Food Chemistry 127 (2011) 735742

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Visible micro-Raman spectroscopy for determining glucose content in beverage industry

I. Delno a, C. Camerlingo b, M. Portaccio c,, B. Della Ventura c, L. Mita c, D.G. Mita c, M. Lepore c
a

Biophysics and Nanoscience Centre, CNISM, Universit della Tuscia, Viterbo, Italy Consiglio Nazionale delle Ricerche, Istituto di Cibernetica E. Caianiello, Pozzuoli, Italy c Dipartimento di Medicina Sperimentale, Seconda Universit di Napoli, Naples, Italy
b

a r t i c l e

i n f o

a b s t r a c t
The potential of Raman spectroscopy with excitation in the visible as a tool for quantitative determination of single components in food industry products was investigated by focusing the attention on glucose content in commercial sport drinks. At this aim, micro-Raman spectra in the 6001600 cm1 wavenumber shift region of four sport drinks were recorded, showing well dened and separated vibrational ngerprints of the various contained sugars (glucose, fructose and sucrose). By proting of the spectral separation of some peculiar peaks, glucose content was quantied by using a multivariate statistical analysis based on the interval Partial Least Square (iPLS) approach. The iPLS model needed for data analysis procedure was built by using glucose aqueous solutions at known sugar concentrations as calibration data. This model was then applied to sport drink spectra and gave predicted glucose concentrations in good agreement with the values obtained by using a biochemical assay. These results represent a signicant step towards the development of a fast and simple method for the on-line glucose quantication in products of food and beverage industry. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 9 April 2010 Received in revised form 7 December 2010 Accepted 1 January 2011 Available online 8 January 2011 Keywords: Glucose quantication Raman spectroscopy Multivariate analysis Industrial drink quality control

1. Introduction Optical methods are nowadays becoming more and more important in food production and quality control (Chao, Kim, & Lawrence, 2008; Kress-Rogers & Brimelow, 2001). This is mainly due to the enormous efforts of many researchers in applying results coming from basic research to specic applications and also to the advances in producing detectors and sources, along with new devices (nanostructured surfaces, hybrid systems and so on) enabling the management of light in samples of industrial interest. A key role in this eld is played by Raman spectroscopy (Chan, 1996; Thygesen, Lokke, Micklander, & Engelsen, 2003), which provides information about composition and molecular structure of samples through the inspection of fundamental vibrations of functional groups. Very recently, the use of Raman spectroscopy has allowed the characterisation of starch and pectin in potato cells (Synytsya, Copkov, Matejka, & Machovic, 2003; Thygesen et al., 2003), of amygdalin in bitter almonds (Micklander, Brimer, & Engelsen, 2002) and the certication of edible oils (Dardenne & Aparicio, 2001; El-Abassy, Donfack, & Materny, 2009). This technique has also resulted to be able to discriminate among different kinds of sugars (Goral & Zichy, 1990) and therefore has been used to differentiate honey from various geographical regions (Good Corresponding author.
E-mail address: marianna.portaccio@unina2.it (M. Portaccio). 0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.01.007

acre, Radovic, & Anklam, 2002). Almost all these studies have been carried out by using near infrared excitation sources to avoid the uorescence emission following visible and/or ultra-violet (UV) excitation, which is typical of many biological samples. The use of infrared radiation (IR) for investigating Raman spectra of biological samples has some drawbacks; the principal one is the lowering of Raman signal intensity, which depends on the fourth power of laser frequency (McCreery, 2000). In addition, these samples usually have a high content of water that shows a high extinction coefcient in this spectral region. Hence, IR radiation can induce a more relevant sample heating, even though the data acquisition times usually employed nowadays can be very short. It should be also noted that powdered samples have been considered in almost all the above-mentioned cases, even if Raman spectroscopy can be successfully adopted to quantify different analytes in aqueous solution (Berger, Itzkan, & Feld, 1997; Dardenne & Aparicio, 2001). In fact, also high concentrations of water dont constitute a limit for Raman spectroscopy owing to very low water Raman signal. This situation is strongly different from IR absorption spectroscopy for which the contribution due to water usually interferes with the signals from other components. Very recently, we have applied micro-Raman spectroscopy with excitation in the visible (visible micro-Raman) to food industry liquid products. By this approach, claried fruit juice composition, with particular attention to pectin, fructose and b-carotene content, has been successfully characterised (Camerlingo et al., 2007).

