high - resolution nuclear magnetic resonance 
 spectroscopy ( nmr ) has 
 become one of the most advantageous techniques for food quality control 
 and authenticity assessment because of many benefits that other techniques 
 do not provide . nmr spectroscopy is nondestructive , 
 selective , and capable of simultaneous detection of a large number 
 of compounds in complex mixtures without separation . this technique not only enables the quantitative determination 
 of principal compounds but also chemometric classification : each spectrum 
 can be treated as a fingerprint . moreover , nmr spectroscopy is the 
 best nontargeted method to use for food control without a priori knowledge 
 about the composition , the origin , or the type of contamination of 
 the samples . previously , nmr and 
 chemometrics were applied in beverage analysis 
 to discriminate fruit juices , beer , or wine , but there is a sparsity of literature on the topic of nmr analysis 
 applied to colas or soft drinks in general . wilson et al . reported how nmr analysis was used in order 
 to identify an unknown soft drink sample . another nmr study was about 
 the nontargeted detection of pesticide residues in soft drinks . nmr spectroscopy was introduced by lachenmeier 
 et al . for the quality control and authenticity assessment of beer 
 in official food control . in the 
 practice of quality control of alcohol - free beverages , we 
 are often confronted with cola samples from questionable origin , which 
 are labeled or sold from tap with premium brand names and are potentially 
 counterfeited . furthermore , we have to control the maximum limits 
 of food additives according to european union law . a large number 
 of methods were published about the simultaneous separation and quantification 
 of sweeteners , additives , and preservatives in soft drinks using reverse - phase 
 high performance liquid chromatography ( hplc ) , uv spectrometry , mass spectrometry 
 ( ms ) , or capillary electrophoresis ( ce ) , but to our knowledge , no study in the literature applied nmr spectroscopy 
 to simultaneously quantify caffeine , artificial sweeteners , preservatives , 
 and colors in the soft drinks . this paper describes the investigation 
 of a combined nmr and chemometric 
 data analysis approach to distinguish different brands of cola drinks . we also explored the potential of high resolution h nmr 
 to allow the identification and the quantification of selected components . the samples were randomly selected 
 from supermarkets in karlsruhe , germany , and in strasbourg , france . acesulfame - k , sodium benzoate , caffeine , vanillin , sodium cyclamate , 
 aspartame , sodium azide , h3po4 , and hcl were 
 purchased from sigma aldrich ( steinheim , germany ) , hydroxymethylfurfural 
 ( hmf ) from acros organics ( geel , belgium ) , koh and kh2po4 from merck ( darmstadt , germany ) , and sulfite ammonia caramel 
 ( e 150d ) from couleur und karamel gmbh ( offenbach , germany ) . the nmr buffer was prepared by dissolving 10.21 g of kh2po4 and 9.75 mg of sodium azide in 50 ml of pure water 
 and then by adjusting the ph to 4.0 with h3po4 or koh . sodium azide was used for the preservation of the samples 
 because this addition was proved to be advantageous , so that even 
 longer storage of samples at room temperature in the autosampler did 
 not lead to significant changes in the spectrum . the soft drinks were 
 degassed by ultrasonication for 10 min , and 800 l of the degassed 
 solution was combined with 100 l of an internal standard ( d2o containing 0.1% of tsp ( sodium salt of 3-(trimethylsilyl)propionic 
 acid - d4 ) . the ph of the mixture was then 
 adjusted to 4.50 by adding 101 l of a buffer ( ph 4.0 ) and 12 
 l of hcl ( 1 m ) or naoh ( 1 m ) using a titration unit btph ( bruker 
 biospin , rheinstetten , germany ) which enabled the very precise adjustment 
 of the ph of all the samples and standard solutions . a 600 l 
 amount of the final solution was poured into an nmr tube for direct 
 measurement . stock solutions with concentrations of 1000 mg / l 
 ( caffeine , aspartame , acesulfame - k , cyclamate , and benzoate ) , of 5 
 g / l ( e 150d ) , of 500 mg / l ( vanillin ) , and of 10 g / l ( hmf ) were prepared 
 in deionized water from the pure substances . by diluting the stock 
 solutions in pure water , 1520 calibration standards were prepared 
 so that the calibration curves were accomplished across the concentration 
 ranges encountered in the soft drinks . the calibration standards were 
 then prepared by the same procedure as that used for the samples ( i.e. , 
 internal standard and buffer additions as well as ph adjustment ) . therefore , 
 the ph was buffered in all samples , so that the differences correspond 
 to true differences in composition of the samples and not to ph effects . the automatic titration unit we have used allows the addition of very 
 small quantities of acid or base ( 1 to 3 l ) in a short time 
 ( 34 min ) . this ensures that the variations of composition 
 caused by dilution are small for the multivariate analysis and that 
 the titration is not time - consuming . the ph for the adjustment was 
 set to 4.5 because this is near the ph of the samples after buffer 
 addition ( about 4.0 ) , and therefore no large dilution has to occur . 
