spectroscopic techniques
Simultaneous Monitoring of Organic Acids and Sugars in
Fresh and Processed Apple Juice by Fourier Transform
Infrared–Attenuated Total Re ection Spectroscopy
JOSEPH IRUDAYARAJ* and JAGDISH TEW ARI
Department of Agricultural and Biological Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802
A combination of Fourier transform infrared spectroscopy (FT-IR)
and chem ometrics was used as a screen ing tool for the determina-
tion of sugars and organic acids such as sucrose, glucose, fructose,
sorbitol, citric acid, and malic acid in processed commercial and
extracted fresh apple juices. Prepared samples of synthetic apple
juice in different constituent concentration ranges were scanned by
attenuated total re ectance (ATR ) accesso ry and the spectral region
in the range between 950 and 1500 cm 2 1 was selected for calibration
model development using partial least squares (PLS) regressio n and
principal com ponent regression (PCR). The calibration models were
successfully validated by high-performance liquid chromatography
(HPLC) m easurements against several commercial juice varieties as
well as juice extracted from different apple varieties to provide an
overall R 2 correlation of 0.998. The present study demonstrates that
Fourier transform infrared spectroscopy could be used for rapid
and nondestructive determination of multiple constituents in com-
mercial and fresh apple juices. Results indicate this approach to be
a rapid and cost-effective tool for routine monitoring of multiple
constituents in a fruit juice production facility.
Index Headings: Fourier transform infrared–attenuated total re ec-
tion; FTIR-ATR ; Chemometrics; Apple juice; High-performance
liquid chromatography; HPLC.
INT RODUCTIO N
The composition 1,2 of apple juice has been an area of
interest for a long time in quality evaluation. Gore 3 com-
pared the quality of juice from a number of leading apple
varieties after pasteurization and storage and concluded
that for apple juice to be most palatable the total solids
content should be greater than 12% and acids greater than
0.5% (e.g., malic). Reducing sugars 4 in apples were de-
termined to be in the range between 6.6 and 1.1%. Hulmi 5
measured the percentage of fructose (4.3 to 8.24%), glu-
cose (1.34 to 1.95%), and sucrose (1.72 to 4.18%), and
also identiŽ ed a few organic acids such as quinic, gly-
colic, succinic, lactic, galacturonic, and citramalic as ap-
ple juice constituents.6
Ion chromatographic analysis of organic acids in fruit
juice has also been reported by Saccani et al.7 The use
Received 13 March 2003; accepted 14 July 2003.
* Author to whom correspondence should be sent. E-mail:
josephi@psu.edu.
0003-7028 / 03 / 5712-1599$2.00 / 0
Volume 57, Number 12, 2003 q 2003 Society for Applied Spectroscop y
of high-performance liquid chromatography (HPLC) for
monitoring sugars and organic acids for quality control
has been reported as well.8 The technique, although ac-
curate, is time consum ing and as of now difŽ cult to im-
plement in an on-line setting. Nondestructive methods
based on high-resolution nuclear m agnetic resonance
(NMR) have shown good correlation in the assessment
of glucose, fructose, and sucrose, along with other com-
ponents.9 Sugar content of intact grapes and sweet cher-
ries has been estimated using NMR by Cho et al.10 These
measurem ents, however, require trained personnel and are
expensive to perform.
The feasibility of optical m ethods such as near-infrared
(NIR) spectroscopy for the evaluation of quality param -
eters such as carbohydrates and pH in apple juice 11 has
shown promise. Although the NIR technique is fast and
well suited for process control applications, a major lim-
itation lies in the intrinsic broad and overlapping bands
encountered in the spectra of sugar components, limiting
its prediction capability in complex mixtures. Infrared
spectroscopy in the mid-infrared (M IR) region with M i-
chelson interferometry has been used to predict sugar
contents in both agricultural and food products. However,
intense water bands in the MIR spectra obstruct the ac-
curacy and sensitivity of quantitative analysis. Efforts to
avoid or minimize errors due to water-absorbing peaks
have been proposed through suitable sample preparation
based on solvent extraction 12 as well as sample accesso-
ries such as attenuated total re ection (ATR) accessories.
Through advanced optics and design, high spectral sig-
nal-to-noise ratio, and throughput from m odern Fourier
transform infrared (FT-IR) spectrometers, detection of in-
dividual constituents as well as subtle compositional dif-
ferences between and among multi-constituent speci-