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As a further step towards a larger use of visible micro-Raman technique in food industry, in this paper we have investigated the possibility to quantitatively determine the concentration of a single component in liquid samples. In particular, we have focused our attention on the determination of the glucose content because it has a key role in food and beverage industry and Raman spectroscopy has been shown to allow its quantication in various matrices (Batsoulis et al., 2005; Berger et al., 1997; Shih & Smith, 2009). As a representative test, different sport drinks have been examined, since their consumption has reached signicant dimensions and they are nowadays a constant element in the diet of different social classes and ages (Amendola, Iannilli, Restuccia, Santin, & Vinci, 2004). The quantitative determination of glucose concentration in commercial untreated samples has been obtained by analysing their Raman spectra by means of the interval Partial Least Square (iPLS) procedure, a multivariate statistical analysis approach that has shown to be very powerful in the analysis of Raman spectra (Hanlon et al., 2000; Delno et al., 2009). The iPLS model has been built by using, as calibration data, Raman spectra obtained from glucose aqueous solutions of known composition, and then it has been applied to sport drink spectra giving glucose concentrations in good agreement with the values obtained by a biochemical assay. The results have demonstrated that visible micro-Raman spectroscopy is a feasible method for glucose quantication in industrial products, such as beverages and fruit juices, without using specic substrates and/or sample preparation procedures. Henceforth, this approach represents a signicant step towards the development of a fast, simple, cost-effective Ramanbased method for glucose quantication in products of food and beverage industry, alternative to expensive, time-, sample- and chemicals-consuming biochemical assays currently used in production and quality control processes.

centration equal to 75% and 50% of A sample. A1 sample was used for obtaining a high-quality Raman spectrum that allowed us to clearly identify the contributions of sport drink main components. By using A2 and A3 samples, we preliminarily tested our experimental technique and data analysis procedure for predicting glucose content in samples more complex than glucose aqueous solutions. All chemicals, including the enzymes for the biochemical assay, were purchased form Sigma (SigmaAldrich, Milano, Italy) and used without further purication. 2.2. Methods 2.2.1. Biochemical assay for glucose determination A biochemical assay was used in order to validate the concentrations obtained with micro-Raman spectroscopy. The enzymatic assay uses glucose oxidase (GOD, EC 1.1.3.4) from Aspergillus niger (154 U mg1). GOD catalyses the oxidation of glucose to gluconic acid according to the scheme:

glucose O2 ! Gluconic acid H2 O2

The resulting hydrogen peroxide is detected by means of a chromogenic oxygen acceptor, composed by phenol and 4-aminophenazone (4-AP), in the presence of horseradish peroxidase (POD, EC 1.11.1.7) (1119 U mg1) according to the reaction (Kaplan, 1987; Trinder, 1969):