 for quantitative analysis , all nmr measurements 
 were performed on a bruker avance 400 ultrashield spectrometer ( bruker 
 biospin , rheinstetten , germany ) equipped with a 5-mm sei probe with 
 z - gradient coils , using a bruker automatic sample changer ( b - acs 120 ) . h nmr spectra were acquired at 300.0 k without sample rotation , 
 and 128 scans and 4 prior dummy scans of 65k points were acquired 
 with a spectral width of 20.0234 ppm , a receiver gain of 28.5 , an 
 acquisition time of 10 s , and a recycle delay of 4 s. water suppression 
 was achieved using the noesy - presaturation pulse sequence ( bruker 
 1d noesygppr1d pulse sequence ) with irradiation at the water frequency 
 during the recycle and mixing time delays . the data were acquired 
 automatically under the control of icon - nmr ( bruker biospin , rheinstetten , 
 germany ) , requiring about 31 min per sample . all nmr spectra were 
 phased , baseline - corrected , and calibrated by the tsp signal at 0.0 
 ppm . the spectral data in the region from 0 
 to 8 ppm were exported to unscrambler x version 10.0.1 ( camo software 
 as , oslo , norway ) . a bucket table was obtained from the complete set 
 of spectra and analyzed by pca . details on the bucketing process of 
 nmr spectra for multivariate data analysis were previously described . we tested several spectral regions for 
 calculation : aliphatic ( 03 ppm ) , midfield ( 36 ppm ) , 
 and aromatic ( 610 ppm ) with 0.01 ppm bucket width . the technique 
 of cross - validation was applied to determine the optimal number of 
 principal components ( pcs ) needed to have robust models : this technique 
 excludes some of the samples , models the remaining samples , and tests 
 the models on the left - out samples , so the significant number of components 
 and the expected prediction error can be estimated . then 
 the data were plotted in a coordinate system defined by two pcs in 
 order to detect the key relationships in the data . separated 
 peaks ( i.e. , peaks not overlapped or interfered with by matrix ) corresponding 
 to each substance were identified and integrated . it was found that 
 in all cases a linear relationship exists between the concentration 
 of the analyte and the ratio between the peak area of the analyte 
 and the internal standard ( tsp ) . , cola samples from germany 
 and france and standard solutions were analyzed several times daily 
 ( intraday , n = 5 ) and over several days ( interday , n = 3 ) . the limit of detection ( lod ) and the limit of quantification 
 ( loq ) were calculated according to the german institute for standardization 
 standard din 32645 using a separate calibration 
 curve ( table 1 ) . the recovery rate was ascertained 
 by adding standard solution at two different concentrations to a real 
 sample . for all calculations , statistical significance was assumed 
 at below the 0.01 probability level . two signals were used depending of 
 the amount of citric acid : for the light sample , the doublet between 
 2.73 and 2.76 ppm was integrated , and for zero samples , the multiplet 
 between 3.03 and 3.11 ppm was integrated . concentrations used for validation 
 are given in parentheses ( expressed in mg / l ) . the nmr spectra of 
 the soft drinks could be classified into two major groups : sugar - containing 
 beverages and sugar - free drinks . figure 1 shows 
 the spectra of normal sugared samples as well as two sugar - free samples 
 designated as  light  and  zero  cola 
 from the same brand . the spectra of sugar - free and normal soft drinks 
 with sugar could be easily differentiated even without pca because 
 of the large peaks of sucrose , glucose , and fructose between 6 and 
 3 ppm . therefore , a separate pca was made for cola drinks with sugar 
 and for sugar - free samples ( so - called light and zero colas ) . cola 
 samples with high quantities of caffeine ( so - called  strong  
 colas ) , caffeine - free , or vanillin - containing drinks were removed 
 from the models as outliers . the type of these special samples could 
 be easily identified by the targeted analysis of the spectra in the 
 aromatic region . figure 2 presents the whole h nmr spectra of a premium - brand cola drink and of a discount - brand 
 cola drink . the differences between the spectra of sugared 
 premium - brand cola and discount - brand cola drinks could not be differentiated 
