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Contents lists available at ScienceDirect

Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol

2 Advances of infrared spectroscopy in natural product research

3 Q1 Christian W. Huck *
4 Institute of Analytical Chemistry and Radiochemistry, CCB – Center for Chemistry and Biomedicine, Innrain 80-82, 6020 Innsbruck, Austria

A R T I C L E I N F O A B S T R A C T

Article history: Natural product’s properties are related to certain classes of compounds such as alkaloids, flavonoids,
Received 1 October 2014 essential oils and others. Traditionally, separation techniques including thin layer chromatography
Accepted 23 October 2014 (TLC), liquid chromatography (LC), gas chromatography (GC) and capillary electrophoresis (CE) even
Available online xxx
hyphenated to mass spectrometry (MS) were used for the elucidation, qualitative and quantitative
analysis of individual compounds.
Keywords: In food industry, spectroscopic investigations using infrared radiation have been used to monitor and
IR spectroscopy
evaluate the composition and quality already since the early sixties. During the last four decades near-
Near infrared
infrared spectroscopy (NIR; 800–2500 nm; 12,500–4000 cm1) has become one of the most attractive
Mid infrared
Imaging spectroscopy and used methods for analysis for the following reasons: it represents a non-invasive analytical tool
Natural products allowing a fast and simultaneous qualitative and quantitative characterization of natural products and
their constituents. Additionally, the development of custom-made hand-held instruments enables in-
field measurement for determining the optimum harvest time.
Attenuated total reflection (ATR) and Fourier transform infrared (FTIR) spectroscopic imaging are
suitable not only for the differentiation of different plant species, but also to distinct various ingredients
within a plant. FTIR spectroscopic microscopy enables molecular imaging of complex botanical samples
and therefore the detection and characterization of the molecular components of biological tissue.
In the present contribution, the principle, technique and methodology of the different infrared
spectroscopic methods are described followed by a discussion of quantitative and qualitative application
possibilities in the field of natural product analysis.
ß 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.

5
6
7 1. Introduction C–H, C–O, C5 5O, N–H and O–H functional groups are excited to 17
perform stretching-, deformation- and scissor-vibrations. In 18
8 Infrared (IR) radiation is the region of the electromagnetic comparison to the mid-infrared (MIR; 4000–400 cm1) region, 19
9 spectrum between the visible (VIS) and the microwave wavelength where only fundamental vibrations (‘‘signatures’’) can be observed, 20
10 (Mcclure, 2003). In near-infrared (NIR) spectroscopy excitation overtones and combinations can be found in the NIR region 21
11 of molecules is accomplished in a wavelength range between containing a manifold of information compared to MIR (Barton, 22
12 750 and 2500 nm, corresponding to a wavenumber range 2002). The result is often a crowded spectrum containing 23
13 between 4000 and 13,000 cm1 (Herschel, 1800). Solid, liquid or overlapping peaks. Although NIR-intensities are 10–1000 times 24
14 gaseous samples can absorb parts of the incoming IR radiation at lower than for the MIR, highly sensitive spectrometers can be built 25
15 specific wavelength resulting in a fingerprint or a spectrum (Blanco through several means including the use of efficient detectors 26
16 and Villarroya, 2002). In this spectral region molecules containing (Mcclure, 2003). The light recorded by the detector contains 27
compositional information, which can be unraveled by a computer 28
to report multiple analyses almost instantaneously. NIR spectros- 29
copy can provide simultaneous, rapid and non-destructive 30
Abbreviations: ATR, attenuated total reflection; CA, cluster analysis; CE, capillary
electrophoresis; CEC, capillary electro chromatography; DR, diffuse reflection; FPA, qualitative and quantitative analysis of major components in 31
focal plane array; FT, Fourier transform; LC, liquid chromatography; MCT, mercury many organic substances (Guggenbichler et al., 2006; Huck-Pezzei 32
cadmium telluride; MEIRS, material enhanced infrared spectroscopy; MIA, et al., 2013; Schönbichler et al., 2014). The NIR spectrum is 33
multivariate image analysis; MLR, multiple linear regression; MVA, multivariate
represented by a huge number of partially overlapping overtones 34
analysis; NIR, near infrared spectroscopy; PCA, principal component analysis; PLSR,
partial least square regression; TCM, Traditional Chinese Medicine; Vis, visible.
and combination vibrations. Additionally to these vibrations, 35
* Tel.: +43 512 507 57304; fax: +43 512 507 57399. scattering effects, instrumental noise and/or sample in-homo- 36
E-mail address: Christian.W.Huck@uibk.ac.at geneities might occur (Mcclure, 2003). As a consequence it is in 37

http://dx.doi.org/10.1016/j.phytol.2014.10.026
1874-3900/ß 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.

