high for samples with high concentrations of metanil yellow and the intensity reduces gradually with decreasing concentration, showing a relative relationship between peak intensity and metanil yellow concentration in the samples. The minimum concentration of metanil yellow for which the peak at 1406 cm´1 was detectable was 1%. The spectral peak position is utilized to identify and distinguish chemicals in a mixture sample, while the intensity of the spectral signal can be utilized to evaluate the chemical concentration in the mixture. Two models, one based on spectral peak intensity at 1406 cm´1 and the other on the ratio of the peak intensity against intensity at a neutral band, were developed for evaluating metanil yellow concentration in turmeric powder. The first model evaluated the intensity of the metanil yellow peak at 1406 cm´1 after fluorescence removal against the metanil yellow concentrations of the samples. Figure6 shows the relation between the metanil yellow’s peak intensity and its actual concentration for samples at seven different concentrations, with a correlation coefficient of 0.93. Foods 2016, 5, 36 10 of 15 Foods 2016, 5, 36 10 of 15 Foods 2016, 5, 36 10 of 15 60

60 R² = 0.93 50 R² = 0.93

y 50 t i s n 40 y e t i t s n n I

40 e t k n a I

e k P

30 a l e a P r 30 l t a c r e t c p e S

20 p S 20

10 10

0 0 0 0 5 5 1010 1515 2020 2525 30 30 35 35 MMeteatnanili lY Yeelllloow Conncceenntrtaratitoino n(% (%) ) Figure 6. Linear relation between FT-Raman spectral peak intensity and corresponding FigureFigure 6. Linear 6. Linear relation relation between between FT FT-Raman-Raman spectral spectral peak peak intensity intensity and and corresponding corresponding sample sample sampleconcentration concentration.. concentration. For the second model, a neutral FT-Raman band—one without any sharp peaks from metanil For the second model, a neutral FT-Raman band—one without any sharp peaks from metanil For the second model, a neutral FT-Raman band—one without any sharp peaks−1 from metanil yellow or turmeric powder—was selected for use with the metanil yellow peak at 1406 cm in´ a1 band yellow or turmeric powder—was selected for use with the metanil yellow peak at 1406 cm −1 in a band yellowratio or turmeric calculation. powder Several— was neutral selected bands for were use tested with the for metanil band ratio yellow calculation. peak at For 1406 the cm mixture in a band ratio calculation.samples, calculation. the Several ratio Several of neutral the neutral intensities bands bands were at these were tested two tested for bands band for was bandratio correlated calculation. ratio calculation. to actual For the metanil Formixture the yellow samples, mixture thesamples, ratioconcentration of the the ratio intensities with of thea much at intensities these lower two co rrelation at bands these wascoefficient. two correlated bands was to correlatedactual metanil to actual yellow metanil concentration yellow withconcentration a much lower with correlationa much lower coefﬁcient. correlation coefficient. 3.3. Evaluation of Metanil Yellow in Mixture by FT-IR 3.3. Evaluation of Metanil Yellow in Mixture by FT-IR 3.3. EvaluationA Nicolet of Metanil 6700 FT Yellow-IR spectrometer in Mixture was by FTutilized-IR to acquire FT-IR spectra in the spectral range of 650 cm−1 to 4000 cm−1. Figure 7 shows the original FT-IR sample spectra, each acquired as an average A Nicolet 6700 FT-IRFT-IR spectrometer was utilized to acquire FT-IRFT-IR spectra in the spectral range of of´ 321 successive scans.´1 Both metanil yellow and turmeric exhibit FT-IR peaks that may be utilized for 650 cm −1 toto 4000 4000 cm cm−1. Figure. Figure 77 shows shows the the original original FT FT-IR-IR sample sample spectra, spectra, each acquiredacquired asas anan averageaverage detecting metanil yellow in mixture with turmeric. The metanil yellow peaks at 997 cm−1 and 1597 cm−1 of 32 successive scans. scans. Both Both metanil metanil yellow yellow and and turmeric turmeric exhibit exhibit FT FT-IR-IR peaks peaks that that may may be utilized be utilized for due to the N=N site (Figure 3a) are not present in the FT-IR spectra of the sample mixtures (Figure ´7).1 for detecting metanil yellow in mixture with turmeric. The metanil yellow peaks at−1 997 cm and−1 detectingSince metanil the peak yellow at 1140 incm mixture−1 (due to with the N=N turmeric. site in metanilThe metanil yellow) yellow shows peaks high intensityat 997 cm in the and sample 1597 cm 1597 cm´1 due to the N=N site (Figure3a) are not present in the FT-IR spectra of the sample mixtures due tospectra the N=N (Figure site 7()Figure and does 3a) notare overlapnot present with inany the peaks FT- IRof spectraturmeric of powder the sample (Figure mixtures 3b), this (FTFigure-IR 7). ´1 (FigureSincepeak the7). peak Sincewas selected at the 1140 peak forcm atidentifying−1 (due 1140 cmto the metanil N=N(due yellow site to the in in N=Nmetanil the mixture site yellow) in metanil sample. shows yellow) high showsintensity high in intensitythe sample in thespectra sample (Figure spectra 7) and (Figure does7 )not and overlap does not with overlap any peaks with anyof turmeric peaks of powder turmeric (Figure powder 3b), (Figure this FT3b),-IR 0 this FT-IR peak was selected for identifying4 metanil yellow in the mixture sample. 1