 by eye because of this high spectral complexity . thus , a chemometric 
 approach , such as pca , was needed to interpret the nmr signals and 
 to uncover hidden properties of the samples such as their type . in 
 our case , the best pca model with regard to classification ability was obtained in the 86 ppm region for the cola samples containing 
 sugar and in the 30 ppm region for the sugar - free samples . figures 3 and 4 show 
 the results of the pca : we could not only differentiate the premium 
 cola brands from the discount brands but clusters from each brand 
 were also clearly separated from each other . the pca scores of the 
 discount brand 6 and 7 were mixed in the same cluster because the 
 beverages of these brands were probably produced in the same factory 
 ( some contract manufacturers conduct commissioned bottling for several 
 discount brands according to our observations ) . h nmr spectra 
 of a normal sugared sample ( a ) compared 
 to two sugar - free samples designated as light ( b ) and zero ( c ) . h nmr spectra of a premium brand cola ( a ) 
 compared 
 to a discount brand cola ( b ) . scatter plot of the pca scores of sugar -containing colas 
 in the 
 aromatic region ( 8.06.0 ppm ) . scatter plot of the pca scores of sugar - free colas in 
 the aliphatic 
 region ( 3.00.25 ppm ) . for the sugar - free samples , the pca scores of brand 
 1 light and 
 brand 1 light without caffeine were represented in the same group 
 in the aliphatic region but the two types could be differentiated 
 by analysis of the aromatic region or by the quantitative analysis . 
 therefore , h nmr combined with chemometrics can be used 
 as a method to discriminate the premium - brand cola samples from the 
 discount - brand cola samples . we also tried to separate the cola drinks 
 by pca according to their origin ( france or germany ) . while the french 
 discount brand 3 was clearly distinguished from the german discount 
 brands , as well as the premium brands , the separation was not as clear 
 inside brand 1 ( a brand that is filled at factories in both countries ) . however , the light and zero types showed a clear difference between 
 countries . in france and germany , different levels of aspartame and 
 acesulfame - k are used , and especially sodium cyclamate is different , 
 which is completely absent in the samples from france . in an 
 investigation by wilson et al . , nmr spectroscopy 
 was used for the identification of unknown samples 
 of soft drink . h nmr spectra of samples of purified caffeine , 
 purified sugar , purified citric acid , and soft drinks from popular 
 brands were collected and compared to the spectrum of the unknown 
 sample for identification . our study confirms the suitability of the 
 approach for a larger sample of commercial beverages . previously , chemometrics combined with other techniques such as a 
 sensor device ( so - called electronic tongue ) were also used for discrimination 
 of cola brands . discrimination of soft 
 drinks was also performed using a colorimetric sensor array and pca . in comparison with these sensors , nmr spectroscopy 
 provides highly reproducible spectra , allows the identification of 
 marker compounds , and presents a high level of chemical discrimination , 
 which enables differentiation between very similar types of samples . moreover , this technique also permits the simultaneous acquisition 
 of structural and quantitative information about a large number of 
 targeted substances in the soft drinks . the nmr range used for quantification 
 and the linearity ranges the high correlation coefficients ( r > 0.99 ) 
 obtained 
 for each calibration graph indicate a good linearity response within 
 the concentration range studied for each compound . no europe - wide legislation exists about maximum limits of caffeine , 
 but the presence of caffeine has to be clearly labeled in drinks containing 
 more than 150 mg / l , according to the european directive 2002/67/ec . figure 5 shows the signal of caffeine in standard 
 solutions and a cola sample . precisions with standards reached 4.2% 
 ( intraday ) and 4.0% ( interday ) and with real samples 1.6% ( intraday ) 
 and 1.5% ( interday ) . we checked that the content of caffeine above 150 mg / l was labeled 
 and that labeling was correct in all instances . in a previous study , 