GOD

H2 O2 phenol 4-AP ! quinone H2 O

The solution absorbance, measured with a spectrophotometer (Perkin Elmer LS 55) at 505 nm, is proportional to the glucose concentration. To use this method we prepared a working reagent solution (WRS) composed of 15U/mL of GOD, 1 U/mL of POD, [phenol] = 0.3 mM and [4-AP] = 2.6 mM in buffer TRIS 100 mM, pH 7.4. Determination of glucose concentration requires two steps: (a) One milli litre of WRS is mixed with 10 lL of standard glucose solution (5 mM). The solution is incubated for 10 min at 37 C, then its absorbance is read at k = 505 nm, providing the reference for our measurements, (b) One milli litre of WRS is mixed with 10 lL of each investigated sample properly diluted with distilled water in order to be sure that its absorbance is linearly proportional to the glucose concentration, as required by BeerLambert law. After incubation for 10 min at 37 C, the absorbance of the sample is read at the above-mentioned wavelength (k = 505 nm). The ratio between the absorbance of the sample and of the standard solution allows us to determine the glucose concentration. 2.2.2. Lyophilising procedure To lyophilise A sample in order to obtain A1 sample, a laboratory-scale freeze-drier (Edwards EF4 Module freeze drier), attached to an Edwards high vacuum pump (Crawley, Sussex, England) was used to process 20 ml of frozen samples at a constant temperature of 48 C and a vacuum pressure of 1.33 103 mbar for 24 h. 2.2.3. Raman spectroscopy measurements Raman spectroscopy provides information about the composition and the molecular structure of samples by inspecting fundamental vibrations of some functional groups. The process of Raman scattering can be viewed as an inelastic scattering process in which the scattered photon is shifted in frequency from the incident photon as it either loses or gains energy from a particular

POD

2. Materials and methods 2.1. Materials High purity glucose powder (>99%) was purchased from RIEDEL (Germany Haen AG) and used without any further treatment. Proper amount of glucose powder was dissolved in distilled water to obtain glucose concentrations [Glu] in the 251050 mM range. Molarity (M) units are used for the concentration. These samples were employed for building and testing the iPLS model. Various commercial (details available on request) sport drinks (A, B, C and D samples) were used for the experimental investigation. Their composition is very similar and is shown in Table 1, whose inspection makes clear that the main components are water and carbohydrates. Small amounts of protein and lipids are also present. From A sample three different samples were obtained. A frozendried sample (A1) was obtained by the lyophilising procedure reported in the next section, and two other samples (A2 and A3) were prepared by dilution with distilled water for a nal drink con-

Table 1 Analytical composition of sport drinks as reported on the labels. Ingredients (g/l) Carbohydrates Protein Lipids Sodium Chloride Potassium Magnesium Sample A 65.7 2 103 0.19 318 103 415 103 80 103 30 103 Sample B 82 0 0 510 103 84 103 52 103 20 103 Sample C 58.3 0 0 520 103 460 103 120 103 50 103 Sample D 100 0 0 n.a. n.a. n.a. n.a.

n.a. stands for not available.

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vibration mode of the molecule. A detailed description of the physics of Raman effect is out of the scope of this paper and can be found elsewhere (see, for instance, Lewis & Edwards, 2001). By combining Raman spectroscopy with microscopy (micro-Raman spectroscopy), qualitative and quantitative information can be obtained in a noninvasive way also from small amount of samples. This method is a very effective tool in food analysis because it is non-destructive and usually does not require special preparation of the sample. The experimental micro-Raman set-up employed for the measurements is shown in Fig. 1. The visible laser source was a He Ne laser operating at a wavelength k = 633 nm, with a maximum nominal power of 17 mW. The laser light was focused on the sample by means of a 50X optical objective (Olympus MPLAN 50X/ 0.75) on a circular area with diameter of 20 lm. The micro-Raman spectrometer was equipped with an optical confocal microscope (Olympus BX40) connected by a 50 lm optical ber to a Jobin Yvon TriAx 180 monochromator equipped with liquid nitrogencooled CCD detector. Three gratings with 300, 600 and 1800 grooves/mm were selectable, allowing a maximum spectral wavenumber resolution of 4 cm1. In this case the 600 grooves/ mm grating was selected. The spectra were acquired using accumulation times ranging from 60 to 600 s by means of a double acquisition process which permits the rejection of spurious peaks due to direct CCD excitations. A drop of the liquid sample was placed on a microscope glass slide with a single well (1 cm large and 0.1 cm depth) suitable for investigating liquid specimens. A cover glass (170 lm thick) was placed on the top of the concavity to avoid sample and optical objective contamination. For each kind of sample, experiments were performed several times and repeated on different drops of the same sample in order to test the reproducibility of the measure. Since our measurements were carried out on liquid samples, they were affected by scattering and optical aberration effects due to water and other scattering elements, which reduce the quality of recorded spectra. Therefore, it was difcult to extract quantitative information from the spectral data (Aarnoutse & Westerhuis, 2005). Nevertheless, using high optical aperture objectives (as in the case of the objective 50X) and appropriately setting the laser focus, a reproducible Raman response was obtained. Even if the total collected signal was decreased when confocal microscopy was