peak was selected for identifying1 metanil yellow in the mixture sample. ) 0 s 4 t i 1 1 n U

. b r a (

e ) c s t n i a n b r U

o . s b b r 4 A a 3 ( 0

6 3 1 7 e 5 4 2 9 c 0 1 1 2 4 1 0 4 n 1 1 1 5 0 a 4 3 b 9 r o s b 4 A 3 0 6 3 1 7 5 4 2 9 0 1 1 2 4 1 0 4 1

1 1 -1 5 Wavelength (cm ) 0 4 3

Figure 7. Original FT-IR spectra of mixture samples for all concentration levels.

Figure 8 shows the representative FTWa-vIRele n spectragth (cm-1) of mixture samples at seven different concentration levels after noise removal using a Savitzky-Golay filter. The spectral peak at 1140 cm−1 Figure 7. Original FT FT-IR-IR spectra of mixture samples for all concentration levels.

Figure8 8shows shows the the representative representative FT-IR FT-IR spectra spectra of of mixture mixture samples samples at at sevenseven differentdifferent concentration levelslevels afterafter noisenoise removalremoval usingusing aa Savitzky-GolaySavitzky-Golay ﬁlter.filter. The The spectral spectral peak peak at at 1140 1140 cm cm´−11

Foods 2016, 5, 36 11 of 15

Foods 2016, 5, 36 11 of 15 is readily visible for sample concentrations from 30% to 5%, but vanishes at 1%. This illustrates that is readily visible for sample concentrations from 30% to 5%, but vanishes at 1%. This illustrates that ´1 the 1140the 1140 cm cmpeak−1 peak can can be be used used toto detectdetect metanil yellow yellow in inturmeric turmeric as low as low as 5%. as 5%.

Foods 2016, 5, 36 0 11 of 15 4 1 1 is readily visible for sample concentrations from 30% to 5%, but vanishes at 1%. This illustrates that

−1 ) the 1140 cm peak s can be used to detect metanil yellow in turmeric as low as 5%. t i n U

. b r 0 a 4 ( 1

1 e c n a b r o 4 ) s 3 s b t 0 i 1 A n U

. b r 2 a 1 (

5 4 4 e 1 9 c n a b r o 4 s 3 b 0 1 A

Wavelength (cm-1) 2

1 5 4 4 1 9 FigureFigure 8. Representative 8. Representative FT-IR FT-IR spectra spectra of mixture samples samples at atallall concentration concentration levels. levels.