Please cite this article in press as: Huck, C.W., Advances of infrared spectroscopy in natural product research. Phytochem. Lett. (2014),
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38 many applications impossible to correctly assign the correspond- R can be converted to the absorbance A(R) at the detector: 100
99
98
39 ing vibration bands. NIR in combination with multivariate   101
40 statistical analysis (MVA) is a powerful combination enabling IR
AR ¼ log10 ¼ log10 R
41 the extraction of the required information from the spectrum I0
42 (Blanco and Villarroya, 2002). The most appropriate chemome-
43 trical procedure is principal component analysis (PCA) for reducing ‘‘Transmittance’’ T, for measurements of light intensities 102
104
103
44 the number of variables facilitating both qualitative and quantita- transmitted through the sample the is defined as 105
45 tive analysis. For establishing suitable quantitative models, a 106
IT
46 selected set of samples has to be analyzed by highly efficient T¼ ¼ 10eA cd
I0
47 reference methods, mainly based on separation technologies
48 including liquid chromatography (LC) (Stöggl et al., 2005, 2004), which provides for the relation 108
107
49 capillary electrophoresis (CE) (Huck et al., 2011), solid-phase   109
50 extraction (SPE) (Sultan et al., 2005) and mass spectrometric (MS) IT
A ¼ eA cd ¼ log10 ¼ log10 T
51 techniques (Najam-ul-Haq et al., 2007; Vallant et al., 2007). I0
52 Attenuated total reflectance (ATR) is a sampling technique used
Corresponding with the transmission or remission spectra the 110
112
111
53 in conjunction with especially mid-infrared spectroscopy. The ATR
respective absorbances can be expressed as: 113
54 technique enables samples to be examined directly in the solid or
55 liquid state without further preparation (TCH_FTIRATR.pdf). There-     114
1 1
56 by, light undergoes multiple internal reflections in a crystal of high AðTÞ ¼ log10 and AðRÞ ¼ log10
T R
57 refractive index. The sample is in contact with the crystal and due to
58 the penetration of IR radiation into the sample an efficient signal-to- ‘‘Transflectance’’ (T) represents a special case of transmission 115
117
116
59 noise (SNR) can be obtained (Schönbichler et al., 2014). and reflection measurement. This mode is often used for in- and 118
60 Another new spectroscopic method, which is generally called on-line measurements of liquids or turbid solutions whereas the 119
61 Fourier transform infrared (FTIR) spectroscopic microscopy for incoming light beam passes the sample, then gets reflected on a 120
62 examination of plant, food and human tissue sections has been non-absorbing mirror substance (in NIRS often Teflon is taken) and 121
63 introduced (Pallua et al., 2011a). This technique represents a powerful is directed to the detector after penetrating the sample a second 122
64 tool in histological characterization and allows the investigation of time. Due to this doubling of the pathlength, even lower 123
65 the spatial distribution of proteins and small molecules within concentrated analytes can be detected (Fig. 1). 124
66 biological systems with high spatial resolution (Pallua et al., 2011b). Taking the absorbance log (1/T) according to Beer’s law 125
67 Imaging enables probing samples under native conditions and offers absorbance is rigorously valid only for non-scattering samples 126
68 new insights into samples without the need for fixation or an (visually clear liquids). In case of solid samples (powders, bulk 127
69 additional marker (Pallua et al., 2011b; Pezzei et al., 2010). samples), which often show strong scattering effects, the absor- 128
70 In the following chapter, technical achievements and data- bance is called ‘‘pseudo absorbance’’ and does not exactly follow 129
71 processing methods in vibrational spectroscopy of natural Beer’s law due to reflections increasing the extinction coefficient 130
72 products are summarized, their efficiencies are discussed and and due to varying path lengths the light takes through the sample 131
73 compared to other more conventional analytical techniques. (Märk et al., 2010). 132
NIR absorption spectra of liquids, solids and semisolids 133
74 2. Methods usually show broad and overlapping bands (in contrast to MIR 134
absorptions). This makes it often hard to correctly assign the 135
75 In this chapter the main principles of near-infrared (NIR), vibrations to the respective chemical bonds. Therefore NIRS can, 136
76 attenuated total reflection (ATR) and imaging/mapping spectros- in most cases, not be used for structural determination of 137
77 copy are discussed. In the following part, selected applications in particular substances, but serves as a fingerprint technique 138
78 the fields of natural product analysis will be examined carefully. revealing physical as well as chemical properties of the samples. 139
Today chemometrics and MVA are commonly used to extract 140
79 2.1. Near-infrared (NIR) spectroscopy useful information from the spectra and to calibrate for 141
quantitative properties. The next section gives an overview of 142
80 ‘‘Near’’ in NIR is related on the position of the electromagnetic available chemometrical, statistical and multivariate techniques 143
81 energy lying next to or near the visible (Vis) energy range. Molecular to process NIR spectra. 144
82 vibrations in the MIR cover absorptions in a range between 400
83 and 4000 cm1 representing the most intense and simplest bands, 2.1.1. Multivariate NIR data analysis 145
84 whereas NIR bands arise in the interval between 4000 and Multivariate data analysis (MVA) based calibration techniques 146
85 12,500 cm1 covering absorptions corresponding to overtones are applied to link the spectral data with target parameters 147
86 and combinations of fundamental vibrations. The basic principle transferred from reference techniques. In most cases, principal 148
87 describing the vibrating system of a diatomic molecule can be component analysis (PCA) based models like principal component 149
88 described by the harmonic and anharmonic oscillator (NIS, 2008). regression (PCR) and partial least squares regression (PLSR) are 150
89 A FT-NIR instrument consists of an e.g. tungsten-halogen light applied. Even linear regression models like multiple linear 151
90 source, interferometer, light-fiber optics, integrating sphere, regressions (MLR) are frequently used. Both spectra interpretation 152
91 sample cell, detector (e.g. PbS) and plotting unit. Light hitting a techniques, band assignments and MVA, are combined into one 153
92 sample can be reflected, scattered and absorbed by its molecules approach to benefit from reliable and problem specific calibrations. 154
93 and partly transvade the sample. The ratio of the portion reflected For the choice of useful signals, wavelength selection and spectra 155
94 and scattered back upon interactions, i.e. the remitted radiation pretreatment are also crucial. Thereby, the operator can choose 156
95 power IR, to the light intensity incident on the sample is defined as between a broad range of useful spectra pretreatments that help 157
96 the ‘‘remittance’’ R increasing the quality of calibrations available (Faber, 1999). 158
97   Especially early contributions by Savitzky and Golay have become 159
IR standard for recent data pretreatment operations (Savitzky et al., 160
R¼
I0 1964). For more detailed information the interested reader is 161