used, this approach improved the readability of micro-Raman signal, limiting the reected light component out-of-focus and, consequently, reducing the noise in the spectra. In addition, by using confocal geometry, extremely small volumes are sampled minimizing scattering and uorescence effects. In our measurements, the confocality pinhole was xed to values in the range of 200 500 lm, depending on the experimental light reection conditions. 2.2.4. Spectra analysis Spectra were preliminarily analysed using the application routines provided by the software package (SpectraMax Software User Guide, JobinYvon Inc., USA) controlling the whole data acquisition system. In details, we preliminarily removed uorescence by using a visual baseline correction based on a 3rd order polynomial t through selected xed points of the spectrum and then we analysed the complex spectra in terms of convoluted Lorentzian shaped vibration modes. Peaks constituting the spectrum were manually selected in order to dene the starting conditions for a best-t procedure. The best-t procedure was then performed to determine convolution peaks with optimised intensity, position and width. Its performance was evaluated by means of the v2 parameter dened, as usual, by the following formula:

Pn Actuali Calculatedi 2

v2

i0

RMSNoise

n f

where the Actual and Calculated are the measured and calculated data, respectively, RMSNoise is the estimated Root Mean Square noise in the Actual data over the tted region, n is to the number of data points in the tted region and f refers to the total number of variables from the peak and baseline functions. Thus, n f is the number of degrees of freedom. The LevenbergMarquardt algorithm was employed to adjust every variable for each peak in an attempt to minimise the v2 parameter. This procedure was extremely useful for correctly determining the peak positions, widths, heights and areas of a set of overlapping peaks. 2.2.5. Multivariate statistical analysis Partial Least Squares methods are devoted to nd a model describing some predicted variables in terms of other observables, that is, to build a regression model between two blocks (data

Fig. 1. Experimental set-up for micro-Raman spectroscopy (see text).

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matrices) X and Y using a latent variable representation of these matrices. These variables are calculated so that they explain the directions of maximum covariance (Boardman, Hui, & Wold, 1981; de Jong, 1993; Esbensen, 2000; Wold & Martens, 1983 ). This enables to use the general equation of all inverse calibration methods as PLS:

3. Results and discussion 3.1. Glucose samples The Raman spectrum in the 5501600 cm1 interval for the highest concentration glucose solution (1050 mM) is shown in Fig. 2 (curve a). The deconvolution procedure described in Section 2.2.4 was applied to the spectrum in the wavenumber range of 5701544 cm1. The iterative tting algorithm converged for a reduced v2 = 1.01 value to a convolution (curve b) of Lorentzian peaks positioned at the wavenumber positions listed in the Table 2. Curve c represents the residual errors for the t procedure. The reported interval represents the ngerprint region and is the most signicant one for the investigated samples. The presence of the characteristic bands of the glucose a anomer at 789, 855, 919, 1336 and 1371 cm1 and those of b anomer at 1073, and 1128 cm1 indicates that glucose in solution is a mixture of the two anomers with predominance of the b conguration, as indicated by their relative intensities (Arboleda & Loppnow, 2000; Cerchiaro, SantAna, Arruda Temperini, & da Costa Ferriera, 2005). Also the peaks at 1401 and 1430 cm1 can be attributed to glucose, as reported in Soderholm, Roos, Meinander, & Hotokka (1999). The contributions at 968, 1036 and 1524 cm1 can be related to some impurities present even in the high purity glucose powder (>99%) employed in the present experiments. The described features obviously appear also in the spectra obtained for the other examined glucose solutions, as witnessed by some representative Raman spectra ([Glu] ranging from 25 to 1050 mM) reported in Fig. 3. The above-mentioned assigned peaks have a height increasing with the increase of the glucose concentration. These results suggest that the glucose content of each solution can be extracted by properly analysing the corresponding Raman spectrum. At this aim, a proper iPLS model has to be built, by means of the above described iPLS method. In Fig. 4 a representative spectrum is reported to show the interval selection procedure for the glucose content evaluation. The wavenumber region was divided into 20 spectral intervals having the same width. Each vertical bar indicates the RMSECV value obtained by the local PLS model for the corresponding interval. The number of latent variables for each model is given at the bottom of the bar. The horizontal dotted line indicates the RMSECV value for the full-spectrum PLS (global) model (RMSECVg). As can be seen, the results for the two intervals spanning the 550 800 and 11501550 cm1 ranges are characterised by a noticeably

Y XB

where X is the data matrix represented by specic observables, Y is the predicted data matrix, and B contains the regression coefcients determined in the calibration step. Different algorithms have been proposed and employed to calculate PLS models (Berger, Koo, Itzkan, Horowitz, & Feld, 1999; Lambert, Pelletier, & Borchert, 2005; Nrgaard et al., 2000). Among the others, interval PLS (iPLS) is a variant of PLS which is particularly promising for the Raman spectra analysis. In fact, it develops local PLS models on (contiguous or non-contiguous) subintervals of the full spectrum (Leardi & Nrgaard, 2004) and, thus, signicant Raman features located in specic intervals could be properly exploited for designing the prediction model. The nal iPLS model is the best among all possible combinations of number and width of spectral subintervals. The use of iPLS model is successful if it overcomes the global PLS model, performed using the full spectrum, that is, when the Root Mean Square Error of Cross-Validation (RMSECV) (Berger et al., 1999; Lambert et al., 2005), dened as

s Pn 2 i1 MeasGlui PredGlui RMSECV n

is lower than the RMSECV global (RMSECVg), representing the RMSECV obtained for global PLS model. For the PLS analysis the samples are usually divided into a calibration set and a validation set. However, if the validation step is performed using full cross validation the splitting of the data set into two subsets can be avoided. In fact, in full cross validation process one sample is left out from the calibration process, followed by the construction of a calibration model using the remaining samples, which is then tested on the sample left out. This procedure is repeated until each sample has been left out once. This means that the validation procedure consists of two looping steps: (i) the model is built with full cross validation; (ii) outlying samples (spike samples) are detected and removed. Then, the model is rebuilt from scratch without the outliers so that the information used to produce them in the previous step is removed from the calibration set. This procedure is repeated until there are no more outliers, provided that the calibration set still contains enough samples to validate the model with good estimates of the root mean-square error. The iPLS method, optimised using full cross validation procedure, was used in the present case for setting up a model able to predict glucose content from Raman spectra of liquid samples, relating glucose content to a selected part of the Raman spectrum (using Eq. (2)). The iPLS model was built using the whole data set (for a total of 120 spectra). The intervals for building the model were selected according to RMSECV. For each solution, the representative spectrum was obtained by averaging various spectra.

919

1073 1128

Raman signal (a.u.)

789

1401 1271 1336 1371 1401 1459

855

968

732

a b c

600
2.2.6. Software The software employed in this work was properly written for Raman analysis purposes and is based on the PLS toolbox 3.5 for MATLAB, from Eigenvector Research, for PCA, cluster analysis and PLS-DA, and the iPLS toolbox for MATLAB, implemented by Lars Noorgard (http://www.models.kvl.dk/source/ipls/).