mens 13 is possible through highly resolved spectra. W hen
the absorption bands do not overlap, simple mathematical
treatments such as Beer–Lambert’s calibration of peak
heights or area with respect to the concentration of a sin-
gle analyte is possible. When multiple analytes are to be
determined and when the absorbing peaks overlap, one
of several multivariate methods such as partial least
squares (PLS) and/or principal component regression
(PC R) are used in conjunction with the spectra to develop
APPLIED SPECTROSCOPY 1599
 F IG . 1. FT-IR spectra of different components relevant to apple juice.
the multi-component calibration models. 14,15 Chemomet-
ric analysis of ATR spectra in the MIR region have been
used to determine sugar and/or ethanol concentrations in
fermentation broths16 as well as in sugar solutions.17
M ultivariate techniques such as PLS and PCR have
proven to be effective for quantitative analysis as well as
for data compression to transform high-dimensional data
with a large number of intercorrelated variates (wave-
numbers) into a reduced new set of variates for further
analysis. A comparative study by Dupuy et al.18 using
different multivariate calibration methods to determine
glucose, fructose, and sucrose in dried fruit juice extracts,
showed that PLS analysis provided the most accurate pre-
dictions. The combination of MIR spectroscopy and mul-
tivariate statistics for the determination of glucose, fruc-
tose, and sucrose in aqueous mixtures was also demon-
strated.19
In this research, Fourier transform infrared spectros-
copy with ATR will be used for simultaneous quantiŽ -
cation of glucose, fructose, sucrose, citric acid, malic
acid, and sorbitol in fresh extracted and processed com-
mercial apple juices. PCR and PLS methods will be used
to develop calibration models for sugar and organic acid
measurem ents using synthetic samples and the m odels
validated by HPLC values.
M ATERIAL S AND M ETH ODS
Sample Preparations. Glucose, fructose, sucrose, cit-
ric acid, m alic acid, and sorbitol were purchased from
Sigma Aldrich Corporation. On the basis of the actual
concentration ranges of sugars and organic acids in fresh
and processed apple juice, 39 synthetic samples were pre-
pared. The prepared samples covered the m inimum and
maximum concentration ranges representative of natural
samples and used for calibration model development. The
Ž ve different brands of processed apple juice chosen in-
clude Meier’s, Tropicana, Mohan M akings, Lichika, and
1600 Volume 57, Number 12, 2003
Chinar, and the six different varieties of fresh apples cho-
sen for juice extraction include Delicious, Golden Deli-
cious, Baldwin, Jonathan, M cIntosh, and Rome Beauty.
The juice was extracted individually using a home juicer
and analyzed for the sugars and organic acids using
HPLC as well as by FT-IR spectroscopy.
Fourier Transform Infrared Spectroscopy. Fourier
transform infrared spectra were measured using a BIO-
RAD 3000 EXCALIBUR spectrometer equipped with a
deuterated triglycine sulfate (DTGS) detector, operating
at 32 cm 2 1 resolution and 0.32 cm per second m irror
velocity. Two hundred and Ž fty-six interferogram s were
co-added before Fourier transformation. The instrument
was allowed to purge for 5 m in with nitrogen gas (grade
1) prior to acquisition of the spectra to m inimize spectral
contribution due to atmospheric carbon dioxide and water
vapor. The sampling station was equipped with an over-
head ATR accessory, comprising transfer optics within
the chamber, through which infrared radiation is directed
to a detachable ZnSe ATR crystal with a 458 parallelo-
gram geometry and 10 internal refractions and m ounted
into a plate with a shallow trough for sample contain-
ment. The depth of penetration, which gives a measure
of the intensity of the resulting spectra, was 1.46 mm.
The reference spectra were recorded using a blank ATR
crystal. Single beam spectra were obtained for all the
samples and ratioed against the background spectra of air
to present the spectra in absorbance units. After each
sample the ATR crystal was thoroughly washed with dis-
tilled water and dried with nitrogen gas and examined by
FT-IR to ensure that no residue from the previous exper-
iment was retained on the crystal surface. The samples
were analyzed in triplicate and the spectra averaged for
further analysis.
H igh-Performance Liquid Chromatography. High-
performance liquid chromatography was perform ed with
a Waters system equipped with a 717 Plus Auto sampler
 F IG . 2. Overlaid spectra of a synthetic m ixture of different components relevant to apple juice.