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Fig. 1. Principles of diffuse reflection, transmission and transflection measurements.

162 referred to the literature (Thurston et al., 2003; Kessler and Kessler, refractive index. ATR uses a property of total internal reflection 188
163 2006; Einax, 2007). resulting in an evanescent wave (Fig. 2). A beam of infrared light is 189
passed through the ATR crystal in such a way that it reflects at least 190
164 2.1.2. Miniaturization of NIR-spectrometers once off the internal surface in contact with the sample. This 191
165 For fast analyses even in the field (e.g. for the determination of reflection forms the evanescent wave which extends into the 192
166 the optimum harvest time) handheld NIR spectrometers show sample. The penetration depth into the sample is typically between 193
167 great promise. Modern miniaturized systems are based on a 0.5 and 2 mm, depending on the wavelength, the angle of incidence 194
168 MEMS (micro-electro-mechanical system) technology offering and the indices of refraction for the ATR crystal and the sample of 195
169 advantages in terms of size, weight, robustness, spectral range interest (Fig. 2) (Fahrenfort, 1961). The beam is then collected by a 196
170 and low-cost manufacturing processes. In such a system, the detector as it exits the crystal. This evanescent principle is only 197
171 reflected light is collected and recombined (using a regular fixed effective if the crystal is made of an optical material with a higher 198
172 grating) onto a single photo detector, a major cost advantage refractive index than the sample being studied. For the analysis of 199
173 compared with diode-array instruments (Sorak et al., 2012). This liquids, pouring a small amount over the surface of the crystal is 200
174 kind of portable NIR analyzer enables an efficient incoming sufficient. Solid samples need to be pressed into direct contact with 201
175 inspection. In contrast to laboratory instrumentation, there is no the crystal. The signal-to-noise ratio (SNR) obtained depends on 202
176 requirement for sampling, a chain of custody, labeling and the number of reflections but also on the total length of the optical 203
177 transportation of the samples and releasing the original container, light path which dampens the intensity. Therefore, a general claim 204
178 but one has to keep in mind that the overall performance is that more reflections give better sensitivity cannot be made. 205
179 below that of a benchtop instruments. Nevertheless, handheld Typical materials for ATR crystals include germanium, KRS-5 and 206
180 spectrometers enable the on-line process inspection enabling a zinc selenide. The excellent mechanical properties of diamond 207
181 dramatic reduction in errors. make it an ideal material for ATR, particularly when studying very 208
hard solids. 209
182 2.2. Attenuated total reflection (ATR) spectroscopy
2.3. Imaging and/or mapping spectroscopy 210
183 Attenuated total reflectance (ATR) is a measurement technique
184 used in conjunction with mainly mid infrared spectroscopy. ATR The first device for infrared micro-spectroscopy was patented 211
185 enables samples to be examined directly in the solid or liquid state (Patent US4877960) in 1989 and the following development 212
186 without further preparation (Tshering Vogel et al., 2010). Thereby, of microprocessor-controlled motorized stages made raster scan 213
187 light undergoes multiple internal reflections in a crystal of high mapping convenient. Functional group maps (images) were 214

Fig. 2. Principle of attenuated total reflection (ATR) spectroscopy.