 a fourier transform infrared ( ftir ) spectroscopic method was developed 
 for quantitative estimation using partial least - squares ( pls ) regression . in contrast to this indirect chemometric ftir 
 approach , caffeine can be more straightforwardly quantified by nmr 
 spectroscopy with a good precision using calibration curves with tsp 
 as internal standard . caffeine concentration was also determined using 
 a microextraction of caffeine on the surface of a fused - silica fiber 
 and gas chromatography with mass spectrometric detection ( gc / ms ) . the practical lod was found at about 2 mg / l , 
 which is close to our value obtained with nmr . another paper described 
 the quantitative determination of caffeine in sugar - free soft drinks 
 on reversed - phase c8 thin - layer chromatography plates using a surface 
 sampling electrospray ionization system with tandem mass spectrometry 
 detection . / electrospray tandem mass spectrometry methods were 
 0.20 ng injected ( 0.50 l ) and 1.0 ng spotted on the plate , 
 respectively . such low limits of detection of caffeine are not possible 
 with nmr spectroscopy but are also not required for the soft drinks , 
 which contain in general around 100 mg / l of caffeine . 400 mhz h 
 nmr spectra of caffeine in standard solutions 
 and the cola sample . in cola drinks , the european directive 94/35/ec 
 sets the maximal 
 concentration of aspartame at 600 mg / l , and we did not detect a concentration 
 above this limit . for the quantification of aspartame , two different 
 multiplets were integrated because of different amounts of citric 
 acid among the sugar - free samples . in the range of 2.76 to 2.73 ppm , 
 the peaks corresponding to aspartame and citric acid can overlap . 
 therefore , this signal was chosen for drinks containing only small 
 quantities of citric acid ( i.e. , the so - called light cola drinks ) . 
 for samples with high quantities of citric acid ( the  zero  
 cola samples ) , the lods of aspartame were 3.5 mg / l for light samples and 
 2.9 mg / ftir with attenuated total reflectance 
 sampling accessory and pls regression was previously used for the 
 rapid determination of aspartame in soft drinks . aspartame was found to average from 0.43 to 0.50 mg / l , 
 similar to that found with our method , and with prediction errors 
 ranging from 2.4% to 5.7% . in contrast with ftir , the quantification 
 of aspartame with nmr spectroscopy does not require the use of chemometrics . benzoate is limited to 150 mg / l ( calculated as free acid ) by the 
 european directive 95/2/ec , and the concentration of benzoate in the 
 soft drinks analyzed did not exceed this limit . according to previous investigations , 
 the analysis of carbonated 
 beverages by capillary electrophoresis ( ce ) enabled the simultaneous 
 determination of aspartame , benzoic acid , and caffeine in 2 min with 
 minimal sample preparation . the lods 
 for caffeine , aspartame , and benzoic acid were 1.6 , 18 , and 4.0 mg / l , 
 respectively . another study compared uv spectrophotometry , liquid 
 chromatography ( lc ) , and ce for the quantification of caffeine , aspartame , 
 and benzoic acid in soft drinks . the 
 lods found were 1.2 , 0.57 , 1.4 mg / l ( lc ) and 1.7 , 0.29 , 2.2 mg / l ( ce ) 
 for caffeine , aspartame , and benzoic acid , respectively . the nmr analysis 
 takes more time ( 31 min ) but allows the simultaneous quantification 
 of seven compounds in one single spectrum with limits of detection 
 similar to that in lc or ce . according to european directive 
 94/35/ec , the concentration of 
 acesulfame - k in soft drinks must not exceed 350 mg / l and the concentration 
 in the cola drinks determined by nmr spectroscopy ranged between 40 
 and 165 mg / l . the lod and loq obtained were 0.75 and 2.5 mg / l , respectively . cyclamate is the sodium or calcium salt of cyclamic acid ( cyclohexanesulfamic 
 acid ) , and its limit was recently lowered from 400 to 250 mg / l ( calculated 
 as free acid ) . cyclamate 
 was not detected in cola drinks from france and was found around the 
 maximum limit in the soft drinks from germany . cyclamate can not be 
 determined with hplc or ce procedures using direct uv detection , as 
 it does not absorb uv light at about 220 nm . however , the simultaneous determination of cyclamate , aspartame , 