Controller with a data acquisition system. Separation of
sugars and organic acids in the Ž ve processed and six
fresh extracted apple juices was carried out using the
Aminex HPX-87H (300 3 7.8 m m) and fast carbohydrate
colum ns (100 3 7.8 mm ). The  ow rate was kept at 0.6
mL/min for the HPX-87H and 1.2 m L/min for the fast
carbohydrate column to prevent base line drift. Sucrose,
glucose, fructose, citric acid, m alic acid, and sorbitol
were m easured using the refractive index and UV detec-
tor. Triplicate injections of all the samples were carried
out to ensure the correct integration of sugars and organic
acids. HPLC results for the sugars and organic acids were
compared against FT-IR results.
Q uantitative Analysis. The GRAMS 32 (Galactic In-
dustries Corporation, Salem, NH) software used for quan-
titative analysis employed the PLS and PCR routines.
Calibration m odels were developed with spectra in ab-
sorbance units using PLS and PCR methods with the
original and Ž rst-derivative transformed spectra. The op-
timum number of factors for calibration was selected on

F IG . 3. FT-IR spectra of selected commercial apple juices.

APPLIED SPECTROSCOPY 1601

TABLE I. Calibration and validation results using PLS and PCR TABLE II. RMSD and R 2 values of the calibration model for syn-
for sugars and organic acids in synthetic samples.a thetic samples.
Calibration Validation PCR PLS
Calibration Factors R 2
SEC R 2
SEP Components RM SD R 2
RMSD R2
PCR 9 0.998 0.662 0.978 2.5 Sucrose 0.0269 0.998 0.0271 0.998
PLS 8 0.898 0.671 0.988 2.2 Glucose 0.0125 0.999 0.0121 0.999
Fructose 0.0269 0.998 0.0271 0.998
a
SEC: standard error of calibration; SEP: Standard error of prediction;
Sorbitol 0.0031 0.997 0.0029 0.997
R 2: correlation coefŽ cient. Citric acid 0.0236 0.996 0.0023 0.996
M alic acid 0.0221 0.998 0.0027 0.998

the basis of the predicted residual sum of square

(PR ESS), which should be m inimized, along with the re- RESULTS AND DISCUSSION
gression, R 2 . The m odel prediction was tested by com-
The FT-IR spectra of sucrose, glucose, fructose, citric
puting the standard error of prediction (SEP) and standard
acid, m alic acid, and sorbitol in Fig. 1 show bands that
error of calibration (SEC), respectively given by:
are well deŽ ned, narrow, and distinct. The vibrational

ÎO (actual 2 predicted )
n mode bands for sucrose, glucose, fructose, citric acid,
2 malic acid, and sorbitol were identiŽ ed pure in solution
SEP 5 i5 1 synthesized from each individual component. The spec-
n tral region between 900 and 1500 cm 2 1 corresponds to

ÎO
n
(actual 2 predicted ) 2
i5 1
SEC 5
n 2 f 21
where actual refers to the concentration of synthetic sam-
ples m easured by an established m ethod such as HPLC,
predicted refers to the concentration computed through
PLS and PCR using spectral data, n is the number of
samples in the calibration set, and f is the number of
factors. The developed calibration m odels were Ž rst
cross-validated in all cases to minimize the risk of over-
Ž tting and then used for testing or validation with the
new data.
the absorption regions of the major components exam-
ined. The range 900 –750 cm 2 1 corresponds to the ano-
meric region that is characteristic of the saccharides. 20
The bands in the region 904 –1153 cm 2 1 are assigned to
C–O and C–C stretching modes while those in the 1474 –
1199 cm 2 1 region are due to O–C–H, C–C–H, and C–O–
H bending vibrational modes. 21
S p ectr a l R egion S electio n . W h en latent variable
methods such as PLS and PCR are employed to build
calibration models, the absorbing regions of the spectra
dictate the number of variables used as input in the mul-
tivariate statistical m odel. The spectral range should in-
clude inform ation on the concentration variation of the
analyte monitored and other m atrix constituents while ex-

F IG . 4. FT-IR spectra of fresh apple juice extracted from selected apple varieties.