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215 obtained from baseline-corrected band regions being representa- Table 1

Overview of prominent bands in IR spectra of complex biological samples such as
216 tive for distinct chemical species in tissue samples. The introduc-
complete cells, tissues or microorganisms in the spectral region from 4000 to
217 tion of Focal Plane Array (FPA) systems, initially uncooled Indium 750 cm1.
218 Gallium Arsenide (InGaAs), for near-IR by Mascott and Lewis
Wave number (cm1) Assignation
219 occurred in 1994, while the subsequent development for mid-IR,
220 using a Mercury Cadmium Telluride (MCT) array by Lewis et al. 3300 Amide A, nN–H of proteins
221 (1995) allowed the fast recording of images. 3100 Amide B, nN–H with 1. Overtone of the
amide I band resonant (Fermi), proteins
3010 nC–H, lipids, cholesterols, esters
222 2.3.1. Instrumental setup 2920 alternatively 2850 nC–H ( CH2, Methyl) antisymmetric/
223 From the instrumental point of view, FTIR spectroscopic symmetric, lipids, proteins, carbohydrates,
224 microscopy is the coupling of a microscope to an IR spectrometer. esters
225 Diffraction, refraction, reflection and absorption effects play a 2956 alternatively 2872 nC–H (CH3, Methyl) antisymmetric/
symmetric, lipids, proteins, carbohydrates,
226 much more important role in micro-spectrometry than in its nucleic acids
227 macroscopic counterpart. Microscopes consist in analogy to optical 1745–1735 nC55O, esters phospholipids and lignin
228 microscopes of the light source (single polychromatic thermal 1695–1620 Amide I-band, proteins
229 source), the splitter (Fourier Transform (FT), tunable filter and 1620–1590 nas COO, pectin; nC55C, lignin
1550 Amide II-band, proteins
230 diffraction grating), the detector (photon detectors, lead sulfide
1515 nC55C, lignin
231 (PbS), indium antimonide (InSb) detectors, uncooled Indium 1450 das CH3 and das CH2, proteins, lipids and
232 Gallium Arsenide (InGaAs) and Mercury Cadmium Telluride lignin
233 (HgCdTe or MCT) detectors) and the optics (fitted to a microscope) 1420 ns COO, pectin
234 (Salzer and Siesler, 2009). Advantages are micro spatial chemical 1400 nC55O of COO-group, fatty acids and amino
acids
235 mapping or imaging of complex heterogeneous samples, high 1370 ds CH3 and ds CH2, proteins, lipids and lignin
236 sensitivity, high selectivity, rapid data acquisition, simple sample 1360–1260 Amide III-band absorptions (predominantly
237 preparation. Furthermore, fully automated examination and C–N stretching) with significant
238 computer enhanced visualization can be accomplished (Gendrin contributions from nCH2 of carbohydrate
residues
239 et al., 2008). The generation of the individual images is based on
1350–1260 nPO2
240 distinct bands in the recorded IR spectra. Some prominent bands of 1310–1240 Amide III-band, proteins
241 complex natural product samples can be deduced from Table 1. 1270 nC–C and nC–O lignin
1250–1220 nP55O symmetric of the PO2 groups,
phospholipids, nucleic acids
242 2.3.2. Measurement technologies
1225 nPO2 asymmetric of nucleic acids,
243 The measurement itself can be carried out in the mapping and/ phospholipids and lignin
244 or imaging mode. In FTIR mapping the IR spectra of the sample are 1185–1120 C–O ring vibrations of nucleic acid ‘‘sugars’’
245 collected sequentially at predefined spatial coordinates. This 1084 nP55O symmetric of the PO2 groups
246 technique presents a convenient, fast and cheap rout for analysis nucleic acids, phospholipids
1000 nC–O, carbohydrates
247 of static samples. Mapping provides information about the spatial
248 distribution of the chemical species within the sample to be nas, asymmetric stretch; ns, symmetric stretch; das, asymmetric deformation bend;
249 obtained. In order to examine different areas of a sample many ds, symmetric deformation bend.
250 separate collections must be made. As an alternative point, line or
251 mapping can be applied: the same side of the sample as the source to record the signal 281
reflected by the sample. This sampling technique allows rapid 282
253
254 1. Point mapping: provides several different areas of a sample to be examination of the distribution of organic compounds on a 283
255 analyzed consecutively; the recorded spectra are not related to complex surface of thick samples. There are two reflection 284
256 each other spatially. measurement sampling techniques in FTIR microscopy: Diffuse 285
257
258 2. Line mapping: defines a series of spectra obtained along one Reflection (DR) and Attenuated Total Reflection (ATR). 286
259 dimension, where chemical changes that occur along this
260 dimension are investigated. 2.3.3. Hyperspectral cube 287
261
262 3. Area mapping: defines a series of spectra to be collected in two FTIR microscopic data often contain a huge amount of data 288
263 dimensions; sampling of large areas requires multiple position- because a single acquisition records thousands of images across 289
264 ing of a sample, collection at each spatial positioning and numerous wavelengths. The obtained hyperspectral images 290
265 much time. contain spatial and spectral information about a sample, where 291
the sample is physically persevered and compartmented into small 292
266 From the sampling technical point of view two modes can be surface or volume areas (referred to as pixels or voxels), each of 293
267 differentiated in FTIR microscopy: transmission (IR beam passes them represented by a full spectrum. Hyperspectral images are 294
268 through the sample) and reflection (IR beam reflects from the often displayed as a three-dimensional (3D) matrix or data cube 295
269 sample surface). Transmission analysis requires the sample to spanning two spatial dimensions (x and y) with a series of 296
270 be partly transparent. The sample can only be a few microns in wavelengths making up the third (spectral) axis. From Fig. 3 a 297
271 thickness, which depends on the molar absorptivity. In many cases model of hyperspectral images can be deduced. 298
272 samples must be placed on substrates transparent to the
273 wavelength range of the probing radiation (e.g. MIR) such as 2.3.4. Spectral treatment 299
274 calcium fluoride (CaF2), barium fluoride (BaF2) or zinc selenide Due to the huge amount of data, the complexity, noise level, 300
275 (ZnSe), otherwise no light might be transmitted to the detector. irregularly shaped baseline contributions must be reduced, 301
276 CaF2 is a material with excellent spectral properties for soft tissue anomalous pixel spectra (outliers), instrumental variations need 302
277 FTIR microscopic studies in the MIR region and is almost insoluble to be eliminated. Methods for visualizing the data either comprise 303
278 in water, which allows post-staining of the tissue. In reflection univariate (classical image representation) and/or multivariate 304
279 measurements the IR beam reflects from the sample surface and analysis, respectively. In univariate analysis constituents can be 305
280 the reflected light is recorded by a detector, which is placed on depicted by taking a slice of the image on a particular relevant 306