 and acesulfame - k was possible in 6 min by ce with capacitively coupled 
 contactless conductivity detection . the 
 relative standard deviation varied in the range of 1.5 6.5% , 
 and the recoveries were between 94% and 108% . in comparison , another study described the 
 separation and simultaneous determination of aspartame , sodium cyclamate , 
 and acesulfame - k by ion chromatography with a suppressed conductivity 
 detector . the lods found were at 0.87 , 
 0.032 , and 0.019 mg / l , respectively . rates of recovery were between 
 98% and 105% , 99% and 104% , and 98% and 103% , respectively . nmr spectroscopy 
 can not give such low lods , which are also not necessary to control 
 the limits . for the sulfite ammonia caramel ( e150d ) , no maximum 
 limit is established 
 by eu law , although usage should be no higher than necessary to achieve 
 the desired purpose . according to the codex alimentarius standard 
 for food additives , the maximum level 
 of sulfite ammonia caramel in water - based flavored drinks is 50  000 
 mg / kg , and we found concentrations of caramel color of about 2000 
 mg / a simultaneous quantification of the class iv caramel , caffeine , 
 aspartame , acesulfame - k , and sodium benzoate in soft drinks was performed 
 by ce in 8 min . for all the food additives , 
 a lod of 5 mg / l was established and a loq of 10 mg / l was obtained . nmr spectroscopy shows similar limits ( except for the caramel color ) 
 and enables the identification and quantification of other compounds 
 such as cyclamate , vanillin , and hmf . for vanillin and hmf , the precision and the recovery are not as 
 satisfactory as for the others compounds because of the small quantities 
 close to the limit of quantification , which are present in the samples . mg / l , respectively , 
 and the loqs were 2.4 and 2.1 mg / l , respectively . no eu limit is established 
 for the concentration of vanillin and of hmf in soft drinks . a method 
 of quantification of hmf by hplc in sugar - containing foods including 
 soft drinks was developed , and the results of hmf varied around 03 
 mg this investigation has shown that h nmr spectra of 
 cola drinks can provide qualitative information about the compositional 
 properties such as the brand and also quantitative information about 
 a number of major components . therefore , nmr spectroscopy combined 
 with pca is a suitable tool for soft drink authentication . moreover we 
 observed that this technique can replace other traditional methods 
 with lower discrimination ability and similar limits of detection 
 and which often focus on the analysis of specific components of the 
 sample . nmr is not yet a common analytical tool used in quality control ; 
 however , the application range has been increasing during the past 
 decade . advantages such as the high level of chemical discrimination , 
 the minimal preparation of the sample , and the diversity of information 
 make the technique interesting and in the future the usage of nmr 
 spectroscopy will certainly increase in routine quality control .
1h nuclear magnetic resonance ( nmr ) spectroscopy 
 ( 400 
 mhz ) was used in the context of food surveillance to develop a reliable 
 analytical tool to differentiate brands of cola beverages and to quantify 
 selected constituents of the soft drinks . 
 the preparation of the samples 
 required only degassing and addition of 0.1% of tsp in d2o for locking and referencing followed by adjustment of ph to 4.5 . 
 the nmr spectra obtained can be considered as  fingerprints  
 and were analyzed by principal component analysis ( pca ) . 
 clusters 
 from colas of the same brand were observed , and significant differences 
 between premium and discount brands were found . 
 the quantification 
 of caffeine , acesulfame - k , aspartame , cyclamate , benzoate , hydroxymethylfurfural 
 ( hmf ) , sulfite ammonia caramel ( e 150d ) , and vanillin was simultaneously 
 possible using external calibration curves and applying tsp as internal 
 standard . 
 limits of detection for caffeine , aspartame , acesulfame - k , 
 and benzoate were 1.7 , 3.5 , 0.8 , and 1.0 mg / l , respectively . hence , 
 nmr spectroscopy combined with chemometrics is an efficient tool for 
 simultaneous identification of soft drinks and quantification of selected 
 constituents .