1602 Volume 57, Number 12, 2003

TABLE III. Predicted concentrations of sugars and organic acids in synthetic samples by PCR. a
Sucrose Glucose Fructose Sorbitol M alic acid Citric acid
SN Ac Pre Ac Pre Ac Pre Ac Pre Ac Pre Ac Pre
1 1.50 1.53 1.00 1.01 0.50 0.50 0.200 0.201 0.200 0.201 0.200 0.200
2 1.55 1.56 1.02 1.03 0.15 0.15 0.205 0.206 0.202 0.203 0.205 0.204
3 1.60 1.59 1.04 1.05 0.20 0.22 0.210 0.209 0.205 0.206 0.210 0.209
4 1.65 1.66 1.06 1.06 0.25 0.26 0.215 0.217 0.210 0.211 0.215 0.214
5 1.70 1.69 1.08 1.09 0.30 0.32 0.220 0.219 0.212 0.214 0.220 0.219
6 1.75 1.75 1.10 1.12 0.35 0.34 0.225 0.226 0.214 0.215 0.225 0.222
7 1.80 1.82 1.12 1.13 0.40 0.41 0.250 0.252 0.216 0.216 0.250 0.255
8 1.85 1.86 1.14 1.15 0.50 0.50 0.260 0.262 0.220 0.221 0.255 0.254
9 1.90 1.92 1.16 1.16 0.60 0.59 0.265 0.266 0.222 0.223 0.260 0.263
10 1.95 1.96 1.20 1.22 0.70 0.70 0.270 0.272 0.230 0.228 0.270 0.272
11 2.00 2.01 1.24 1.27 0.80 0.77 0.275 0.276 0.235 0.237 0.275 0.276
12 2.05 2.06 1.29 1.28 0.90 0.88 0.280 0.281 0.245 0.247 0.280 0.282
13 2.10 2.13 1.32 1.34 1.00 1.01 0.285 0.284 0.260 0.255 0.288 0.281
14 2.15 2.15 1.36 1.35 1.10 1.13 0.290 0.293 0.265 0.264 0.290 0.293
15 2.20 2.22 1.40 1.43 1.20 1.21 0.295 0.294 0.275 0.270 0.294 0.293
16 2.50 2.57 1.45 1.46 1.30 1.32 0.300 0.305 0.280 0.281 0.300 0.301
17 2.60 2.64 1.48 1.46 1.40 1.42 0.303 0.302 0.285 0.286 0.302 0.303
18 2.65 2.66 1.50 1.50 1.50 1.52 0.305 0.304 0.290 0.294 0.307 0.308
19 2.70 2.69 1.55 1.56 1.60 1.65 0.308 0.307 0.300 0.301 0.308 0.307
20 2.75 2.77 1.60 1.61 1.70 1.73 0.310 0.313 0.302 0.303 0.309 0.308
21 2.80 2.83 1.65 1.65 1.80 1.82 0.312 0.311 0.303 0.304 0.310 0.310
22 2.85 2.86 1.70 1.71 1.90 1.93 0.315 0.316 0.306 0.307 0.317 0.319
23 2.90 2.93 1.74 1.72 1.95 1.94 0.318 0.319 0.310 0.311 0.318 0.316
24 2.95 2.95 1.80 1.81 2.00 2.03 0.320 0.322 0.315 0.316 0.322 0.321
25 3.00 3.02 1.84 1.85 2.05 2.03 0.325 0.326 0.320 0.321 0.330 0.332
26 3.05 3.06 1.88 1.89 2.10 2.12 0.330 0.332 0.325 0.327 0.332 0.333
27 3.10 3.12 1.90 1.92 2.15 2.15 0.335 0.337 0.330 0.329 0.340 0.339
28 3.15 3.15 1.95 1.96 2.20 2.23 0.340 0.341 0.335 0.337 0.345 0.342
29 3.20 3.21 2.00 2.02 2.30 2.33 0.350 0.353 0.340 0.338 0.350 0.351
30 3.35 3.27 2.05 2.04 2.35 2.34 0.360 0.361 0.350 0.354 0.360 0.364
31 3.30 3.35 2.10 2.12 2.40 2.42 0.370 0.372 0.355 0.353 0.370 0.373
32 3.35 3.36 2.15 2.16 2.50 2.52 0.380 0.384 0.360 0.361 0.380 0.381
33 3.40 3.42 2.18 2.19 2.60 2.62 0.390 0.393 0.370 0.372 0.390 0.394
34 3.50 3.53 2.20 2.22 2.70 2.71 0.400 0.402 0.390 0.395 0.400 0.402
35 3.60 3.64 2.25 2.26 2.75 2.73 0.415 0.416 0.400 0.401 0.415 0.414
36 3.70 3.75 2.30 2.31 2.80 2.81 0.420 0.422 0.410 0.414 0.430 0.433
37 3.80 3.82 2.35 2.36 2.85 2.86 0.440 0.443 0.420 0.421 0.450 0.455
38 3.90 3.94 2.38 2.38 2.90 2.93 0.450 0.451 0.440 0.444 0.470 0.474
39 4.00 4.02 2.40 2.41 3.00 3.00 0.460 0.462 0.455 0.453 0.500 0.502
a
Ac 5 Actual values, Pre 5 Predicted.