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Fig. 3. Model of the hyperspectral cube.

307 wavenumber. In order to analyze the spectral and spatial hypericin, 4000–7192 and 7292–10,000 cm1 for rutoside and 347
308 information contained in an image various techniques also known 4000–10,000 cm1 for hyperoside. With the exception of hyper- 348
309 as multivariate image analysis (MIA) have been introduced (Salzer forin NIR delivered generally better calibration models for all 349
310 and Siesler, 2009). In contrast to univariate image analysis MIA the calibrated parameters. This work demonstrates that NIR and 350
311 uses all of the information contained in the hyperspectral image. ATR-IR are suitable methods for the fast quantitative analysis of 351
312 With this technique unlabeled infrared spectral data can be unknown commercial preparates of St. John’s wort – for extracts as 352
313 separated into different clusters based. Clustering also of FTIR well as for herbs. For qualitative analysis deploying PCA models 353
314 microscopic data can be performed such that spectra within the it could be achieved to separate between samples containing 354
315 same cluster are as similar as possible and spectra in different Kushenol H and Kushenol G and samples not containing these 355
316 clusters are as dissimilar as possible where different types of cells substances. This method can be applied for the fast, direct 356
317 may be separated within biological tissue. Clustering techniques identification of products containing herbs with Chinese prove- 357
318 include hierarchical clustering, k-means (KM) clustering and fuzzy nience, which might not be acceptable in some cases according to 358
319 C-means clustering (Pallua et al., 2011b, 2012). the DAC (Huck-Pezzei et al., 2013). 359

320 3. Selected applications in natural product analysis 3.1.2. NIR-TLC 360

The use of near infrared (NIR) spectroscopy hyphenated to thin- 361
321 In the following section selected applications in natural product layer chromatography (TLC) was demonstrated for analysis of 362
322 analysis with special focus on phytomics, metabolomics, proteo- methoxylated flavones in a phytomedicine. Thereby, the following 363
323 mics and food quality are introduced and discussed. two objectives were achieved: first, a method for rapid, qualitative 364
identification of five methoxylated flavones, denoted G1, G2, G3, 365
324 3.1. Phytomics G4, and G5, was achieved, in order of their RF values in normal- 366
phase TLC, and, second, a quantitative model for analysis of G4 367
325 3.1.1. St. John’s wort (30 ,40 ,50 -trimethoxyflavone), the compound most representative of 368
326 In a recent study, ATR-MIR and NIR were conducted to evaluate Primula veris flowers in phytomedicine, was established. To 369
327 their suitability for two purposes in the analysis of St. John’s wort provide appropriate reference analytical data for building the 370
328 products (Huck-Pezzei et al., 2013). In the first step the suitability multivariate cluster and partial least-squares regression (PLS) 371
329 to use these vibrational spectroscopic techniques for fast and non- model, TLC was performed on alumina with n-hexane–ethyl 372
330 invasive quantitative analysis of main ingredients was checked. acetate 70:30 (v/v) as mobile phase. Forty-four spectra of eleven 373
331 For the calibration of the NIR system a HPLC-method was independent phytomedicine samples were analyzed with five 374
332 conducted (Huck et al., 2006). In the following step the efficiency scans to generate a qualitative cluster model based on PCA that 375
333 of a NIRS method to distinguish between St. John’s wort and enabled differentiation between G1 and G5 on the basis of 376
334 products derived there from with provenience from Europe and their methoxylation pattern (Fig. 4). This PLS model, in the 377
335 China was evaluated. This method can now be used as an easy and calibration range between 0 and 1000 mg L1, enabled quantifica- 378
336 efficient tool to identify adulterations. Following the polynomial tion of G4 with a standard error of cross validation (SECV) 379
337 order (first number in the brackets) and the number of smoothing <54.61 mg L1. The possibility of conducting qualitative and 380
338 points (second number in brackets) of the Savitzky Golay quantitative analysis simultaneously by use of this method 381
339 derivatives are summarized: NIR 1st derivative (1/3), NIR 2nd revealed NIRS to be an efficient alternative to conventional modes 382
340 derivative (2/3), NIR 3rd derivative (3/5), ATR-IR 1st derivative (2/ of detection used for analysis of G1–G5, especially in phytome- 383
341 13), ATR-IR 2nd derivative (2/3) and ATR-IR 3rd derivative (3/15). dicines (Mattle et al., 2010). 384
342 The wavenumber ranges utilized for calibrations for ATR-IR were
343 650–1185 and 2801–3670 cm1 for hyperforin, 650–3655 cm1 3.1.3. Antioxidative capacity 385
344 for hypericin, 650–1803 and 2446–3659 cm1 for rutoside and In a previous study near-infrared (NIR) and attenuated-total- 386
345 650–1786 and 2809–3659 cm1 for hyperoside. The ones for reflectance infrared (ATR-IR) spectroscopy techniques in hyphen- 387
346 NIR are 4000–10,000 cm1 for hyperforin, 4000–7192 cm1 for ation with partial least squares (PLS) regression were utilized to 388