800 1000 1200 1400 -1 Wavenumber shift (cm )

1600

Fig. 2. Raman spectrum (6001600 cm1 spectral region) for a 1050 mM glucose solution sample (line a) and the corresponding deconvolution in Lorentzians (lines b). The main peak centre positions are labelled. Plot c refers to the result of the difference between the raw experimental data and the convolution of Lorentzian components (residual errors from the t). The acquisition time was 600 s.

I. Delno et al. / Food Chemistry 127 (2011) 735742 Table 2 Main peaks in Raman spectra of 1050 mM glucose solution and Sample A1 in the range 7001500 cm1 and tentative assignment in agreement with Arboleda & Loppnow, 2000; Cerchiaro et al., 2005; Soderholm et al., 1999 and Engelsen, http:// www.models.kvl.dk. Assignments Fructose Sucrose Other a-anomer a-anomer Fructose a-anomer Other Other b-anomer b-anomer Fructose Glucose a-anomer a-anomer Glucose Glucose Glucose; fructose Other

739

250 200

RMSECV

Peak wavenumber (cm1) for glucosea

Peak wavenumber (cm1) for sample A1b 634 710 742 789 836 867 918

150 100 50 0

789 855 919 968 1036 1073 1128 1271 1336 1371 1401 1430 1459 1524
a b

1065 1128 1264 1332 1375

600

800

1000

1200
-1

1400

Wavenumber shift (cm )

Fig. 4. Cross-validated prediction performance (RMSECV) for specic interval models (bars) and for the ve PLS-component full-spectrum model (dotted line) on the investigated Raman spectra plotted together with the corresponding normalised mean spectrum. The italic numbers on the bars indicate the optimal number of PLS components used in each interval model.

1456

see Fig. 2. see Fig. 7.

Raman signal (a.u.)

1050 mM 525 350 175 130 125 105 50 25

shown and in the lower panels corresponding results for the 11081155 cm1 interval are presented. For each calibration curve a good linear dependence is observed. The nal iPLS model was then built using both the selected intervals and employed for predicting glucose content from Raman spectra of glucose aqueous solutions used as unknown set. In Fig. 6, the glucose concentration given by the nal iPLS model is reported as a function of the measured concentration. The predicted value is in very good agreement with the measured one for each sample. The goodness of the procedure is witnessed by a linear tting with an angular coefcient equal to 1.004 0.008 and a correlation coefcient of 0.991. 3.2. Sport drink samples In Fig. 7, the Raman spectrum (curve a) of the A1 sample (the aliquot of A sample that underwent the lyophilising procedure) is shown together with the results of the deconvolution procedure. In this case, the iterative tting algorithm converged for a reduced v2 = 1.94 to the convolution of Lorentzian peaks reported in Fig. 7 (curve b). Curve c represents the residual errors for the t procedure. By inspecting Fig. 7, it comes clear that the glucose peaks already evidenced in Raman spectra of aqueous glucose solutions are observed in A1 sample spectrum along with other peaks typical of the sample (see Table 2). According to literature, they can be assigned to fructose and sucrose, thus indicating the presence of these sugars in the sample. In particular, peaks at 634, 867, 1264 and 1456 cm1 can be assigned to fructose according to Cerchiaro et al., 2005. The peak at 710 cm1 can be ascribed to sucrose (Engelsen, www.models.kvl.dk) while the peak at 742 cm1 has to be attributed to some other unknown components. Raman spectra of A, A2 and A3 samples are shown in Fig. 8 and the main peaks are indicated and labelled. Even though the signal intensities and signal-to-noise ratio of the spectra are worse than those of the spectrum reported in Fig. 7, the main peaks are clearly evident, as, for instance, those assigned to a and b anomers of glucose. It has to be underlined that the 918 and 1128 cm1 peaks, that fell in the intervals selected for building iPLS model for glucose quantication, are clearly detectable in sport drinks Raman spectra, resulting to be well distinguished by the specic features of other liquid sample components. This suggests that the built iPLS model can be used for the quantitative evaluation of glucose content in the investigated sport drinks. When iPLS analysis, using the previously selected intervals (913961 and 11081155 cm1), is per-