cluding regions dominated by noise or other artifacts that
might be incorporated into the m odel. Proper choice of
the region can also minimize the problem of nonlinearity
in the data. Suitable spectral regions for the constituents
of interest can be identiŽ ed by computing the correlation
spectra at ever y wavelength corresponding to the con-
centration of sugars and organic acids. Regions that show
a high positive or negative correlation are selected, while
regions with low or no correlation could be ignored. For
this application, the spectra in the region between 950
and 1500 cm 2 1, most likely due to sugars and organic
acids, had the highest correlation and hence was selected
for analysis.
Q uantitative Analysis of Sugars and Organic Acids.
Multivariate statistics are useful in spectral analysis be-
cause it facilitates the simultaneous inclusion of multiple
spectral intensities, which greatly improves the precision
and predictive ability of the analysis. In this work, PLS

TABLE IV . Comparison of PCR analysis of spectra with HPLC.

Sucrose Glucose Fructose Sorbitol Citric Malic
Name IR HPLC IR HPLC IR HPLC IR HPLC IR HPLC IR HPLC
Meier’s 2.42 2.45 1.30 1.33 1.00 1.02 0.203 0.203 0.311 0.31 0.21 0.215
Tropicana 2.38 2.41 1.21 1.25 1.20 1.29 0.237 0.234 0.321 0.32 0.21 0.216
Mohan M. 2.21 2.24 1.30 1.33 1.31 1.21 0.205 0.207 0.322 0.327 0.20 0.203
Lichica 2.33 2.36 1.50 1.52 1.22 1.23 0.209 0.210 0.420 0.421 0.29 0.296
Chinar 2.49 2.51 1.40 1.39 1.55 1.60 0.216 0.220 0.254 0.259 0.21 0.215
Delicious 2.23 2.23 1.34 1.33 1.25 1.25 0.202 0.204 0.254 0.254 0.22 0.223
Golden D. 2.34 2.34 1.40 1.42 1.29 1.29 0.264 0.265 0.255 0.256 0.250 0.255
Baldwin 2.36 2.36 1.29 1.28 1.23 1.23 0.288 0.287 0.254 0.255 0.23 0.232
Jonathan 2.23 2.25 1.55 1.57 1.29 1.29 0.211 0.212 0.265 0.266 0.21 0.216
McIntosh 2.00 2.05 1.66 1.66 1.30 1.30 0.241 0.241 0.231 0.234 0.23 0.232
Rome Beauty 2.09 2.08 1.02 1.02 1.45 1.45 0.251 0.254 0.231 0.233 0.236 0.239