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Fig. 4. NIR-TLC for qualitative and quantitative analysis of methoxylated flavones in a phytomedicine.

389 determine the antioxidant capacity of Primulae flos cum calycibus quality, not only to determine the exact origin of the products and 415
390 samples. Folin–Ciocalteu (FC), ferric ion reducing antioxidant their unsophisticatedness but as well their quality, analysing 416
391 power (FRAP), 2,2-diphenyl-picrylhydrazyl (DPPH), 2,20 -azino-bis- different distinct parameters. One of these quality parameters of 417
392 (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) milk is the total concentration of protein. A part of the total protein 418
393 and cupric reducing antioxidant capacity (CUPRAC) assays were concentration in milk is made up by the phosphoprotein casein, 419
394 performed as reference methods. Different spectral pretreatments more precisely a mixture of different types of casein (a, b and k). By 420
395 such as standard normal variate (SNV), 1st or 2nd derivative, were using titanium dioxide nanoparticles as a carrier material, casein 421
396 applied to remove scattering effects. For all assays, cross and test- was highly selectively extracted via a bidentate stable complex 422
397 set validations were performed. The ability of the two spectro- directly from different milk samples following an optimized 423
398 scopic techniques to replace the five assays was evaluated and protocol and analyzed using near-infrared (NIR) spectroscopy in 424
399 compared. The standard error of prediction (SEP) and the ratio diffuse reflection mode. This protocol is based on the principle of 425
400 performance deviation (RPD) were determined and corrected for MEIRS (material enhanced infrared spectroscopy), a work pub- 426
401 the imprecision of the reference data to obtain the respective lished by Petter et al. on the highly selective analysis of LDL and 427
402 SEPcorr and RPDcorr values. In general, NIR demonstrated advan- HDL in human serum samples (Fig. 5) (Petter et al., 2009). 428
403 tages over ATR-IR spectroscopy and resulted best for the ABTS Calibration models of solutions with different casein concentra- 429
404 assay (R2: 0.94, RPDcorr: 4.66; test-set validation). Also with ATR-IR tions (serial dilution) in a range between 250 and 1750 mg l1 and 430
405 spectroscopy, the best prediction power was obtained for the ABTS 20,000 and 50,000 mg l1 were developed using multivariate 431
406 assay (R2: 0.94, RPDcorr: 4.10; test-set validation). The feasibility of methods, including partial least squares regression (PLSR). The 432
407 vibrational spectroscopy as a fast and simple tool to replace wet lower concentration range ensured the systems sensitivity toward 433
408 chemistry assays for the measurement of the antioxidant capacity small changes in the casein concentration giving evidence for the 434
409 of Primulae flos cum calycibus samples was demonstrated feasibility to screen real samples in the ensuing step. The 435
410 (Schönbichler et al., 2014). calibration model developed for the casein concentrations from 436
250 to 1750 mg l1 gave a SECV of 112.7, a Bias-value of 1.1887, a 437
411 3.2. Metabolomics R2 of 0.97188 and an RPD-value of 4.45 (Fig. 5). Furthermore the 438
casein content of different milk samples from diverse countries 439
412 Quality control of milk and derived products grew more including (South-Tyrol [Italy], Hungary, France, Germany and 440
413 important over the last years, not only for the consumers but also Austria) was quantitatively determined, as it is one of the major 441
414 for the producers. There is a strong demand, according to seals of aims and challenges to highlight the quality of milk samples from 442

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Fig. 5. Quantitative analysis of LDL/HDL in human serum, casein in milk products by selective formation of a bidentate casein-titanium oxide complex.