600

800

1000

1200

1400
-1

1600

Wavenumber shift (cm )

Fig. 3. Raman spectra (6001600 cm1 spectral region) of water glucose solutions at different glucose concentrations (range 201050 mM). Spectra obtained with an integration time of 600 s.

worse (that is, higher) RMSECV compared to RMSECVg. On the contrary, some intervals in the 8001150 cm1 range have a RMSECV lower than RMSECVg, thus, being more representative of glucose content than the full spectrum. According to RMSECV metric, two intervals were selected: 913961 cm1 and 11081155 cm1, including one a anomer peak (that located at 919 cm1) and a b anomer peak (the one located at 1128 cm1), respectively. The outlined intervals were used for building the iPLS model, validated by the cross-validation procedure. The results are shown in Fig. 5, where the intervals selected for the model (highlighted in the left panels of the gure) are reported, along with the corresponding calibration curves (right panels). Each calibration curve shows the relationship between the true glucose concentration (Measured [Glu]), that is, the nominal concentration of each solution, and the value of the same parameter predicted by applying the local iPLS model (Predicted [Glu]). In particular, in the upper panels results obtained by considering the 913961 cm1 range are

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600

800

1000

1200
-1

1400

Wavenumber shift (cm )

Raman signal (a.u.)

600

800

1000

1200
-1

1400

Wavenumber shift (cm )

Fig. 5. Selected intervals used in the iPLS model (left panels) for Raman spectrum and the corresponding predictions obtained using single intervals (right panels). Numbers represent the different spectra.

1100
1065

1000 900

836 867 918

Predicted [Glu] (mM)

Raman signal (a.u.)

800 700 600 500 400 300 200 100 0 0 100 200 300 400 500 600 700 800 900 1000 1100

710 742

1332 1375

634

1264

1456

r=0.991

1128

a b c

600

800

1000

1200
-1

1400

1600

Wavenumber shift (cm )

Fig. 7. Raman spectrum (6001600 cm1 spectral region) of sample A1, that is, of the aliquot of sample A that underwent the liophylisation procedure (line a) and the corresponding deconvolution in Lorentzians (lines b). The main peak centre positions are labelled. Plot c refers to the result of the difference between the raw experimental data and the convolution of Lorentzian components (residual errors from the t). The acquisition time was 600 s.

Measured [Glu] (mM)

Fig. 6. Predicted concentration of glucose (Pred [Glu]) as a function of the nominal ones (Measured [Glu]) as obtained by using the iPLS model.

formed on the spectra of Fig. 8, a glucose concentration of 136 15, 115 14 and 72 6 mM is obtained for A, A2 and A3 samples, respectively. The highest value is in good agreement with the result of enzyme assay that gives a value of 144 8 mM (see A sample, Table 3) for the undiluted sample. Also for the other samples, the use of iPLS procedure allowed us to obtain an estimation of glucose concentration that is in good agreement with results of biochemical assays (see Table 3). Accordingly, a glucose content of 84 14% and 53 6% with respect to that of the A sample is ob-

tained for A2 and A3 samples, respectively, comparing well with the nominal dilution values (75% and 50%, respectively). Representative Raman spectra of other commercial sport drinks, that is, of B, C, and D samples are shown in Fig. 9. The main peaks already seen for A sample are still evident. In particular, the 918 and 1128 cm1 peaks, that were selected for building the iPLS model for glucose quantication are clearly observed. When the previously described

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1332 1456 1370

741

836 634 920

1065 1128

350 300

1264

Raman spectroscopy Biochemical assay

Raman Intensity (a.u.)