APPLIED SPECTROSCOPY 1603

TABLE V. Comparison of PLS analysis of spectra with HPLC.
Sucrose Glucose Fructose Sorbitol Citric Malic
Nam e IR HPLC IR HPLC IR HPLC IR HPLC IR HPLC IR HPLC
Meier’s 2.40 2.40 1.30 1.32 1.02 1.02 0.205 0.207 0.312 0.314 0.212 0.215
Tropicana 2.40 2.45 1.20 1.25 1.23 1.20 0.237 0.239 0.322 0.324 0.213 0.216
Mohan Making 2.21 2.20 1.30 1.34 1.32 1.33 0.205 0.208 0.324 0.329 0.205 0.207
Lichica 2.33 2.34 1.56 1.52 1.24 1.22 0.209 0.211 0.423 0.422 0.290 0.296
Chinar 2.47 2.46 1.40 1.41 1.53 1.52 0.219 0.220 0.256 0.257 0.215 0.218
Delicious 2.34 2.37 1.33 1.34 1.24 1.24 0.202 0.207 0.252 0.253 0.220 0.222
Golden Delicious 2.33 2.34 1.45 1.48 1.23 1.24 0.264 0.266 0.256 0.259 0.251 0.252
Baldwin 2.36 2.36 1.30 1.33 1.23 1.23 0.284 0.284 0.258 0.257 0.238 0.239
Jonathan 2.23 2.27 1.52 1.53 1.29 1.29 0.219 0.220 0.264 0.266 0.215 0.214
McIntosh 2.01 2.05 1.67 1.66 1.30 1.30 0.245 0.248 0.231 0.233 0.231 0.234
Rome Beauty 2.08 2.09 1.02 1.023 1.46 1.46 0.251 0.254 0.232 0.235 0.232 0.233

and PCR m ethods have been employed to develop mul-
tivariate calibration m odels from the thirty-nine prepared
samples for predicting sugars and organic acid contents.
Figure 2 depicts the family of spectra of the synthetic
mixtures used for calibration and provided in Table II. In
order to m odel the system without over-Ž tting the con-
centration data, a cross-validation m ethod, based on leav-
ing out one sample at a time, was used. The (R 2) corre-
lation and the number of factors used in the respective
calibration models were 0.998 and 9 for PCR and 0.898
and 8 for PLS (Table I). The standard error of calibration
and the standard error of predication were also calculated
and are presented in Table I. The root mean square de-
viation (RM SD), an indicator of the average error in the
analysis, and the square of the correlation coefŽ cient (R 2 ),
an indicator of the degree of Ž t by PCR and PLS for the
individual constituents, are presented in Table II. The
RM SD and R 2 values for all of the constituents studied
were consistent and show excellent predictive ability by
both the m odels. The predicted concentrations by one of
the methods, PCR, along with actual values are listed in
Table III for reference.
The FT-IR procedure and the calibration models de-
veloped were tested on processed apple juice from Ž ve
different commercial brands (Meier’s, Tropicana, Mohan
Makings, Lichica, and Chinar) and fresh juice from six
different varieties of apple (Delicious, Golden Delicious,
Baldwin, Jonathan, McIntosh, and Rome Beauty) for the
different sugars and acids. The ATR spectra of processed
apple juice from different brands and fresh apple juice
from different apple varieties in the range between 950
and 1500 cm 2 1 are shown in Figs. 3 and 4, respectively.
The concentrations of sucrose, fructose, glucose, sorbitol,
citric acid, and m alic acid in the processed and extracted
fresh apple juice predicted by PCR and PLS methods
compare favorably well with data from the HPLC meth-
od, as presented in Tables IV and V, respectively. Both
the PLS and PCR calibrated m odels were consistent in
the prediction of the m ultiple analytes in the real samples,
demonstrating the potential of the developed m ethod as
a routine quality control tool in industries.
CONCLUSION
The infrared technique appears to be a suitable m ethod
for accurate and rapid determination of sugar and organic
components in fresh and processed apple juice. For im-
proved prediction, calibration needs to be perform ed on
real samples rather than synthetic m ixtures so that the
contribution of all the components in apple juice can be
accounted for to improve prediction. The approach also
demonstrates that the selected constituents in apple juice
can be determined without any physical or chemical sep-
aration. The results are highly promising and have the
potential to be developed into a routine procedure for the
determination of multiple constituents without sample
preparation in quality control. The suitability of this pro-
cedure for on-line analysis is being explored using a
 ow-through ATR accessory.
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1604 Volume 57, Number 12, 2003