443 the alpine region by bordering its geographical origin. It can be amount of carbohydrates, nucleic acids and phospholipids com- 472
444 demonstrated that this novel method – combining a selective pared to periblem or rhizoderm tissue. 473
445 casein enrichment method with near-infrared spectroscopy and
446 PCA analysis – can be successfully implemented in order to 3.4. Food quality 474
447 distinguish between milk samples from different countries.
Farming in the Alps with its difficult and therefore necessarily 475
448 3.3. Proteomics costly conditions of production is increasingly pressurized by 476
globalization and market liberalization. To be able to compete 477
449 The capability of spectroscopic imaging to accurately reproduce against cheaper sustainable producing competitors, it is important 478
450 tissue histology of Urtica dioica root tissue was demonstrated by to point out the added value in order to achieve adequate prices 479
451 Pallua et al. (2011a). Before IR spectroscopic imaging, the optical and to remain competitive in the face of cheaper suppliers. In 480
452 image displayed in Fig. 6(A) is collected from a tissue section recent years it became possible for North Tyrol and South Tyrol to 481
453 measured by FTIR imaging with a nominal lateral resolution of incorporate the added value of regionality, origin and quality into 482
454 25 mm  25 mm per pixel for each spot and directly compared with customers purchasing decisions. A central role in this marketing 483
455 the images constructed from chemical maps. The optical image strategy is played by seals of approval, which affirmed both the 484
456 shows different tissue types: plerom tissue, periblem tissue and origin and quality of alpine products. In the next step it is 485
457 rhizoderm tissue. The chemical map (Fig. 6(B)) of the absorption at important for the marketing strategy to sustain brand loyalty as 486
458 1662–1645 cm1, which is commonly attributed to amid-I proteins well as consumer’s trust in these seals. It becomes necessary for 487
459 represents a homogeneous distribution in all tissue types. The producers and retailers to use state-of-the-art methods in order to 488
460 chemical map of the absorption at 1591–1528 cm1, which is prevent false declarations of the products. ‘‘OriginAlp’’ is an 489
461 attributed to amid-II proteins, is shown in Fig. 6(C). This FTIR imaging Interreg IV project funded by the European Union, which involves 490
462 result clearly demonstrates a very high absorption of this band in the cross-border cooperation between Austria (North and East Tyrol) 491
463 periblem and rhizodermis region and indicates that this tissue types and Italy (South Tyrol). The project’s aim is establishing efficient 492
464 produces a high amount of amid-II proteins. Fig. 6(D) depicts a and fast analytical methods for quality and origin control of 493
465 chemical map generated by integrating the area under the band regional alpine food products (De Benedictis et al., 2012). 494
466 absorption at 1138–988 cm1, which is commonly attributed to In the following, this subchapter is divided into an apple, 495
467 carbohydrates, nucleic acids and phospholipids. The chemical map meat and cheese part, three typical products from the alpine 496
468 of the absorption at 1160–1140 cm1 correlates well with the area. Authentication, typicality and traceability are discussed by 497
469 morphology of the plerom region and indicates that this tissue type investigating the efficiency of NIR spectroscopy to discriminate 498
470 produces a high amount of the mentioned ingredients. These between different species on one and hand and between different 499
471 observations lead to the fact that the plerom tissue produces a high geographic origins on the other. 500

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Fig. 6. IR spectroscopic imaging study to depict the distribution of proteins in Urtica dioica.

501 3.4.1. Apple spectrum. It was found that a growing height above the mean sea 519
502 In a first experiment the efficiency of a novel surface scanning level having a main influence related to the successful determina- 520
503 system (Fig. 7) was tested for the discrimination of different tion of the origin as apples from higher regions had a more intense 521
504 species (De Benedictis et al., 2012; Schmutzler and Huck, 2014). coloring. From the obtained PCA plot the possibility to separate 522
505 Therefore, spectra of 8 Pink Lady and 11 Golden Delicious apples samples cultivated in the alpine area from all other countries can 523
506 were recorded. Data treatment using Savitzky–Golay first deriva- be deduced. The samples from different countries are thereby not 524
507 tive enabled the separation of both species with the first principal that clearly separated from each other. A possible explanation 525
508 component explaining 77%, the second 18% of the total variance. for this is that all influences on the apples result in changes to 526
509 In the following investigation the separation of GD apples from their spectra. 527
510 the alpine area against other regions was enabled employing
511 this optimized surface scanning technique, which lead to cluster 3.4.2. Meat 528
512 formation. This cluster formation clearly demonstrates that it is Since the meat scandal in 2013, several investigations employ- 529
513 possible to separate similar apple samples according to their origin ing different analytical techniques including NIRS, for the detection 530
514 by NIRS. The chemical reason, why different geographical origins of offal adulteration in meat, were carried out (Zhao et al., 2014; 531
515 are resulting in different spectra recorded with NIRS, can hardly Morsy and Sun, 2013). The Tyrolean ‘‘Jahrling’’ (yearling) is young 532
516 be clearly investigated by NIRS as it is impossible to take all cattle from sucker cow and a typical product of the alpine area with 533
517 influences, including chemical compounds and physical parame- high quality. The animals are grown appropriate to the species 534
518 ters into consideration, which all have effects on the measured on farms corresponding to their natural conditions. They are feed 535

Fig. 7. Surface scanning technique for the analysis of apples (Schmutzler and Huck, 2014).