Measured [Glu] (mM)

250 200 150 100 50

A2

A3
600 800 1000 1200
-1

1400

1600

Raman Shift (cm )

Fig. 8. Representative Raman spectra (6001600 cm spectral region) of sample A, A2 and A3 as obtained using an integration time 600 s. Main peaks are indicated and labelled.
1

0
A A2 A3 B C D

Sample
Fig. 10. Measured glucose concentration for commercial beverage samples as obtained by iPLS analysis of Raman spectra and by biochemical assay.

Table 3 Glucose concentration values for different sport drink samples as evaluated from Raman spectra and enzymatic assay. Samples A A2 A3 B C D Glucose content from Raman spectra (mM) 136 15 115 14 72 6 280 50 123 8 290 40 Glucose content from enzymatic assay (mM) 144 8 108 7 72 5 240 9 125 5 250 10

the error affecting the iPLS prediction, it is worth noting that for four over the six examined cases the error bar is comparable with the one affecting results of biochemical assay. 4. Conclusions Glucose content in aqueous solutions and in commercial beverages was determined by applying the iPLS method for the analysis of their Raman spectra, obtained by employing a visible laser source and a micro-Raman apparatus. To obtain useful Raman spectra from liquid heterogeneous samples is often difcult and many samples are therefore dried before being measured. Furthermore, it is necessary to employ more expensive laser sources and detectors in the infrared region in order to reduce uorescence effects. In the present case, the use of confocal geometry enabled us to obtain informative Raman spectra by means of a less expensive visible light apparatus. In addition, by using confocal geometry extremely small volumes are sampled minimizing scattering and uorescence effects. These results demonstrate that visible light micro-Raman spectroscopy in combination with appropriate multivariate methods can be successfully adopted to quantitatively determine glucose contents in liquid samples without any treatment. In particular, the iPLS method allowed us the use of very simple samples for calibration since we were interested in the concentration determination of a single component. Obviously micro-Raman spectroscopy together with the i-PLS method can be employed for analysing more than one substance at a time in a heterogeneous compound using appropriate calibration samples avoiding the use of different expensive biochemical assays. References
Aarnoutse, P. A., & Westerhuis, J. A. (2005). Quantitative Raman reaction monitoring using the solvent as internal standard. Analytical Chemistry, 77, 12281236. Amendola, C., Iannilli, I., Restuccia, D., Santin, I., & Vinci, G. (2004). Multivariate statistical analysis comparing sport and energy drinks. Innovative Food Science and Emerging Technologies, 5, 263267. Arboleda, R. H., & Loppnow, G. R. (2000). Raman spectroscopy as a discovery tool in carbohydrate chemistry. Analytical Chemistry, 72, 20932098. Batsoulis, A. N., Siatis, N. G., Kimbaris, A. C., Alissandrakis, E. K., Pappas, C. S., Tarantilis, P. A., et al. (2005). FT-Raman spectroscopic simultaneous determination of fructose and glucose in honey. Journal of Agricultural and Food Chemistry, 53, 207210. Berger, A. J., Itzkan, I., & Feld, M. (1997). Feasibility of measuring blood glucose concentration by near infrared Raman spectroscopy. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 53, 287292.

836 742 918

1065 1128

1332 1264 1370 1456

Raman Intensity (a.u.)

D
600 800 1000 1200
-1

1400

1600

Raman Shift (cm )

Fig. 9. Representative Raman spectra (6001600 cm1 spectral region) of sample B, C and D as obtained using an integration time 600 s. Main peaks are indicated and labelled.

procedure is performed on spectra of Fig. 9, a glucose concentration of 280 50, 123 8 and 290 40 mM is obtained for B, C and D samples, respectively. Also for these samples a satisfactory agreement with the results of the biochemical assay is obtained, as shown in Fig. 10, where all the results on sport drink glucose concentration are summarised. The small differences between biochemical and iPLS predictions outlined for all the samples ensures that iPLS analysis is able to feasibly evaluate the content of a single component (glucose, in the present case) by the analysis of Raman spectra including also ngerprints of various components. As far as

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