−1 -1 TheThe relation relation between between the the 1140 1140 cm cm´1 peakW a intensityv intensityelength (cm ) and and metanil metanil yellow yellow concentration concentration was was evaluated. For all sample spectra, baseline drift was removed using the Multiplicative Scatter evaluated. For all sample spectra, baseline drift was removed using the Multiplicative Scatter Correction (MSC)Figure method. 8. Representative The spectral FT-IR spectrasignal ofafter mixture baseline samples drift at all removal concentration was levels.utilized to develop Correction (MSC) method. The spectral signal after baseline drift removal was utilized to develop concentration evaluation models. The relation between the 1140 cm−1 peak intensity and metanil yellow concentration was concentrationSimilar evaluation to the FT-Raman models. analysis, two models were developed for evaluation of metanil yellow evaluated. For all sample spectra, baseline drift was removed using the Multiplicative Scatter Similarconcentration to the using FT-Raman the FT analysis,-IR spectra. two The models first model were developed correlated the for spectral evaluation peak of intensity metanil atyellow Correction (MSC) method. The spectral signal after baseline drift removal was utilized to develop concentration−1 using the FT-IR spectra. The first model correlated the spectral peak intensity at 1140concentration cm with actual evaluation metanil models. yellow concentrations. The result shows that the spectral peak intensity ´1 −1 1140at cm 1140with Similarcm actualis tohighly the metanil FT correl-Ramanated yellow analysis, with concentrations. metanil two models yellow were Theconcentration developed result shows for (correlation eva thatluation the ofcoefficient spectral metanil peakyellow of 0.88). intensity ´1 at 1140Theconcentration cm secondis model highly using was correlated the developed FT-IR with spectra. using metanil Thespectral first yellow band model concentrationratio. correlated The spectral the (correlation spectral band peakratio coefficient intensitywas obtained at of 0.88). The secondusing1140 the cm model spectral−1 with was actual peak developed metanil intensity yellow usingat 1140 concentrations. spectral cm−1 and band theThe spectralresult ratio. shows The intensity spectralthat the of spectral a band neutrally peak ratio intensitypositioned was obtained usingspectral theat 1140 spectral band cm−1— is peakone highly where intensity correl theated spectraat with 1140 metanil are cm flat´1 yellow and and the lackconcentration spectral sharp peaks intensity(correlation for metanil of coefficient a neutrally yellow of and0.88). positioned for turmeric or its other constituents. Several such bands were selected and tested. The best result was spectralThe band—one second model where was the developed spectra areusing flat spectral and lack band sharp ratio. peaks The spectral for metanil band ratio yellow was and obtained for turmeric obtainedusing theusing spectral the band peak ratio intensity of 1140 at 1140cm−1 cmagainst−1 and 966 the cm spectral−1. It can intensity be seen of in a theneutrally Figure positioneds 3a,b and 8 or its other constituents. Several such bands were selected and tested. The best result was obtained thatspectral the spectral band— bandone whereat 966 thecm −1 spectra does not are represent flat and lackany sharpchemical peaks in the for sample metanil and yellow has and no sharp for ´1 ´1 usingpeaks. theturmeric band The or ratio ratio its other ofwas 1140 co correlatednstituents. cm against with Several the 966 such actual cm bands concentration. Itwere can selected be seen of and metanil in tested. the Figures yellow The best 3 ina,b result the and samplewas8 that the ´1 spectralmixtures.obtained band The at using 966correlation the cm banddoes coefficient ratio notof 1140 represent of cmthe− 1band against any ratio 966 chemical method cm−1. It wascan inthe befound seen sample toin bethe and0.95, Figure hashighers 3 noa,b than sharpand that8 peaks. The ratioforthat the was spectralthe spectral correlated peak band intensity with at 966 the method,cm− actual1 does as not concentrationshown represent in Figure any ofchemical9. metanil in the yellow sample in and the has sample no sharp mixtures. The correlationpeaks. The coefficient ratio was correlatedof the band with ratio the methodactual concentration was found ofto metanil be 0.95, yellow higher in the than sample that for the mixtures. The correlation coefficient of the band ratio method was found to be 0.95, higher than that spectral peak intensity1 method,8 as shown in Figure9. for the spectral peak intensity method, as shown in Figure 9. R² = 0.95 15 18

R² = 0.95 o

i 12 t 15 a R

d n a

B 9

o l

i 12 t a a r t R c

e d p n S a

6B 9

l a r t c e p 3S 6

0 3 0 5 10 15 20 25 30 35 Metanil Yellow Concentration (%) 0 0 5 10 15 20 25 30 35 Metanil Yellow Concentration (%)

Figure 9. Linear relation between FT-IR spectral peak intensity and sample concentration. Foods 2016, 5, 36 12 of 15

4. Conclusions This study utilized FT-Raman spectroscopy and FT-IR spectroscopy techniques for the detection of metanil yellow in turmeric powder. FT-Raman and FT-IR spectra of metanil yellow, turmeric, and curcumin were acquired and analyzed. The FT-Raman spectral peak at 1406 cm´1 and FT-IR spectral peak at 1140 cm´1 were selected as representative peaks for metanil yellow. FT-Raman and FT-IR spectral analysis of sample mixtures of metanil yellow in turmeric at eight different concentrations showed that the FT-Raman method was able to detect metanil yellow at 1% concentration, while detection by the FT-IR method was limited to 5% concentration. For both FT-IR and FT-Raman analysis, two models were developed to evaluate metanil yellow concentration. The first model simply correlated a single peak intensity (FT-Raman 1106 cm´1 and FT-IR 1140 cm´1) to the actual sample concentration. The second model used a ratio of peak intensity and intensity at a neutral band. The band ratio model performed better for the FT-IR analysis (correlation coefficient of 0.95), while the peak intensity model was superior (correlation coefficient of 0.93) for the FT-Raman analysis. The results show that FT-Raman and FT-IR spectroscopy can be utilized for detection of chemical contaminants in food powders such as carcinogenic metanil yellow in turmeric.

Author Contributions: S.D. and K.C. conceived and designed the experiments; S.D. performed the experiments, J.Q. and M.K. helped in the experiment; S.D., W.S., and K.C. analyzed the data; S.D. and D.C. wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

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