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Fig. 8. (a) 2d- and (b) 3d-factor plot for the discrimination of yearling (Alps origin) from meat of typical other originating countries.

536 mainly with breast milk, later on with hay and grass. The way of reamed cheese samples were recorded over a wavenumber range 581
537 natural breeding and feeding of the cattle in the Alps is the secret of between 4000 and 10,000 cm1 in diffuse reflection mode. In 582
538 the excellent meat quality that is retrieved. The meat is young, order to generate reproducible spectroscopic data at least 583
539 tender, juicy and lightweight (low fat content) making it very easy 3 charges of each type of cheese were taken into consideration. 584
540 to digest. A Tyrolean yearling is nine to twelve months old. The Corresponding reflection spectra were transformed into the 585
541 meat combines all the positive properties of veal and the classic absorption spectra, which showed strong absorbing water bands 586
542 beef being dark pink, tender and fine grained. The fine marbling at 5000 cm1 (combination band) and 7000 cm1 (overtone) and a 587
543 guarantees the outstanding taste and juiciness. One important second overtone of carbonyl (5620–5950 cm1). In the next step 588
544 quality feature is the optimal maturation. All farmers, where the the efficiency of NIR was proven in order to see whether it is 589
545 Tyrolean young cattle is growing, are partners of the Austrian sensitive for the whole cheese or if the differentiation results only 590
546 Agricultural Marketing (AMA) high quality initiative. The animals according to the fat content. The recorded two-dimensional factor 591
547 and their growing conditions including the environmental plot enables a clear differentiation between 8% (Aschbacher), 45% 592
548 parameters (meadows and fields) are checked on a regular basis. (Alpkäse), 50% (Stilfser and Bergkäse) fat cheese with a separated 593
549 Only through the proper management of the sustainable protec- cluster containing Stilfser and Bergkäse, which shows that the 594
550 tion of the cultural landscape and the yearling itself it is possible to technique is not only sensitive monitoring the fat content but also 595
551 guarantee this high meat quality over time. For the consumer it is allows to separate between different types of cheese in accor- 596
552 of importance that with the yearling a quality controlled food with dance with reported previous findings (Holroyd and Review, 597
553 known origin can be bought. 2013). This distinction was achieved by employing three principal 598
554 In order to establish a NIR based methodology for the components with the first explaining 73%, the second 18% and the 599
555 geographic authentication 50 yearling samples with differences third 6% of the total variance. 600
556 in breed and different local origin within the alpine area were
557 analyzed toward young beef samples of approximately the same 4. Conclusions 601
558 age four typical beef producing countries. For all animals a data
559 sheet containing details about gender, feeding, fat class, altitude Near-infrared (NIR) and attenuated total reflection (ATR) 602
560 etc. was provided. All samples were minced and temperature was spectroscopy offer new fast, non-invasive analytical strategies for 603
561 set at 10 8C prior spectral measurements recording at least 64 scans the quality control of natural products. Imaging and mapping 604
562 per sample. Data treatment using baseline and multiplicative techniques enable a detailed insight into the biochemical composi- 605
563 scattering correction (MSC) enabled the separation according to tion of a single plant and/or human tissue sample. Miniaturization 606
564 the geographical origin with the first principal component will be of high importance in order to be able to carry out 607
565 explaining 80%, the second 11% of the total variance employing measurements directly onto the field during ripening of medicinal 608
566 NirCal software as depicted in the 2d- (Fig. 8a) and 3d-score plot plants on one hand, on the other hand direct measurement at severe 609
567 (Fig. 8b). The obtained cluster also shows some partial overlap with points in food industry should be enabled by specially designed 610
568 samples originating from Sweden. The reason therefore must be miniaturized sample probes. The development of hand-held NIR 611
569 investigated more detailed by the analysis of the fatty acid profile imaging devices might give great promise. These methods are time- 612
570 employing gas chromatography–mass spectrometry (GC–MS). saving (fast), non-invasive and enable the determination of a 613
manifold of parameters simultaneously making them more and 614
571 3.4.3. Cheese more attractive especially in comparison to traditional applied 615
572 In the previous chapter we had already reported about the separation methods. 616
573 possibility to discriminate between different fractions of milk
574 including casein, fat and whey (De Benedictis et al., 2012). Isotopic Acknowledgements 617
575 mass spectrometric investigation of the isotope ratios d13 C and
576 d15N showed that these fractions give a direct hint on the The authors want to thank the Ministry for Health, Family and Q2618
577 geographic origin of milk samples even for the differentiation Youth (Vienna, Austria) (Novel Analytic Tools for Quality Control in 619
578 between closely related regions (Scampicchio et al., 2012). Based Traditional Chinese Medicine, Project No. 80855) and the Interreg 620
579 on these findings the feasibility of NIR determining species IV initiative of the European Union (project ‘‘OriginAlp’’) for 621
580 and origin was checked as described in the following: spectra of financial support. 622

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